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The “Bipartite” Structure of the First Genome of Ampelomyces quisqualis, a Common Hyperparasite and Biocontrol Agent of Powdery Mildews, May Point to Its Evolutionary Origin from Plant Pathogenic Fungi
Powdery mildews are among the most important plant pathogens worldwide, which are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. The taxonomy of the genus Ampelomyces is unresolved, but well-supported molecular operational taxonomic units were repeatedly defined suggesting that the genus may include at least four to seven species. Some Ampelomyces strains were commercialized as biocontrol agents of crop pathogenic powdery mildews. However, the genomic mechanisms underlying their mycoparasitism are still poorly understood. To date, the draft genome of a single Ampelomyces strain, designated as HMLAC 05119, has been released. We report a high-quality, annotated hybrid draft genome assembly of A. quisqualis strain BRIP 72107, which, based on phylogenetic analyses, is not conspecific with HMLAC 05119. The constructed genome is 40.38 Mb in size, consisting of 24 scaffolds with an N50 of 2.99 Mb and 96.2% completeness. Our analyses revealed "bipartite" structure of Ampelomyces genomes, where GC-balanced genomic regions are interspersed by longer or shorter stretches of AT-rich regions. This is also a hallmark of many plant pathogenic fungi and provides further evidence for evolutionary affinity of Ampelomyces species to plant pathogenic fungi. The high-quality genome and annotation produced here provide an important resource for future genomic studies of mycoparasitisim to decipher molecular mechanisms underlying biocontrol processes and natural tritrophic interactions.
# Introduction
Powdery mildews (Erysiphaceae), common obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. As powdery mildews are themselves parasites, Ampelomyces spp. are also considered as hyperparasites [bib_ref] The role of hyperparasites in host plant -parasitic fungi relationships, Kiss [/bib_ref] [bib_ref] The role of hyperparasitism in microbial pathogen ecology and evolution, Parratt [/bib_ref]. The natural tritrophic interactions between host plants, powdery mildews, and Ampelomyces Significance Mycoparasitic fungi belonging to the genus Ampelomyces attack powdery mildews, which are important plant pathogenic fungi worldwide. Despite their ecological significance, and importance as biocontrol agents of crop pathogenic powdery mildews, limited genetic resources are available for Ampelomyces. The first high-quality assembly and annotation of the A. quisqualis genome produced here will provide an invaluable resource for genomic studies of mycoparasitism and points to possible evolutionary origin of Ampelomyces from plant pathogenic fungi.
spp. have been intensively studied in the field from an ecological context [bib_ref] Intracellular mycoparasites in action: interactions between powdery mildew fungi and Ampelomyces, Kiss [/bib_ref] [bib_ref] Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic..., Kiss [/bib_ref] [bib_ref] A hyperparasite affects the population dynamics of a wild plant pathogen, Tollenaere [/bib_ref] [bib_ref] Host phenology and geography as drivers of differentiation in generalist fungal mycoparasites, Pintye [/bib_ref] [bib_ref] Variation and correlations between sexual, asexual and natural enemy resistance life-history traits..., Numminen [/bib_ref]. Some Ampelomyces strains have been commercialized as biocontrol agents of crop pathogenic powdery mildews [bib_ref] Interactions between fungi and other microbes, Boddy [/bib_ref] [bib_ref] Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are..., Legler [/bib_ref]. The taxonomy of the genus Ampelomyces is unresolved, but well-supported molecular operational taxonomic units (MOTUs) were defined suggesting that the genus may include at least four to seven species [bib_ref] Genetic diversity of Ampelomyces mycoparasites isolated from different powdery mildew species in..., Liang [/bib_ref] [bib_ref] Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic..., Park [/bib_ref] [bib_ref] Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic..., Kiss [/bib_ref] [bib_ref] Existence of different physiological forms within genetically diverse strains of Ampelomyces quisqualis, Angeli [/bib_ref] [bib_ref] No indication of strict host associations in a widespread mycoparasite: grapevine powdery..., Pintye [/bib_ref] [bib_ref] Green fluorescent protein transformation sheds more light on a widespread mycoparasitic interaction, N Emeth [/bib_ref] [bib_ref] Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: their phylogeny..., N Emeth [/bib_ref]. All strains belonging to the genus Ampelomyces are hyperparasites of powdery mildews just like the type species A. quisqualis. The name A. quisqualis has been applied to phylogenetically diverse Ampelomyces hyperparasites belonging to different MOTUs [bib_ref] Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic..., Park [/bib_ref] [bib_ref] Existence of different physiological forms within genetically diverse strains of Ampelomyces quisqualis, Angeli [/bib_ref] , which highlights the need for a taxonomic reassessment of this binomial [bib_ref] Biology and biocontrol potential of Ampelomyces mycoparasites, natural antagonists of powdery mildew..., Kiss [/bib_ref] [bib_ref] Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are..., Legler [/bib_ref]. Experiments with Ampelomyces transformants exhibiting the green fluorescent protein (GFP) indicated that these hyperparasites cannot thrive as saprobes; living powdery mildew colonies constitute their primary niche .
The GFP study has established a framework for a molecular genetic toolbox for Ampelomyces strains , which was later applied in a gene knock-out project [bib_ref] Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: their phylogeny..., N Emeth [/bib_ref]. In terms of genomic resources, these hyperparasites are largely unexplored: to date, the draft genome of a single Ampelomyces strain, designated as HMLAC 05119, has been released [bib_ref] 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of..., Haridas [/bib_ref]. The strain was isolated from an undetermined powdery mildew infecting Youngia japonica in China . The transcriptome of another Ampelomyces strain, CNCM I-807 or M-10, which was commercialized as the active ingredient of the AQ10 Biofungicide product in the United States and the European Union [bib_ref] Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are..., Legler [/bib_ref] , is the only other genomic resource available to date for the genus Ampelomyces. The AQ10 strain was isolated from an undetermined powdery mildew infecting Catha edulis in Israel [bib_ref] Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are..., Legler [/bib_ref]. The analysis of its transcriptome during the early and the late stages of parasitism revealed the upregulation of some genes related to toxin biosynthesis, together with other potentially mycoparasitism-related proteins such as secreted proteases and putative virulence factors during mycoparasitism [bib_ref] Transcriptional reprogramming of the mycoparasitic fungus Ampelomyces quisqualis during the powdery mildew..., Siozios [/bib_ref]. However, approximately 50% of the Ampelomyces transcripts did not point to any known protein sequences [bib_ref] Transcriptional reprogramming of the mycoparasitic fungus Ampelomyces quisqualis during the powdery mildew..., Siozios [/bib_ref]. This may indicate that a part of the Ampelomyces proteome is unique, or this type of mycoparasitism has not been studied in sufficient detail in other interfungal parasitic relationships.
Here, we present a high-quality, annotated hybrid draft genome assembly of A. quisqualis strain BRIP 72107. Our phylogenetic analysis presented here revealed that BRIP 72107 is not conspecific with HMLAC 05119, the only Ampelomyces strain with a known genome [bib_ref] 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of..., Haridas [/bib_ref]. BRIP 72107 is, however, conspecific with the commercial AQ10 strain, and both belong to the MOTU that has included many Ampelomyces strains newly isolated from the field in diverse studies in China, Europe, Japan, the United States, and Korea [bib_ref] Genetic diversity of Ampelomyces mycoparasites isolated from different powdery mildew species in..., Liang [/bib_ref] [bib_ref] Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic..., Park [/bib_ref] [bib_ref] Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic..., Kiss [/bib_ref] [bib_ref] No indication of strict host associations in a widespread mycoparasite: grapevine powdery..., Pintye [/bib_ref] [bib_ref] Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: their phylogeny..., N Emeth [/bib_ref]. Therefore, this genome will be useful to decipher molecular mechanisms underlying both biocontrol processes and natural tritrophic interactions.
# Results and discussion
## Genome assembly and annotation
Ampelomyces quisqualis strain BRIP 72107 was assembled into 24 scaffolds with a total assembly size of 40,378,121 bp and genome completeness of 96.2% [fig_ref] Table 1: Genome Statistics for Ampelomyces Strains Sequenced to DateStrain BRIP 72107 was sequenced... [/fig_ref]. Of the total of 3,786 Dothideomycetes Benchmarking Universal Single-Copy Orthologs (BUSCOs) searched, BRIP 72107 included 3,641 complete (96.2%), 16 fragmented (0.4%), and 129 missing (3.4%) BUSCOs. Based on a genome size of 42 Mb estimated by Jellyfish, and a total of 8.8 and 8 Gb of sequence data generated by MinION and Illumina MiSeq platforms, respectively, we estimate a genome coverage of 400Â. A combination of ab initio and evidence-based gene modeling with two additional rounds of gene predictions after training SNAP in Maker pipeline resulted in 22,470 predicted exons within 10,439 genes (including 7,255 evidence-supported gene models), including 4,203 with 3'-UTRs and 4,345 with 5'-UTRs.
## Bipartite genome of a. quisqualis
The genome of BRIP 72107 has a bimodal GC content, due to the presence of gene-rich, GC-balanced regions interspersed by long or short stretches of AT-rich, gene-sparse regions [fig_ref] FIG. 1: -The first 1,400,000 bp of BRIP 72107 contig-4 is demonstrated here as... [/fig_ref]. This phenomenon was observed in almost all the assembled contigs, with AT-rich regions varying in size from 15 to 270 kb. The "bipartite" feature of the genome is also a hallmark of many plant pathogenic fungi that is hypothesized to arise when duplicated DNA, such as transposons, undergoes C to T transitions by the process of repeat-induced point mutation [bib_ref] OcculterCut: a comprehensive survey of AT-rich regions in fungal genomes, Testa [/bib_ref]. This substantial proportion of repetitive and AT-rich regions has been proposed to result in the "two-speed" evolution of these genomes, where genes located close to the AT-rich regions (mostly secreted proteins) have higher rates of evolution [bib_ref] The two-speed genomes of filamentous pathogens: waltz with plants, Dong [/bib_ref]. Recent analyses revealed a surprisingly close phylogenetic relationship between Ampelomyces mycoparasites and plant pathogens such as Parastagonospora nodorum and Leptosphaeria maculans [bib_ref] 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of..., Haridas [/bib_ref] , and the Ampelomyces genus is classified in the Leptosphaeriaceae family that includes many plant pathogens and other plant-associated species. These results, together with the bipartite genome structures of all these fungi, may point to the evolutionary origin of Ampelomyces from plant pathogenic fungi.
# Phylogenetic analysis
Phylogenetic analysis of 28 Ampelomyces strains based on the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA), including the 5.8S rRNA gene, revealed a high level of diversity within the genus Ampelomyces, with nine well-supported MOTUs [fig_ref] FIG. 1: -The first 1,400,000 bp of BRIP 72107 contig-4 is demonstrated here as... [/fig_ref] , which highlighted the need for a taxonomic reassessment of the genus Ampelomyces. Strain BRIP 72107 belonged to the strongly supported clade/MOTU 1, together with the commercial strain AQ10, whereas HMLAC 05119 was part of MOTU 4. This indicated that the two strains are not conspecific. Another strain, 94013, commercialized as Q-fect in Korea [bib_ref] Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic..., Park [/bib_ref] , also belonged to MOTU 4. Previous phylogenetic analyses of Ampelomyces strains also concluded that the genus consists of multiple species [bib_ref] Genetic diversity of Ampelomyces mycoparasites isolated from different powdery mildew species in..., Liang [/bib_ref] [bib_ref] Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic..., Park [/bib_ref] [bib_ref] Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic..., Kiss [/bib_ref] [bib_ref] Existence of different physiological forms within genetically diverse strains of Ampelomyces quisqualis, Angeli [/bib_ref] [bib_ref] No indication of strict host associations in a widespread mycoparasite: grapevine powdery..., Pintye [/bib_ref].
# Materials and methods
## Sample collection and culturing
Strain BRIP 72107 was isolated from Golovinomyces bolayi infecting Cestrum parqui collected in Toowoomba, Queensland, Australia. The strain is available from the Queensland Plant Pathology Herbarium (BRIP), which includes a large collection of living fungal and bacterial strains, in addition to herbarium specimens. The isolation process was done as described by [bib_ref] Genetic diversity of Ampelomyces mycoparasites isolated from different powdery mildew species in..., Liang [/bib_ref]. The identity of strain BRIP 72107 as Ampelomyces was confirmed via sequencing the nrDNA ITS region using universal primers ITS1-F [bib_ref] ITS primers with enhanced specificity for basidiomycetes -application to the identification of..., Gardes [/bib_ref] and ITS4 [bib_ref] Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, White [/bib_ref]. Four-week-old mycelia of BRIP 72107 grown in potato dextrose broth were lyophilized overnight. One hundred milligrams of lyophilized mycelia were flash-frozen in liquid nitrogen and ground with stainless steel beads (2.8 mm diameter; Sigma-Aldrich) in a FastPrep-24 (MP Biomedicals, Australia) at 6.5 m/s for 30 s and stored at À80 C until DNA extraction.
## Dna and rna extraction
For long-read sequencing, high-molecular weight (HMW) DNA was extracted using a chloroform/isoamyl alcohol extraction method with an isopropanol precipitation as described by [bib_ref] Purification of high molecular weight genomic DNA from powdery mildew for long-read..., Feehan [/bib_ref]. Briefly, ground mycelia were lysed in 700 ml lysis buffer (potassium metabisulfite 0.25 M, Tris 0.2 M pH 7.5, ethylenediaminetetraacetic acid 50 mM, NaCl 2 M, 2% CTAB, ddH 2 O) pre warmed to 65 C with 300 ml 5% Sarcosyl prewarmed to 65 C and incubated for 30 min at 65 C. DNA was isolated by chloroform/isoamyl alcohol extraction and subsequent isopropanol precipitation. RNase Genome completeness for the two genomes was determined based on benchmarking universal single-copy orthologs (BUSCOs) [bib_ref] BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs, Simão [/bib_ref] against the dothideomycetes_odb10 database. [bib_ref] OcculterCut: a comprehensive survey of AT-rich regions in fungal genomes, Testa [/bib_ref] to classify genome segments into (10 mg/ml) treatment was performed for 2 h at 37 C and finally the DNA was cleaned with AMPure XP beads (Beckman Coulter). HMW DNA was purified using the Qiagen Genomic-tip 20/G kit according to manufacturer's instructions and quantified using the Qubit v.3.0 fluorometer (ThermoFisher Scientific, Australia). Quality ratios were checked using the Denovix DS-11 series (Life Science Technologies) and the integrity was assessed through electrophoresis on a 0.8% agarose gel containing 1:20,000 GelRed (Biotium, Australia), then stored at À20 C. For Illumina MiSeq sequencing, DNA was extracted from lyophilized fungal mycelia using a DNeasy Plant Mini Kit (Qiagen, Australia) according to manufacturer's instructions, except for the final step where DNA was eluted in 10 mM filter-sterilized Tris-HCl (pH 8.5).
Total RNA from fresh fungal mycelia flash-frozen and ground in liquid nitrogen was extracted using an RNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions using the purification of total RNA from plant cells and tissue and filamentous fungi. The final product was checked via agarose gel electrophoresis and quantified using a Qubit v.3.0 fluorometer (ThermoFisher Scientific, Australia) and submitted to the Australian Genome Research Facility (Melbourne, Australia) for total mRNA sequencing.
## Genome sequencing
Long-read sequencing was performed using Oxford Nanopore Technology (ONT). A MinION library was constructed from 1,000 ng DNA using a Genomic DNA by Ligation kit (SQK-LSK109; ONT, Oxford, United Kingdom) according to the standard protocol. The library was loaded onto a MinION FLO-MIN 106 R9.4.1 flow cell and sequenced for 39 h. Read quality statistics were assessed using Nanoplot v.1.28.2 on Galaxy Australia Portal [bib_ref] The Galaxy platform for accessible, reproducible, and collaborative biomedical analyses: 2018 update, Afgan [/bib_ref]. For Illumina short-read sequencing, library preparation was conducted on 150 ng DNA using an Illumina DNA Prep kit and Nextera DNA CD Indexes (Illumina, Singapore) according to manufacturer's instructions. The library was sequenced on an Illumina MiSeq platform using a 600-cycle paired-end V3 reagents kit. Read quality statistics were assessed using FastQC v.0.11.8 (Andrews 2010) on Galaxy Australia Portal.
## Read preparation, genome assembly, and annotation
All raw data were screened and filtered for bacterial contamination using Kraken v.2.1.1 [bib_ref] Kraken: ultrafast metagenomic sequence classification using exact alignments, Wood [/bib_ref].
Adapter removal from Illumina reads was conducted using BBduk from the BBmap suite v.36.86 [bib_ref] The MaSuRCA genome assembler, Zimin [/bib_ref] was used. Raw Illumina reads without barcode-removal and quality filtering were used for MaSuRCA assembly as recommended by the developer. The completeness of the genome assembly was evaluated via Benchmarking Universal Single-Copy Orthologs (BUSCO) v.1.2 [bib_ref] BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs, Simão [/bib_ref]. Genome statistics of the generated assembly were compared with that of HMLAC 05119 [bib_ref] 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of..., Haridas [/bib_ref] using Quast v.2.0.5 [bib_ref] QUAST: quality assessment tool for genome assemblies, Gurevich [/bib_ref]. The program OcculterCut v.1.1 [bib_ref] OcculterCut: a comprehensive survey of AT-rich regions in fungal genomes, Testa [/bib_ref] was used to scan the genome of A. quisqualis strain BRIP 72107, Ampelomyces sp. strain HMLAC 05119 [bib_ref] 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of..., Haridas [/bib_ref] , and Leptosphaeria maculans strain v.23.1.3 [bib_ref] Effector diversification within compartments of the Leptosphaeria maculans genome affected by repeat-induced..., Rouxel [/bib_ref] to determine their percent GC content distribution.
Transcriptome assembly was conducted using Trinity v.2.10.0 [bib_ref] Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data, Grabherr [/bib_ref]. Genome annotation was conducted using Maker v.2.31.9 [bib_ref] MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes, Cantarel [/bib_ref]. A repeat library was generated with RepeatModeler v.2.0.1 and repeats were masked prior to annotation. A first round of RNA-evidenced gene prediction was conducted using Maker. The resulting annotation was used to produce a hidden Markov model (HMM) profile for A. quisqualis, which was further refined with a second round of SNAP training and used for the final annotation [bib_ref] MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes, Cantarel [/bib_ref].
## Phylogenetics analyses
To depict the molecular diversity within the genus Ampelomyces and phylogenetic relationship of strains BRIP 72107 and HMLAC 05119, a Bayesian phylogram was constructed using MrBayes v.3.2.4 [bib_ref] MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large..., Ronquist [/bib_ref] based on the GTRþIþG nucleotide substitution model selected using PAUP v.4.0b10 [bib_ref] The phylogenetic handbook: a practical approach to DNA and protein phylogeny, Swofford [/bib_ref] and MrModeltest v.2.3. [bib_ref] MrModeltest v2. Program distributed by the author, Nylander [/bib_ref]. ITS sequences of reference isolates were obtained from NCBI GenBank database. An ITS sequence was not available for strain HMLAC 05119; therefore, ITS sequence of BRIP 72107 was used as a query against its published genome (GenBank accession number: VOSX00000000.1) to extract the ITS region of HMLAC 05119 and include in the phylogenetic analysis. distinct AT-rich and GC-balanced regions. The percentage values shown on the left and right sides of the vertical blue lines indicate the percentage of the genome classified as AT-rich and GC-balanced, respectively. (E) The majority rule consensus Bayesian phylogram inferred from the internal transcribed spacer sequences of the nuclear ribosomal DNA and the intervening 5.8S region (TreeBASE no. 28185). Tip labels include Ampelomyces strain number, the powdery mildew host, the plant host, and the country of origin. NCBI GenBank accession numbers used to construct the tree are provided in parentheses. The tip labels in bold represent Ampelomyces strains with available whole genome assemblies: BRIP 72107 sequenced in the current study and HMLAC 05119 [bib_ref] 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of..., Haridas [/bib_ref]. Bayesian posterior probability values are shown at the branches. The tree is rooted to Phoma herbarum strain CBS 276.37. The scale bar represents nucleotide substitutions per site.
[fig] FIG. 1: -The first 1,400,000 bp of BRIP 72107 contig-4 is demonstrated here as an example showing GC-balanced regions interspersed by longer or shorter stretches of AT-rich regions (A). The GC content distribution of Ampelomyces spp. (B and C) show striking similarity to the plant pathogenic fungus, Leptosphaeria maculans (D). Vertical blue lines show the GC cut-off points selected by OcculterCut [/fig]
[table] Table 1: Genome Statistics for Ampelomyces Strains Sequenced to DateStrain BRIP 72107 was sequenced in the current study. HMLAC 05119 was obtained from the JGI Genome Portal(Haridas et al. 2020). [/table]
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Gender Differences in Predictors and Long-Term Mortality of New-Onset Postoperative Atrial Fibrillation Following Isolated Aortic Valve Replacement Surgery
Purpose: Postoperative atrial fibrillation (POAF) after coronary artery bypass grafting (CABG) has been associated with increased risk of death in women but not in men. We aimed to explore predictors and long-term mortality in POAF following isolated aortic valve replacement (AVR) surgery in men and women. Methods: This study included 379 severe aortic stenosis patients with no prior atrial fibrillation (AF) who underwent isolated AVR surgery. We used multiple logistic regression to investigate independent gender-specific predictors of new-onset POAF, and we performed Kaplan-Meier (KM) to determine the impact of POAF in long-term mortality according to gender. Results: Advanced age and coronary artery disease prevalence were higher among POAF patients in both genders. On multiple analysis, increased postoperative peak lactate was independently associated with POAF in men, while lower mean aortic valve gradient was associated with POAF in women. Area under the curve (AUC) for the model was 0.77 [0.68-0.86] and 0.69 [0.60-0.78] for men and women, respectively. At 4-year follow-up, POAF was linked to increased risk of death in men but not in women. Conclusion: In severe aortic stenosis, factors associated with POAF and its impact on mortality differed between genders, with an increased risk of death observed only in men.
# Introduction
Atrial fibrillation (AF) remains the most common complication after cardiac surgery, [bib_ref] New onset postoperative atrial fibrillation and early anticoagulation after cardiac surgery. Scand..., Maaroos [/bib_ref] which occurs more frequently in valvular than in coronary artery bypass grafting (CABG) surgery (38-64% versus 17-33%). [bib_ref] Atrial natriuretic peptide predicts impaired atrial remodeling and occurrence of late postoperative..., Yilmaz [/bib_ref] [bib_ref] Postoperative atrial fibrillation and mortality after coronary artery bypass surgery, Villareal [/bib_ref] [bib_ref] Primary isolated aortic valve surgery in octogenarians, Ferrari [/bib_ref] Aortic stenosis is the most common valvular disease, [bib_ref] Atrial natriuretic peptide predicts impaired atrial remodeling and occurrence of late postoperative..., Yilmaz [/bib_ref] with increasing incidence due to aging demographics, particularly in women, that negatively affects the quality of life and survival. [bib_ref] Conventional aortic valve replacement in 2005 elderly patients: a 32-year experience, Langanay [/bib_ref] [bib_ref] Gender differences in incidence and in-hospital outcomes of surgical aortic valve replacement..., López-De-Andrés [/bib_ref] Aortic valve replacement (AVR) surgery still remains the gold standard for severe symptomatic aortic valve stenosis, improving quality of life and overall survival. [bib_ref] Primary isolated aortic valve surgery in octogenarians, Ferrari [/bib_ref] [bib_ref] Conventional aortic valve replacement in 2005 elderly patients: a 32-year experience, Langanay [/bib_ref] Although several studies have explored the influence of gender in outcomes after cardiac surgery, most of these focused on CABG. Female gender has been reported as a risk factor in several cardiovascular diseases, namely with regard to mortality and postoperative complications, including increased hospital stay and in-hospital morbidity, such as postoperative atrial fibrillation (POAF). [bib_ref] Being an elderly woman: is it a risk factor for morbidity after..., Basaran [/bib_ref] [bib_ref] Early and midterm outcomes in female patients undergoing isolated conventional coronary surgery, Erguneş [/bib_ref] Little is known about gender-specific POAF independent predictors in aortic stenosis. Stratifying POAF risk according to gender could improve POAF prediction and tailor patient management. In this study, we aimed to explore whether POAF-related factors differ by gender and how POAF influences long-term mortality in both genders.
# Materials and methods
This study aimed to compare the risk factors for POAF for each gender; study the survival between POAF and postoperative sinus rhythm (POSR) in men and women.
## Study population
Between January 2014 and December 2015, 1353 consecutive patients who were submitted to isolated AVR surgery were retrospectively analyzed. We excluded (1) reoperations and emergent cases and patients with other concomitant procedures, including CABG, aorta or other valvular surgery; (2) documented history of endocarditis or previous permanent or paroxysmal AF; (3) severe aortic regurgitation and more than mild mitral or tricuspid valve disease. Ultimately, we included 379 patients with aortic stenosis and a balanced distribution between genders (194 (51.2%) were women). This study was approved by the institution's ethics committee and data confidentiality was assured.
## Data collection
Patients had an echocardiography up to 6 months prior to surgery; all indexed measurements are normalized to body surface area (BSA). Smoking was considered when patients reported being active smokers or past smokers within less than a year. Median follow-up time for this cohort was 38 months. All gathered data were introduced in a coded database. Prolonged length of stay (LOS) was defined as the time of hospitalization above the 75th percentile. Hemodynamic instability was considered when inotropic support was needed during the postoperative period. Reported in-hospital outcomes included in-hospital mortality, stroke, myocardial infarction, cardiac arrest, atrioventricular block, and infection.
Primary outcomes were long-term all-cause mortality and occurrence of new-onset AF during the postoperative period. POAF was monitored by telemetry during the first 48 h post-surgery and afterwards by electrocardiogram (ECG) if an arrhythmic pulse was detected. It was assessed through telemetry and ECG recordings and doctors and nurses records. POAF was defined both electro-physiologically and clinically as recommended by the American Association for Thoracic Surgery: ECG recordings which demonstrate characteristics AF findings for at least 30 seconds or for the entirety of the ECG if shorter than 30 seconds; clinically significant AF which requires treatment with rate or rhythm control agents, anticoagulants or increases hospital stay. 9)
# Statistical analysis
IBM SPSS Statistics version 25 was used for all statistical analysis. Categorical variables were presented as percentages and continuous variables as mean and standard deviation. Categorical variables were analyzed using chi-squared test or Fisher's exact test when appropriate and continuous variables were analyzed with t-test for independent samples. Multiple analysis was performed using a logistic regression model through a stepwise approach to fit the model and find independent POAF predictors in both genders; included variables were tested for collinearity and sample size was considered when defining the final multivariate model. The area under the curve (AUC) of a receiving operating characteristic (ROC) curve was used to test the predictive value of the model in each gender group. Survival analysis was conducted using the Kaplan-Meyer (KM) method; Log-Rank and Breslow tests, and Cox Regression were used to evaluate risk factors affecting survival.
# Results
## Patient demographics
Patient characteristics are listed in [fig_ref] Table 1: Patient characteristics [/fig_ref]. Women were significantly older than men, with a mean age at operation of 72.8 (±9.7) and 68.5 (±10.5), respectively (p <0.001). Symptoms at presentation were similar between genders, including angina, syncope, and New York Heart Association (NYHA) functional class. The percentage of smoking men was much higher when compared to women, with 63% vs 7% (p <0.001) of patients being active smokers or ex-smokers within less than a year.
Other cardiovascular risk factors were equally distributed among genders (diabetes, hypertension, and dyslipidemia). Pulmonary hypertension, chronic lung disease, cerebrovascular disease, and presence of neoplasia did not differ between men and women. Although surgical coronary artery disease was an exclusion criterion, some patients had non-surgical coronary artery disease, which was similar between groups (9% vs 14.9% in women and men, respectively, p = 0.089). Most patients had preserved ejection fraction (88.8% vs 87.6%, p = 0.601) and mild mitral and tricuspid valve disease were equally distributed. Female patients had more severe disease at presentation, with increased indexed left atrium diameter and septum thickness, as well as maximum and mean aortic valve gradient; the percentage of low gradient female patients (mean gradient <40 mmHg) was lower and had a trend toward significance (1.6% in women vs 5.3% in men, p = 0.078). Similarly, aortic valve area was significantly smaller in women (0.71 ± 0.18 vs 0.79 ± 0.19 cm 2 , p <0.001). Surgical measured lactate peak serum levels and bypass time were significantly increased in women when compared to men (mean ± SD 3.0 ± 0.9 vs 2.5 ± 1.0 mmol/L, p <0.001 and 91.3 ± 27.6 vs 98.4 ± 28.7 min, p = 0.019). Aortic cross-clamp time presented a similar trend to the time of bypass, with 65.6 ± 19.1 min in women and 71.3 ± 20.6 min in men (p = 0.008).
## Hospital outcomes
In-hospital mortality occurred in 2.6% of women and 1.1% of men (p = 0.450). Stroke and myocardial infarction did not differ among genders, representing 1.1% vs 2.2% and 1.1% vs 0.5% of cases, respectively (p = 0.443 and p = 1.000). Conduction disturbances such as atrioventricular block occurred in 7.4% of women and 5.5% of men (p = 0.446), with the need for pacemaker in 3.2% and 2.2% of cases, respectively (p = 0.751). Cardiac arrest was documented in four female patients (2.1%, p = 0.123). Hemodynamic instability during and after surgery, assessed by the need for inotrope administration, had a high prevalence, and was similar between genders (47.6% vs 48.9%, p = 0.803). Documented infection was similar between genders and occurred in 5.3% of women and 4.9% of men (p = 0.590) and prolonged LOS had a prevalence of 23.9% and 20.3% in women and men, respectively (p = 0.414). At discharge, amiodarone medicated patients represented 12.3% of women and 13.2% of men (p = 0.805) and warfarin was prescribed in 52.6% of women and 72.5% of men (p <0.001).
## Impact of poaf in hospital outcomes by gender
POAF had an overall occurrence of 41.2%, with 50% of cases occurring until the 2nd postoperative daymedian (IQR) of 2 (1)-overall and between genders. POAF did not differ between women and men, presenting an occurrence of 45.0% vs 39.2% (p = 0.263), respectively. The arrhythmia was either self-limited (34%) or pharmacologically treated with amiodarone (57.1%) or electric cardioversion or both (9%). There were no differences between genders regarding POAF management, being self-limited in 34.1% of women vs 33.8% of men; pharmacologically treated in 55.3% vs 59.2% and treated with electrical cardioversion or both in 10.6% vs 7% (p = 0.725). Prolonged LOS was significantly different between POSR and POAF patients in both genders-18.2% vs 31.7% (p = 0.035) for women and 11.8% vs 35.4% (p <0.001) for men. However, infection remained non-significant for each gender and between POSR and POAF (6.8% vs 3.7%, p = 0.516, in women; 3.6% vs 7.1%, p = 0.423 in men). As with the case of infection, atrioventricular block remained similar between genders and rhythm group-5.8% vs 9.8% in women, p = 0.315 and 5.5% vs 5.7% in men, p = 1.000. Although warfarin was prescribed more frequently in men (see above), in POAF patients, there were no documented differences between genders-warfarin prescribed in 53.4% of women vs 57.6% of men (p = 0.629) and amiodarone in 26.0% vs 32.2% (p = 0.436). On the other hand, warfarin prescription was significantly higher in POSR men (51.5% vs 81.0%, p <0.001), with no differences in amiodarone prescription (2.1% vs 2.9%, p = 0.716).
## Poaf-related factors by gender univariate analysis
Concerning POAF gender-related factors, patients who developed POAF were generally older in both groups: in women, the mean age was 75 ± 7.5 in those who developed POAF and 71 ± 11.2 in women who remained in SR (p = 0.015); in men, POAF had a mean age of 72.3 ± 9.0 vs 66.2 ± 10.6 (p <0.001). Moreover, coronary artery disease presented as a risk factor for POAF in both groups: 13 (16.9%) vs 3 (3.1%), p = 0.003, in women; 16 (23.5%) vs 10 (9.5%), p = 0.012, in men.
However, mean aortic gradient was a protective factor for POAF only in women (51.8 ± 13.0 vs 56.1 ± 15.1,
[formula] p = 0.041). [/formula]
The presence of tricuspid valve disease was associated with increased risk of POAF only in male patients (20 (38.5%) vs 17 (21.0%), p = 0.028). Similarly, chronic lung disease was associated with POAF exclusively in men-23 POAF patients (33.8%) vs 17(17.2%), p = 0.013.
Medication did not influence the outcome in either gender. Results for POAF-related factors in men and women are shown in [fig_ref] Table 2: Univariate analysis ACEI/ARB [/fig_ref]. Corresponding univariate logistic regression analysis and odds ratios (OR) are depicted in [fig_ref] Table 3: Logistic regression [/fig_ref].
# Multivariate analysis
In women, we identified as independent predictors of POAF advanced age [fig_ref] Figure 1: Area under the ROC curve for the multivariate logistic regression model in... [/fig_ref]. Results referring to the previous model in both genders are shown in [fig_ref] Table 3: Logistic regression [/fig_ref].
# Mortality analysis
Median follow-up time for the whole cohort was 38 months, with men and women presenting 37 and 39 months of follow-up, respectively. KM curves for both genders were similar and the Log Rank test was nonsignificant (p = 0.736), therefore not suggesting any difference in long-term mortality between genders [fig_ref] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for... [/fig_ref]. At 12 months, female patients had a cumulative survival of 94.8%, while for men was 96.8%. At 48 months of follow-up, both gender groups showed a cumulative survival of 93%. [fig_ref] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for... [/fig_ref]
## Impact of poaf on mortality by gender
Concerning KM curves between POSR and POAF patients, women had no differences when comparing both groups, with a slight superposition between curves (non-significant Breslow test, p = 0.749). [fig_ref] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for... [/fig_ref] At 12 months of follow-up, women who remained in sinus rhythm during the postoperative period had a 97.1% survival probability, whereas women with POAF presented 95.3% cumulative survival. At 24 months, women presented a survival probability of 94.2% and 94.1%, respectively. At 36 months, there was a slight inversion of the KM curves, with POSR women showing a cumulative survival of 94.2% and POAF women 92.7% [fig_ref] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for... [/fig_ref]. Men, however, had a significant difference between POSR and POAF groups (Log Rank test, p = 0.024), showing a divergence in the KM curves as early as the first year of follow-up (99.1% vs 94.4% of cumulative survival) [fig_ref] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for... [/fig_ref]. At 24 months, POSR and POAF male patients presented 97.3% and 90.1% probability of survival and at 36 months, survival shifted to 95.8% and 88.7%, respectively. At 48 months, men with POAF had a cumulative survival of 86.4%, while men in the POSR group maintained the 95.8% survival probability [fig_ref] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for... [/fig_ref] Thus, POAF predicted mortality only in men, with a HR (95% CI) of 3.551 [1.093-11.532], p = 0.035.
# Discussion
In this study, at surgery, women presented more severe aortic valve disease than men, including worsened ventricular re-modeling, with an increased indexed interventricular septum thickness and left atrium diameter. Coronary artery disease, even though non-surgical, and advanced age were predictors of POAF for both genders. The latter emerged as an independent predictor in the multivariate analysis. The remaining risk factors for POAF were gender-specific, with chronic lung disease showing an association with POAF in men, although non-significant in the multivariate analysis. Other valvular disease, although mild, was increased only in male POAF patients. Peak postoperative lactate was an independent predictor of POAF exclusively in men, while higher mean aortic gradient represented an independent protective factor in women. The multivariate model presented significant differences in performance for predicting disease according to gender-a classical prediction model based on clinical, echocardiographic and surgical parameters appears to perform well in men, although lacking efficacy in female patients. Mortality does not differ between genders, either in-hospital or long term. Although POAF occurs similarly in men and women, only in men POAF occurrence predicts long-term mortality. Of the tested variables as potential risk factors for mortality, a reduced ejection fraction associated with an adverse outcome only in women.
In this cohort, POAF had an overall occurrence of 41.2%, with 50% of cases occurring until the 2nd postoperative day, which agrees with previous studies, although the arrhythmia varies with patient characteristics, definition of the arrhythmia and methods of heart rhythm monitoring. 10) Accordingly, reported POAF peak occurrence is between the second and fourth days post-surgery, with most cases appearing in the first 5 days and only a few after the first week. [bib_ref] Atrial natriuretic peptide predicts impaired atrial remodeling and occurrence of late postoperative..., Yilmaz [/bib_ref] [bib_ref] ESC Guidelines for the management of atrial fibrillation developed in collaboration with..., Kirchhof [/bib_ref] There is conflicting evidence to whether women are more prone to POAF after CABG than men. Alam et al [bib_ref] Association of gender with morbidity and mortality after isolated coronary artery bypass..., Alam [/bib_ref] found women have less risk of developing POAF, although it has been reported no difference in risk or increased risk. [bib_ref] Being an elderly woman: is it a risk factor for morbidity after..., Basaran [/bib_ref] [bib_ref] Atrial fibrillation in elderly patients after coronary artery bypass grafting; gender differences..., Kokkonen [/bib_ref] In this study of isolated aortic valve patients, it was observed no difference in POAF occurrence between men and women.
Several clinical and echocardiographic POAF predictors have been identified, such as age, obesity, pulmonary and arterial hypertension, mitral valve disease, left atrium dilation, left atrium volume index, atrial natriuretic peptide (ANP), and subclinical hypothyroidism, with age being the most reproducible risk factor among studies, as confirmed in this study. [bib_ref] Atrial natriuretic peptide predicts impaired atrial remodeling and occurrence of late postoperative..., Yilmaz [/bib_ref] There is little evidence on predictors according to gender, and this cohort suggests some potential differential risk factors for POAF in patients submitted to isolated AVR surgery. This study underlines the importance of stratifying patients taking gender into consideration.
Patients who develop AF in the postoperative period are at greater risk of thromboembolic events, infections, including pneumonia, respiratory failure, renal failure, intensive care unit readmission, prolonged LOS, increased overall costs, and 30-day mortality. [bib_ref] Postoperative atrial fibrillation and mortality after coronary artery bypass surgery, Villareal [/bib_ref] [bib_ref] Predicting newonset postoperative atrial fibrillation in cardiac surgery patients, Tran [/bib_ref] In this cohort of patients, prolonged LOS was in fact different between POSR and POAF patients. On the other hand, there were no differences concerning in-hospital outcomes between these groups in each gender, although events were sparse and thus difficult to identify differences objectively. Numerous studies in CABG surgery have reported higher hospital mortality in female patients than in their male counterparts, [bib_ref] Association of gender with morbidity and mortality after isolated coronary artery bypass..., Alam [/bib_ref] [bib_ref] Coronary artery bypass grafting in South Asian patients: impact of gender, Khan [/bib_ref] although mid-and long-term mortality appears to be similar in both gender groups. [bib_ref] Early and midterm outcomes in female patients undergoing isolated conventional coronary surgery, Erguneş [/bib_ref] [bib_ref] Gender differences in outcomes after hospital discharge from coronary artery bypass grafting, Guru [/bib_ref] There is some evidence to suggest that between 5 and 6 years after CABG surgery, the results are the same or might be better for female patients, as suggested by the work of Abramov et al. with the increase in female late survival after CABG. 17) Ahmed et al. recruited 1114 women and 3628 men submitted to CABG surgery and evaluated morbidity and mortality for a follow-up of 7.9 years. The study concluded women had increased long-term cardiac mortality even though similar all-cause mortality. [bib_ref] Female sex as an independent predictor of morbidity and survival after isolated..., Ahmed [/bib_ref] Similarly to CABG surgery, women have a higher preoperative risk than men in combined valvular and CABG surgery, [bib_ref] Sex-specific long-term outcomes after combined valve and coronary artery surgery, Doenst [/bib_ref] representing an independent risk factor for post-surgery morbidity and mortality. [bib_ref] Genderrelated differences in morbidity and mortality during combined valve and coronary surgery, Ibrahim [/bib_ref] Likewise, in AVR surgery, female gender predicts increased in-hospital and 30-day all-cause mortality. [bib_ref] Sex differences in the utilization and outcomes of surgical aortic valve replacement..., Chaker [/bib_ref] [bib_ref] The influence of gender on in-hospital clinical outcome following isolated mitral or..., Wong [/bib_ref] [bib_ref] Sex-related differences in 2197 patients undergoing isolated surgical aortic valve replacement, Elhmidi [/bib_ref] Concerning long-term mortality, Elhmidi et al. reported no influence of gender in late mortality in a cohort of 2197 patients submitted to isolated AVR surgery. Several studies agree with this finding, suggesting gender does not influence long-term outcomes in AVR. [bib_ref] Sex-specific long-term outcomes after combined valve and coronary artery surgery, Doenst [/bib_ref] [bib_ref] Effect of gender on treatment and outcomes in severe aortic stenosis, Hartzell [/bib_ref] In this cohort of patients, although no difference in postoperative mortality was observed between genders, female patients did show an increased preoperative risk, with worse disease, and longer surgical timings (bypass time) as well as worse response to surgery (peak lactate). Concerning overall long-term mortality, men and women had similar survival probabilities at a maximum follow-up of 52 months, which concurs with what is observed in the literature. Furthermore, several studies have implicated POAF in short-and long-term mortality, which suggests the need to monitor this group of patients closely. [bib_ref] New-onset atrial fibrillation predicts long-term mortality after coronary artery bypass graft, El-Chami [/bib_ref] In this cohort, POAF had a trend toward significance as a risk factor for long-term mortality (data not shown). Furthermore, POAF after CABG has been reported as a predictor of long-term mortality in female patients but not in males. [bib_ref] Gender difference in the long-term clinical implications of new-onset atrial fibrillation after..., Lee [/bib_ref] The present study suggests POAF as a predictor of long-term mortality in men but not in women, in patients submitted to isolated AVR surgery, which does not agree with the available literature in CABG. This finding does not appear to be related to baseline characteristics as both men and women with POAF are older and have higher comorbidity rates; moreover, other risk factors tested by cox regression in men did not influence mortality. It has been suggested men are more prone to develop AF than women, although the basis for these differences remain unclear. Men have increased expression of relevant repolarizing ion channel subunits, which might decrease repolarization time and thus abbreviate atrial refractoriness and promote re-entry. [bib_ref] Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts, Gaborit [/bib_ref] In this cohort, only POAF in men predicts permanent AF during the first year; in women, POAF does not predict AF (data not shown). This suggests POAF in men represents either electrical and/or structural re-modeling as an arrhythmia-developing substrate, although indexed left atrium diameter does not predict POAF in either gender in this cohort. Conversely, women could develop a more benign POAF with transitory inflammation as the main risk factor for the arrhythmia, thus not predicting established AF at follow-up. Inflammation is a recognized pathway for POAF, with C-reactive protein, IL-6 and IL-2 being implicated in the development of the arrhythmia; treatment with anti-inflammatory agents has been reported to decrease POAF occurrence. [bib_ref] Role of inflammation in atrial fibrillation pathophysiology and management, Harada [/bib_ref] Thus, POAF could predict mortality in men because it predicts established AF only in this gender. Men could have to be more aggressively treated to prevent permanent AF and related morbidity and mortality, although more studies are necessary to confirm this hypothesis and explore other mechanisms of gender-related differences in AF pathophysiology.
POAF is a known risk factor for permanent AF, as well as morbidity and mortality, including thromboembolic events. [bib_ref] New-onset atrial fibrillation predicts long-term mortality after coronary artery bypass graft, El-Chami [/bib_ref] There are several studies on the prevention (β-blockers, colchicine, etc.) and early treatment of POAF although not according to gender. [bib_ref] New onset postoperative atrial fibrillation and early anticoagulation after cardiac surgery. Scand..., Maaroos [/bib_ref] [bib_ref] Colchicine for primary prevention of atrial fibrillation after openheart surgery: systematic review..., Lennerz [/bib_ref] This study suggests men might benefit more from the prevention of the arrhythmia than women, although only all-cause mortality differed between men and women who presented POAF. Other outcomes, as increased hospital stay and immediate postoperative morbidity, were similar between genders. In addition, these results could potentially translate to transcatheter aortic valve implantation (TAVI), as new-onset post-procedure AF occurs in up to 32% of cases and, although some studies do not report differences between men and women, disparities in long-term all-cause mortality between genders are still debatable. [bib_ref] Gender-related differences on short-and long-term outcomes of patients undergoing transcatheter aortic valve..., Katz [/bib_ref] This cohort is retrospective, which is a limitation of this study. POAF definition is based on written records which could under-detect our main outcome. Still, our prevalence reports are similar to existing literature. Furthermore, larger cohorts are necessary to confirm these results and increase evidence on gender differences in both predictors of POAF and POAF impact on mortality.
# Conclusion
Although larger cohorts are needed to confirm these results, this study underlines the importance of stratifying patients according to gender and finding genderspecific prediction models for POAF. Patients who develop new-onset AF following isolated AVR surgery need to be closely monitored, although mortality appears to be increased only in men.
[fig] Figure 1: Area under the ROC curve for the multivariate logistic regression model in men and women [/fig]
[fig] Figure 2: KM curves and respective survival probabilities between POSR and POAF patients for women (A) and men (B). (C) KM curves between women and men. KM: Kaplan-Meier; N: number of patients at follow-up; POAF: postoperative atrial fibrillation; POSR: postoperative sinus rhythm; SP: survival probability [/fig]
[table] Table 1: Patient characteristics [/table]
[table] Table 2: Univariate analysis ACEI/ARB: angiotensin converting enzyme inhibitor/angiotensin receptor blocker; NYHA: New York Heart Association Functional Classification; PSAP: pulmonary systolic arterial pressure [/table]
[table] Table 3: Logistic regression [/table]
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A novel planar optical sensor for simultaneous monitoring of oxygen, carbon dioxide, pH and temperature
The first quadruple luminescent sensor is presented which enables simultaneous detection of three chemical parameters and temperature. A multi-layer material is realized and combines two spectrally independent dually sensing systems. The first layer employs ethylcellulose containing the carbon dioxide sensing chemistry (fluorescent pH indicator 8-hydroxy-pyrene-1,3,6-trisulfonate (HPTS) and a lipophilic tetraalkylammonium base). The cross-linked polymeric beads stained with a phosphorescent iridium(III) complex are also dispersed in ethylcellulose and serve both for oxygen sensing and as a reference for HPTS. The second (pH/temperature) dually sensing system relies on the use of a pH-sensitive lipophilic seminaphthorhodafluor derivative and luminescent chromium(III)-activated yttrium aluminum borate particles (simultaneously acting as a temperature probe and as a reference for the pH indicator) which are embedded in polyurethane hydrogel layer. A silicone layer is used to spatially separate both dually sensing systems and to insure permeation selectivity for the CO 2 /O 2 layer. The CO 2 /O 2 and the pH/ temperature layers are excitable with a blue and a red LED, respectively, and the emissions are isolated with help of optical filters. The measurements are performed at two modulation frequencies for each sensing system and the modified Dual Lifetime Referencing method is used to access the analytical information. The feasibility of the simultaneous four-parameter sensing is demonstrated. However, the practical applicability of the material may be compromised by its high complexity and by the performance of individual indicators.
# Introduction
Optical sensors proved to be reliable analytical tools for monitoring various chemical and biological species. They are only minimally invasive, are free of electromagnetic interferences, can be miniaturized rather easily and are highly versatile in their formats, which include planar optodes, sensor paints, fiber-optic (micro)sensors, and dispersible nanosensors. In many important applications, simultaneous monitoring of several parameters is highly desirable. Notably, optical sensors provide a unique possibility of multiplexing since light can carry various information simultaneously (e.g., light intensity, decay time, polarization etc.). It is not surprising that the sensors enabling simultaneous monitoring of several parameters attract considerable interest and became very popular recently. They are represented by two different types. The array-type sensors combine individual sensor spots, fiber-optic sensors, or sensing particlesbrought in close proximity. Such sensors usually possess limited resolution. On the other side, true multi-analyte sensors enable simultaneous monitoring of several parameters in exactly the same point. A variety of dually sensing materials were reported recently, most of them designed for simultaneous monitoring of one chemical parameter (mostly oxygen, but also carbon dioxide or pH) and temperature [bib_ref] Correcting lifetime measurements for temperature, Coyle [/bib_ref]. In fact, all optical sensors are cross-sensitive to temperature and these effects should be compensated for if temperature is not kept constant. Several sensors for simultaneous monitoring of pH and oxygen and carbon dioxide and oxygen were also reported. Recently, Wolfbeis and co-workers reported a triple sensor for simultaneous monitoring of oxygen, pH, and temperature. The sensing material relied on three types of permeation-selective microbeads mixed together in a hydrogel matrix. The three probes are excited with a violet LED and possess three distinct emissions which are separated with help of optical filters.
To the best of our knowledge, no optical sensor for simultaneous measurement of more than three parameters was reported so far. In this contribution, we will describe a novel sensing material suitable for simultaneous monitoring of four parameters including oxygen, carbon dioxide, pH, and temperature. In contrast to the sensor array (consisting of three microoptodes and a thermocouple) Paratrend® from Biomedical Sensors Ltd., capable of monitoring the same four analytes, our true multi-analyte sensor is manufactured as a planar optode and can also be used for imaging purposes. The same principle can be used to design optical sensors for other analytes.
## Experimental
# Materials
Tetraoctylammonium hydroxide (TOAOH, 20% in methanol), sodium 8-hydroxy-pyrene-1,3,6-trisulfonate (HPTS), ethylcellulose (EC, ethoxyl content of 49%), seminaphthorhodafluor decyl ester (SNARF-DE, chromoionophore XIII) were obtained from Aldrich (www.sigmaaldrich.com). Styrene, divinylbenzene, sodium dodecyl sulfate (SDS) were bought from Fluka (www.sigmaaldrich.com). 2,2′-Azabis(2methyl propionitrile) (AIBN) and aluminum oxide (Brockmann I) were purchased from Fisher Scientific (www.at. fishersci.com). The components for the preparation of silicone rubber: vinyl-terminated polydimethylsiloxane (DMS-V31, viscosity 1000 cSt.), methylhydrosiloxanedimethylsiloxane copolymer (HMS-301), tetravinyltetramethyl cyclotetrasiloxane (SIT 7900.0) and platinum divinyltetramethylsiloxane complex (PC075) were obtained from ABCR (www.abcr.de). Silica gel particles (LiChro-sorb® Si60, Ø 5 μm) were acquired from Merck (www. merck.de). 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) and sodium chloride were bought from Roth (www.carl-roth.de). The polyurethane hydrogel (available under the name HydroMed™ D4) was from AdvanSourse biomaterials (www.advbiomaterials.com). Poly(ethylene glycol terephthalate) support (Mylar®) was from Goodfellow (www. goodfellow.com). Nitrogen, oxygen, synthetic air and 5% carbon dioxide in nitrogen (all of 99.999% purity) were obtained from Air Liquide (www.airliquide.at).
Preparation of the iridium(III) acetylacetonato-bis-(3-(benzothiazol-2-yl)-7-(diethylamino)-coumarin) (Ir(C S ) 2 acac)and microcrystalline powder of chromium(III)-activated yttrium aluminum borate (Cr-YAB, Ø 2.6±0.5 μm)is reported elsewhere. The HPTS(TOA) 3 ion pair was prepared from HPTS and tetraoctylammonium chloride, analogously to the procedure described previously.
Preparation of the oxygen-sensing beads Prior to use, styrene and divinylbenzene were passed through a column packed with aluminum oxide in order to remove the inhibitors. 850 μl of styrene, 1556 μl of divinylbenzene, 22 mg of the oxygen indicator Ir(C S ) 2 (acac) and 2 g of chloroform were dispersed in 80 ml of water containing 220 mg of SDS. The emulsion was prepared with help of a homogenizer D-1 (ART modernelabortechnik, www.miccra. com) and was further ultrasonicated for 5 min. The emulsion was placed in a 250-ml three-necked flask and deoxygenated for 20 min by blowing nitrogen through the flask under vigorous stirring. Then, 50 mg of AIBN were added and the emulsion was heated up to 90°C. It was stirred overnight over nitrogen at this temperature. The resulting dispersion of the microbeads was centrifuged at 400 rpm in order to remove large aggregates. The remaining beads were precipitated with ethanol and were washed twice with acetone and three times with ethanol to remove the surfactant and the dye adsorbed on the surface. The beads were dried in the Petri dish and homogenized in the agate mortar.
Preparation of the multi-analyte sensor 1. CO 2 /O 2 layer: 50 mg of the oxygen-sensing beads and 20 mg of EC49 were dispersed/dissolved in 400 mg of ethanol. In the second vial, 2.5 mg of HPTS(TOA)and 80 mg of EC 49 were dissolved in the mixture of 1 g of tetrahydrofurane and 600 mg of ethanol. Carbon dioxide was purged through the "cocktail" and 100 μl of methanolic TOAOH solution was added. The two "cocktails" were mixed together and were knife-coated onto a dust-free polyester support using a 75-μm spacer to result in ∼6-μm-thick sensing layer after solvent evaporation. 2. Silicone isolation layer: the "cocktail" of silicone primers was obtained by mixing 400 mg of the vinylterminated PDMS, 16 μl of methylhydrosiloxanedimethylsiloxane copolymer, 3 μl of tetravinyltetramethyl cyclotetrasiloxane, and 5 μl of the platinum complex catalyst in 400 mg of hexane. The "cocktail" was coated onto the CO 2 /O 2 layer (25 μm spacer) and left to polymerize at room temperature for 20 min. Then, silica gel particles (Ø 5 μm) were spread on the partly polymerized surface and the material was left at ambient air for another 2 h. The excess of the particles was removed mechanically. 3. pH/temperature layer: 150 mg of hydrogel D4 and 150 mg of Cr-YAB were dissolved/dispersed in the mixture of 900 mg of ethanol and 100 mg of water. Three hundred microliters of the stock solution of SNARF-DE in ethanol (1 mg⋅ml −1 ) was added. The "cocktail" was knife-coated on the silicone layer (75 μm spacer) to result in ∼10 μm thick sensing layer after solvent evaporation. In order to prevent poisoning by acidic gases and prolong the sensor lifetime, the planar optodes were stored at −18°C in a sealed plastic bag filled with 100% carbon dioxide.
Measurements Emission spectra were acquired on a Hitachi F-7000 fluorescence spectrometer (www.hitachi.com) equipped with a red-sensitive photomultiplier R 928 from Hamamatsu (www.hamamatsu.com). Absorption spectra were measured on a Cary 50 UV-Vis spectrophotometer (www.lzs-concept.com). The size of the beads was estimated from the photographic images acquired on a Zeiss Axiovert 25 CFL microscope (Zeiss, www.zeiss.de) using a SensiCAM camera (cooled CCD-chip, 640×480 pix, www.pco.de).
Interrogation of the multi-analyte sensor was performed using a home-made device which is schematically represented in . The device consists of two separate detection channels each of them equipped with a photomultiplier module (H9306-02 from Hamamatsu, www. sales.hamamatsu.com) and an LED. A 450 nm LED (Roithner, www.roithner-laser.com) combined with a short-pass BG-12 filter (Schott, www.schott.com) is used for excitation in the O 2 /CO 2 channel and the emission is filtered through the HC 562/40 band-pass filter (Analysentechnik, www.ahf.de). Excitation in the pH/temperature channel is performed with a 605 nm LED (Roithner) combined with a HC 620/52 band-pass filter (Analysentechnik) and the emission is filtered through an RG-695 long-pass filter from Schott. The LEDs are sinusoidally modulated using a two-phase lock-in amplifier (SR830, Stanford Research Inc., www.thinksrs.com). The interrogation in each channel is performed at two different modulation frequencies: 12 and 22 kHz in the O 2 /CO 2 channel and 1.5 and 4.5 kHz in the pH/temperature channel. A four-armed optical fiber bundle (LEONI Prinz Fiber Optics GmbH, www.leoni-fiber-optics.com) is used to guide the excitation light to the sensor and to guide back the emission.
The pH was adjusted to the desired value using CHES, MES, and MOPS buffers. The pH of the buffer solutions was controlled by a digital pH meter (InoLab pH/ion, WTW GmbH & Co. KG, www.wtw.com) calibrated at 25°C with standard buffers of pH 7.0 and 4.0 (WTW GmbH & Co. KG, www.wtw.com). The buffers were adjusted to constant ionic strength (IS=0.2 M) using sodium chloride as the background electrolyte. Temperature was controlled by a cryostat ThermoHaake DC50. At least three independent measurements were performed to obtain a calibration curve.
# Results and discussion
General considerations In order to demonstrate the feasibility of simultaneous monitoring of four parameters with a Schematic representation of the home-made device and the optical arrangement for interrogation of the multi-analyte sensor single sensor, we chose the three analytes (carbon dioxide, oxygen, and pH) describing many metabolic processes to be combined with the detection of temperature. Evidently, determination of these parameters is of much importance in many areas of science and technology, for example in biology, marine chemistry, biotechnology, environmental monitoring, and medicine. Temperature was an obvious choice since all optical chemosensors are affected by temperature and should be corrected for these effects if temperature is not kept constant. In fact, the combination of these parameters was realized in the Paratrend® sensor from Biomedical Sensors Ltd. The sensor represented an array of three fiber-optic sensors (for oxygen, carbon dioxide and pH) combined with a thermocouple for temperature compensation. Due to the large number of analytes and rather small dimensions (Ø 0.5 mm) the sensor was quite complex in manufacturing. On the contrary, the multi-analyte sensor technology seems to be rather attractive since many identical sensor spots can be easily prepared from the planar sensors even if the design of such a sensor is rather complex.
Evidently, the sensor architecture is expected to become increasingly sophisticated with the increasing number of analytes that need to be analyzed simultaneously. In other words, a multi-analyte sensor cannot be a simple combination of the individual probes for several reasons. First, permeation selectivity should be insured. For example, a carbon dioxide optical probe (which relies on the use of a fluorescent pH indicator) should not be influenced by pH of the analyzed solution. Second, interferences originating from reabsorption of the emitted light or from Föster resonance energy transfer should be avoided. To avoid FRET spatial isolation of the indicators is necessary. This can be realized by using a multi-layer approach or by immobilizing the sensor chemistries in nano-or microbeads. Third, photophysical properties of the indicators are crucial for their choice since separation of the signals from individual indicators is required. This can be achieved either spectrally (for the indicators having substantially different excitation and emission spectra) or via the decay time. Due to the limited amount of luminescent indicators available, it is extremely difficult to choose more than two pairs of probes with adequate characteristics (sensitivity, selectivity, chemical stability, photostability) and substantially different spectral properties. Evidently, a combination of the spectral separation and the separation by the decay time is obvious for designing a multi-analyte sensor. Finally, signal referencing is necessary in order to obtain a reproducible sensing material. For example, the luminescence decay time is a self-referenced parameter, but the fluorescence intensity is not. It depends on the number of parameters such as intensity of the light source and sensitivity of the photodetector, scattering in the sensor, turbidity and coloration of the probe, etc. Therefore, the luminescence intensity is usually referenced either ratiometrically or using the so called dual lifetime referencing technique. As was demonstrated previously, the long-lived luminescence cannot only be used for referencing of the short-lived fluorescence, but also to sense an additional analyte.
Sensor design and materials used shows the chemical structures and the response of the 4 probes chosen for combination in the multi-analyte sensor. As can be seen, the individual probes enable monitoring of the analytes in physiologically relevant range. They are also adequate for determination of the same analytes in sea water. A crosssection of the multi-analyte sensor is schematically shown in [fig_ref] Figure 3: Cross-section of the multi-analyte sensor [/fig_ref]. The sensor consists of the two sensing layers (CO 2 /O 2 and pH/temperature) which are spatially isolated with help of the silicone layer. The first layer is designed to enable simultaneous sensing of carbon dioxide and oxygen. The carbon dioxide sensing chemistry is based on the established systemand includes the lipophilic ion pair of 8-hydroxy-pyrene-1,3,6-trisulfonate and tetraoctylammonium cation (HPTS(TOA)and an excess of the lipophilic base TOAOH (converted into hydrocarbonate in presence of carbon dioxide) which are dissolved in ethyl cellulose (49% w/w of the ethoxyl groups). Such composition is known to result in rather sensitive sensors. To produce less sensitive sensors (e.g., for blood analysis) tetraoctylammonium hydroxide may be substituted by tetrabutylammonium hydroxideand the EC 49 by EC 46 (46% w/w of the ethoxyl groups) which is known to have lower solubility of carbon dioxide.
A phosphorescent iridium(III) coumarin complex Ir (C S ) 2 acacwas the oxygen indicator of choice because of its high brightness and good spectral compatibility to HPTS. Simple addition of the oxygen indicator into ethylcellulose is undesirable since (1) basic tetraoctylammonium hydrocarbonate may affect the stability of the iridium(III) complex, and (2) photostability of HPTS is expected to deteriorate dramatically due to the photoinduced formation of singlet oxygen. Therefore, the oxygen indicator was embedded into highly cross-linked polymeric microbeads. These polymeric beads are prepared by the emulsion polymerization of styrene and divinylbenzene and virtually do not swell in common organic solvents. This insures that the indicator remains inside the beads when they are dispersed in the ethylcellulose/tetrahydrofurane/ethanol "cocktail". Additionally, as will be demonstrated below, oxygen sensitivity is significantly enhanced compared to the polystyrene-based materials. Typical dimensions of the beads (estimated from the microscopic images) are 3-8 μm. The beads of 3-5 μm in diameter are usually adequate to produce a highly homogeneous sensor layer. Although the larger beads (Ø 6-8 μm) are expected to exceed the thickness of the layer, this effect is not visible when interrogating a sensor spot with an optical fiber of several millimeters in diameter.
Evidently, an additional protecting layer is required for the CO 2 sensor to be usable in aqueous solutions. Ionic species (particularly protons or hydroxide ions) can diffuse inside and influence the calibration plots by interacting with the sensor components. A hydrophobic silicone rubber seems to be an excellent choice to ensure the permeation selectivity. In fact, polydimethylsiloxanes possess very high permeability to gases but do not let the ionic species through. Additionally, apolar polydimethylsiloxane is an extremely bad solvent for virtually any dye. Therefore, the diffusion of the indicators from the CO 2 /O 2 layer into the pH/temperature layer or in the reverse direction becomes impossible. A serious problem arising here is very poor adhesion of the hydrophilic hydrogel layer (pH/temperature) on the highly hydrophobic silicone. Initial attempts were focused on hydrophilization of the silicone surface by plasma treatment in the atmosphere of air which is a widespread method of polymer surface modification. Unfortunately, it was found that even using the moderate settings the CO 2 /O 2 sensing layer was severely affected by the modification resulting in irreproducible drift in the calibration plots. Therefore, the surface modification was performed in the following manner. First, the mixture of the silicone primers and the catalyst was diluted with hexane (which does not swell the CO 2 /O 2 layer) to adjust the thickness of the layer, and the composition was spread onto the CO 2 /O 2 layer and was allowed to partly polymerize. Then, silica gel beads (Ø 5 μm) were spread on the surface of the silicone to be entrapped there. Thus, the beads not only increased the overall surface of the silicon layer but also improved the hydrophilicity of the layer. It should be noted here that neither the monolayer of silica beads dispersed on the surface of the silicon layer nor the crosslinked oxygen-sensitive beads contained in the first layer prevent the excitation light from reaching the pH/temperature layer and the emission from this layer from reaching the detector. Some light scattering caused by the beads does not necessarily reduce the luminescence but can even enhance it similarly to the effect produced by scattering TiO 2 particles dispersed in the optical sensors.
Finally, a third pH/temperature sensing layer relied on a lipophilic pH indicator dissolved in the polyurethane hydrogel (HydroMed™ D4) and the chromium(III)-doped yttrium aluminum borate phosphor particles dispersed in the same material. The phosphor serves as a reference for the pH indicator and as a temperature probe. It is important that the hydrogel (which has been widely used in optical pH sensors)is permeable for all the analyzed species including protons, oxygen and carbon dioxide. Considering possible alternatives to Cr-YAB other thermographic phosphors (e.g., rubi), in principle, can be used in the multi-analyte sensor. However, Cr-YAB shows superior temperature sensitivity compared to other phosphors. As will be demonstrated in the following, spectral compatibility of the probe with the pH indicator and the red LED is essential for application in the multi-analyte sensor so that Cr-YAB is an evident choice here. Notably, other thermographic phosphors are not excitable with red light. Chromium(III)-doped yttrium aluminum garnet represents an exception (the excitation spectrum is similar to that of Cr-YAB) but its brightness is very low.
Spectral properties of the probes and optical setup depicts the absorption and emission spectra of the probes as well as the emission spectra of the LEDs and the transmittance spectra of the optical filters. As was mentioned above, a fluorescent and a phosphorescent probe can be combined together, the latter serves also as a reference for the fluorescent one. Therefore, the fourparameter sensor can be viewed as a combination of two dual sensors which are spectrally independent on each other. Both HPTS in its basic form and the Ir(C S ) 2 acac are efficiently excitable with a 450 nm LED . Notably, the absorption maximum of HPTS in its acidic form (in the presence of CO 2 ) is located at ∼400 nm so that this form of the dye is not excitable with the blue LED. On the other hand, the blue LED does not excite the pH indicator in the hydrogel layer, and the Cr-YAB is excitable only to a minor extent . Both the pH indicator in its basic form and the temperature phosphor are excitable with the red 605-nm LED. Notably, neither the acidic form of the pH indicator nor the components of the CO 2 /O 2 sensing system are excitable with the red LED. Two additional filters for the emission ensure complete separation of the luminescence signals from both systems . A band-pass interference filter (542-582 nm) is used for the CO 2 /O 2 system and completely eliminates the emissions of the pH indicator and the temperature phosphor which can be excited either directly with the blue LED or indirectly (reabsorption of the light emitted by the CO 2 /O 2 system). Finally, a longpass RG 695 filter is used to isolate the emissions of SNARF-DE and Cr-YAB.
Method A modified Dual Lifetime Referencing methodwas used to obtain the unbiased signals in each pair of probes. Measurement at two modulation frequencies (12 and 22 kHz for the CO 2 /O 2 sensor and 1.5 and 4.5 kHz for the pH/temperature sensor) provides the decay times for the phosphorescent component (in our case of the oxygen indicator and of the temperature probe, respectively) which are independent on the amount of fluorescence:
[formula] t ¼ 1 2p f 2 1 À f 2 2 AE ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi f 2 2 À f 2 1 À 4 cotΦ 2 Á f 2 Á f 2 1 À cotΦ 1 Á f 1 Á f 2 2 À Á cotΦ 2 Á f 2 À cotΦ 1 Á f 1 ð Þ q 2 cotΦ 2 Á f 2 Á f 2 1 À cotΦ 1 Á f 1 Á f 2 2 À Á 0 @ 1 Að1Þ [/formula]
where Φ 1 and Φ 2 are the overall phase shifts at modulation frequencies f 1 and f 2 .
Once the decay times of the long-lived component are known, the phase shifts for this component can be calculated (Φ phos =arctan(2π⋅f⋅τ)) and the following equa-tion be used to access the amplitude of the fluorescent indicator (A fluor ):
[formula] cotΦ ¼ cotΦ phos þ 1 sin Φ phos Á A fluor A phosð2Þ [/formula]
The amplitude of the phosphorescent indicator (A phos ) is determined from the calibration plots obtained for luminescence intensity: A phos =I phos ⋅dm, where dm is demodulation.
It should be mentioned here that although we used the frequency domain method, the sensor can also be interrogated in the time domainand used for 2D imaging of the analyte distribution.
Apparatus The measuring unit is composed of 2 PMTs, a lock-in amplifier and a 4-armed optical fiber. Evidently, the current interrogation set-up is not compact which may be inconvenient for practical applications. A much more compact device can be constructed if the PMTs are substituted by photodiodes. Further, the four-armed optical fiber can be replaced with a two-armed fiber combined with a dichroic mirror. As far as the excitation sources are concerned, the two LEDs can be replaced by a single dually emitting blue/red LED or an RGB-LED in which two colors (blue and red) can be used for interrogation of the sensor. In case of commercially available dually emitting LEDs (see e.g., www.pur-led.de) the emission wavelengths (λ max 465 and 625 nm) are not completely identical to those used in the current set-up but they are still adequate for interrogation of the probes. It should be mentioned here that in case of dually emitting or RGB LEDs the use of a color subtraction filter along with a long-pass filter is likely to be necessary in order to eliminate the long-wavelength emission component of the blue light. Such filters (absorbing the light between 500 and 615 nm) are commercially available (see e.g., www. edmundoptics.com). [fig_ref] Figure 5: Calibration plots for the temperature probe in the multi-analyte sensor [/fig_ref] shows the calibration plots for temperature. As can be observed, the decay time of the temperature probe calculated according to Eq. 1 is influenced neither by oxygen nor by carbon dioxide. This demonstrates that carefully selected optical properties of the indicators, excitation sources and optical filters completely eliminate possible optical cross-talk from the O 2 /CO 2 system. On the other hand, pH of the solution significantly affects the temperature calibration plots [fig_ref] Figure 5: Calibration plots for the temperature probe in the multi-analyte sensor [/fig_ref]. Since the temperature probe itself is completely inert to pH changes, the origin of this cross-sensitivity originates entirely from the calculation algorithm. In fact, Eq. 1 is valid only for indicators having strictly mono-exponential luminescence decay and a deviation from this will result in error in decay time determination. As one can observe, the calculated decay time decreases at higher pH, i.e., when the amount of fluorescence increases. Based on this observation, decreasing of the amount of the pH indicator relative to the temperature probe may minimize this cross-talk. Nevertheless, since the pH is determined as well, the cross-talk can be easily compensated for. This can be performed, e.g., with the help of a 3D calibration plot shown in [fig_ref] Figure 5: Calibration plots for the temperature probe in the multi-analyte sensor [/fig_ref] which describes the dependency of the calculated decay time on both temperature and pH. Evidently, several iterations are required to obtain the correct temperatures and pH values. It should also be mentioned here that if the sensor is interrogated in time domain, the short-lived fluorescence is eliminated completely after a short delay and cannot affect the luminescence decay of the long-lived temperature probe. Therefore, no cross-talk to pH is expected to be observed with this method. Spectral properties of the materials and optical components used in the multi-analyte sensor: a the absorption spectra of the pCO 2 and pO 2 probes; b the emission spectra of the same probes; c the absorption spectra of the pH and temperature probes; and d the emission spectra of the same probes. For convenience, the emission spectra of the LEDs and the transmittance spectra of the optical filters are also shown within on same scale
## Temperature sensing
Sensing of pH [fig_ref] Figure 6: Temperature dependence of the calibration plots of the pH probe in the... [/fig_ref] shows the calibration plots obtained for the pH probe at different temperatures. The amplitude of the pH probe (A fluor ) represents a referenced parameter which does not depend on such settings as the intensity of excitation light, PMT voltage etc. Evidently, knowing temperature is essential here, since the phase angle of the temperature probe Φ phos and the amplitude of the same probe A phos in Eq. 2 are determined by the luminescence decay time. It is also evident that temperature has pronounced influence on the acidity constant of the pH indicator and the pK a decreases as the temperature increases. Therefore, it is absolutely essential to know temperature for precise determination of pH with the luminescent sensors. The pK a value of the pH probe at 20°C was found to be 7.7. Considering this value, the sensor retains sufficient dynamics both at pH intervals of 6.5-7.5 and 7.5-8.5, and thus, is potentially suitable for physiological measurements and for applications in marine science, respectively.
It should be mentioned here that optical pH sensors are known to be cross-sensitive to ionic strength. This crosssensitivity can vary from very high (in case of highly charged indicatorsto virtually negligible. Crosssensitivity to ionic strength also is influenced by the nature of polymer and typically is higher for the polymers bearing charged groups rather than for neutral ones such as hydrogel D4. Previously, we demonstrated that in case of seminaphthorhodafluor decylester, which is also used in this work, the cross-sensitivity to ionic strength is very low. In fact, the change in the apparent pK a value does not exceed 0.1 units on going from 0.1 to 0.72 mM. Since the ionic strength does not influence the performance of the carbon dioxide, oxygen, and temperature probes, the overall effect on the performance of the multi-analyte sensor is virtually negligible. demonstrates the calibration plots obtained for the oxygen probe in the multi-analyte sensor. As can be seen, the calculated decay times of the oxygen probe are not influenced by the pH of the analyzed solution. Carbon dioxide partial pressure does not influence the calculated decay time of the oxygen probe either which confirms that Eq. 1 works very well in this case. On the other hand, temperature affects the decay times of the phosphorescent indicator (τ 0 =10.05, 9.93, and 9.58 μs at 5, 20, and 50°C, respectively) and has a pronounced effect on the Stern-Volmer plots . This behavior is typical for luminescent oxygen indicators dissolved in polymeric matrices. As can be observed, the Stern-Volmer plots are not linear which indicates localization of the indicator in two regions having substantially different microenvironments. The so called "two-site model" proposed by Demas and co-workers is commonly used to describe such behavior: 3D software using the following empirical equation: τ=a+b⋅(T)+c⋅(T) 2 +d⋅(pH)+e⋅(pH) 2 +f⋅ (pH), where a-f are numerical coefficients where f and 1−f are the fractions of the total emission for the first and the second component, respectively, and K SV 1 and K SV 2 are the Stern-Volmer constants for each component. We used the simplified equation (the K SV 2 is assumed to be 0) to fit the decay time plots. Despite the fact that Eq. 3 has physical meaning only for the ratio of luminescence intensities and not for the decay times, the fit was found to be adequate (correlation coefficient>0.998). The coefficient f is calculated to be 0.76±0.02 for all the temperatures and the K SV constant are 0.150, 0.194, and 0.25 kPa −1 for 5, 20, and 40°C, respectively. Notably, the sensitivity to oxygen for the iridium(III) coumarin embedded into the cross-linked beads is significantly higher than for the same dye in polystyrene(K SV =0.043 kPa −1 ; τ 0 /τ=1.59 at 25°C and 19.8 kPa) which is likely to be due to higher free volume in the crosslinked beads.
## Oxygen sensing
[formula] I I 0 ¼ t t 0 ¼ f 1 þ K 1 SV O 2 ½ þ 1 À f 1 þ K 2 SV O 2 ½ð3Þ [/formula]
Carbon dioxide sensing The calibration curves for carbon dioxide sensing are shown in [fig_ref] Figure 8: Calibration plots for carbon dioxide probe in the multianalyte sensor [/fig_ref]. Evidently, the calibration is not influenced by pH. However, the amplitude of the carbon dioxide probe was found to be affected by oxygen. Since oxygen has no effect on the fluorescence intensity of the HPTS(TOA)it is evident that Eq. 2 is not fully adequate in this case. In fact, Φ phos depends on the decay time of the oxygen indicator and A phos depends both on its decay time (which is used to calculate the demodulation) and luminescence intensity. The latter parameter should be determined independently for the oxygen probe alone . The potential errors arise from the fact that the above frequency domain algorithm allows calculating only the average decay times of the oxygen probe which is only a rough approximation. However, some deviation from mono-exponential behavior is likely in the presence of oxygen and this effect is not easily predictable. Therefore, it is easier to empirically compensate the cross-talk to oxygen since its partial pressure is known. It is also quite evident that temperature dramatically affects the sensitivity of the carbon dioxide [fig_ref] Figure 8: Calibration plots for carbon dioxide probe in the multianalyte sensor [/fig_ref] which decreases when temperature is increased. Such behavior of "plastic" carbon dioxide sensors is well documented in the literaturebut is rarely compensated for. In terms of sensitivity, the sensor is suitable for monitoring of atmospheric carbon dioxide albeit with rather low sensitivity (A 0 /A=1.06 and 1.18 at 20 and 5°C, respectively). Further improvement in sensitivity of the HPTS-based sensors is rather problematic so that other fluorescent indicators with higher pK a values may be needed. Indeed, as was demonstrated by Mills and Chang for the triphenylmethane dyes the sensitivity of the plastic carbon dioxide sensors improves with increase in pK a values. Unfortunately, the fluorescent pH indicators with pK a values higher than that of HPTS are not readily available. On the other hand, the sensitivity of the sensor can be reduced (to make it more adequate for measurements of blood gases) by using ethylcellulose with lower ethoxyl content and by using less bulky quaternary ammonium bases.
Response times One of the potential problems associated with the multi-layer sensors is their longer dynamic response. shows the response and recovery curves for varying pO 2 , pCO 2 , and pH. As can be seen, the additional pH/temperature layer and silica gel beads significantly increase the response and the recovery times for oxygen and carbon dioxide compared to the dual pO 2 / pCO 2 sensor. In fact, t 95 values (time needed for 95% of the signal change to occur) were found to be 10 s (0 kPa O 2 → 21 kPa O 2 ) and 12 s in the reverse direction for the dual sensor and 19 and 22 s, respectively, for the multi-analyte sensor. The sensor responds slower to carbon dioxide and the t 95 values are 35 s (0 kPa CO 2 →2.5 kPa O 2 ) and 122 s in the reverse direction for the dual sensor and 49 and 274 s, respectively, for the multi-analyte sensor. Much slower recovery times for carbon dioxide are explained by high sensitivity of the sensor so that even small remaining concentrations of CO 2 still produce significant drop in the amplitude.
Considering the response of the multi-analyte sensor to pH, the diffusion in the outer pH/temperature layer is not influenced by the O 2 /CO 2 layer so that the sensor responds virtually identical to the dual sensor. The t 95 values were found to be 12 s for changing from pH 5.5 to 8.5 and 34 s in the reverse direction.
Due to the physical nature of the process which does not involve the diffusion of the analytes the response of the multi-analyte sensor to temperature can be, in principle, very fast. Evidently, it is limited by the time needed for the sensor to warm or to cool. Therefore, this value will depend on the thickness of the Mylar support (which significantly exceeds the thickness of the sensing layers) but also on the form and diameter of an optical fiber used.
Chemical and photochemical stability Evidently, any multi-analyte sensor is expected to inherit the problems associated with the individual probes. Therefore, chemical and photochemical stability of the indicators should always be considered. For example, all optical carbon dioxide sensors are irreversibly poisoned by some acidic gases such as HCl. The sensors show cross-sensitivity to other acidic gases, e.g., SO 2 or H 2 S. When slowly oxidized by oxygen, these species may irreversibly poison the sensor. As a consequence, the carbon dioxide sensors are also crosssensitive to sulfite and sulfide ions present in solution. Therefore, to avoid poisoning in the lab atmosphere the sensors are best stored in a sealed pack filled with carbon dioxide. On the other side, the stability of the pH indicator SNARF-DE deteriorates at pH>10 due to hydrolysis of the ester bond. These limitations should be always considered. The oxygen and the temperature probes are much more robust chemically but for the former photobleaching can be a critical issue as discussed in the following. Moreover, the photobleaching of the other probes may be enhanced in the multi-analyte sensor, e.g., due to production of singlet oxygen. Consequently, the probes located in the proximity Response of the multi-analyte sensor to changes in pO 2 (a), pCO 2 (b) and pH (c). The response to all analytes is measured in aqueous solutions at RT to the pO 2 probe (in our case HPTS(TOA)for carbon dioxide measurements) may show much faster photobleaching. provides an example how photobleaching affects the calibration of the pO 2 /pCO 2 dual sensor (and, similarly, the multi-analyte sensor). As can be seen, photobleaching affects the luminescence amplitude to a much higher extent than the luminescence phase shift. This indicates that the photobleaching rates of both probes are similar and the ratio of the two luminescences does not change significantly. The iridium(III) coumarin complex still shows slightly faster bleaching rates than HPTS(TOA)since the overall phase shift decreases. As expected, the sensor drift depends on the overall irradiation time. The drift is rather fast if continuous irradiation is performed. In case of 200 ms irradiation (sufficient for acquiring phase shifts at two modulation frequencies) for each 6 s the drifts are much slower. Finally, virtually no drift is observed after 2.5 h if the acquisition is performed once in 50 s. It is also evident that photobleaching is much faster in presence of oxygen which confirms that singlet oxygen plays an important role in photobleaching pathways. Therefore, addition of singlet oxygen scavengers or quenchersmay significantly reduce the photobleaching rates but possible effects on the sensor chemistries (e.g., quenching) should be considered. On the first glance, it is also evident that the use of more photostable indicators will reduce the drifts caused by photobleaching. However, this is only true if the photostability of both indicators is improved to a similar extent. Significant improvement in the photostability of a single probe is expected to have a negative effect since the bleaching will result in faster change of luminescence ratio.
In case of the multi-analyte sensor, the temperature probe is virtually inert to photobleaching and it does not generate singlet oxygen so the drift in the calibration is determined by the photostability of the pH indicator. Singlet oxygen produced by the oxygen probe does not affect the pH indicator since the diffusion ways are too long and the lifetime of singlet oxygen is rather short. However, considering other sensor designs (e.g., when all the probes are contained in a single layer) the bleaching by photosensitized singlet oxygen is expected to affect all the probes.
# Conclusions
A quadruple sensor is reported for the first time and the proof of concept is demonstrated. The sensor is capable of monitoring important chemical parameters and temperature which allows the other three probes to be compensated for the temperature effects. Two spatially separated sensing layers (pO 2 /pCO 2 and pH/temperature) are interrogated independently by using a pair of LEDs and emission filters. The effect of photobleaching on the overall amplitude and overall phase shift (f= 12 kHz) for the dual pO 2 /pCO 2 sensor excited with the 605-nm LED. Conditions: 1 0 kPa O 2 , 0 kPa CO 2 ; 2 0 kPa O 2 , 2 kPa CO 2 ; 3 12 kPa O 2 , 0 kPa CO 2 ; 4 12 kPa O 2 , 2 kPa CO 2 ; 5 20 kPa O 2 , 0 kPa CO 2 pCO 2 and pH, respectively) and a probe with a long-lived luminescence (for pO 2 and temperature, respectively) which also serves as a reference for the fluorescent probe. The decay times of the luminescent probes are extracted from measurements at two modulation frequencies. The interrogation algorithms are adequate but some empirical correction is necessary presumably due deviation of the luminescence decay profiles from mono-exponential law. It is found that an additional outer pH/temperature layer roughly doubles the response times of the multi-analyte sensor to oxygen and carbon dioxide compared to the response of the dual pO 2 / pCO 2 sensor. Photobleaching of the probes is found to alter the calibration plots and should be taken in consideration. Particularly, photodegradation of both pO 2 and pCO 2 probes is much faster in presence of oxygen. The multi-analyte sensor can be suitable for monitoring of chemical parameters in marine research and for analysis of blood gases and other applications are certainly possible.
[fig] Figure 3: Cross-section of the multi-analyte sensor (not to scale) [/fig]
[fig] Figure 5: Calibration plots for the temperature probe in the multi-analyte sensor: a at varying oxygen and carbon dioxide content; b at varying pH. The luminescence decay times are calculated using Eq. 1 (modulation frequencies 1.5 and 4.5 kHz). The fit of the 3D dependency is performed in the [/fig]
[fig] Figure 6: Temperature dependence of the calibration plots of the pH probe in the multi-analyte sensor (modulation frequency f=1.5 kHz, air saturation, 0% CO 2 ) [/fig]
[fig] Figure 8: Calibration plots for carbon dioxide probe in the multianalyte sensor: left at varying pO 2 and pH; right at varying temperature sensor [/fig]
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A Novel Temporary Anchorage Device Aided Sectional Mechanics for Simultaneous Orthodontic Retraction and Intrusion
Closing spaces on light wires with inadequate knowledge and inappropriate mechanics can cause a "roller coaster" effect leading to an improper occlusion. Current knowledge of biomechanics, along with the incorporation of TADs, has made this process less challenging and more predictable. Resistance to sliding is considered the most prominent inhibitor of space closure in archwireguided space closure or sliding mechanics, in turn delaying treatment duration considerably. In our case, resistance to sliding, primarily binding of the wire in the bracket slot, was nullified with the use of loop and sectional mechanics. This case report is aimed at showcasing the successful treatment of a young lady with a novel clinical setup to retract the canines into the premolar extraction space and simultaneously retract and intrude the anterior segment using sectional archwires and TADs in just under nine months. The current setup with sectional wires and TADs produced an uprighting, and an intrusive effect on the upper incisors during space closure. Additionally, the anchorage design avoided any significant change in the vertical dimension during sagittal correction of the Class II malocclusion. The occlusal plane remained almost stable with good amount of uprighting of the lower incisors following lower space closure too. The use of good biomechanical principles helped us achieve all the treatment goals and objectives in a very short period of time.
# Introduction
Closing edentulous sites in orthodontics is a challenging task. Using light wires with inadequate knowledge and inappropriate mechanics in this task can cause a "roller coaster" effect leading to an improper occlusion. Current knowledge of biomechanics, along with the incorporation of temporary anchorage devices (TADs), has made this process less challenging and more predictable.
"Friction" has received much attention in orthodontics, especially when it comes to space closure. Resistance to sliding is considered the most prominent inhibitor of space closure in archwire-guided space closure or sliding mechanics. Resistance to sliding is caused due to three components. These are (1) classical friction, (2) binding, and (3) notching.
Binding and notching have been proved to play an essential role in the resistance to sliding. It is clear from the current literature that resistance to sliding is an unavoidable concern in archwire-guided tooth movement or sliding mechanics, especially in closing extraction spaces and in turn prolongs the treatment duration considerably.
Segmented arch mechanics was proposed to have bettercontrolled tooth movement than archwire-guided tooth movement and is multifactorial, as reflected in the literature. Segmented arch mechanics suggests dividing the dental arch into three major segments (especially in extraction cases). These three segments are one anterior (incisors and canines) and two posteriors (teeth posterior to the extraction site). The canines are retracted first into the extraction spaces, followed by retraction of the incisors. This technique is aimed at minimizing posterior teeth movement forward, also known as maximum anchorage . Unfortunately, this technique also prolongs treatment duration due to its twostep nature.
This case report is aimed at showcasing the successful management of the spaces required to improve the esthetics and function of an adult female through the simultaneous retraction and intrusion of the whole anterior segment. This technique was achieved through the retraction of the canines with the posterior teeth and TADs as anchorage, while the four incisors were retracted and intruded using the TADs combined with sectional archwires in just under nine months.
## Diagnosis and treatment planning
A 25-year-old female patient presented to our office with prior braces. On clinical examination, she presented with a convex profile, vertical growth pattern, slight gummy smile, extracted teeth 1.4 and 2.4, noncoincident midlines, increased overjet of 8 mm, and a deep bite. Canines were retracted halfway through on a 0.016″ nickel-titanium (NiTi) wire on 0.022″ slot MBT prescription brackets.
The patient had brought along her pretreatment records along for evaluation. Initial pretreatment extraoral records revealed a convex profile with incompetent lips. Pretreatment intraoral records revealed an overjet of 5 mm, visible buccal corridors, and narrow maxillary arch with Class II canines on the left and the right. Root stumps in relation to 4.6 and spaces between her lower anterior teeth were present.
Radiographic evaluation of the initial pretreatment records revealed a slightly retrognathic mandible (SNB, 78.88°), steep mandibular plane angle (FMA, 28.94°) with proclined upper and lower incisors (U1-SN, 109.45°; IMPA, 101.07°). Tooth numbers 2.4 and 3.6 had undergone prior endodontic treatmentand.
2.1. Treatment Objectives. The patient had been undergoing orthodontic treatment for two years already before visiting our practice. The patient expressed psychological frustration with the existing treatment due to its duration and the mounting pressure for her need to leave the country in the next nine months. She stressed it enough that she was not willing to continue her orthodontic treatment elsewhere and would have the appliance removed, whether completed or not.
With the patient's wishes in mind, we decided to fabricate realistic goals and objectives, which were to correct the severe deep bite, close the residual spaces on the upper and lower arches to achieve ideal overjet and overbite, achieve Class I canine relationships, and achieve a pleasant profile. One of the objectives was also to maintain space for an extra premolar prosthesis in the 4 th quadrant as there was insufficient time to close the molar space.
## Treatment alternatives.
Three alternatives were constructed in order to achieve the treatment objectives successfully.
(a) The first option would be to bond the second molars and relevel and align the arches followed by using an intrusion arch to correct the deep bite. Then, the canine retraction could be continued on a heavy stainless steel archwire followed by anterior retraction using loop or sliding mechanics by using the posterior segment as anchorage (b) The second option was to bond the second molars and use an intrusion arch to correct the deep bite followed by protraction of the canines. Then, two TADs could be placed between the upper second premolars and first molars (1.5-1.6, 2.5-2.6) followed by en masse retraction by sliding mechanics (c) The third option was to design a mechanotherapy which would allow simultaneous canine retraction using a section of wire with a closing loop along with simultaneous anterior intrusion and retraction using sectional wires and three TADs. Two of which would be placed between the upper first and second molars (1.6-1.7, 2.6-2.7) and the third between the two upper central incisors (1.1-2.1)
After careful consideration of all the three treatment options, the third option was finalized due to the given time constraints and was thoroughly explained to the patient. Posterior segments consisted of a segment of 0:019 ″ × 0:025 ″ SS extending from the first molar to the canines on either side. A closing loop was fabricated on the segment just distal of the canines.
Three TADs (1:6 × 8 mm) were inserted, two of them were placed between the upper first and second molars (1.6-1.7, 2.6-2.7) and the third between the two upper central incisors (1.1-2.1).
## Activation.
Elastic traction using a power chain from the posterior TADs was directed to the hooks fabricated on the anterior segment of wire just distal to the lateral incisor brackets in order to retract the anterior segment. The force was measured to be 150 grams per side using a Dontrix force gauge. Elastic traction using an elastic thread was applied from the anterior miniscrew to the sectional archwire between the two incisors in order to prevent the uncontrolled tipping, which would be a direct result of the posterior traction. At the same time, this would help intrude the anterior segment eventually correcting the deep bite. The force for intrusion was measured to be 60 grams.
The posterior segment was activated by cinching the wire posteriorly distal to the first molar and anteriorly mesial to the canines, thereby activating the closing loop, eventually 2
Case Reports in Dentistry retracting the canines. A passive ligature was tied from the posterior TADs to the second premolar brackets for indirect anchorage in order to prevent molar mesialization during canine retraction. In six months, the anterior segment and the canines were retracted, and the deep bite was also corrected. The lower arch was stabilized passively with a 0:019″ × 0:025″ SS wire from the third month itself as the arch had leveled and the minor spaces had closed early in the treatment.
## 2.3.3.
Leveling and Alignment. Following space closure, the arch was releveled using a 0:017 ″ × 0:025 ″ NiTi archwire for two months. The wire was tightly engaged in the bracket slots, and a power chain was placed from the upper first molar to the first molar on the other side to prevent space opening. This helped in paralleling the roots without opening up spaces.
## Finishing and retention.
Finishing was done on a 0:019 ″ × 0:025 ″ SS with light settling elastics following which the brackets were debonded. The anterior TAD was not removed and still kept in place to aid in the retention.
Essix retainers were planned on the upper and lower arches with the addition of a lingual button embedded in the upper Essix between the two central incisors. The patient was informed to wear the retainers for 24 hours for the next 2 years. In addition, she was instructed to attach a light intermaxillary elastic (3/16 ″ , 2 Oz) from the anterior TAD to the button on the Essix at night for the next two years to retain the intrusion of the anterior segment.
# Results
A balanced profile with an esthetic and pleasing smile was achieved along with harmony between the upper and lower lips, lip competence, and bilateral Class I canine relationships. The dental midlines were corrected, and no muscle or joint problems had developed during the treatment.
A panoramic radiograph taken after debonding showed acceptable root angulations with no evidence of root resorption, and stable bone levels.
Posttreatment cephalometric analysis showed that the sagittal jaw relationship improved while facial height remained almost constant. There was a marked decrease in the upper and lower incisor inclination (IMPA, 97.48°; U1-SN, 100.76°) . The overjet was corrected, addressing the patient's initial complaint of protrusive teeth and protruding lips.
# Discussion
Poor choice of dental procedures, incorrect treatment indication, adoption of hazardous treatment strategies, inadequate treatment performance, wrong estimation of treatment time, not changing treatment plan when necessary, and not establishing good communication with the patient are failures that may significantly affect outcomes, quality, and stability of correction. Moreover, these factors delay the treatment by a significant amount of time.
Retracting teeth on a light, flexible, round NiTi wire with large forces causes a phenomenon called "roller coaster" effect. The "roller coaster" effect is observed when a wire Case Reports in Dentistry of low strength such as NiTi archwire is used for canine retraction. NiTi does not have the stiffness to remain rigid when a retracting force such as an elastic chain is stretched from the molar to the canine. The molar and premolar crowns tend to tip mesially and extrude distally. The flexible NiTi then bends gingivally and, as a result, tends to tip the canine crown distally. The orientation of the canine bracket when the crown tips distally tend to extrude the incisors and deepen the bite. The use of proper biomechanics to overcome the iatrogenic side effects and satisfactorily complete the case is paramount in such situations.
In our case, prior use of high forces to retract the upper canines bilaterally on a 0.016 ″ NiTi wire resulted in such "roller coaster" effects. In the recent decade, many studies have shown a surge in the usage of TADs as direct anchorage for correction of transmigrated lower canines, upper 5 Case Reports in Dentistry canine impactions, and molar uprightingwith the aid of sectional mechanics to nullify the unwanted side effects which accompany the use of archwire-guided mechanics or the usage of opposing dental components as anchorage.
The current clinical biomechanical setup would be more of a customization of the required mechanics to achieve the ideal treatment objectives. Since our main problem was time underscored by the demands and duration of pre-paring the anchorage, segregating the maxillary arch into 3 segments (1.6-1.3; 1.2-2.2; 2.3-2.6) was needed in order to prudently negate the demands of time, anchorage, and force vectors required to achieve the ideal treatment objectives. By performing this, the canines could be positioned in a Class I relationship by the independent utilization of the second premolars, and first molars reinforced with indirect anchorage from the posterior TADs as anchorage. The incisors could Case Reports in Dentistry be retracted and intruded with the use of TADs, which could provide an ideal anchorage to support absolute anchorage demands and provide the ideal force vector required to correct the overjet and gingival show.
In the presented case, resistance to sliding, primarily binding of the wire in the bracket slot, was nullified with the use of loop and sectional mechanics. Power chains from the TADs posteriorly delivered forces of 150 grams to the anterior hooks per side, and the elastic thread from the third TAD (between the upper central incisors) delivered 60 grams of intrusive force to the anterior segment.
The advantage of this setup was that force delivered onto the anterior segment was completely utilized in the retraction and simultaneous intrusion unlike in archwire-guided mechanics wherein most of the force is unaccounted for and is probably lost in binding and notching.
Canine retraction was performed using a segment of 0:019 ″ × 0:025 ″ SS with closing loops distal to the canines with appropriate antirotational bends to prevent the distobuccal rotation of the canines.
Evidence has suggested that anterior intrusion is very hard to retain. Therefore, it was decided to maintain the third TAD (between the upper central incisors) in place during the retention period, and the patient was instructed to wear 3/16 ″ 2 Oz elastics from the TAD to the lingual button incorporated in the Essix between the central incisors at night time during the retention phase.
In our case, this setup with sectional wires and TADs produced an uprighting, and intrusive effect on the upper incisors during space closure (9°reduction in the U1-SN). Additionally, the anchorage design avoided any significant change in the vertical dimension during sagittal correction of the Class II malocclusion and. The occlusal plane remained almost stable (2°increase). There was a good amount of uprighting on the lower incisors (4°d ecrease in the IMPA) following lower space closure. The apt use of sound biomechanical principles helped us achieve all the treatment goals and objectives in under nine months.
# Conclusion
We should always have in mind our limitations and the importance of proper orthodontic training while treating patients. This clinical biomechanical setup of sectional archwires with TADs helped us achieve all the treatment goals (canine retraction, retraction, and intrusion of the anterior segment) in a short period of time.
## Conflicts of interest
The authors declare that they have no conflicts of interest.
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Acuphagia on the Obsessive-Compulsive Spectrum in an Adolescent Male
Background. Pica is a condition that is commonly missed in childhood. This condition occurs worldwide and is considered normal in children from ages 18-to-36 months. It is also commonly seen in pregnant women due to associated nutritional deficiencies. Acuphagia is a subtype of pica which has been briefly described in the literature. Its classification has been speculated to belong on a spectrum of obsessive-compulsive disorders (OCD). This case involves Mr. C, a 16-year-old male with a history of depression, anxiety, and ten previous intentional foreign body ingestions involving sharp objects such as needles, forks, and thumbtacks. He states that he recently ate a nail and denies any current obsessions. He was admitted from a local involuntary receiving facility due to decreased bowel movements in the last week. Learning points and recommendations for practitioners are described.
## Case
Mr. C is a 16-year-old male with a history of depression, generalized anxiety disorder, obsessive-compulsive disorder (OCD), and ten previous intentional foreign body ingestions typically involving sharp objects such as needles, forks, and thumbtacks. He states that he recently ate a nail and denies any current obsessions. He was admitted from a local involuntary receiving facility due to decreased bowel movements in the last week. He usually stools 1-2 times per week but cannot recall the last time he stooled. He states that he has general abdominal discomfort, but it is not sharp or severe. He describes the discomfort "all over" but rubs the periumbilical areas when discussing symptoms. He otherwise denies any vomiting, diarrhea, back pain, or difficulty with urinating. He states that he had "low-grade" fevers at the outside facility. He likes to eat regular, well-varied meals. He takes Colace inconsistently and has never taken MiraLAX daily. Abdominal X-rays were performed and most recently revealed "distention of multiple segments of the colon and concern for ileus." In addition, 16 mm metal density resem-bling a nail was noted to have progressed to the transverse colon. His vitals are stable (Tmax: 100.1; HR: 100 bpm; BP: 131/75 mmHg; RR: 16 bpm; O2 stats 100% in RA). He was given Tylenol 500 mg, PO, for 05/10 abdominal pain.
Of note, the patient denied any recent self-injurious behavior and denies any suicidal or homicidal ideations. He states that he is being escalated to a residential facility in another state. Previous similar episodes have required extraction by GI, including his most recent admission where he ingested a part of the IV.
He is allergic to bananas and takes the following medications: Protonix 40 mg PO qDay; Seroquel 400 mg PO qDay, Klonopin 1 mg PO at bedtime, and Prozac 20 mg PO qDay. He does not forget to take medications. His past surgical history includes three episodes of gastroscopy and esophagoscopy a year ago. His family history is noncontributory. He does not smoke tobacco products or use recreational drugs, but occasionally drinks alcohol and smokes tobacco.
His labs are within expected limits. His review of systems is positive for constipation and abdominal pain in all quadrants. On physical exam, his abdomen is soft, nontender, and nondistended, with no swelling. On this admission, the objects were passed without surgical intervention the following day, and the patient was successfully transferred back to his involuntary facility.
# Discussion
This case is lends support to the literature which suggests that pica is on a spectrum of OCD related disorders. However, it differs from other studies in that this patient has a type of pica termed acuphagia. This subtype involves the involuntary urge to eat sharp objects such as knives or needles. Acuphagia in someone with anxiety leading to ileus has been undocumented. This case is significant in that he reported that his behavior was an involuntary attempt to not feel anxious or depressed. He also mentioned that he was tired of being at the outside facility and hoped to be transferred to our inpatient unit (which only serves adults). Other cases which have speculated that pica is on a spectrum of OCD involve more typical features such as eating paint to soothe anxiety. Acuphagia is unique because one would expect eating sharp objects to make anxiety worse. This gives credit to the idea that all forms of pica may be on a continuum of OCD and anxiety disorders more globally.
Pica is a condition that is commonly missed in childhood. The term is derived from "Pica pica," which is the Latin word for brown-billed magpie. This bird eats indiscriminately, and as a result, will eat foreign objects. This condition occurs worldwide and is considered normal in children from ages 18 to 36 months. It is also commonly seen in pregnant women due to associated nutritional deficiencies. However, it is uncommon in nonpregnant adults. In fact, the DSM-5 explains that eating at least one or more objects for nonnutritive, nonfood substances over a period of one month is enough for a diagnosis of pica in adults. In this case, the patient had ingested over 10 objects within the past year.
Acuphagia is a subtype of pica which has been briefly described in the literature. Its classification is controversial as it has been speculated to belong to a spectrum of OCD. It is worth mentioning that documented cases of acuphagia are scarce. Silvermandescribed one of the first patients in 1973. In this case, the patient was a 2-year-old girl who had a craving for metal objects. This was possibly due to a dietary deficiency, and once her lack of appetite was treated, she was cured of pica after treatment. This is the typical clinical course of pica and documented cases of eating sharp, metallic objects remains uncommon in older patients.
A more recent case by in 2007 described a 22-year-old man in Nigeria presented with vomiting after meals, cough, weakness, inability to walk, and swelling of the legs and face. The authors attribute poverty, isolation, neglect, and loneliness as triggers for his "bizarre" behavior. A case from 2018 involved a young woman with a significant history of schizophrenia presenting with acuphagia. She also ingested several sharp objects which were removed surgically. Our case expands upon those reported to suggest a possible linkage between acuphagia, OCD, and perhaps psychiatric illness more generally.
It is worth noting that Mr. C's acuphagia persists despite his current psychiatric treatment. He has had many inpatient visits over the past few years and is on a stable regime of medications for his depression and OCD. He has also never had a psychotic episode. The connection between OCD and other mood disorders is well studied. In OCD, the behavior is typically irrational at resolving the preceding obsessions. Common themes involve handwashing, touching objects, or possibly hoarding behavior. However, a growing body of evidence demonstrates that these behaviors typically provide a sort of self-calming of internal anxieties. Interestingly, this does not apply to the current case. Eating sharp objects such as nails and needles is inherently anxiety inducing. In this case, the patient's "compulsions" did not alter his underlying anxiety and depression which were being managed with psychotropic medications.
## Guidelines recommendations and learning points.
Little is known about the course of acuphagia, and there are currently limited formal guidelines on the management of this disorder. Current guidelines focus exclusively on causes due to an underlying nutritional deficiency or behavioral therapy for those on the autism spectrum. They emphasize a thorough assessment including a basic metabolic workup. Behavioral recommendations include environmental arrangements (e.g., locks on cabinets), teaching alternative skills (e.g., throwing the objects away), and redirection. Due to the safety risk of acuphagia specifically, these guidelines are incomplete in altering evaluation and management. These limitations warrant further investigation of this unique clinical phenomenon. Adapted guidelines for impulse control and safety suggests the following:
(i) Educating caregivers to arrange home environments with similar safety precautions as inpatient units (e.g., finger foods) (ii) Helping the client identify impulses and notifying the caregiver or sublimating with a safer activity (iii) Engagement in constructive activities to distract the client such as physical activity (iv) Enrollment in mindfulness and acceptance therapy
In conclusion, acuphagia may be a distinct psychological disorder from other forms of pica described in the DSM-5. In this case, it was unrelated to suicidal or homicidal ideations, anxiety, depression, or OCD. It has also been documented in patients with schizophrenia. While there is debate for pica to belong on a spectrum of OCD related disorders, this case suggests that the etiology may be something else. Regardless of classification, it seems to have persisted while his anxiety and depression was well controlled on psychotropic medications. Addressing nutritional needs is the first-line treatment for pica in other populations including pregnant women and children. However, in our case, his nutritional needs were being met, and his mental health disorders were controlled. This suggests that acuphagia may require a unique treatment or higher level of care. Further research into the
Conflicts of InterestThe authors declare that they have no conflicts of interest.
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Integrative multiomics analysis of human atherosclerosis reveals a serum response factor‐driven network associated with intraplaque hemorrhage
1. This multiomics analysis identified a cardiovascular signature in carotid atherosclerotic lesions, which provides excellent stratification of low-/highrisk carotid plaques. 2. This study highlights the advantages of multiomics analysis in terms of model robustness, biological significance, and clinical translatability. 3. The prediction model pointed to an SRF-regulated disease network providing valuable new insights that expedite the design of targeted intervention in plaque rupture.AbstractBackground: While single-omics analyses on human atherosclerotic plaque have been very useful to map stage-or disease-related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale-intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model.Methods:In this study, we compared protein and gene makeup of lowversus high-risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage,This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method. Results: We identified a protein-gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA − PDGFRα + fibroblast-like cell content (p = 2.4E-05) andArg1 + macrophage content (p = 2.2E-04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia-1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts. Conclusions: In conclusion, our integrative omics study has identified an intraplaque hemorrhage-associated cardiovascular signature that provides excellent stratification of low-from high-risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF-regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.K E Y W O R D Scarotid atherosclerosis,
## Foundation, grant/award numbers: introduction
Atherosclerosis and associated cardiovascular diseases remain one of the leading causes of death worldwide. [bib_ref] Global and regional prevalence, burden, and risk factors for carotid atherosclerosis: a..., Song [/bib_ref] In the clinically relevant stage, atherosclerotic lesions may transition from a stable, low-risk to an unstable phenotype, at high risk of causing acute ischemic events, such as myocardial infarction and ischemic stroke. [bib_ref] Pathology of the vulnerable plaque, Virmani [/bib_ref] Histopathology and experimental studies and more recently, genomewide association studies (GWAS) and extensive omics searches have considerably enhanced our understanding of this disease's pathogenesis. [bib_ref] Progress and challenges in translating the biology of atherosclerosis, Libby [/bib_ref] So far, the latter studies have largely relied on global profiling of mRNA expression or protein abundance in plaque tissue and were primarily designed to dissect differential expression, leading amongst others to the identification of plaque-contained biomarkers correlating with future cardiovascular events (e.g., osteopontin). [bib_ref] Identification of two genes potentially associated in iron-heme homeostasis in human carotid..., Ayari [/bib_ref] [bib_ref] Local atherosclerotic plaques are a source of prognostic biomarkers for adverse cardiovascular..., De Kleijn [/bib_ref] [bib_ref] Prediction of ischemic events on the basis of transcriptomic and genomic profiling..., Folkersen [/bib_ref] For instance, based on mRNA expression data from the Biobank of Karolinska Endarterectomies (BiKE) cohort, [bib_ref] Gene expression signatures, pathways and networks in carotid atherosclerosis, Perisic [/bib_ref] were able to identify novel gene networks operating in the atherosclerotic plaque. [bib_ref] The use of network analyses for elucidating mechanisms in cardiovascular disease, Diez [/bib_ref] A limitation of such global approaches is that they often fail to reveal detailed information about a particular cellular component in plaques. Laser-capture microscopy 9,10 and single-cell RNA sequencing-based analysis of atherosclerotic plaque macrophages [bib_ref] Single-cell RNA-seq reveals the transcriptional landscape and heterogeneity of aortic macrophages in..., Cochain [/bib_ref] [bib_ref] Atlas of the immune cell repertoire in mouse atherosclerosis defined by single-cell..., Winkels [/bib_ref] [bib_ref] Transcriptome analysis reveals nonfoamy rather than foamy plaque macrophages are proinflammatory in..., Kim [/bib_ref] bypassed this pitfall. These studies allowed assessment and deconvolution, respectively, of the expression profiles of relevant plaque-contained subsets, and for the latter confirmed the strong proinflammatory nature of non-foamy plaque macrophages. [bib_ref] Transcriptome analysis reveals nonfoamy rather than foamy plaque macrophages are proinflammatory in..., Kim [/bib_ref] However, for indepth dissection of critical processes in plaque, mere transcriptional profiling may fall short incorporating net biosynthesis and protein activity, whereas proteomics profiling alone will bias toward most abundant, rather than most relevant proteins, implying that both scales have serious shortcomings. [bib_ref] Investigating the correspondence between transcriptomic and proteomic expression profiles using coupled cluster..., Rogers [/bib_ref] Moreover, protein and mRNA profiles are notorious for their rather poor mutual correlation, [bib_ref] Comparative analysis of proteome and transcriptome variation in mouse, Ghazalpour [/bib_ref] [bib_ref] Methods for the integration of multiomics data: mathematical aspects, Bersanelli [/bib_ref] complicating the translation of genomics findings to clinical practice. Therefore, it is important to interrogate plaques by multiple omics and to evaluate the integrated model for biological and/or clinical implications. [bib_ref] Multi-omics approaches to disease, Hasin [/bib_ref] To this end, significant efforts have already been made, and a few recent studies have illustrated the potential of such approaches. [bib_ref] Novel multiomics profiling of human carotid atherosclerotic plaques and plasma reveals biliverdin..., Matic [/bib_ref] [bib_ref] Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques, Langley [/bib_ref] [bib_ref] Altered metabolism distinguishes high-risk from stable carotid atherosclerotic plaques, Tomas [/bib_ref] Applying differential expression and intersect analyses [bib_ref] Novel multiomics profiling of human carotid atherosclerotic plaques and plasma reveals biliverdin..., Matic [/bib_ref] and/or linking omics with genetics data, [bib_ref] Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques, Langley [/bib_ref] [bib_ref] Altered metabolism distinguishes high-risk from stable carotid atherosclerotic plaques, Tomas [/bib_ref] these studies largely ignored mutual interaction of gene and/or proteins, and, for the former, did not truly integrate the data scales in a multivariate manner. [bib_ref] Integration of omics: more than the sum of its parts, Buescher [/bib_ref] Therefore, in the current study we set out to integrate the protein, peptide, and mRNA makeup of carotid artery lesions from the Maastricht Human Plaque Study (MaasHPS) [bib_ref] Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to..., Goossens [/bib_ref] of symptomatic carotid endarterectomy (CEA) patients. We interrogated a total of 42 carotid artery lesions, based on the presence/absence of intraplaque hemorrhage (IPH), a hallmark of plaque stability and event risk. [bib_ref] Intraplaque haemorrhages as the trigger of plaque vulnerability, Michel [/bib_ref] [bib_ref] Composition of carotid atherosclerotic plaque is associated with cardiovascular outcome: a prognostic..., Hellings [/bib_ref] By integrative machine learning [bib_ref] Lê Cao K-A. mixOmics: an R package for 'omics feature selection and..., Rohart [/bib_ref] we were able to identify a composite disease stage classifier, the performance of which was benchmarked against single-omics derived classifiers using only mRNA expression or protein/peptide abundance and validated in several independent plaque datasets (GEO accession numbers: GSE28829, GSE43292, and GSE21545).
# Methods
## Sample collection and classification
Atherosclerotic plaque samples obtained during carotid endarterectomy from 24 symptomatic patients were collected. The endarterectomy specimens were cut into parallel, transverse segments of 5-mm thickness. Each alternating segment was snap-frozen in liquid nitrogen and stored at −80 - C, their flanking segments fixed for 24 h in formalin, decalcified for 4 h before processing and embedding in paraffin for histological evaluation. Hematoxylin-eosin (H&E) stained plaque tissue was macroscopically preclassified for plaque stage before omics experiments. Segments were stratified into non-IPH and IPH groups according to the absence or presence of IPH. For each CEA specimen and patient, two samples (one non-IPH and one IPH) were collected for omics experiments (n = 24 at the start for each). However, one IPH and four non-IPH samples did not pass the QC test and were excluded from analysis (see the following sections). Simultaneously with the microarray performance for the omics experiments, but prior to bioinformatic analysis, tissue was sectioned further for additional staining and computer-aided quantitative measurement of plaque IPH. Based on this indepth reinspection, the three experienced pathologists (MJAPD, JCS, MJJG) agreed to remove one ambiguous "non-IPH" sample, showing small but surfacedetached luminal fibrin clot, as this could represent either a surgery artefact or bona fide IPH. In three allegedly "non-IPH" samples, quantitative morphometry detected minor IPH (0.43%, 0.40%, 0.33%, respectively, data not shown), which was overlooked in the preclassification. These three were recategorized as IPH after inspection by the pathologists. Due to the recategorization of samples from the non-IPH to the IPH group, three pairs of the total 26 samples of the IPH group were from three patients, respectively. To explore the potential confounder effects of this adjustment, we performed hierarchical clustering of the samples based on the plaque traits (measured as described in Section 2.2). This analysis convincingly showed that there is no patient-specific heterogeneity among the samples, as well as that plaque phenotype is dominant over sample origin (data not shown). The final cohort for this study therefore included transcriptomics, proteomics, and peptidomics for 16 non-IPH and 26 IPH plaques (see ,B for flow scheme of cohort buildup). For detailed information on the patient cohort definition, see .
## Morphometry and immunohistochemistry
All H&E slides were photographed at a 12.5× magnification and examined digitally using Leica Q500MC software. Total tissue, media, cap, necrotic core, hemorrhage, and luminal thrombus area were measured on H&E. Plaque size was calculated by subtracting the medial and luminal thrombus area from that of the total carotid tissue. Necrotic core and hemorrhage area were quantified relative to the plaque size. Cap thickness was measured at three to 15 positions throughout the entire cap region, at regular intervals. In addition, we measured the relative area of the following histological features: CD31 + endothelial cell content (% of total plaque area), CD3 + T-cell content (% of total plaque area), CD68 + macrophage content (% of total plaque area), iNOS + (M1) macrophage content (% of CD68 + macrophage area), Arg1 + (M2) macrophage content (% of CD68 + macrophage area), collagen content (% Sirius red of total plaque area), αSMA + smooth muscle cell (SMC) content (% of total plaque area), αSMA -PDGFRα + fibroblast-like cell content (% of total plaque area), and calcification (% Alizarin red of total plaque area). These histological features are denoted as plaque traits, their distribution and statistical differences between paired non-IPH and IPH plaques are shown in . Moreover, a correlation heatmap was created to explore the relationships between the plaque traits [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref].
For the MFAP4 immunohistochemistry for validation purpose, 4-μm thick sections were cut from 35 plaques (13 non-IPH, 22 IPH plaques) left available from the total 42 plaques. Deparaffinization and rehydration was performed using xylene and graded ethanol. Endogenous peroxidase F I G U R E 1 Workflow of carotid sample collection and data analysis. (A) Entire carotid endarterectomy specimen was cut in parallel 5-mm thick slices, snap-frozen in liquid nitrogen, and stored until use. Every second slice was sectioned. After H&E staining, sections were categorized using the criteria of Virmani et al., [bib_ref] Lessons from sudden coronary death, Virmani [/bib_ref] and were further classified histologically based on the presence/absence of IPH, resulting in 16 non-IPH and 26 IPH plaques for omics analysis. Workflow for single-and multiomics integrative sPLS-DA analyses are shown in the orange dashed square. The integrative sPLS-DA prediction model was obtained by connecting transcriptomics, proteomics, and peptidomics as an entirety; m: number of samples; n1, n2, n3: number of features. (B) Flow scheme of the MaasHPS cohort build-up and the criteria for sample exclusion. In total, 42 samples (16 non-IPH and 26 IPH plaques) were included in this study was blocked with 0.3% H 2 O 2 for 15 min, followed by heatinduced antigen retrieval in a microwave for 10 min in a low pH target retrieval solution (K8005, DAKO, Agilent Technologies). After cooling down for 20 min, sections were incubated overnight at 4 - C with polyclonal rabbit anti-human MFAP4 antibody (1:1000, NBP2-30439: Novus Biologicals). After washing in TBS, sections were incubated for 1 h at room temperature in Brightvision poly-HRP anti-rabbit IgG (ImmunoLogic, VWR KDPVR55 HRP). After washing in TBS, staining was developed using the chromogen 3,3′-diaminobenzide (DAB, DAKO K3468) for 5 min, and sections were counterstained with Mayer's hematoxylin (VWR International B.V., Amsterdam, The Netherlands), dehydrated, mounted, and cover slipped. Sections were digitized using the Pannoramic 1000 scanner (3DHistech Ltd.) at 20× magnification. Morphometric analysis was done using the free software package QuPath (v0.2.3) to measure percentage area positivity/total area.
## Sample preparation for transcriptomics and proteomics/peptidomics
Snap-frozen omics segments were pulverized and 5-20 mg of material was subjected to transcriptomic, proteomic, and peptidomic analysis.
RNA was isolated by guanidium thiocyanate extraction, followed by further purification using the Nucleospin RNA II kit (Macherey-Nagel GmbH & Co. KG). RNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). RNA quality and integrity were determined on the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Pure RNA samples that had an RNA Integrity Number (RIN) greater than 6.0 and a sample purity A260/280 ratio greater than 1.8 were taken for transcriptomic analysis (45 out of 48 samples). One analysis for a non-IPH plaque had failed, therefore in total four unqualified non-IPH samples were excluded from the microarray experiment.
Proteomic analysis was carried out by utilizing the 8-plex iTRAQ reagent for relative quantification. Reference sample used for relative quantification was created by pooling the redundant specimens derived from the equal portions of the samples for omics experiments. Pulverized tissue samples (5-20 mg) were homogenized in a Covaris E100 ultrasonic homogenizer using 400-μl lysis buffer of 6 M guanidium hydrochloride, 1% Triton-114, 50 mM triethylammonium bicarbonate (TEAB), 50 mM dithiothreitol (DTT), and protease inhibitor tablets (Roche, one-fourth tablet in 10 ml buffer). The homogenate was centrifuged for 15 min at 4000 × g to remove insoluble debris. DTT content of the homogenate was raised to 100 mM and protein reduction was completed through incubation at 70 - C for an hour. Alkylation was completed by adding 0.5 M iodoacetamide and followed by a 30-min incubation at room temperature. The excess alkylating reagent was quenched by addition of 12 μl of 1 M DTT. The protein content of the homogenate was recovered on a poros R1 column using a Vision Chromatography Station (Applied Biosystems). Proteins were eluted with 70% isopropanol/30% formic acid solvent mixture and dried down in a Speedvac.
## Omics data collection
Biotinylated cRNA was prepared using the Illumina Total-Prep RNA Amplification Kit (Ambion, Inc., Austin, TX, USA) according to the manufacturer's specifications starting with 100 ng total RNA. Per sample 750 ng of cRNA was used for hybridization. Hybridization and washing were performed according to the Illumina standard assay procedure. Scanning was performed on the Illumina Bead-Station 500 (Illumina, Inc., San Diego, CA, USA), while image analysis and extraction of raw expression data were done using Illumina Beadstudio v3 Gene Expression software with default settings (no background subtraction and no normalization). Transcripts were measured by Illumina HumanRef-8 v2.0 expression BeadChip. Proteins were resuspended in 2 M freshly prepared urea, 1 M TEAB, 1% n-octyl-glucoside buffer and digested with trypsin added at 1:20 w/w ratio to the sample, for 4 h at 37 - C. Digestion was stopped by increasing the temperature to 95 - C for 5 min. Digested samples were labeled by the 8-plex iTRAQ reagents following the manufacturer's protocols (Applied Biosystems) using a sample digest quantity that represents approximately 40-μg protein content. Samples were labeled with the reagents yielding the m/z 114, 115, 116, 118, 119, 121 reporter fragments in the MS/MS scans. Reference samples were labeled with the 113 and 117 reagents. iTRAQ labeled reactions were quenched by the addition of 1 M ammonium bicarbonate.
Eight samples (three non-IPH, three IPH, and two replicates for QC and reference) constituting an iTRAQ mix were combined, desalted, and fractionated by strong cation exchange (SCX) chromatography using an Agilent 1200 instrument. Eight SCX fractions were injected for HPLC, and resolved over a 90-min gradient of 5% solvent B (10% H2O/90% ACN/0.1% TFA) to 38% B (solvent A: 95% H2O/5% ACN/0.1% TFA). The elution volume was collected onto a plate for mass spectrometric analysis by matrix-assisted laser desorption ionization (MALDI) as 10-s intervals, using 10 mg/ml of alpha-cyano-4hydroxycinnamic acid in 50%-50% acetonitrile-water as the matrix. Each HPLC run was represented as a 500-spot array on the MALDI plate. These plates were analyzed on an AB4800 mass spectrometer (MDS/SCIEX, Concord, ON, Canada).
Peptide quantification was carried out by calculating the average ion intensity ratios relative to the m/z 113 and 117 peaks. Protein ratios were determined as the medians of all peptide ratios matching to the same protein.
Peptide sequences were identified from MS/MS fragmentation spectra using the Mascot search engine (Matrix Science, UK) and the SwissProt database. The whole proteomic/peptidomic analysis was carried out following the same protocols as in a previous plasma proteomics study for cardiovascular disease. [bib_ref] Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI..., Juhasz [/bib_ref] Experimental details and parameters used in this analysis can be found from the paper given above. Once all the study samples were analyzed, the complete peptide set was remapped to a minimum protein set, whereafter proteins and peptides identified were mapped to genes or gene families. The list of protein and peptide entries used in the multiomics integration analysis can be found in .
The corresponding microarray profile of one IPH sample with failed MS measurement (due to technical reasons) was removed. The final MaasHPS cohort therefore included transcriptomics for 16 non-IPH and 26 IPH plaques, with successful proteomics and peptidomics profiling from the same samples.
## Data preprocessing
A total of 22,184 human transcripts and variants as defined by the NCBI Reference Sequence (RefSeq) were analyzed. Transcriptomic data were analyzed using the R Bioconductor lumi package (v2.38.0). First, we performed a variance stabilizing transformation, which is incorporated in the lumi package. Then, the robust spline normalization (RSN) algorithm in the lumi package was applied to normalize the data. As low-variance genes and noise expression will not only reduce the effectiveness of subsequent clustering and machine learning but also slow down computations, we selected the top 10,000 most variable genes from a total pool of 17,759 unique detectable genes, for further analysis. Measured values of the abundance of 1330 proteins and 4736 peptides were normalized using a procedure based on Vandesompele et al. [bib_ref] Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple..., Vandesompele [/bib_ref] Intrinsic to the analysis methodology, datasets were showing a considerable rate of missing values (26.45% and 38.94%, respectively). To reduce the noise and bias affected by features with sub-and perithreshold abundance, we discarded all features with ≥50% missing values. For the remaining features, missing values were imputed by k-nearest neighbors (k-NN) imputation (k = 7) in an unsupervised manner, as this proved superior to other methods in a pilot analysis. [bib_ref] Missing value estimation methods for DNA microarrays, Troyanskaya [/bib_ref] Finally, 10,000 genes, 943 proteins, and 2637 peptides were detectable in the 42 samples (16 non-IPH vs. 26 IPH) and were used for further analysis. Genes, proteins, and peptides were mapped based on the HUGO Gene Nomenclature Committee (HGNC) symbols.
## Single-and multiomics data analysis
For single-omics analysis of the transcriptomic, proteomic, and peptidomic data, we deployed sparse partial least squares-discriminant analysis (sPLS-DA, or single sPLS-DA), 29,30 a multivariate methodology which allows sample classification and feature selection by projecting the data into a lower dimensional space in a supervised manner. While applying this method on omics datasets, which typically have a high-dimensional feature space with limited sample size, this algorithm implements LASSO 31 to limit the number of features used in the model so as to reduce the curse of dimensionality. To enable classification based on multiblock datasets, derived from transcriptomic, proteomic, and peptidomic analysis of plaques, we deployed integrative sPLS-DA using DIABLO provided in the mixOmics R package (http://mixomics.org/, v6.8.5), [bib_ref] Lê Cao K-A. mixOmics: an R package for 'omics feature selection and..., Rohart [/bib_ref] a powerful package shown to extract biologically relevant signatures from multiple omics data by maximizing the sum of the covariance between all pairs of latent components from each dataset and projecting the different omics data into a common space. [bib_ref] Novel multivariate methods for integration of genomics and proteomics data: applications in..., Günther [/bib_ref] Compared with other multiomics machine learning algorithms, this method has several advantages. First, it allows full integration of multiomics data by extracting common information from different omics layers for classification. Second, it enables model-embedded feature selection, which is a great help for identifying disease-specific novel biomarkers. Third, this method provides clear and interpretable visualization, facilitating downstream exploration and interpretation by biologists. The inherent characteristics [bib_ref] Partial least squares for discrimination, Barker [/bib_ref] and the successful applications of sPLS-DA on several microarray data [bib_ref] Prediction of clinical outcome with microarray data: a partial least squares discriminant..., Pérez-Enciso [/bib_ref] [bib_ref] Tumor classification by partial least squares using microarray gene expression data, Nguyen [/bib_ref] suggest the suitability of using this method in our study.
## Parameter optimization
SPLS-DA requires extensive optimization of both the number of components and the number of features for constructing each component. In brief, the number of testing components was set from one to six, as in most cases, the prediction performance deteriorates rapidly when the number of the components increase above five. Then, the number of features to be tested for constructing the sPLS-DA component was configured. For single sPLS-DA on transcriptomics, proteomics, and peptidomics, a range of features from three to 300 (from three to 30 in steps of three, from 30 to 60 in steps of six, from 60 to 150 in the step of 15, and from 150 to 300 in the step of 30) were set for each component. For integrative sPLS-DA, to achieve the comparable number of selected features, the range of testing features here for each component and each omics was set from 10 to 100 (from 10 to 50 in steps of five, from 50 to 100 in steps of 10, in total 14 candidates per component per omics). Specifically, for integrative sPLS-DA, a design matrix representing how close the datasets should be connected to each other needs to be configured. According to Singh et al.'s suggestion, [bib_ref] DIABLO: an integrative approach for identifying key molecular drivers from multiomics assays, Singh [/bib_ref] we configured the design matrix C as the correlation between omics datasets as 0.1, and the correlation between omics dataset and outcome dummy matrix as 1.
Optimal parameters can be derived in one step from the model with the best classification in cross-validation. A series of single and integrative sPLS-DA prediction models were obtained from each combination of these parameter settings, and their performances were tested under stratified five-fold cross-validation with 1000 random repeats. Considering the unbalance of the sample phenotypes, to fairly reflect the classification performance of training models, in this tuning phase we used balanced error rate (BER), which takes the average of the errors on each class to evaluate the classification error. The optimal parameters can be concluded from (for integrative sPLS-DA, data not shown) and are listed in . The performance of the single and integrative sPLS-DA models with the given optimal parameters was evaluated by accuracy and area under the curve of receiver operating characteristic (AUC) under stratified five-fold cross-validation with 10,000 random repeats .
## Feature selection
Inspired by the method used in a previous multiomics study, [bib_ref] Novel multivariate methods for integration of genomics and proteomics data: applications in..., Günther [/bib_ref] to ensure that selected features had broader significance beyond the single best prediction model, we performed stratified sampling with replacement generating 10,000 incomplete copies of the full dataset; each copy covered 80% of the original data, and phenotype distribution was identical to that of the original dataset. We then ran single-and multiomics analysis on each copy, selecting the overall highest ranking features by aggregating the loading weight value (i.e., the importance to the model) of each feature from each copy to obtain the final feature subset. The optimal parameters were used to determine the number of the selected features.
To simplify the downstream analysis, we assembled gene, peptide, and protein identifiers of selected features into a final feature set, unifying the identifiers into the corresponding gene symbol and excluding duplications. The final selections used for overrepresentation analysis, transcriptional regulatory analysis, and validation entailed 291 and 283 gene features, for single and integrative sPLS-DA, respectively. The full lists of the selected features are presented in , sorted by the overall feature importance from high to low.
## 2.9
Bioinformatic analyses
## Differential gene expression analysis
We used the function lmFit() provided in the limma R package (v3.42.2) for differential expression analysis on preprocessed transcriptomics between IPH and non-IPH groups. Positive log2 fold change (Log2FC) values indicate genes have higher expression in IPH group, and vice versa. Results of differential expression analysis for the 17,759 unique detectable genes can be found in 2.9.2 Gene set overrepresentation analysis (GSOA)
We performed GSOA using the R packages clusterProfiler 37 (v3.12.0) and ReactomePA 38 (v1.28.0) identifying biological functions of the selected gene sets. Three categories of GSOA datasets were queried: Gene Ontology (Biological Process GOBP, Molecular Function GOMF, and Cellular Component GOCC), Reactome Pathway Database (REACTOME), and Kyoto Encyclopedia of Genes and Genomes (KEGG). All genes covered by this Illumina microarray platform and all detectable proteins/peptides in the MS experiments were used as the background. Benjamini-Hochberg adjusted p-values were calculated as a cutoff to avoid presenting the false discovery of significant terms. The full list of GSOA results for both single and integrative sPLS-DA can be found in .
## 2.9.3
Transcriptional regulation analysis iRegulon 39 (v1.3), a Cytoscape plugin, was used to map transcription factors (TFs) driving a gene network. The selected features by integrative sPLS-DA were set as input in iRegulon for TF discovery. By setting the default parameters, we have a broad search space including 9713 position weight matrices (PWMs) and 1120 ChIP-seq tracks. The putative regulatory region was set to 20 kb centered around transcription start sites (TSS). Top five TFs were ranked by normalized enrichment score (NES) and visualized as a transcriptional regulatory network.
## Validation cohorts and approach
The predictive power of genes from the multiomics signature was validated in the following independent human carotid endarterectomy cohorts on microarray gene expression: GSE28829 40 (n = 29; 13 early and 16 advanced plaques) and GSE43292 4 (n = 64; 32 plaque-free artery segments and 32 atheromatous plaques). The prediction performance of the gene lists obtained from integrative sPLS-DA components 1-4 (MULTI 1-4, n = 283) and single sPLS-DA components 1-2 (SINGLE, n = 291, for peptidomics only component 1) were tested by logistic regression (solver = "lbfgs"), support vector machine (SVM, kernel = "rbf," gamma = "auto") and decision tree (default settings) with stratified five-folds crossvalidation and 1000 random repeats provided in the scikitlearn machine learning package (v0.24.1) in Python. As the integrative sPLS-DA component 1 (MULTI 1, n = 22) was dominant in our model, we also compared the performance of the MULTI 1 signature with that of MULTI 1-4 signature from the complete four-component model. Classification performance using full data (FULL) was evaluated as a baseline. Predictive performance was evaluated by two measures: AUC and accuracy.
## Bike verification
BiKE cohort patient inclusion for CEA surgery and enrolment in the biobank, clinical data, sample processing and large-scale datasets have been described in details previously. [bib_ref] Gene expression signatures, pathways and networks in carotid atherosclerosis, Perisic [/bib_ref] [bib_ref] Novel multiomics profiling of human carotid atherosclerotic plaques and plasma reveals biliverdin..., Matic [/bib_ref] In this study, the classification performance of MULTI 1 was also validated on transcriptomics (n = 137; 127 carotid plaques and 10 normal arteries) and proteomics (n = 36; 18 carotid plaques and 18 adjacent, healthy, matched segments) from the BiKE cohort. Within the BiKE, two subcohorts were defined. For the first, we compared non-atherosclerotic tissues (normal; n = 10 for transcriptomics, n = 18 for proteomics) versus atherosclerotic arteries (plaques; n = 127 for transcriptomics, n = 18 for proteomics), based on their morphological and histological characteristics (HIST). For the second, carotid plaque tissues were classified as asymptomatic (n = 40 for transcriptomics, n = 9 for proteomics) versus symptomatic (n = 87 for transcriptomics, n = 9 for proteomics) based on the patient's clinical presentation (CLIN). For both subcohorts, we tested the classification performance of the identified multiomics signature MULTI 1 on transcriptomics and proteomics, respectively, using logistic regression with the same setting mentioned above for GSE28829 and GSE43292. Gene set enrichment analysis (GSEA) was performed on the signature MULTI 1 against the genes and proteins from BiKE transcriptomics and proteomics, with the genes/proteins ranked by log2 fold change based on the comparisons of HIST and CLIN.
# Statistical analysis
Component-component and component-trait associations were measured by Pearson's correlation coefficients with p-values. Classification performances are presented as the mean ± standard deviation (SD). Distribution of plaque traits and IHC-based MFAP4 quantification for paired plaques are presented as box and whisker plots with the first quartile, median, third quartile, and the largest or smallest values within 1.5 times the interquartile range above the third quartile or below the first quartile, respectively. Statistical significance between groups was evaluated using two-tailed Wilcoxon rank-sum test (for nonnormally distributed data) or Student's t-test (for normally distributed data). For the MaasHPS data, paired statistical testing was performed. Shapiro-Wilk test was used for the normality test. Statistical significance between the classification performance was analyzed by Student's t-test. Significance level is denoted by *p-value < .05, **p-value < .01, ***p-value < .001, ****p-value < .0001. Statistical analyses were performed in R (v3.6.3).
# Results
In this study, we deeply phenotyped low-versus highrisk human carotid artery atherosclerotic lesion segments, as defined by the absence or presence of IPH, building an integrated gene/protein classification model for IPH. For this, we interrogated a carotid endarterectomy cohort (MaasHPS) by transcriptomic (microarray), proteomic, and peptidomic analysis (both LC-MALDI-MS/MS). Flanking sections were taken for detailed histological examination of cellular and acellular plaque composition and progression stage 41 , resulting in 42 CEA samples (16 non-IPH vs. 26 IPH, from 24 patients (see Materials and methods section and ,B for cohort build-up scheme). After preprocessing, a total of 10,000 genes, 943 proteins, and 2637 peptides were analyzed according to the workflow described in . For clarity sake, mRNAs/genes, proteins, and peptides from omics data are termed features, and a set of features is termed signature.
## Single-omics spls-da
First, we built single sPLS-DA models based on the transcriptomic, proteomic, and peptidomic datasets, separately. The optimal parameters for model building can be found in and . As is evident from [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] , all three models were able to segregate IPH from non-IPH plaque at high predictive performance, with accuracies of approximately 0.9 and AUCs of >0.95 ; in fact, nearly 90% of samples could be correctly classified by all three omics datasets separately. Next, we constructed heatmaps for the three omics layers, respectively, depicting the differential expression of signature members by IPH versus non-IPH plaque [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] and . Component 1 gene, protein, and peptide signatures showed strong differential expression between plaques with or without IPH. This is in support of the discriminative power of component 1 [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] , and suggests that the first component is the dominant factor in the prediction model. Next, we mapped the distribution of the selected feature sets [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] , based on the Pearson's correlation coefficient between the latent components (i.e., components 1 and 2) and the selected features. [bib_ref] Visualising associations between paired 'omics' data sets, González [/bib_ref] For both components, selected features were distributed differently, centering toward the extreme ends of component 1, and being more diffusely distributed for component 2. Thus, unlike component 2, component 1 features showed pronounced mutual interaction, suggesting involvement in a coordinated biological process. GSOA of the selected signatures for the first, decisive component revealed a clear overrepresentation of fibroblast and extracellular matrix-associated biological processes [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] , . In contrast, but expected based on the diffuse distribution in [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] ,C, component 2 did not offer deeper insights into hemorrhage relevant processes. Of note, even for component 1, the level of overrepresentation was rather low, precluding firm conclusions based on single sPLS-DA only.
## Multiomics spls-da
In order to establish a more comprehensive and robust model of IPH, we sought to analyze plaque transcriptomic, proteomic, and peptidomic data in an integrated manner by integrative sPLS-DA using the optimized parameters in . The resulting prediction model provided excellent discrimination of non-IPH versus IPH plaque , and performed at least equally good as all of the single-omics models, in terms of accuracy and AUC . The features of the first, but not second to fourth components, showed overt differential expression between non-IPH and IPH plaque and , regardless of their origin (i.e., gene, protein, peptide). Furthermore, the selected proteins and peptides in showed considerable overlap (12 out of 20, . Also, for each of the other three components, part of the selected proteins and peptides are overlapping , implying the contribution of the proteomics and pep-tidomics to the multiomics model are to some extent similar but not identical. Cross-component correlation analysis confirmed the strong mutual correlation of component members from the three omics layers, whereas cross-interaction across the components was very weak, underpinning the high level of independence of the four components of our model. To add biological meaning to the prediction model, we set out to identify cofunctional gene/protein/peptide clusters, correlating with each of the four components of the integrative model . Several clusters were observed, suggesting a very strong mutual correlation between genes, proteins, and peptides. This points to coordinate function and/or regulation of the cluster members. GSOA of the gene, protein, and peptide members underlying the integrated classification model showed a clear cardiovascular signature , with marked overrepresentation of extracellular matrix organization (components 1 and 3), lipid metabolism (component 2), and immune response terms (component 4). Interestingly, components 1 and 3 showed considerable functional overlap, despite their low interdependence. Of note, the first component of the multiomics model was very comparable to that of the single-omics models, and showed a significant level of overlap . To validate the GSOA findings, we correlated the four components with several important plaque traits, such as IPH, fibrosis, and macrophage presence , . The dominant first component was highly correlated with plaque size (correlation = 0.74, p = 1.8E-08) and hemorrhaged plaque area (correlation = 0.48, p = 1.4E-03), as expected, as well as with αSMA -PDGFRα + fibroblastlike cells (correlation = 0.64, p = 2.4E-05), Arg1 + healing macrophage phenotype (correlation = −0.62, p = 2.2E-04), and collagen content of non-IPH plaque (correlation = 0.72, p = 3.6E-03), concordant with the GSOA findings . Indeed, plaque size, hemorrhage area, collagen content, and αSMA -PDGFRα + cell content were significantly correlated with each other [fig_ref] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics [/fig_ref] , possibly explaining the correlations between component 1 and these plaque traits Genes, proteins and peptides, and TFs are depicted in different shapes; gene-level differential expression (log2 fold change) is shown by green-red gradient. The top five regulatory basis, we performed regulatory analysis by iRegulon to identify TFs driving this signature set, and built a regulatory network of the IPH signature encompassing mRNAs, protein, peptides, and in silico-identified TFs. As can be seen from , all the 22 nonredundant members of component 1 were targeted by the top five enriched TFs. MRNA of the highest ranked TF, serum response factor (SRF), was significantly and sharply downregulated in hemorrhaged plaque regions, mirroring the effects seen on most of the SRF connected network members. SRF signaling is tightly dependent on its cofactor, where myocardin family members drive locomotion and cell adhesion activities, while T-cell factors promote proliferation. Hypergeometric testing using the reference dataset of Xie's study [bib_ref] MKL1/2 and ELK4 co-regulate distinct serum response factor (SRF) transcription programs in..., Xie [/bib_ref] showed a clear overrepresentation of SRF-targeting network members in megakaryoblastic leukemia-1/2 (MKL1/2) regulated SRF-dependent genes (p-value = 2.7E-03), but not in ETS-domain protein 4 (ELF4) regulated SRF-dependent genes . This points to the former as the dominant signaling pathway in non-IPH plaque, promoting actin cytoskeleton organization, locomotion, and cell adhesion regulated by SRF in plaque.
One of the SRF-regulated features with the strongest downregulation in IPH compared to non-IPH, at gene expression level , was microfibril-associated glycoprotein 4 (MFAP4), which has been described to localize in vascular wall extracellular matrix fibers and to be involved in neointima formation. [bib_ref] MFAP4 promotes vascular smooth muscle migration, proliferation and accelerates neointima formation, Schlosser [/bib_ref] Immunohistochemical validation of MFAP4 presence in plaque sections from the same cohort (MaasHPS) indeed confirmed its reduced abundance in hemorrhaged plaques . We also examined the MFAP4 gene expression and protein abundance in BiKE cohort, which showed a downregulation in atherosclerotic arteries compared to normal or adjacent tissues, as well as in symptomatic patients compared to asymptomatic patients . Moreover, based on GSEA, we observed significant negative NES of the SRFregulated network members in BiKE cohort for the comparisons of HIST and CLIN, suggesting the general downregulation of the network members in carotid plaques compared with normal or adjacent tissues, and in symptomatic patients compared with asymptomatic patients
## Independent validation of findings from multiomics analyses
Finally, we assessed whether the IPH signature was able to discriminate low-from high-risk plaques in two independent mRNA cohorts in the public domain: GSE28829 40 (n = 29; mRNA; early vs. advanced plaque) and GSE43292 4 (n = 64; mRNA; intact arterial tissue vs. atheroma). Although the cohort setup differs from our cohort, carotid advanced plaque (GSE28829) and atheroma (GSE43292), both largely represent unstable plaque phenotypes (thin cap fibroatheroma and beyond). The main difference is in the control tissue with even lower risk than our nonhemorrhaged stable plaque: that is, early-stage lesion (low risk) and nonlesioned arterial tissue, in the two validation cohorts, respectively, versus nonhemorrhaged stable plaque in our study, but we argued that our signature may still be valid for these broader disease trajectories.
Indeed, in both cohorts, the signature genes from integrative sPLS-DA component 1 (MULTI 1) were differentially expressed between control versus diseased artery . Of note, the first component of the multiomics model (MULTI 1) by itself already performed surprisingly well in stratifying the two plaque phenotypes for GSE28829, albeit not as good as the complete four-component gene set (MULTI 1-4, and . Apparently, the first component is dominant in stratifying the validation cohorts, with a complementary contribution of components 2-4. Validation in GSE43292 did not show major differences in predictive power of the SINGLE, MULTI 1, and MULTI 1-4 gene sets, with overall high performance (AUCs >0.8) for all gene sets tested by logistic regression and SVM and . Decision tree classifiers performed less impressive than the other two classifiers . Here, the MULTI 1-4 gene set appeared to be superior to the other models , possibly owing to the structure of the decision tree classifier, in which the splitting nodes can incorporate the independent features from distinct multiomics components. Finally, we validated performance of the MULTI 1 signature in the BiKE cohort study, which includes both high-powered transcriptomics (n = 127) and proteomics datasets (n = 36) from CEA tissue. The MULTI 1 feature set performed well on both the transcriptomics and proteomics in segregating advanced plaque from healthy carotid tissue (histological, HIST; see . Interestingly, while both involved hemorrhaged plaques, our signature was even able to stratify plaques from symptomatic versus asymptomatic patients based on their proteomics at a reasonable classification performance (AUC = 0.80), and to a lesser extent transcriptional profile (AUC = 0.57; clinical, CLIN; see . Indeed, all the MULTI 1 members overlap with MULTI 1-4 and SINGLE , suggesting the MULTI 1 signature derived from the dominant component of the multiomics model is essential for plaque classification.
# Discussion
Elucidation of critical processes in the transition of lowrisk into high-risk rupture-prone plaque in humans will pave the way for early diagnosis of, and targeted intervention in atherosclerosis-related cardiovascular diseases.
Here we have deployed integrative analysis of transcriptomic, proteomic, and peptidomic expression/abundance profiles of human carotid artery plaque to build a multitethered prediction model for distinguishing low-from high-risk carotid artery lesions. The significance of this model was corroborated in multiple independent plaque cohort studies. Finally, based on this model, we constructed an SRF-driven regulatory gene/protein network overrepresented in extracellular matrix remodeling and interaction terms, which could serve as a starting point for the design of a targeted intervention. Single-omics studies on human atherosclerotic plaque by us (this study, Goossens et al., Eijgelaar et al.) [bib_ref] Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to..., Goossens [/bib_ref] [bib_ref] Equivalence testing in microarray analysis: similarities in the transcriptome of human atherosclerotic..., Eijgelaar [/bib_ref] and others [bib_ref] Identification of two genes potentially associated in iron-heme homeostasis in human carotid..., Ayari [/bib_ref] [bib_ref] Local atherosclerotic plaques are a source of prognostic biomarkers for adverse cardiovascular..., De Kleijn [/bib_ref] [bib_ref] Prediction of ischemic events on the basis of transcriptomic and genomic profiling..., Folkersen [/bib_ref] [bib_ref] The use of network analyses for elucidating mechanisms in cardiovascular disease, Diez [/bib_ref] have already shown the power of these high-throughput approaches for biomarker discovery and mechanistic studies. For instance, Ayari and Bricca have identified the CD163/HO-1 axis to be associated with ironheme homeostasis in atherosclerotic plaque, using differential expression analysis on microarray data extracted from 68 carotid atheroma specimens. [bib_ref] Identification of two genes potentially associated in iron-heme homeostasis in human carotid..., Ayari [/bib_ref] However, singleomics approaches intrinsically fail to capture the complex-ity of biological systems as a whole, providing useful but incomplete information. [bib_ref] The challenges of integrating multiomic data sets, Palsson [/bib_ref] [bib_ref] Data integration in the era of omics: current and future challenges, Gomez-Cabrero [/bib_ref] Moreover, genomic and peptide/protein expression/abundance in plaque were seen to show poor mutual correlation. [bib_ref] Comparative analysis of proteome and transcriptome variation in mouse, Ghazalpour [/bib_ref] Consequently, genomics findings are not directly translatable to (pre)clinical application, while protein-based models generally are rather sparse, biased toward high-abundance proteins and lacking their regulatory context. This combined with the observation that both domains display distinct but complementary correlations with relevant clinical traits pleads in favor of integrative multiomics analysis on atherosclerosis to have the best of both worlds, producing a robust, translatable model of disease progression.
This study illustrates the benefits of truly integrative omics analysis in terms of accuracy, visibility, and model interpretability. Our multiomics prediction model achieved similar, if not higher, accuracy in stage classification as a single-omics model. The prediction power of the selected features was successfully validated on several independent cohorts comparing healthy artery or early (stable) plaque versus advanced (unstable) plaque or atheroma. Even, the selected features performed reasonably well in stratifying plaques from symptomatic versus asymptomatic patients at protein level (BiKE proteomics), although plaques in both groups were having an advanced (unstable) phenotype. However, the true merit of integrative analysis is in the robustness of the model, its biological significance, and the direct clinical translatability of network-contained proteins. Significances of overrepresented terms in the multiomics signature outperformed those of the single-omics signature, due to the strong mutual interaction of signature members per component. Interestingly, while the dominant component of the single-and multiomics models showed considerable overlap, the auxiliary second to fourth components of the single-and multiomics models differed substantially, with those of the latter being much more coherent and biologically meaningful. The dominant component in the multiomics model was overrepresented in collagen metabolism and macrophage inflammation terms, reflecting peri-rupture modeling processes. The auxiliary components alluded to additional CVD relevant terms, such as lipid and lipoprotein metabolism, neutrophil responses and, again, tissue metabolism, mirroring early responses to hemorrhage and/or rupture. Additionally, we found that the BLVRB, which had been previously proposed by Matic performance of MULTI 1 feature subsets on BiKE cohort in distinguishing normal (n = 10 for transcriptomics; n = 18 for proteomics) versus plaque (n = 127 for transcriptomics; n = 18 for proteomics) based on histological characteristics (HIST), or asymptomatic (n = 40 for transcriptomics; n = 9 for proteomics) versus symptomatic (n = 87 for transcriptomics; n = 9 for proteomics) based on clinical events (CLIN). For B and C, results were measured by accuracy and AUC using logistic regression, with stratified five-fold cross-validation and 1000 random repeats. Results are presented as mean ± SD. (D) Overlapping between feature sets SINGLE, MULTI 1, and MULTI 1-4 et al. [bib_ref] Novel multiomics profiling of human carotid atherosclerotic plaques and plasma reveals biliverdin..., Matic [/bib_ref] to define IPH, was also contained in our multiomics model (component 4).
Literature search for the MULTI 1 elements indicated an overall relationship (15 of the total 22) to vascular smooth muscle function and inflammation in cardiovascular diseases . Interestingly, the regulatory network based on the dominant component, identified SRF as the driving factor in peri-rupture remodeling. Despite the fact that a small media could not be excluded from the dissected tissue during the CEA surgery, the lack of correlation between the dominant component and the media size indicated the SRF-driven network was only associated with intimal not medial processes related to IPH. SRF is a MADS family type TF, which acts by binding CArG box motifs. The relevance of SRF for cardiovascular health is well documented. [bib_ref] Restricted inactivation of serum response factor to the cardiovascular system, Miano [/bib_ref] [bib_ref] HERP1 inhibits myocardininduced vascular smooth muscle cell differentiation by interfering with SRF..., Doi [/bib_ref] In cardiac and vascular smooth muscle cells, it was seen to drive phenotypic switch by regulating contractile gene programs. [bib_ref] Myocardin: a novel player in atherosclerosis, Xia [/bib_ref] In endothelial cells, SRF is essential for VEGF-induced cell signaling and angiogenesis, and thus endothelial dysfunction. [bib_ref] SRF selectively controls tip cell invasive behavior in angiogenesis, Franco [/bib_ref] Moreover, SRF mediates cellular lipid and glucose responses by controlling LXRB gene expression, modulating these metabolic sensors. [bib_ref] Elk1 and SRF transcription factors convey basal transcription and mediate glucose response..., Nilsson [/bib_ref] While a direct role in ischemic heart disease is likely, experimental data to underpin this notion is lacking.
Mechanistically, SRF transcriptional responses depend critically on its coactivator. The major coactivator classes are T-cell factors (e.g., TCF21, 53 ELK1/2), which are responsible for renin-angiotensin-aldosterone system (RAAS)activated mechanosensing, and myocardin-family members (e.g., MRTF), which transduces mothers against decapentaplegic homolog (SMAD) and Rho-associated kinase responses. [bib_ref] Rho/Rho-associated kinase signal regulates myogenic differentiation via myocardinrelated transcription factor-A/Smad-dependent transcription of..., Iwasaki [/bib_ref] As pointed out by competition between T-cell and myocardin family members for SRF will skew its responses toward antagonistic proliferative and contractile programs of gene expression, respectively. [bib_ref] SRF co-factors control the balance between cell proliferation and contractility, Gualdrini [/bib_ref] Our data seem to plead for MKL1/2-SRF signaling as the major dysregulated axis in IPH plaque, suggesting that remodeling responses will prevail.
Although our integrative multiomics approach holds promise, it has a number of limitations. First, this study was based on a moderately dimensioned cohort study containing CEA tissues from male patients and may therefore only partly reflect the population's diversity in disease risk profile as well as pathogenesis. The fact that we were able to confirm the validity of our selected genes in multiomics model in several other cohorts, however, indicates that our findings have a wider scope. Second, the multiomics approach was only based on genes, proteins and peptides. Incorporation of metabolomics and lipidomics into the multiomics signature may further strengthen the model and deepen our insight into key processes in this clinically relevant stage transition. [bib_ref] Integration of expression data in genome-scale metabolic network reconstructions, Blazier [/bib_ref] [bib_ref] More is better: recent progress in multiomics data integration methods, Huang [/bib_ref]
# Conclusions
In conclusion, our study underpins the added value of integrative multiomics analysis to a single-omics study of human atherosclerosis. Comparing a single-and multiomics analyses of human carotid atherosclerotic plaque, we showed that while both approaches perform excellently in segregating low-from high-risk plaques, the latter provides much deeper insight into critical processes taking place prior to or just after plaque rupture in humans. Moreover, our integrative multiomics approach revealed an SRF-driven gene/protein network, associated with this phase transition, which could guide efforts to the design of new treatments to promote non-expansive plaque healing.
## A c k n o w l e d g m e n t s
## C o n f l i c t o f i n t e r e s t
The authors declare that there is no conflict of interest.
[fig] F I G U R E 2: Single-omics sPLS-DA on transcriptomics, proteomics, and peptidomics. (A) Two-component sample distribution (16 non-IPH and 26 IPH) for single-omics sPLS-DA on plaque transcriptomics (gene), proteomics (protein), and peptidomics (peptide), respectively. For visualization purpose, two components were included in the plot for peptidomics. (B) Heatmap shows expression level of the selected genes (comp1, n = 150; comp2, n = 54) from transcriptomics. Expression values were z-normalized per row. (C) Selected features by single sPLS-DA on the three omics are plotted, respectively (n = 204 for genes; n = 111 for proteins; n = 3 for peptides), according to their Pearson's correlation coefficient to the components 1 and 2. (D) GSOA on standardized gene set of single sPLS-DA. For each component, six highly ranked overrepresented terms are selectively shown F I G U R E 3 Multiomics sPLS-DA on transcriptomics, proteomics, and peptidomics. (A) Two-component sample distribution for multiomics sPLS-DA on plaque transcriptomics (gene), proteomics (protein), and peptidomics (peptide). Non-IPH and IPH plaques are denoted in skyblue and coral, samples from different omics layer (16 non-IPH and 26 IPH for each omics layer) are denoted by red, blue, and green borders. Considering the high consistency between the components of the three omics layers in the multiomics model (see C), three [/fig]
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Split-hand/feet malformation: A rare syndrome
Split-hand/split-foot malformation (SHFM) is mainly inherited as an autosomal dominant trait with incomplete penetrance and characterized by malformation of the limb involving the central rays of the autopod. It presents with a deep median cleft of the hand and/or foot, aplasia/hypoplasia of the phalanges, metacarpals, and metatarsals. Pathogenic mechanism is a failure to maintain signaling from the median apical ectodermal ridge. Without this signaling, cells of the underlying progress zone stop proliferation and differentiation which in turn results in defects of the central rays. We describe a case of SHFM in 10-year-old boy.
# Introduction
Split-hand/split-foot malformation (SHFM) is also known as ectrodactyly. It is a malformation of the limb involving the central rays of the autopod and presenting with a deep median cleft of the hand and/or foot, aplasia/hypoplasia of the phalanges, metacarpals, and metatarsals. It can occur as part of a syndrome or isolated entity. The incidence of SHFM is around 1 in 90,000 live births. [bib_ref] Cleft hand: Classification, incidence, and treatment. Review of the literature and report..., Barsky [/bib_ref] SHMS expresses in two ways, one is nonsyndromic, where the isolated involvement of limbs occurs and other is associated anomalies known as syndromic form.We report a nonsyndromic case of SHFM.
## Case report
A 10-year-old boy, second child of physically normal parents, presented with deformed hands and feet since birth. In hands, bilateral syndactyly, aplasia of the phalanges and metacarpals was seen in . The X-ray of the hand [ ] showed absence of first metacarpal and multiple phalanges in both the hands. Both the feet had deep midline cleft [ and b] and syndactyly. X-ray showed absence of multiple metatarsals and phalanges in both the feet. There were no other dysmorphic features. Anthropometric measurements were within normal limits. Physical and systemic examination were normal. Developmentally appropriate for the age. He was a product of nonconsanguineous marriage and term normal delivery with no significant perinatal events. The index case was second in birth order. There was no facial dysmorphism in the patient. The two siblings of the index case were physically normal. The probable inheritance pattern in our case is autosomal recessive as no other sibling or other family members are affected.
# Discussion
SHFM is developed due to a failure of median apical ectodermal ridge activity, which leads to increased cell death or reduced cell proliferation. Defects in genes among antero-posterior signaling (Shh), proximo-distal signaling (Tp63, Dlx5/6) or dorsoventral signaling (Lrp6, Dkk1), can also cause SHFM. [bib_ref] Pathogenesis of split-hand/split-foot malformation, Duijf [/bib_ref] SHFM is commonly inherited as autosomal dominant mode with reduced penetrance. Some cases have been reported to have been inherited as autosomal recessive and X-linked forms. [bib_ref] Evidence for autosomal recessive inheritance of split hand/split foot malformation: A report..., Gül [/bib_ref] [bib_ref] X-chromosomally inherited split-hand/split-foot anomaly in a Pakistani kindred, Ahmad [/bib_ref] Six types SHFM have been described till now [ [fig_ref] Table 1: Types of split hand/foot malformation SHFM [/fig_ref] ], among which type I is most common variety. Chromosomal rearrangement leads to association of ectrodactyly with other abnormalities. Disorders associated with ectrodactyly are ectrodactyly-cleft palate syndrome, ectrodactyly-ectodermal dysplasia-clefting syndrome, ectrodactyly-fibular aplasia/hypoplasia syndrome, ectrodactyly-ectoder mal dysplasia-macular dystrophy syndrome, and ectrodactyly-polydactyly. [bib_ref] The London dysmorphology database, Winter [/bib_ref] SHFM may need surgical treatment to the improve function and appearance. Parents should be counseled regarding the possibility of recurrence of the disease in the future siblings.
## Financial support and sponsorship
Nil.
## Conflicts of interest
There are no conflicts of interest.
[fig] Figure 2, Figure 1: X-ray showing aplasia/hypoplasia of the phalanges (a), metacarpals and metatarsals (b) (a) Median clefts of feet and (b) syndactyly of hands b a [/fig]
[table] Table 1: Types of split hand/foot malformation SHFM: Split-hand/foot malformation; AD: Autosomal dominant; AR: Autosomal recessive; XLR: X-linked recessive [/table]
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When choice becomes limited: Women’s experiences of delay in labour
Choice and patient involvement in decision-making are strong aspirations of contemporary healthcare. One of the most striking areas in which this is played out is maternity care where recent policy has focused on choice and supporting normal birth. However, birth is sometimes not straightforward and unanticipated complications can rapidly reduce choice. We draw on the accounts of women who experienced delay during labour with their first child. This occurs when progress is slow, and syntocinon is administered to strengthen and regulate contractions. Once delay has been recognized, the clinical circumstances limit choice. Drawing on Mol's work on the logics of choice and care, we explore how, although often upsetting, women accepted that their choices and plans were no longer feasible. The majority were happy to defer to professionals who they regarded as having the necessary technical expertise, while some adopted a more traditional medical model and actively rejected involvement in decision-making altogether. Only a minority wanted to continue active involvement in decision-making, although the extent to which the possibility existed for them to do so was questionable. Women appeared to accept that their ideals of choice and involvement had to be abandoned, and that clinical circumstances legitimately changed events.
# Introduction
Choice and patient involvement in decision-making are strong aspirations of contemporary healthcare [bib_ref] Towards a history of choice in UK health policy, Greener [/bib_ref] [bib_ref] Knights, knaves or pawns? Human behaviour and social policy, Grand [/bib_ref] -embodied within policy discourse and codes of conduct for professionals [bib_ref] Patient and Public Involvement in Health: The Evidence for Policy Implementation. London:..., Department [/bib_ref]. The move towards delivering person-centred carehas been endorsed through policy and professional statements emphasising its centrality to the delivery of good care. An increased emphasis on paying attention to and displaying suitable respect for patients' values and preferences is framed as an important guard against the dangers of paternalism and autocratic practice on the part of professionals [bib_ref] Empty ethics: The problem with informed consent, Corrigan [/bib_ref]. Indeed, it has been argued that patients are now being primed for action rather than passivity [bib_ref] Actors, patients and agency: A recent history, Armstrong [/bib_ref].
However, attempts to translate these principles into practice reveal several potential problems. This article is primarily concerned with two and the potential interaction between them. First, the extent to which individual patients really welcome choice and actively wish to engage in making decisions about who provides their care, where and using what treatments or interventions is not clear, and there is some evidence that the continuing asymmetry within clinician-patient interactions is co-constructed between the two parties rather than being a simple matter of professional dominance [bib_ref] On the remarkable persistence of asymmetry in doctor/patient interaction: A critical review, Pilnick [/bib_ref]. Policies emphasising choice tend to position patients in particular ways and in relation to other actors, most obviously professionals [bib_ref] Towards a history of choice in UK health policy, Greener [/bib_ref]. In doing so, they can serve to offer up preferred identities for patients [bib_ref] Governing the ethical consumer: Identity, choice and the primary care medical encounter, Mcdonald [/bib_ref] , with choice often being argued to be based on a rational consumer model that may not always be well-suited to the healthcare context. As a result, many have argued that (at least some) choice models seem to ask rather a lot of patients, and it is not always clear that they want to take on this work [bib_ref] The indeterminacy of choice: Political, policy and organisational implications, Clarke [/bib_ref] [bib_ref] Towards a history of choice in UK health policy, Greener [/bib_ref].
Second, decisions about which course of treatment or which intervention to pursue take place in a range of clinical circumstances. While some are undoubtedly amenable to patients being involved in decisions about their care (e.g. conditions for which different treatment options exist, but each carries different benefits and risks), there are others in which, it can be argued, patient involvement in decision-making is either not possible or not desirable. Indeed, some would argue that seeking to involve patients in these contexts is inappropriate and does not give sufficient credit to professional knowledge or training. Emergency situations raise important challenges to involvement in decision-making because the options available, as well as the time that would be needed to discuss them, can be extremely limited. Contemporary health empowerment discourses have been critiqued for their tendency to ignore or obscure the complex forms of dependence that characterise many healthcare experiences and situations [bib_ref] Informing health? Negotiating the logics of choice and care in everyday practices..., Henwood [/bib_ref] , and research has shown that even relatively well-established processes such as the taking of consent can be challenging in these circumstances [bib_ref] Why do women consent to surgery, even when they do not want..., Dixon-Woods [/bib_ref].
As well as being important in their own rights, the potential interconnections between these two critiques of choice warrant consideration. Perhaps the most obvious connection is the contrast between patients who may say they want choice in the abstract but then find this an unwelcome burden, or simply inappropriate, when actually ill or injured. In exploring this tension, we turn to the work of Annemarie, who argues that, while they can sometimes be complementary, patient choice and good care are much more often at odds with each other. For Mol, the ideal of patient choice carries with it a whole set of assumptions (a 'logic') that acts as a mode of organising and interacting, of understanding and of distinguishing between good and bad outcomes. She argues that the 'logic of choice' assumes professionals limit themselves to presenting facts which the patient assesses in order to make his or her choice of desired outcome, and the professional then uses appropriate techniques to deliver this. However, deciding to do something is rarely enough to actually achieve it, and central to Mol's critique is the idea that the 'logic of choice' unhelpfully focuses attention on discrete end products. This is unhelpful because, she argues, care is better understood as an interactive and often open-ended process that is shaped and re-shaped depending on its results. What is or is not achievable in any particular care context cannot always be known or clearly set out in advance, but rather is contextual and changeable; in care, time twists and turns and there is no crucial moment when all facts are known. By focusing on 'end products', the logic of choice oversimplifies the relationship between means and ends. It is for these reasons that Mol argues the 'logic of choice' is in tension with the 'logic of care', and that the latter is preferable in a great many situations. She concludes her work by calling for further explorations of how these two logics 'interfere' with each other in specific healthcare contexts:
That the logic of choice and the logic of care are so profoundly different begs the question as to what happens when these two modes of thinking and acting get mixed together -as they do in real life. The possible interferences are many. This is precisely what this article seeks to explore.
## Choice in maternity care
We take as our focus maternity care, a setting in which competing discourses about the most appropriate way to care for and support labouring women are well established [bib_ref] Childbirth embodiment: Problematic aspects of current understandings, Walsh [/bib_ref] and different options are (at least in theory) open to women [bib_ref] Women's childbirth preferences and practices in the United States, Miller [/bib_ref]. While we acknowledge there are some important differences between maternity care and the diabetes care context within which Mol's work developed, we believe it is appropriate to use her work here. While childbirth itself does not, of course, equate with illness, the women in our study had all experienced a particular complication (explained below) which, under current guidance, is managed medically.
Within the United Kingdom, a range of organisations are influential in shaping maternity care, including both professional bodies (such as the Royal College of Obstetricians and Gynaecologists and the Royal College of Midwives) and consumer groups (such as the National Childbirth Trust), with the National Institute for Health and Care Excellence (NICE) responsible for producing evidence-based guidance. Recent UK policy and practice in this area has focused on choice , for example, over place of birth (the four options in the UK context being home birth, a freestanding midwife-led unit, a midwife-led unit alongside a hospital or a hospital obstetric unit or 'labour ward'), and women are invited to develop a 'birth plan' [bib_ref] Sheila Kitzinger's letter from England: Birth plans, Kitzinger [/bib_ref] [bib_ref] Birth plans: The good, the bad and the future, Lothian [/bib_ref] during pregnancy in order to record their preferences. The emphasis is therefore increasingly placed on women engaging in a process of information-seeking about the choices available and subsequently making decisions that best fit their preferences. This can be illustrated in the UK context by reference to the 'Pregnancy Planner' -a National Health Service (NHS)-provided online resource for pregnant women which explains a birth plan as follows:
A birth plan is a record of what you would like to happen during your labour and after the birth. You don't have to create a birth plan but if you would like one your midwife will be able to help. Discussing a birth plan with your midwife will give you the chance to ask questions and find out more about what happens in labour. It also gives your midwife the chance to get to know you better and understand your feelings and priorities.In principle, this would seem a good idea as there is evidence that the involvement in decision-making can improve women's birth experiences and lead to better physical and emotional outcomes [bib_ref] Alternative versus conventional institutional settings for birth, Hodnett [/bib_ref] , and also that place of birth can impact women's birth experiences [bib_ref] The impact of birthplace on women's birth experiences and perceptions of care, Overgaard [/bib_ref]. Pregnant women may vary enormously in their preferences -some women highly value easy and quick access to medical technology and welcome interventions such as an epidural for pain relief, others prefer to approach labour more 'naturally' and prefer to have little or no pain relief or other medical intervention [bib_ref] Splitting bodies/selves: Women's concepts of embodiment at the moment of birth, Lupton [/bib_ref].
The offer of choice in maternity care is typically based on the assumption that women are 'low risk', that there are options available and that the risk is comparable between the choices. However, birth is often not straightforward and unanticipated complications can rapidly reduce the scope for choice and possibly meaningful involvement in decisionmaking [bib_ref] The best laid plans? Women's choices, expectations and experiences in childbirth, Malacrida [/bib_ref]. The best laid plans may not be achievable in practice, and choice suggests an element of equipoise of outcome that may not always reflect reality. For example, it is common for women who begin their labours in midwifeled units to be transferred to obstetric units, especially for first pregnancies [bib_ref] Transfers of women planning birth in midwifery units: Data from the birthplace..., Rowe [/bib_ref]. Transfers may take place for clinical reasons (such as concerns for mother and/or baby) but can also happen if women decide to pursue an intervention not available in a midwife-led unit, such as an epidural for pain relief.
## Delay during labour
The particular clinical focus of this article is delay during labour, in which contractions are either not frequent and/or strong enough for labour to progress. Once in established labour (regular painful contractions and progressive cervical dilation from 4 cm), assessment of progress includes cervical dilation. Delay is suspected if dilatation of less than 2 cm in 4 hours occurs and confirmed if progress of less than 1 cm is found 2 hours later. To facilitate progress during this 2-hour period, the woman will be encouraged to mobilise, consider hydration (e.g. a sports drink) and discuss appropriate and effective pain relief. If her membranes are still intact, artificial rupture will be advised.
If delay is confirmed, transfer to obstetric-led care takes place (if not already the case), and the use of syntocinon (a synthetic form of the hormone oxytocin) is recommended to increase the strength and frequency of contractions . The safety of mother and baby is routinely assessed by more intense monitoring by the midwife and obstetrician, and this normally includes support and effective pain relief, monitoring of the strength and frequency of contractions, the woman's observations and fluid balance. Electronic foetal heart monitoring is routinely offered to detect signs of foetal hypoxia, should they occur. Progress is re-assessed after 4 hours of syntocinon and a decision made about birth.
Having delay during labour confirmed therefore has several implications for how the woman's labour and birth progress. First, women who have chosen to begin their labour elsewhere (i.e. at home or in either type of midwife-led unit) will be transferred to an obstetric unit. Second, the need to ensure adequate pain relief means the majority of women in this situation have an epidural inserted [bib_ref] Standard or high dose oxytocin for nulliparous women with confirmed delay in..., Kenyon [/bib_ref]. Given that preferences about pain relief are commonly occurring features of birth plans [bib_ref] Anesthesia and analgesia-related preferences and outcomes of women who have birth plans, Pennell [/bib_ref] , recommendations to have an epidural are likely often not to fit well with what women had planned, but may nevertheless be welcomed given that their labour is now prolonged and they are likely to be extremely tired. Third, while evidence suggests that, in its current regimens, the use of syntocinon can shorten labour by about 2 hours, it also shows it will not ultimately change the mode of birth, that is, women who would have had a caesarean will still ultimately do so . High-dose regimens have not been fully evaluated, but may reduce the likelihood of a caesarean and increase the likelihood of spontaneous vaginal birth [bib_ref] High dose versus low dose oxytocin for augmentation of delayed labour, Mori [/bib_ref] [bib_ref] High-dose vs low-dose oxytocin for labor augmentation: A systematic review, Wei [/bib_ref].
While we acknowledge the important debates around the systemic or structural context and how this may be shaping care practices within the birth setting (e.g. the potential medicalisation of what many would regard as a natural process and the shifting patterns of professional involvement in birth), these are not the central focus of this article. Similarly, while we acknowledge those who argue that delay in labour may be more complex than a 'mechanical' physiological problem [bib_ref] Beyond evidence-based medicine: Complexity and stories of maternity care, Downe [/bib_ref] , it is not our intention in this article to consider the appropriateness of the medical interventions these women experienced.
Relatively little is known about the experiences of women who become delayed in labour, and the evidence that does exist presents a mixed picture. For example, two UK-based studies using questionnaires found that intervention for delay was not necessarily viewed as negative by women [bib_ref] Dysfunctional labour: A randomised trial, Blanch [/bib_ref] [bib_ref] A prospective study of women's views of factors contributing to a positive..., Lavender [/bib_ref] , while two more recent small-scale interview studies from Scandinavia have suggested that experiencing delay can be problematic and lead women to need particular support from health professionals [bib_ref] Experiences of non-progressive and augmented labour among nulliparous women: A qualitative interview..., Kjaergaard [/bib_ref] [bib_ref] Some Swedish women's experiences of prolonged labour, Nystedt [/bib_ref].
In this article, we focus on how women experience having delay in labour confirmed, and the subsequent interventions that follow from this. In particular, we explore how women understand, and come to terms with, their labours not progressing as they would have wished, how they experience a reduction in choice about birth options and what this reveals about the relative importance of choice compared to other outcomes.
# Methods
Women were recruited as part of a pilot study comparing high-and low-dose syntocinon for delay in labour, led by SK [bib_ref] Standard or high dose oxytocin for nulliparous women with confirmed delay in..., Kenyon [/bib_ref]. Women were only eligible for the study when they had delay confirmed and had already opted to receive syntocinon (usual care constituted the low-dose arm of the study). The pilot study was based in three English maternity units and recruited 94 women over a period of 7 months between November 2010 and May 2011. All women who took part in the pilot study were invited to take part in this interview study. Women were invited to interview 2 weeks after birth (alongside the receipt of other pilot study-related follow-up such as questionnaires). Women received a material including an invitation letter, a Participant Information Leaflet and an interview reply slip, which they returned if they were willing to be interviewed. Reply slips were returned to the pilot study office and forwarded to NA, who led the qualitative element. She recorded women's characteristics using data supplied by the pilot study office (study site, mode of birth and pain relief) and un-blinded to reveal allocation to study arm (low or high dose). Women were contacted, given the opportunity to ask any questions, and an interview time and venue were arranged. Written consent was taken at the interview itself. The interviewer (see Acknowledgements) remained blinded to study arm allocation throughout.
We had planned to sample purposively to include women from the low-and highdose arms; women who had had a caesarean section, instrumental or spontaneous vaginal birth; and women who did or did not have an epidural during labour. However, a relatively low response rate meant that all women who agreed to be interviewed were followed up. In total, 19 women responded and 18 were interviewed (we lost contact with the remaining woman). Although we were unable to sample purposively in these circumstances, the final sample was diverse. The sample characteristics are shown in [fig_ref] Table 1: Interview sample characteristics [/fig_ref].
A semi-structured topic guide with broad areas was developed from a literature review, discussions within the project team and input from our consumer representative (see Acknowledgements). While this was used to guide the interviews, the emphasis was on encouraging women to discuss their own perspectives freely.
All interviews were audio-recorded and transcribed verbatim, with permission from participants. The purpose of the interviews was to explore women's understandings of the study and the information-giving and consent-taking processes adopted (not reported here; see [bib_ref] Standard or high dose oxytocin for nulliparous women with confirmed delay in..., Kenyon [/bib_ref] , as well as their views and experiences of labour and birth.
Data from the interviews were analysed using the constant comparative method, assisted by NVivo 8 software. Transcripts were read in detail and open codes were initially applied line-by-line to the data. The open codes were then incrementally grouped into organising categories or themes. These categories were modified and checked constantly and further open codes were incorporated as analysis proceeded. The categories and their specifications (the coding scheme) were then programmed into the software. The coding scheme was used to process the data set systematically by assigning each section of text to a category, according to the category specifications. The pilot study, including this interview study element, was reviewed and given a favourable opinion by the Leicester, Northampton and Rutland 1 NHS Research Ethics Committee (reference 10/H0406/30).
## Findings
## Choices had been made, but had to be revisited
While there are, of course, some circumstances in which women's options about labour and birth may be limited by clinical factors, the exclusion criteria for recruitment to the pilot study meant that only women who had nothing in their medical history to make them 'high risk' (such as gestational diabetes, existing maternal or foetal disease or concern, previous uterine surgery or vaginal bleeding in the pregnancy of clinical significance) were eligible for recruitment [bib_ref] Standard or high dose oxytocin for nulliparous women with confirmed delay in..., Kenyon [/bib_ref]. This meant that all options were initially open for these women, and that they were therefore ideally placed to engage in the kind of information-seeking and decision-making processes embodied within the current policy discourse around maternity care.
The majority of women interviewed did indeed report having planned how they wanted their labour and birth to be to some degree, including where they wanted to give birth and what kind of pain relief they would like to use. The emphasis was most commonly on wanting 'as natural a birth as possible': I went to the birthing centre. I tend to worry quite a lot about, erm, clinical aspects, I don't like needles and I don't like that sort of environment. It worries me a bit and makes me a bit anxious. (Participant 1) I really didn't want to have anything to try and combat the pain … it's just I wanted to try and have as natural a birth as possible without pain relief, rather than intervention like that. As previously explained, having delay in labour confirmed necessitates a transfer to an obstetric unit if the woman has begun her labour in any other setting as this is the only place in which syntocinon can be administered. It is also likely that more pain relief will be advised, and the possibility of a caesarean section becomes more likely. As it became clear that the anticipated progress through labour was not being made, the possibility that plans would have to change was naturally upsetting and women commonly reflected in their interviews on the 'ideal' labour and birth that they felt they had lost: I first went to the birthing centre as opposed to the delivery suite. That was my ideal labour, would have been deliver in the birthing centre … I probably went up to the ward about quarter past nine and then I didn't really like it. [the doctor] was saying how do I feel about having an epidural [prior to syntocinon] and they went through the pros and cons of the epidural and I decided that I was going to have one and to be honest, I'd already thought all the way through that I was kind of against it and I did kind of want, I would have loved a water birth. There came a point at which choice receded As explained above, the exclusion criteria for the pilot study meant that only 'lowrisk' women were recruited. Before labour commenced, therefore, all options were open for these women, but, as time went on, the scope for choice diminished. Corporeal realities began to set the agenda. On the whole, although they often found it upsetting, women were accepting of the fact that the plans they had made were no longer feasible.
Fundamental to this widespread acceptance was a recognition on the part of women themselves that they were not making the anticipated progress through labour. In many cases, the women were acutely aware that their body was not doing what it needed to in order to progress the labour as their uterine contractions and cervical dilatation patterns were being regularly monitored by midwives. Even if concerns were not immediately conveyed to the woman, the midwife's language or behaviour could suggest to them that things were not going as would be hoped [bib_ref] The swan effect in midwifery talk and practice: A tension between normality..., Scamell [/bib_ref].
The cervix needs to reach full dilatation (10 cm) before the second stage of labour can begin and the baby be born -this is often referred to colloquially as reaching 'the magic 10'. Women typically focused in on the number of centimetres of cervical dilation they had reached and were aware that they were not where they needed to be:
So it got to the stage where I was dilated at five centimetres but then that just stuck, nothing changed. [fig_ref] Table 1: Interview sample characteristics [/fig_ref] We got to eight centimetres ok and then examined me again and part of the cervix wasn't dilating further although most of it was but part of it wasn't and so they said 'ok, we'll give it another couple of hours', so we gave it another couple of hours and then examined again, it was still the same. This focus on talking in terms of the number of centimetres of cervical dilation reached appeared to serve as a form of objective measurement of their progress through the first stage of labour. The numbers reported to them by health professionals following examinations were accepted seemingly without question, and women could themselves appreciate that there was a discrepancy between where they were and where they needed to be. Repeat examinations that produced the same figures led them to accept that their progress had become 'stuck'. It was at this point that women commonly began to draw distinctions between what they had hoped would happen during their labour and birth and what their situation actually was:
In my ideal world I would have just had a water birth but that wasn't feasible so I was open to suggestions. (Participant 2)
## Coping with diminishing choice
Having accepted that their labour and birth were not progressing as they would have wished, there were differences in the ways in which women talked about how they went about approaching their revised circumstances and the degree to which they wished to stay involved in decision-making. The majority of women interviewed were ultimately willing to cede control of decision-making about how their labour progressed to the health professionals caring for them. In many ways, this was similar to entering the sick role in that they appeared to accept that they no longer knew what needed to happen and were happy to defer to those they regarded as having the technical expertise required to manage the changed situation effectively and make decisions on their behalf [bib_ref] The sick role and the role of the physician reconsidered, Parsons [/bib_ref] [bib_ref] Parsons revisited: From the sick role to …?, Williams [/bib_ref] :
I have no knowledge of birth, I'm not a midwife, I have no knowledge, so I think I was very much open to suggestion and open to what they were saying to me and always felt that whatever they were saying was always going to be in mine or baby's best interests anyway. There appeared to be some kind of 'tipping point' at which the situation changed from being about what their preferences would be from a range of possible options (as it had been for place of birth, type of pain relief, etc.) to one in which the possibilities were much reduced and were being driven by clinical necessity rather than patient preference. Importantly, women who adopted this approach accepted the legitimacy of this change as it was being presented to them -typically because they trusted the health professionals caring for them: I did feel that I had control over what was going on, what was happening, up until the point where they said we've got to get baby out and, at that point, I just thought 'well whatever these guys think they need to do now'. At that point I was happy really to trust whatever they were saying to me … I was fairly trusting in the people that were advising me or sort of telling me what the options were. (Participant 5) This is not to say that these women were always necessarily particularly happy with what ultimately happened to them (e.g. having a caesarean), but they did accept the legitimacy of health professionals' assessments of their situation. The fact that their baby may be at risk added an extra layer of complexity -there was such a lot at stake and there did not seem to be any other option. This can be seen in the two data extracts below, both from women who ultimately gave birth via caesarean section: Combined with their acceptance of the legitimacy of these professionals' technical expertise, the women were also very aware that their resources were rapidly depletingthey were very tired, frequently in a great deal of pain, and often affected to some degree by the effects of pain relief. They had reached a point at which they were happy to let someone else take charge and manage the situation: By this point I was exhausted and the birth plan had well and truly, could have been ripped up anyway. Some women went further and actively tried to reject any kind of involvement in making decisions about what to do. While the women discussed above were happy to defer ultimate decision-making to health professionals, this smaller group went beyond this and actively sought to detach or remove themselves from the situation completely. While they of course could not do this bodily, they talked about mentally or emotionally seeking to withdraw from the situation as a coping mechanism and just wait for it all to be over: I realised there's no way of getting away from this [situation] and just thinking 'I just need to pretend I'm dead', like just completely take myself out of the whole situation and just shut down to get through it. (Participant 9) It sounds very strange but I almost wasn't very interested, you know … I was happy to just let everybody else worry about what was happening. So deep seated was these women's desire to absent themselves that, in these cases, even involvement at the level of being asked to sign consent forms (e.g. for a caesarean section) was experienced as an unwelcome intrusion. In common with work exploring the role of patient consent in emergency surgical situations, the seeking of consent was experienced as problematic and interpreted as largely tokenistic or ritualistic rather than having any real meaning or significance: I definitely wasn't in the frame of mind you'd normally get someone to consent for something in. Only a small minority of women reported having been keen to stay involved in the decision-making process and being unhappy if they felt they were being left out. In the extract below, one such woman recounts how she challenged health professionals who, she felt, were leaving her out of important discussions:
[formula] DID [/formula]
They were talking [about a possible] caesarean and I did actually pipe up and say 'you're talking about me in a room, talk to me, if caesarean is what you're considering, then I want to be part of that decision-making' and at which point, the, she was a surgeon, I presume she was a consultant as well came back and said 'I am sorry, force of habit' and we discussed the options. In contrast to those women who were happy to defer to health professionals who they regarded as having the necessary technical expertise to manage the situation on their behalf, for this woman the principle of maintaining involvement and being meaningfully consulted on what might have to happen retained its importance. These few women are an interesting exception, and it is debatable whether they really were involved in making decisions in any meaningful way as, given the clinical circumstances, the options were really very limited.
# Discussion
Choice and patient involvement in decision-making are strong aspirations of contemporary healthcare, but translating these somewhat abstract principles into practice is often far from straightforward, and it has been argued that politicians have stoked up choice as something which doctors and patients often do not recognise and/or cannot achieve [bib_ref] Towards a history of choice in UK health policy, Greener [/bib_ref] [bib_ref] Discriminating customers, responsible patients, empowered users: Consumerism and the modernisation of health..., Newman [/bib_ref]. In this article, we sought to explore how these ideals of choice and patient involvement in decision-making may be disrupted by unanticipated complications that can rapidly limit the extent to which either is likely to be achievable in practice. By drawing on the accounts of women who experienced delay during labour with their first child, we have examined how these women experienced the sometimes rapid reduction in the potential for them to exercise choice about how their labour and birth progressed. We situated our work in the context of Annemariework on the inherent tension she sees between the 'logic of choice' and the 'logic of care'.
We have demonstrated how, although they often found it upsetting, women commonly accepted that the choices and plans they had made about how they wanted their labours and births to be were no longer feasible as their labours became 'abnormal'. Women appeared to accept that the ideal of making choices that fitted with their values and preferences had to be abandoned, and that clinical circumstances legitimately changed events. The majority were willing to defer to clinical staff who they regarded as having the necessary technical expertise, while some women actively rejected any involvement in decision-making altogether appearing not even to want to be kept informed. Only a minority sought to continue an active role in decision-making, although it is not clear to what extent this was actually possible.
What is noticeable is that, for many women, the plans they had made were let go fairly easily -they 'went out the window' or were 'ripped up'. These were women having their first child, so there is some recognition on their part that, with the benefit of hindsight, they had been 'planning in the dark' as they had little idea what labour and birth would be like and their ideal hopes and expectations were often very different to their actual experiences [bib_ref] More in hope than expectation: A systematic review of women's expectations and..., Lally [/bib_ref] [bib_ref] Anesthesia and analgesia-related preferences and outcomes of women who have birth plans, Pennell [/bib_ref]. In situations such as this in which corporeal realities set the agenda, and the women accepted that the safety of either themselves or their baby was potentially at risk, ideals of patient choice and involvement in decision-making appeared to be readily abandoned and were sometimes completely inverted. The group of women who sought to detach themselves as fully as possible from the situation adopted a very passive patient role and could be understood as wanting to be 'rescued' by health professionals. While 'informed consent' was sought for each intervention, women talked about these interventions as 'not being an option' -there did not seem to be a choice to be made anymore.
It is interesting to reflect on how and why women were apparently so able to reconcile themselves to their changed circumstances and be prepared to accept that the choices and plans they had made could not now be followed. Ashas suggested, the 'logic of care' is characterised by fluidity and as an interactive and on-going process which is shaped and re-shaped depending on its results. What seems to be the case here is that the slow progress of their labours had given women time to adjust to, and accept, the fact that their birth would not be as they had planned or would have liked. This longitudinal aspect, combined with the apparent objectivity of the degree of cervical dilation measured and reported at each examination, perhaps meant that these women were more able to accept their changed situation than if it had been presented to them completely out of the blue and with no prior warning.
Having accepted the legitimacy of having to do things differently to how they may have liked or planned, and that corporeal realities were now setting the agenda, the priority for these women very firmly became the safe delivery of their baby, rather than their own preferences and choices about labour and birth. It seems, then, that the emphasis switches very clearly from what they may want to what their baby needs. What does this tell us about the relative importance of choice compared to other outcomes? It would appear that, in this context at least, choice becomes framed as associated with the mother and for her benefit, and that, while this may be nice to have where possible, it does not come above the safety of the baby. When the latter appears to be at risk, the former loses any significance.has argued that one of the problems with the 'logic of choice' is that it focuses on discrete end products that, it assumes, are all deliverable by health professionals. Before their labours began, the majority of women we interviewed had made choices about how they wanted their labours and births to be, typically this meant a 'natural' process with minimal (ideally no) medical intervention. The 'end product' here was the birth itself. The unanticipated complications they experienced served to make them re-evaluate what the important 'end product' actually was, and they subsequently focused on a safe and healthy baby that must be achieved through any means necessary.
While the current socio-cultural context can be argued to make adopting an apparently passive patient role difficult [bib_ref] Consumerism, reflexivity and the medical encounter, Lupton [/bib_ref] , the women in this study successfully managed the transition from being active choosers to relying on doctors' judgement. While the contemporary discourses and policy statements about choice and patient involvement position patients and professionals as equal partners, it is clear that the vast majority of women in this study were very willing to adopt instead an asymmetrical relationship with those caring for them [bib_ref] On the remarkable persistence of asymmetry in doctor/patient interaction: A critical review, Pilnick [/bib_ref] , preferring to place their trust in professionals to make choices and decisions in their best interests. Women talked very clearly about recognising and respecting the technical knowledge and expertise they regarded these professionals as possessing. They were prepared to put their trust in them with the expectation that whatever they did would be in the best interests of both them and their babies. Indeed, while discourses of choice may be argued to offer up preferred identities to patients [bib_ref] Governing the ethical consumer: Identity, choice and the primary care medical encounter, Mcdonald [/bib_ref] , in this context at least the discourse around motherhood was also a powerful force -a 'good mother' relies on the doctor's expertise to keep her baby safe from harm [bib_ref] Women's childbirth preferences and practices in the United States, Miller [/bib_ref] rather than pursuing her preference for a vaginal birth when the doctor tells her that her baby is showing signs of distress and a caesarean section is needed.
If choice appears to be something of a 'red herring' in this context, then what can we learn from our data about what is important to women and how they can best be supported during this type of event? The importance of aspiring to offer choice and involvement in decision-making must be balanced with the need to keep other possible outcomes always in mind. Ashas argued, the 'logic of care' places the fluidity and uncertainty of care centre-stage; the 'logic of choice', in contrast, fails to do so. While not so evident in our study, evidence from elsewhere shows that many women who have emergency caesarean sections have not thought about the possibility antenatally [bib_ref] Women's views on the impact of operative delivery in the second stage..., Murphy [/bib_ref]. Knowing about, and being prepared for, what may potentially happen if things do not go to plan may be important in helping women cope in these circumstances.
This study has some important limitations. First, all of the mothers and babies in the pilot study, and therefore eligible for recruitment to the interview element, were healthy after birth and the views and experiences of others with poorer outcomes may be different. Second, the data on which this article is based are drawn from interviews conducted after the event and not on direct observation of these women and their encounters with health professionals during their labours and births. Third, due to the lower than anticipated response rate, the sample size is smaller than we would ideally have liked, although the sample is heterogeneous and includes women from the low-and high-dose arms of the pilot study; those who had a caesarean section, instrumental or spontaneous vaginal birth; and those who did or did not have an epidural during labour. Furthermore, analysis showed that the demographic characteristics of the interview sample did not differ significantly from the wider pilot study population.
Notwithstanding these limitations, this study makes an important contribution to understanding how women who have been offered, and engaged with, the aspirations of choice and involvement in decision-making cope with experiencing a clinical situation in which choices rapidly recede. Their acceptance of the changed circumstances as legitimately limiting choice, combined with the willingness of many to defer to the technical knowledge and expertise of professionals, clearly demonstrates that, in this context at least, the importance attached to choice diminishes rapidly in favour of other outcomes. To return tocall for more empirical work on the interferences that happen when the 'logic of choice' and the 'logic of care' get mixed together in real life, our work has shown that the latter can very easily displace the former and be accepted as the preferred and most appropriate mode by all concerned. Deborah [bib_ref] Consumerism, reflexivity and the medical encounter, Lupton [/bib_ref] has argued that in interactions with doctors, patients may pursue both consumerist and passive patient subject positions simultaneously and variously due to the complex and changeable nature of healthcare. Even those supportive of a consumerist model generally would, she argues, place their trust in doctors to make decisions on their behalf on some occasions. As we highlighted in our introduction to this article, contemporary health empowerment discourses tend to ignore or obscure the complex forms of dependence that characterise many healthcare experiences and situations [bib_ref] Informing health? Negotiating the logics of choice and care in everyday practices..., Henwood [/bib_ref]. Yet, dependency is a central feature of much illness experience and works against the full taking-up of a consumerist approach -patients cannot always be Health 21 (2) ideal-type consumers [bib_ref] Consumerism, reflexivity and the medical encounter, Lupton [/bib_ref]. The challenging role for health professionals is to effectively bring about a smooth transition from the 'logic of choice' into the 'logic of care' -to do so in a person-centred way that affords people dignity, compassion and respect and offers them personalised care and support . The accounts of women we interviewed for this study suggested that, in the vast majority of cases, they believed this had been achieved. We have already highlighted as a limitation of this work that it is based solely on the accounts of women shortly after the event and not on observations in real time. Future observational work of this kind would be very valuable in further understanding how health professionals working in such contexts can best manage this transition.
[table] Table 1: Interview sample characteristics. [/table]
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Understanding Dyslexia in the Context of Developmental Language Disorders
Purpose: The purpose of this tutorial is to discuss the language basis of dyslexia in the context of developmental language disorders (DLDs). Whereas most studies have focused on the phonological skills of children with dyslexia, we bring attention to broader language skills. Method: We conducted a focused literature review on the language basis of dyslexia from historical and theoretical perspectives with a special emphasis on the relation between dyslexia and DLD and on the development of broader language skills (e.g., vocabulary, syntax, and discourse) before and after the identification of dyslexia. Results: We present clinically relevant information on the history of dyslexia as a language-based disorder, the operational definitions used to diagnose dyslexia in research and practice, the relation between dyslexia and DLD, and the language abilities of children with dyslexia. Conclusions: We discuss 3 clinical implications for working with children with dyslexia in school settings: (a) Children with dyslexia-with and without comorbid DLDs-often have language deficits outside the phonological domain; (b) intervention should target a child's strengths and weaknesses relative to reading outcomes, regardless of diagnostic labels; and (c) those who have dyslexia, regardless of language abilities at the time of diagnosis, may be at risk for slower language acquisition across their lifetime. Longitudinal studies are needed to assess multiple language skills early, at the time of the diagnosis of dyslexia, and years later to better understand the complex development of language and reading in children with dyslexia.
Purpose: The purpose of this tutorial is to discuss the language basis of dyslexia in the context of developmental language disorders (DLDs). Whereas most studies have focused on the phonological skills of children with dyslexia, we bring attention to broader language skills. Method: We conducted a focused literature review on the language basis of dyslexia from historical and theoretical perspectives with a special emphasis on the relation between dyslexia and DLD and on the development of broader language skills (e.g., vocabulary, syntax, and discourse) before and after the identification of dyslexia. Results: We present clinically relevant information on the history of dyslexia as a language-based disorder, the operational definitions used to diagnose dyslexia in research and practice, the relation between dyslexia and DLD, and the language abilities of children with dyslexia. Conclusions: We discuss 3 clinical implications for working with children with dyslexia in school settings: (a) Children with dyslexia-with and without comorbid DLDs-often have language deficits outside the phonological domain; (b) intervention should target a child's strengths and weaknesses relative to reading outcomes, regardless of diagnostic labels; and (c) those who have dyslexia, regardless of language abilities at the time of diagnosis, may be at risk for slower language acquisition across their lifetime. Longitudinal studies are needed to assess multiple language skills early, at the time of the diagnosis of dyslexia, and years later to better understand the complex development of language and reading in children with dyslexia.
A lthough the term dyslexia is familiar to most of the lay public, there is no consensus on precise diagnostic criteria. Most definitions of dyslexia agree on primary inclusionary criteria, including marked difficulties with word reading, decoding, and spelling as evidenced by low accuracy and/or fluency on standardized assessments. There is also a general agreement that these difficulties should be inconsistent with or "unexpected" in consideration of other aspects of development, including general intellectual abilities (American Psychiatric Association [APA], 2013; [bib_ref] A definition of dyslexia, Lyon [/bib_ref] [bib_ref] Defining dyslexia, Tunmer [/bib_ref]. For example, children with hearing or vision impairment or with neurodevelopmental syndromes or who have had a prior head injury may experience reading and spelling difficulties as a result, but they would not be considered to have dyslexia. Some definitions further specify that poor instruction should be ruled out as a cause of reading and spelling difficulty [bib_ref] A definition of dyslexia, Lyon [/bib_ref]. In research and practice, the operationalization of these inclusionary and exclusionary criteria varies widely, leading to sizeable variation in estimated prevalence rates-from as low as 3% to as high as 20% of the population [bib_ref] Incorporating RTI in a hybrid model of reading disability, Spencer [/bib_ref].
One source of confusion concerns perceptions about the oral language abilities of children with dyslexia. On the one hand, dyslexia has been described as a "languagebased" disorder for many years; such descriptions have been focused primarily on phonological deficits as a core feature of dyslexia [bib_ref] A definition of dyslexia, Lyon [/bib_ref] [bib_ref] Demystifying the " D" word: Why and how the term dyslexia should..., Moats [/bib_ref]. On the other hand, there is less clarity about the extent to which other aspects of language development, such as vocabulary, syntax, and discourse, are affected in individuals with dyslexia. Although one line of research has established that dyslexia and developmental language a University of South Carolina, Columbia disorder 1 (DLD; are separate disorders that frequently co-occur [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] , some experts have suggested that the presence of DLD would make word reading difficulties no longer "unexpected" and therefore should exclude a child from the classification of dyslexic [bib_ref] Reading disability defined as a discrepancy between listening and reading comprehension: A..., Badian [/bib_ref] [bib_ref] Cross-disciplinary dialogue about the nature of oral and written language problems in..., Silliman [/bib_ref] [bib_ref] Incorporating RTI in a hybrid model of reading disability, Spencer [/bib_ref] [bib_ref] Defining dyslexia, Tunmer [/bib_ref]. In this article, we consider the language basis of dyslexia from a historical and theoretical perspective drawing from pertinent empirical work. We discuss the overlap of dyslexia and DLD and their relative frequency, followed by clinical implications and directions for future research.
Defining Dyslexia as a "Language-Based" Disorder When William Berlin first introduced the term dyslexia in 1887, he used it to describe adult patients who had reading problems as a result of cerebral disease, and the disorder was conceptualized within the general class of aphasias [bib_ref] Historical perspectives on dyslexia, Richardson [/bib_ref]. The first published case study of a developmental reading disorder was written by W. Pringle Morgan, who used the term congenital word blindness, in 1896. Morgan's description of "Percy," a 14-year-old boy with severe reading difficulty, bears striking resemblance to the current characterizations of children with dyslexia: "He has been at school or under tutors since he was 7 years old, and the greatest efforts have been made to teach him to read, but, in spite of this laborious and persistent training, he can only with difficulty spell out words of one syllable…. I may add that the boy is bright and of average intelligence in conversation. His eyes are normal…and his eyesight is good. The schoolmaster who has taught him for some years says that he would be the smartest lad in school if the instruction were entirely oral" [bib_ref] Hidden language impairments in children: Parallels between poor reading comprehension and specific..., Morgan [/bib_ref]. Subsequent articles by James [bib_ref] Four cases of congenital word-blindness occurring in the same family, Hinshelwood [/bib_ref] reported six cases of children with congenital word blindness across two generations of a single family, providing suggestive evidence of a genetic component that is consistent with modern-day evidence [bib_ref] Oral language deficits in familial dyslexia: A meta-analysis and review, Snowling [/bib_ref]. Approximately 30 years after Morgan's first case was reported, Samuel Orton examined over 1,000 children in the state of Iowa to determine the prevalence of word blindness, finding that one in 10 children had marked difficulty with reading words. Orton observed that many of these children had a history of oral language problems, and he was one of the first to frame dyslexia as part of a larger set of DLDs. Since those foundational studies, dyslexia has been referred to by many other terms such as visual agnosia for words, psycholexia, strephosymbolia, and specific reading disability [bib_ref] A brief history of time, phonology, and other dimensions of developmental dyslexia, Wolf [/bib_ref].
Contemporary researchers have confirmed Orton and Morgan's notion of dyslexia as a language-based disorder [bib_ref] Predicting dyslexia from kindergarten: The importance of distinctness of phonological representations of..., Elbro [/bib_ref] [bib_ref] Dyslexia as a phonological deficit: Evidence and implications, Snowling [/bib_ref] , based primarily on deficits in the phonological domain. In a 1989 article entitled "Defining Dyslexia as a Language Based Disorder," Hugh Catts stated, "Dyslexia is a developmental language disorder that involves a deficit(s) in phonological processing. This disorder manifests itself in various phonological difficulties as well as a specific reading disability" ; see also [bib_ref] Defining dyslexia as a developmental language disorder: An expanded view, Catts [/bib_ref]. Explicitly labeling dyslexia as a language-based disorder was, in part, a strong and direct response to the misperception that dyslexia is a visually based disorder (cf. American Academy of Pediatrics, 2009). It is noteworthy that Hinshelwood had also presented strong arguments against a visual deficits explanation for word blindness as early as 1900. The primary phonological deficit associated with dyslexia negatively impacts the specificity at which sounds are stored and recalled in words as well as an individual's ability to manipulate sounds in words and connect sounds to letters to read words. There is now an abundance of evidence that children with dyslexia, on average, perform poorly on tasks that involve phonology including phoneme awareness, word and nonword repetition, and word retrieval (see review by [bib_ref] Specific reading disability (dyslexia): What have we learned in the past four..., Vellutino [/bib_ref].
As we have reviewed, dyslexia is defined as a difficulty with word level reading and spelling skills, which are in turn caused by phonological deficits. However, being a good reader involves more than only reading the words on a page. As conceptualized in the simple view of reading ; see also [bib_ref] Unique and common effects of decoding and language factors in predicting reading..., Foorman [/bib_ref] , reading comprehension is the product of accurate and efficient word reading and language comprehension. The language comprehension component (sometimes called "linguistic comprehension" or "listening comprehension") encompasses all of the linguistic knowledge and skills required for a listener to comprehend a text if it was read aloud, including vocabulary and semantic processing, syntax, inferencing, and discourse. In contrast to the large amount of evidence for phonological deficits in children with dyslexia, the status of their broader language abilities in these domains outside phonology is less clear. Many studies have reported that, in addition to phonological deficits, children with dyslexia also have weaknesses in other aspects of language including vocabulary, morphology, syntax, and discourse, often before the onset of formal reading instruction (e.g., [bib_ref] Language basis of reading and reading disabilities: Evidence from a longitudinal investigation, Catts [/bib_ref] [bib_ref] Very early language deficits in dyslexic children, Scarborough [/bib_ref] [bib_ref] Family risk of dyslexia is continuous: Individual differences in the precursors of..., Snowling [/bib_ref]. However, two factors complicate the determination of language (dis)abilities in children with dyslexia. The first is variation in how the definition of dyslexia is operationalized for diagnosis. The second is variation in the time of onset of oral language difficulties. Noting the time of 1 onset is important because reading difficulties can themselves cause slower language development, as much of language is learned via reading experience [bib_ref] Early reading acquisition and its relation to reading experience and ability 10..., Cunningham [/bib_ref] [bib_ref] Distinguishing cause from effect-Many deficits associated with developmental dyslexia may be a..., Huettig [/bib_ref].
## Operationalizing the definition of dyslexia
Morgan's (1896) description of Percy was the first documented case of childhood dyslexia, and it included multiple characteristics present in contemporary definitions of dyslexia [bib_ref] A definition of dyslexia, Lyon [/bib_ref] : (a) a severe difficulty learning to read, despite (b) normal vision, (c) adequate instruction, and (d) average intelligence. Given these characteristics, as well as the boy's ability to learn from oral instruction, the reading problem is quite "unexpected" (cf. [bib_ref] A definition of dyslexia, Lyon [/bib_ref]. However, how is this "unexpected" deficit operationalized in the diagnosis of dyslexia, and how do language skills outside the domain of phonology factor in? Although Morgan's description of Percy noted strong oral language abilities, that characteristic does not appear in most contemporary definitions of dyslexia (but see [bib_ref] Defining dyslexia, Tunmer [/bib_ref].
Traditionally, an IQ achievement discrepancy approach was used to operationalize dyslexia definitions for diagnosis for educational or research purposes. Under this approach, children were considered to have dyslexia when their word reading skills, as measured by norm-referenced measures of word reading speed or accuracy, were "discrepant" from their intelligence [bib_ref] The external validity of age-versus IQ-discrepancy definitions of reading disability: Lessons from..., Pennington [/bib_ref] [bib_ref] Prevalence of reading disability in boys and girls, Shaywitz [/bib_ref]. Under this approach, it was assumed that the IQ score was an indicator of a child's potential, and a word reading score that fell significantly below an IQ score was viewed as evidence that the child was not performing at his or her full potential. Also under this approach, IQ was often quantified by a full-scale IQ that was a composite of both verbal and nonverbal IQ scores. Thus, under this approach, children with broad language deficits were less likely to qualify for a dyslexia diagnosis than children with normal language abilities because children with broad language deficits would be unlikely to achieve a high verbal IQ score. Instead, children with IQ scores commensurate with their word reading deficits were often referred to as "garden variety" poor readers, and it was believed that that they would not experience the same benefit from reading interventions as children with dyslexia .
The IQ achievement discrepancy model fell out of favor for several reasons. First, there were statistical issues: The size of the observed discrepancy would depend on the tests used (i.e., some word reading and IQ tests were easier than others), and because of regression to the mean (i.e., extreme scores are statistically more likely to be preceded or followed by less extreme scores), children with high IQs were more likely to qualify as dyslexic than children with low IQs [bib_ref] Psychometric approaches to the identification of LD: IQ and achievement scores are..., Francis [/bib_ref]. In addition, because reading requires formal instruction, it could take several years for test scores to suggest a "significant" discrepancy between IQ and reading achievement [bib_ref] Intelligence testing and the discrepancy model for children with learning disabilities, Fletcher [/bib_ref] , often delaying access to interventions. Finally, there was a lack of evidence that reading profiles were different between discrepant and nondiscrepant poor readers [bib_ref] IQ is irrelevant to the definition of learning disabilities, Siegel [/bib_ref] , and both groups were able to improve their reading skills when provided an evidence-based intervention [bib_ref] Toward distinguishing between cognitive and experiential deficits as primary sources of learning..., Vellutino [/bib_ref].
As an alternative to the IQ discrepancy approach, a somewhat more liberal approach to diagnosing dyslexia has been to use an IQ cutoff to rule out low cognitive abilities with no stipulation of a discrepancy between IQ and word reading abilities [bib_ref] Differentiating between difficult-to-remediate and readily remediated poor readers: More evidence against the..., Vellutino [/bib_ref] [bib_ref] The doubledeficit hypothesis and difficulties in learning to read a regular orthography, Wimmer [/bib_ref]. In practice, this meant that children with dyslexia had low word reading in the presence of "normal" intelligence. Although both verbal and nonverbal IQ scores have been used with this approach (e.g., [bib_ref] Syntactic comprehension in reading and listening: A study with French children with..., Casalis [/bib_ref] [bib_ref] Multiple stimulus presentation yields larger deficits in children with developmental dyslexia: A..., Zoccolotti [/bib_ref] , most current diagnostic criteria for dyslexia quantify adequate cognition using only nonverbal IQ measures and a liberal cutoff that does not qualify the child as being "cognitively impaired," for example, within 2 SDs of the mean (e.g., [bib_ref] Word learning deficits in children with dyslexia, Alt [/bib_ref]. Relative to the IQ discrepancy approach, the IQ cutoff approach provides a greater opportunity for children with language deficits beyond the domain of phonology to be identified as having dyslexia because it does not require that a child have a high verbal IQ.
As the field grappled with how to operationalize "average intelligence" in the diagnostic criteria for dyslexia, the importance of "adequate instruction" also came into the forefront. An influential study by focused on first-grade students with poor word reading abilities. When these children were provided one semester of high-quality, evidence-based reading instruction, the majority of them showed substantial improvement, such that they were no longer considered poor readers. The smaller group of children that did not respond to treatment showed poorer phonological skills before the onset of instruction than those who did respond. The authors recommended that only those who do not respond to high-quality, evidencebased reading instruction should be considered reading disabled, whereas the others may have demonstrated initially low reading scores due to experiential or instructional deficits. On the basis of the results of this study and others like it (Al [bib_ref] Characteristics of children who are unresponsive to early literacy intervention: A review..., Otaiba [/bib_ref] [bib_ref] Individual differences in response to early interventions in reading: The lingering problem..., Torgesen [/bib_ref] [bib_ref] What time may tell: Towards a new conceptualization of developmental dyslexia, Wolf [/bib_ref] , the reauthorization of the federal special education law in 2004 (Individuals with Disabilities Education Improvement Act, 2004; PL 108-446) allowed for identification of learning disabilities based on a student's failure to respond to scientifically based instruction. The diagnosis of dyslexia then became less important for public schools using this approach because it was a failure to respond to intervention, rather than a specific diagnostic label, that led to special education services. However, children meeting the standard criteria for dyslexia would still be identified for these services if they were not making adequate progress in response to evidence-based instruction in the regular education system. Research that has examined predictors of response to instruction has shown that children with broader language deficits, including problems with vocabulary and grammar, tend to show poorer responses to instruction than children whose language difficulties are restricted to phonology (Al [bib_ref] Who are the young children for whom best practices in reading are..., Otaiba [/bib_ref] [bib_ref] Reading with vocabulary intervention: Evaluation of an instruction for children with poor..., Duff [/bib_ref] [bib_ref] Effects of supplemental early reading intervention at 2-year follow up: Reading skill..., Vadasy [/bib_ref] [bib_ref] Young children at risk of literacy difficulties: Factors predicting recovery from risk..., Whiteley [/bib_ref].
## The relationship between dld and dyslexia
To some, the characterization of dyslexia as a language-based disorder may be confusing in light of another prominent language disorder, DLD. Children with DLD have an unexpected deficit in language abilities despite adequate environmental stimulation and cognitive abilities with no neurological impairments [bib_ref] Phase 2 of CATALISE: A multinational and multidisciplinary Delphi consensus study of..., Bishop [/bib_ref] ; National Institute of Deafness and Other Communication Disorders, 2017). These children may have language deficits across multiple dimensions of language-phonology, morphology, syntax, vocabulary, and pragmatics-but operational definitions often require deficits in more than one language domain [bib_ref] Phase 2 of CATALISE: A multinational and multidisciplinary Delphi consensus study of..., Bishop [/bib_ref] [bib_ref] Prevalence of specific language impairment in kindergarten children, Tomblin [/bib_ref]. Although DLD is recognized as a persistent disorder with negative impacts on literacy, academic progress, and employment opportunities (Nippold, [bib_ref] Expository discourse in adolescents with language impairments: Examining syntactic development, Mansfield [/bib_ref] [bib_ref] Language profiles and literacy outcomes of children with resolving, emerging, or persisting..., Snowling [/bib_ref] [bib_ref] Adult psychosocial outcomes of children with specific language impairment, pragmatic language impairment..., Whitehouse [/bib_ref] , evidence suggests that a large proportion of children who qualify as having DLD are either not identified or are identified in later school grades, based on problems with reading comprehension [bib_ref] Language deficits in poor comprehenders: A case for the simple view of..., Catts [/bib_ref] [bib_ref] Estimating familial loading in SLI: A comparison of direct assessment versus parental..., Conti-Ramsden [/bib_ref] [bib_ref] Hidden language impairments in children: Parallels between poor reading comprehension and specific..., Morgan [/bib_ref] [bib_ref] Prevalence of specific language impairment in kindergarten children, Tomblin [/bib_ref]. It has been argued that parents and teachers may be more aware of problems with speech articulation and word reading than problems with understanding and producing oral language [bib_ref] Identifying children at risk for language impairment or dyslexia with group-administered measures, Adlof [/bib_ref] [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] [bib_ref] Hidden language impairments in children: Parallels between poor reading comprehension and specific..., Morgan [/bib_ref] [bib_ref] Cross-disciplinary dialogue about the nature of oral and written language problems in..., Silliman [/bib_ref].
There are clear parallels between the definitions of dyslexia and DLD. First, they both involve a deficit that is "unexpected" given the absence of intellectual disabilities, perceptual deficits, or other medical explanations for the observed deficits. Second, they both stipulate adequate environmental stimulation. In the case of dyslexia, the unexpected deficit is in word reading, and adequate stimulation is appropriate instruction in reading. In the case of DLD, the unexpected deficit is in overall language development, and adequate stimulation is human language interactions. Interestingly, there has been a recent surge of advocacy in the United States to raise awareness about dyslexia (Ward-Lonergan & Duthie, 2018), and internationally to raise awareness of DLD [bib_ref] RALLI: An Internet campaign for raising awareness of language learning impairments, Bishop [/bib_ref] , but this advocacy is generally conducted in parallel with relatively little attention to co-occurrences.
If dyslexia is a language-based disorder, then do all children with dyslexia have DLD? Although the question appears to be straightforward, the varied criteria used to diagnose dyslexia have made answering this simple question complex. G. M. [bib_ref] On the "specifics" of specific reading disability and specific language impairment, Mcarthur [/bib_ref] pooled study samples from prior research to examine the proportion of children receiving services for DLD or dyslexia who would meet diagnostic criteria for both disorders. They found that 55% of children with dyslexia could be classified as having DLD, and 51% of children with DLD could be classified as having dyslexia. Furthermore, all but 10% of children with dyslexia scored below average on standardized language assessments, and all but 20% of children with DLD scored below average on reading measures. These findings raised questions about whether dyslexia and DLD were different manifestations of the same disorder [bib_ref] Developmental dyslexia and specific language impairment: Same or different?, Bishop [/bib_ref] [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref]. Perhaps, the diagnostic label assigned to a child experiencing reading or language difficulties was simply a reflection of the practitioner assigning it (e.g., school psychologist vs. speech-language pathologist).
In a 2004 review of the literature, Bishop and Snowling proposed that a partial distinction between DLD and dyslexia should be maintained, stating, "It is important to distinguish children with relatively pure phonologically based reading problems from those with more global oral language impairments" (p. 862). They proposed a two-by-two model crossing phonological deficits against broader, nonphonological language skills (e.g., morphology, vocabulary, and syntax). As shown in , they hypothesized that phonological deficits underlie both dyslexia and DLD, but the two disorders would be differentiated on the basis of broader language skills. Whereas children with DLD would show deficits in both phonological and nonphonological language skills, skills outside the phonological domain would be relatively intact for children with dyslexia. Thus, in [bib_ref] Developmental dyslexia and specific language impairment: Same or different?, Bishop [/bib_ref] model, most children with DLD should have dyslexia, because of presumed underlying phonological deficits, but not all children with dyslexia would have DLD. [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] tested [bib_ref] Developmental dyslexia and specific language impairment: Same or different?, Bishop [/bib_ref] partial distinction hypothesis and two competing hypotheses, which we refer to here as the "phonological deficit severity" hypothesis and the "distinct disorders" hypothesis. The phonological deficit severity hypothesis (see proposed that phonological deficits underlie both DLD and dyslexia, but these phonological deficits are more severe in children with DLD and have negative impacts on the development of broader language skills. Under the phonological deficit severity hypothesis, all children with DLD should have phonological deficits that lead to dyslexia. The distinct disorders hypothesis (see posited that DLD and dyslexia are fully distinct and separate disorders that frequently co-occur, with dyslexia characterized by phonological deficits and DLD characterized by language deficits outside the phonological domain. The key difference between this hypothesis and [bib_ref] Developmental dyslexia and specific language impairment: Same or different?, Bishop [/bib_ref] partial distinction hypothesis is that the distinct disorders hypothesis predicted that some children with DLD-that is, those without dyslexia-would have phonological skills in the normal range. [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] had three important strengths present in few prior or subsequent studies. First, the study involved over 500 children who were drawn from a population-based sample who had participated in an epidemiologic study of language impairment. In contrast, most other studies have involved clinically referred samples, which likely include participants with more severe deficits and potentially more overlap between DLD and dyslexia. Second, [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] assessed reading and language skills in the same children from kindergarten through eighth grade. The DLD diagnosis was determined by kindergarten language scores, and children meeting the criteria for dyslexia were identified at the second, fourth, and eighth grades. In contrast, most other studies have examined a single time point, making it difficult to disentangle language problems that may have been caused by reading difficulties. Third, [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] used seven different methods to classify children as having dyslexia when examining the overlap between DLD and dyslexia: IQ discrepancy models based on (a) full-scale IQ and (b) nonverbal IQ, which did not require low achievement (such that children with word reading abilities in the normal range who still showed a discrepancy from IQ would be classified as dyslexic); IQ discrepancy models based on (c) full-scale IQ plus low achievement and (d) nonverbal IQ plus low achievement; and IQ cutoff models based on (e) full-scale IQ, (f ) nonverbal IQ, and (g) low word reading without reference to intelligence. [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] found that 17%-36% of children with kindergarten DLD also met criteria for dyslexia in the second through eighth grades, depending on the criteria used to diagnose dyslexia. The lowest rates of overlap were observed when dyslexia was diagnosed using a full-scale IQ discrepancy formula (17.0%-18.8% overlap), and the highest rates of overlap were observed for the low achievement definition with no reference to IQ (31.0%-35.6% overlap). Using IQ discrepancy and low achievement criteria, 14%-19% of children with dyslexia in the second through eighth grades also met the criteria for DLD. Although the rates of overlap were significantly higher than would be expected by chance, they were considerably lower than the rates of overlap that had been reported in prior studies involving clinically referred or convenience samples. In fact, in this population-based sample, the majority of children with DLD did not have dyslexia and the majority of children with dyslexia did not have DLD.
In follow-up analyses, [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] found that the vocabulary, morphology, and syntax deficits of children with DLD without dyslexia were just as severe as those of children with both DLD and dyslexia, which indicated that the phonological deficit associated with dyslexia did not translate to more severely impaired language skills in general. . Three hypotheses about the relation between dyslexia and developmental language disorder (DLD) tested by [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref]. Panel a depicts the phonological severity deficit hypothesis [bib_ref] Toward an understanding of developmental language and reading disorders, Kamhi [/bib_ref] , in which both dyslexia and DLD are caused by phonological deficits, with more severe phonological deficits leading to deficits in nonphonological domains. This hypothesis was rejected because of the existence of numerous children who showed deficits in vocabulary, grammar, and discourse, despite good skills in phonology. Panel b depicts the partial distinction hypothesis [bib_ref] Developmental dyslexia and specific language impairment: Same or different?, Bishop [/bib_ref] , in which all children with DLD show poor phonology (and therefore poor word reading), but in addition, they also have deficits in the other aspects of language, including vocabulary, grammar, and discourse. This hypothesis was rejected because of the existence of numerous children who met the standard diagnostic criteria for DLD but did not have poor phonology or poor word reading. Panel c depicts the fully distinct hypothesis, in which dyslexia and DLD are fully distinct disorders, with different underlying deficits. This model was supported by data from a large sample of children drawn from an epidemiologic study investigating the prevalence of DLD [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] and has been supported in numerous follow-up studies (e.g., [bib_ref] Identifying children at risk for language impairment or dyslexia with group-administered measures, Adlof [/bib_ref] [bib_ref] Dyslexia and specific language impairment: The role of phonology and auditory processing, Fraser [/bib_ref] [bib_ref] Phonological deficits in specific language impairment and developmental dyslexia: Towards a multidimensional..., Ramus [/bib_ref]. Children who are referred to in studies as "poor comprehenders" display poor reading comprehension despite adequate word reading abilities. Studies indicate that approximately one third of poor comprehenders met the standard diagnostic criteria for DLD [bib_ref] Morphosyntax in poor readers, Adlof [/bib_ref] [bib_ref] Language deficits in poor comprehenders: A case for the simple view of..., Catts [/bib_ref] [bib_ref] Hidden language impairments in children: Parallels between poor reading comprehension and specific..., Morgan [/bib_ref]. The remaining two thirds exhibited moderate deficits in vocabulary, syntax, and discourse, although they did not qualify as having DLD.
On the other hand, children with dyslexia, with or without DLD, consistently showed difficulty with phonologically based tasks, including phonemic awareness and nonword repetition. Taken together, these results indicated that phonological deficits were more closely associated with dyslexia than with DLD. It is notable that, in the Catts et al. sample, children with both DLD and dyslexia were more likely to have received clinical services in the primary grades, although their language skills were not more severely impaired compared with their peers with DLD without dyslexia. This finding provided additional evidence for the hypothesis that clinically referred samples overrepresent the overlap between DLD and dyslexia.
Considering the three hypotheses for the frequent overlap between children meeting criteria for DLD and dyslexia, [bib_ref] Are specific language impairment and dyslexia distinct disorders, Catts [/bib_ref] concluded that the evidence best supported the distinct disorders hypothesis. The phonological deficit severity hypothesis was ruled out by the existence of numerous children with DLD without dyslexia. The fact that children with dyslexia, with or without DLD, consistently showed difficulty with phonologically based tasks, whereas those with DLD without dyslexia showed relatively mild and transient difficulties, was contrary to the predictions of [bib_ref] Developmental dyslexia and specific language impairment: Same or different?, Bishop [/bib_ref] partial distinction hypothesis.
Many subsequent studies have provided converging evidence for the existence of these distinct subgroups [bib_ref] Identifying children at risk for language impairment or dyslexia with group-administered measures, Adlof [/bib_ref] [bib_ref] Word learning deficits in children with dyslexia, Alt [/bib_ref] [bib_ref] Impairment in non-word repetition: A marker for language impairment or reading impairment?, Baird [/bib_ref] ; De Groot, Van den Bos, [bib_ref] Rapid naming and phonemic awareness in children with reading disabilities and/or specific..., De Groot [/bib_ref] [bib_ref] Specificity and characteristics of learning disabilities, Eisenmajer [/bib_ref] [bib_ref] Dyslexia and specific language impairment: The role of phonology and auditory processing, Fraser [/bib_ref] [bib_ref] Reading comprehension in children with specific language impairment: An examination of two..., Kelso [/bib_ref] [bib_ref] What do diagnostic test data tell us about differences in the profiles..., Kim [/bib_ref] [bib_ref] Phonological processing deficits in specific reading disability and specific language impairment: Same..., Mcarthur [/bib_ref] [bib_ref] Phonological deficits in specific language impairment and developmental dyslexia: Towards a multidimensional..., Ramus [/bib_ref]. With the exception of [bib_ref] Identifying children at risk for language impairment or dyslexia with group-administered measures, Adlof [/bib_ref] and, all studies involved clinically referred or convenience samples, and most studies involved participants from a wide age range (e.g., 7-12 or 6-16 years) measured at a single time point. Onlyfollowed children longitudinally beginning in preschool, but both DLD and dyslexia determinations were made at the age of 9 years. Across these samples, children with DLD displayed a range of word reading abilities: Some children with DLD exhibited severe word reading deficits consistent with criteria for dyslexia, whereas others showed average or aboveaverage word reading skills, similar to their typically developing peers. Likewise, children with dyslexia showed a range of language abilities with some severe enough to warrant a diagnosis of DLD.
In summary, current evidence suggests that dyslexia and DLD are distinct disorders, which frequently co-occur. The wide range of co-occurrence observed across studies (17%-71%) is likely due to sampling differences (clinically referred samples vs. those from epidemiological studies of the general population) and time point of the diagnosis of dyslexia and language impairment (at the same time or language impairment diagnosed earlier than dyslexia). Studies that draw from the general population and that diagnose DLD before formal schooling provide the strongest evidence because they avoid bias for comorbidity from clinically referred sampling and they avoid the impact of dyslexia on language skills through decreased reading experience.
## Language abilities in children with dyslexia
Although research supports the conclusion that dyslexia and DLD are two separate disorders that frequently co-occur, some studies also suggest that children with dyslexia who do not have DLD may still present with relatively weak language skills compared with typically developing peers [bib_ref] Identifying children at risk for language impairment or dyslexia with group-administered measures, Adlof [/bib_ref] [bib_ref] Phonological deficits in specific language impairment and developmental dyslexia: Towards a multidimensional..., Ramus [/bib_ref]. For example,examined speech and language skills of children who met criteria for dyslexia and/or DLD at the age of 9 years. As a group, children with dyslexia who did not meet the criteria for DLD still showed significantly poorer vocabulary, sentence repetition, and syntactic comprehension than typically developing children, although their standard scores were within normal limits. However, other studies evidence a range of language skills in children with dyslexia who do not have DLD, with group means that are not significantly different from the typically developing controls [bib_ref] Specificity and characteristics of learning disabilities, Eisenmajer [/bib_ref] [bib_ref] Dyslexia and specific language impairment: The role of phonology and auditory processing, Fraser [/bib_ref]. In some studies, group means and standard deviations for children with dyslexia but not DLD suggest that many individuals display above-average standardized language scores (e.g., above the 50th percentile; [bib_ref] Word learning deficits in children with dyslexia, Alt [/bib_ref] [bib_ref] Rapid naming and phonemic awareness in children with reading disabilities and/or specific..., De Groot [/bib_ref] [bib_ref] What do diagnostic test data tell us about differences in the profiles..., Kim [/bib_ref]. As discussed previously, almost all of these studies have involved clinical samples with relatively wide age ranges and have examined language and word reading abilities concurrently at a single point in time. This makes it difficult to determine whether the observed language deficits in children with dyslexia were present before the onset of reading instruction or whether they are a result of limited reading experience (see [bib_ref] Early reading acquisition and its relation to reading experience and ability 10..., Cunningham [/bib_ref] [bib_ref] Distinguishing cause from effect-Many deficits associated with developmental dyslexia may be a..., Huettig [/bib_ref].
A recent study by attempted to overcome this issue by examining word learning abilities in second-grade children with dyslexia who did not have DLD. In this study, the mean Core Language standard score on the Clinical Evaluation of Language Fundamentals-Fourth Editionwas 99.96 (SD = 8.75) for the students with dyslexia, and the mean Expressive Vocabulary Test-Second Editionstandard score was slightly above average (M = 103, SD = 11). Despite their strong oral language and expressive vocabulary scores, when presented with opportunities to learn novel words, the children with dyslexia showed poor word learning compared with typically developing peers, especially apparent when learning the phonological aspects of words (i.e., their sounds and sound combinations in expressive and receptive tasks). Interestingly, they also had difficulty on a few visually based word-learning tasks, but note that all tasks involved some aspect of phonology.
## Preschool language abilities in children with dyslexia
Even in carefully controlled studies of school-aged children with dyslexia, it is difficult to determine if subpar language abilities in children with dyslexia were impacted by the phonological deficit central to dyslexia (most language tasks involve some phonology) and/or were a consequence of dyslexia (children with dyslexia read less, and reading text is an avenue for increasing language skills once children begin to read [bib_ref] Early reading acquisition and its relation to reading experience and ability 10..., Cunningham [/bib_ref] [bib_ref] Distinguishing cause from effect-Many deficits associated with developmental dyslexia may be a..., Huettig [/bib_ref]. Therefore, studies that examine broader language skills before formal reading instruction can be especially informative.
Studies of children with a family history of dyslexia are particularly useful for examining preschool language skills in children with dyslexia. As noted by [bib_ref] Oral language deficits in familial dyslexia: A meta-analysis and review, Snowling [/bib_ref] , children in these studies are recruited before they begin formal schooling, typically at birth, which allows for an examination of early language skills before receipt of reading instruction and before the impact of reading on language development. In addition, these studies avoid clinical bias because the reading outcome is not known when children are enrolled in the study. This is in contrast to a large proportion of studies that recruit children with an existing diagnosis of dyslexia, who are likely to be more severely affected. Third, these studies can be more efficient than a population-based longitudinal study because using this method yields a good number of children with dyslexia. This is because a child who has a family history of dyslexia (i.e., a parent or sibling is diagnosed with dyslexia) has approximately a 50% chance of also having dyslexia. In contrast, very large samples from the healthy population are required to include a similar number of children with dyslexia.
For the purpose of examining preschool language skills of children with dyslexia, we reviewed the studies included in Appendix B of Snowling and Melby-Lervåg's (2016) recent meta-analysis. In these studies, children with and without a family history of dyslexia were recruited and tested on cognitive-linguistic tasks before formal reading instruction and then tested again in the early school grades to determine who met criteria for dyslexia and who did not. This provides a helpful way to know which early skills were associated with having dyslexia and which were instead associated with having a family history of dyslexia. We focused specifically on the 24 studies that involved alphabetic languages; within that sample, 12 studies examined language skills outside the phonological domain and compared them between the reading outcome groups [bib_ref] The roles of family history of dyslexia, language, speech production and phonological..., Carroll [/bib_ref] [bib_ref] Predicting dyslexia from kindergarten: The importance of distinctness of phonological representations of..., Elbro [/bib_ref] [bib_ref] Precursors of literacy delay among children at genetic risk of dyslexia, Gallagher [/bib_ref] [bib_ref] Impaired non-speech auditory processing at a pre-reading age is a risk-factor for..., Plakas [/bib_ref] [bib_ref] Very early language deficits in dyslexic children, Scarborough [/bib_ref] [bib_ref] Early syntactic development of dyslexic children, Scarborough [/bib_ref] [bib_ref] Longitudinal study of speech timing in young children later found to have..., Smith [/bib_ref] [bib_ref] Early phonological and lexical markers of reading disabilities, Smith [/bib_ref] [bib_ref] An exploratory study of the development of early syllable structure in reading-impaired..., Smith [/bib_ref] [bib_ref] Children at family risk of dyslexia: A follow-up in adolescence, Snowling [/bib_ref] [bib_ref] Language development, literacy skills, and predictive connections to reading in Finnish children..., Torppa [/bib_ref] [bib_ref] Dutch children at family risk of dyslexia: Precursors, reading development, and parental..., Van Bergen [/bib_ref]. We highlight four key findings, the first two of which are also provided in Snowling and Melby-Lervåg's meta-analysis. First, on average, children with a family history of dyslexia showed early and persistent deficits in phonology compared with their peers with no family history, but not all of them developed dyslexia [bib_ref] Oral language deficits in familial dyslexia: A meta-analysis and review, Snowling [/bib_ref]. Second, as a group, children with a family history of dyslexia who developed dyslexia were more severely impaired in the phonological domain of language and in broader language domains (e.g., vocabulary, grammar) compared with their peers with and without a family history who did not develop dyslexia (e.g., [bib_ref] The roles of family history of dyslexia, language, speech production and phonological..., Carroll [/bib_ref] [bib_ref] Precursors of literacy delay among children at genetic risk of dyslexia, Gallagher [/bib_ref] [bib_ref] Impaired non-speech auditory processing at a pre-reading age is a risk-factor for..., Plakas [/bib_ref] [bib_ref] Very early language deficits in dyslexic children, Scarborough [/bib_ref] [bib_ref] Early syntactic development of dyslexic children, Scarborough [/bib_ref] [bib_ref] Children at family risk of dyslexia: A follow-up in adolescence, Snowling [/bib_ref] [bib_ref] Language development, literacy skills, and predictive connections to reading in Finnish children..., Torppa [/bib_ref]. Third, in comparison with the numerous tasks used to obtain detailed profiles of skills in the phonological domain of language (e.g., [bib_ref] Predicting dyslexia from kindergarten: The importance of distinctness of phonological representations of..., Elbro [/bib_ref] [bib_ref] Impaired non-speech auditory processing at a pre-reading age is a risk-factor for..., Plakas [/bib_ref] [bib_ref] An exploratory study of the development of early syllable structure in reading-impaired..., Smith [/bib_ref] , relatively few tasks were used to measure broader language skills in most individual studies (but see, e.g., [bib_ref] Children at family risk of dyslexia: A follow-up in adolescence, Snowling [/bib_ref] [bib_ref] Language development, literacy skills, and predictive connections to reading in Finnish children..., Torppa [/bib_ref]. Across studies, receptive vocabulary was the most commonly studied nonphonological language task. Fourth, no studies considered whether and/or what proportion of children who did go on to have dyslexia also had comorbid DLD, and only two studies assessed broader language skills (using measures of sentence recall and vocabulary) at the time of the dyslexia diagnosis [bib_ref] The roles of family history of dyslexia, language, speech production and phonological..., Carroll [/bib_ref] [bib_ref] Children at family risk of dyslexia: A follow-up in adolescence, Snowling [/bib_ref]. Both of those studies provided evidence that those with a family history of dyslexia who went on to have dyslexia had poorer broader language skills than their peers with a family history who did not go on to have dyslexia. However, judging from the small effect sizes that represent the mean differences between groups with and without dyslexia on language measures administered before and at the time of the dyslexia diagnosis, it is likely that some but not all children with dyslexia would also qualify as having a DLD.
A final note is that few studies directly compared children with dyslexia who had a family history of dyslexia with children with dyslexia without a family history of dyslexia. [bib_ref] The roles of family history of dyslexia, language, speech production and phonological..., Carroll [/bib_ref] caution against assuming that all dyslexic children-with and without a family historyare the same. Future studies could further clarify the complex relationship between language development and dyslexia by including children with dyslexia sampled both from families with known history and from the general population and using multiple measures of language, including phonological and broader language tasks both before and at the time of the dyslexia diagnosis.
## Clinical implications
In light of the surge in advocacy surrounding dyslexia and DLD (see [bib_ref] RALLI: An Internet campaign for raising awareness of language learning impairments, Bishop [/bib_ref] [bib_ref] Phase 2 of CATALISE: A multinational and multidisciplinary Delphi consensus study of..., Bishop [/bib_ref] [bib_ref] The state of dyslexia: Recent legislation and guidelines for serving school-age children..., Ward-Lonergan [/bib_ref] , it is important that researchers, practitioners, and the public are aware that dyslexia and DLD are distinct but often co-occurring disorders. Although the exact rates of co-occurrence will depend on the specific diagnostic criteria used for both dyslexia and DLD, it is likely that at least half of the children who are identified with reading disabilities in schools or clinics will have co-occurring DLD (G. M. [bib_ref] On the "specifics" of specific reading disability and specific language impairment, Mcarthur [/bib_ref]. In addition, many children with dyslexia who perform within normal limits on standardized language assessments may have subclinical language deficits that warrant monitoring and educational accommodations. As described in the next section, there are numerous questions that remain to be answered by future research. Despite these questions, the evidence we have reviewed points to several important clinical implications for individuals in school settings.
First, although many SLPs are aware that children on their caseloads may have reading difficulties, they (and other special education providers) may not be fully aware that children with identified dyslexia (or a specific reading disability) often have language needs outside the phonological domain. Children with dyslexia, by definition, have difficulties with word reading. However, as we have reviewed, many children with dyslexia will also struggle with other aspects of language that affect reading comprehension (likewise, children with DLD, by definition, struggle with language comprehension; many also struggle with word reading, and most will struggle with reading comprehension; see . Current assessment frameworks that are used to determine whether a child meets diagnostic criteria for dyslexia and related special education services in the US public schools do not explicitly require that oral language skills beyond phonological awareness be assessed. It is important for SLPs and other school personnel to advocate for the assessment of language skills across multiple domains during the evaluation process and for those skills to be monitored over time. Assessing multiple domains of language would include assessment of phonology, orthography, morphology, semantics, syntax, and discourse processing. Ideally, a thorough investigation of each domain would include both receptive and expressive tasks.
Second, regardless of the specific diagnostic label, intervention should target a child's strengths and weaknesses across all domains of language because they all impact reading comprehension. It is beyond the scope of this article to discuss specific intervention approaches, but we point readers to other sources that recommend and describe evidence-based instruction that explicitly and systematically teaches children phonological awareness, sound-letter associations, orthographic patterns, morphological awareness, vocabulary, syntactic awareness, and narrative and expository text structures (e.g., Al [bib_ref] Elementary grade intervention approaches to treat specific learning disabilities, including dyslexia, Otaiba [/bib_ref] [bib_ref] Assisting students struggling with reading: Response to Intervention and multi-tier intervention for..., Gersten [/bib_ref]. Collaboration between multiple service providers, including classroom teachers, speechlanguage pathologists, reading specialists, and other special educators, can help ensure that these domains are effectively addressed for all students [bib_ref] SLP-educator classroom collaboration: A review to inform reason-based practice, Archibald [/bib_ref] [bib_ref] Important constructs in literacy learning across disciplines, Foorman [/bib_ref]. Interprofessional education may be helpful for facilitating a successful collaboration between these varied service providers in addressing students' language and literacy needs [bib_ref] The knowledge and perceptions of prospective teachers and speech language therapists in..., Wilson [/bib_ref].
Third, those who have dyslexia, regardless of language abilities at the time of diagnosis, are at risk for slower language acquisition and slower growth of world knowledge across their lifetime, as a result of reduced reading experience, a phenomenon known as the Matthew effect. To a large extent, the vocabulary, complex syntax, and general world knowledge that are acquired by adolescents and adults are acquired from texts [bib_ref] Early reading acquisition and its relation to reading experience and ability 10..., Cunningham [/bib_ref] [bib_ref] Distinguishing cause from effect-Many deficits associated with developmental dyslexia may be a..., Huettig [/bib_ref]. The most important line of defense to prevent Matthew effects is to provide high-quality, evidence-based reading intervention as early as possible. However, compensatory techniques that build the child's exposure to rich text and create opportunities to acquire world knowledge may also help to mitigate the risk of Matthew effects (see [bib_ref] From deficit remediation to capacity building: Learning to enable rather than disable..., Rappolt-Schlichtmann [/bib_ref]. For example, students can be encouraged to listen to audiobooks, which provide exposure to the same advanced language structures without the requirement of the child to do the heavy lifting of decoding. [bib_ref] The effects of audiobooks on the psychosocial adjustment of pre-adolescents and adolescents..., Milani [/bib_ref] found that children with dyslexia who were provided audiobook versions of their school textbooks showed a significant improvement in reading skills and a significant reduction in emotional or behavioral problems (as measured by parent report) over a 5-month period, relative to a control group who received only printed texts. The authors hypothesized that the audiobooks may have enhanced students' independence, therefore leading to the reduction in emotional and behavioral problems. In addition to compensatory techniques such as audiobooks, educators can also cultivate a lifelong love for reading and learning by helping children find books that match their interests and expand their knowledge of the world around them.
## Directions for future research
Studies of children with a family history of dyslexia suggest that more severe oral language deficits in the preschool years are associated with a higher likelihood of having dyslexia in the school grades [bib_ref] Oral language deficits in familial dyslexia: A meta-analysis and review, Snowling [/bib_ref]. However, on the basis of the family history studies we reviewed, which are quite comprehensive longitudinal studies of language and dyslexia, it remains unclear to what extent that early oral language deficits persisted in the school grades in children with dyslexia. We hypothesize that deficits in broader language skills such as vocabulary, morphology, and syntax may show peaks and valleys during development (cf. [bib_ref] Connecting early language and literacy to later reading disabilities: Evidence, theory, and..., Scarborough [/bib_ref] in children with dyslexia, depending on the time of assessment. Mild language deficits may appear to be remediated or compensated in the early school years as children benefit from high-quality oral language input with the onset of schooling. In later school grades, when more vocabulary and complex syntactic structures are acquired through reading experience, children with dyslexia may show Matthew effects, in which broader language skills show slower growth compared with peers without dyslexia due to less reading experience (D. [bib_ref] The influence of reading on vocabulary growth: A case for a Matthew..., Duff [/bib_ref] [bib_ref] Individual differences in reading development: A review of 25 years of empirical..., Pfost [/bib_ref] [bib_ref] Children at family risk of dyslexia: A follow-up in adolescence, Snowling [/bib_ref]. Testing this hypothesis will require a longitudinal study that assesses multiple language skills early, at the time of the diagnosis of dyslexia, and years later.
In addition to the need for longitudinal studies that track language development across multiple domains before, during, and after the onset of dyslexia, there is also a need for more research to understand the mechanisms by which dyslexia and DLD manifest both separately and together in specific children. There is clear evidence that both genetic and environmental factors contribute to these disorders [bib_ref] Language growth and genetics of specific language impairment, Rice [/bib_ref] and that the neurobiological profiles of dyslexia and DLD are different (C. [bib_ref] Anatomical risk factors that distinguish dyslexia from SLI predict reading skill in..., Leonard [/bib_ref]. There is also some evidence that different genetic components may be involved in dyslexia than DLD [bib_ref] Distinct genetic influences on grammar and phonological shortterm memory deficits: Evidence from..., Bishop [/bib_ref]. However, it is still the case that studies more frequently ignore the co-occurrence of dyslexia and DLD than account for it in their design or analyses. Accounting for this co-occurrence is of pivotal importance, so that the conclusions drawn about one disorder are not confounded by the unknown presence of the other disorder in the participant sample. There is also a need to attend more closely to factors that contribute to risk and resilience for students with dyslexia and/or DLD [bib_ref] Socio-emotional and cognitive resilience in children with reading disabilities, Haft [/bib_ref] [bib_ref] From deficit remediation to capacity building: Learning to enable rather than disable..., Rappolt-Schlichtmann [/bib_ref] , including but not limited to malleable environmental factors such as child-caregiver interactions around language and literacy and school instructional practices.
# Conclusions
In this article, we presented three clinical implications for working with children dyslexia in school settings: (a) Children with dyslexia-with and without comorbid DLDs-often have language deficits outside the phonological domain (in addition to core deficits in the phonological domain); (b) intervention should target a child's strengths and weaknesses relative to reading outcomes, regardless of diagnostic labels; and (c) those who have dyslexia, regardless of language abilities at the time of diagnosis, may be at risk for slower language acquisition across their lifetime. Future studies should follow the children at risk for dyslexia over time to assess multiple language skills early, at the time of the diagnosis of dyslexia, and years later to better understand the complex development of language and reading in children with dyslexia.
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AcknowledgmentsThe research reported in this article was supported, in part, by funding from National Institutes of Health Grants R03DC013399 (PI: Adlof ) and R03DC9667 (PI: Hogan). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Electrochemical Behavior of Quinoxalin-2-one Derivatives at Mercury Electrodes and Its Analytical Use
Derivatives of quinoxalin-2-one are interesting compounds with potential pharmacological activity. From this point of view, understanding of their electrochemical behavior is of great importance. In the present paper, a mechanism of electrochemical reduction of quinoxalin-2-one derivatives at mercury dropping electrode was proposed. Pyrazine ring was found to be the main electroactive center undergoing a pH-dependent two-electron reduction process. The molecule protonization of nitrogen in the position 4 precedes the electron acceptance forming a semiquinone radical intermediate which is relatively stable in acidic solutions. Its further reduction is manifested by separated current signal. A positive mesomeric effect of the nonprotonized amino group in the position 7 of the derivative III accelerates the semiquinone reduction yielding a single current wave. The suggested reaction mechanism was verified by means of direct current polarography, differential pulse, cyclic and elimination voltammetry, and coulometry with subsequent GC/MS analysis. The understanding of the mechanism was applied in developing of analytical method for the determination of the studied compounds.
# Introduction
The identification and determination of trace amounts of biologically active substances and their metabolites represent one of the most important problems of contemporary analytical chemistry. Extra attention is paid to the compounds exhibiting bacteriostatic, virostatic, and cancerostatic effects. These include compounds capable of interactions (via intermolecular hydrogen bonds) with binding sites of nucleic acids intervening in their primary functions [bib_ref] Energetics of echinomycin binding to DNA, Leng [/bib_ref] [bib_ref] Echinomycin inhibits chromosomal DNA replication and embryonic development in vertebrates, May [/bib_ref]. This phenomenon can be used in targeted cancer therapy, especially if tumor cells exhibit resistance towards conventional chemotherapeutic treatment.
The parent quinoxaline-2-one has become one of the active components of the preparation whose activity against HIV-virus was tested [bib_ref] Homoacyclovir analogues of unnatural bases and their activity against hepatitis B virus, El Ashry [/bib_ref]. Natural quinoxaline analogues, such as quinomycin antibiotics (equinomycin) and triostin-es, belong among well-known compounds with confirmed virostatic and bacteriostatic activity [bib_ref] Studies on quinoxaline antibiotics. 1. General properties and the producing strains, Kuroya [/bib_ref]. Similar effects can also be expected for synthetic 3 substituted quinoxaline-2ones. Some of their analogues are used (with certain limitations) in agriculture and veterinary medicine, others as fluorescent derivative agents in HPLC [bib_ref] Peroxydicarbonatemediated oxidation of N-(ortho-aryloxyphenyl) and N-(ortho-arylaminophenyl)aldimines, Leardini [/bib_ref].
Much of the complex quinoxaline metabolism remains to be understood. A partial approximation of the biotransformation mechanism can be achieved using the electrochemical charge-transfer model in which the electrode can be considered a simplified biological receptor.
Few references can be found regarding the electrochemical behavior of 3 substituted quinoxaline derivatives [bib_ref] Ricerche polarografiche su sostanze eterocicliche. 2, Furlani [/bib_ref] [bib_ref] Polarography of 3-alkylquinoxalones. 7. Polarography of heterocyclic compounds, Pflegel [/bib_ref]. Two-electron irreversible polarographic reduction of 3methylquinoxalin-2(1H)-one has been reported to proceed in the pH range of 0-14. A radical intermediate forming in the first step of the electroreduction in strongly acidic media can either receive an electron and a proton in the second step or form a N,N -dimer dependently on quinoxalinone concentration [bib_ref] Ricerche polarografiche su sostanze eterocicliche. 2, Furlani [/bib_ref] [bib_ref] Polarography of 3-alkylquinoxalones. 7. Polarography of heterocyclic compounds, Pflegel [/bib_ref]. The aim of this paper is to contribute to the explanation of electrochemical behavior of quinoxaline-2one derivatives (I-III, [fig_ref] Figure 1: Chemical structures of 3-methylquinoxalin-2 [/fig_ref] at the mercury electrode, and to identify both the intermediate and final products of their electrochemical reduction.
## Experimental
## Instrumentation.
A Radelkis OH 102 polarograph (Radelkis, Budapest) in a three-electrode arrangement with dropping mercury electrode (DME, t = 3.6 s, m = 1.98 mg/s), auxiliary platinum and reference-saturated calomel electrode (SCE) was used for the recording of DC curves. An Eco-Tribo Polarograph with pencil mercury microelectrode (Polaro-Sensors, Prague) was used for the DC-tast polarography, differential pulse (DP) voltammetry (potential scan rate 20 mV/s, modulation amplitude -50 mV), adsorptive stripping (AdSV) and cyclic voltammetry (CV) measurements. The same reference and auxiliary electrodes were used as those described above.
The computation of data filtering and elimination equations was performed using self-made macro in Microsoft Office Excel v. 2003 (Microsoft Corporation, USA).
Controlled potential coulometry measurements were carried out using an OH 404 analyzer (Radelkis, Budapest) equipped with a mercury pool cathode (26.4 cm 2 ). The SCE was used as a reference. The platinum auxiliary electrode was placed in the anodic compartment separated with a frit.
Before the analysis, all samples were deaerated with a nitrogen stream for 10 min.
For GC/MS identification of coulometric reduction products, an HP 6890 chromatograph with an HP 7683 injector and an Agilent 5973 N mass spectrometer (all Agilent, Palo Alto, USA) were used. Chromatographic conditions: HP-5 MS column (30 m × 0.25 mm × 0.25 μm), helium as a carrier gas (99.998%, SIAD, Bergamo, Italy), flow-rate 0.9 mL/min. Programmed temperature was 50- C/2 min-10- C/min up to 300- C and 5 min isothermically.
Spectrophotometric data were acquired by a Philips PU 8755 UV/VIS spectrophotometer (Cambridge, UK) and a Beckman DU 7500 spectrophotometer (Fullerton, USA), using quartz cuvettes.
A calibrated inoLab pH Level 1 equipped with a SenTix 41 combined electrode (WTW, Weilheim, Germany) was used for pH measurements.
## Chemicals and reagents.
Standards of quinoxaline-2(1H)-one derivatives were synthesized and purified at the Department of Organic Chemistry, Palacky University, Olomouc. The purity (>97%) of the standards obtained was verified by checking the melting points, IR spectrometry (KBr technique) and by HPLC-MS.
Stock methanolic solutions (1 mM) were further diluted by methanol if needed. In cases when analyte concentrations exceeded 1 μM, a methanol addition (10%, v/v) was necessary to maintain homogeneous solution because of the low water solubility of the studied compounds.
Britton-Robinson buffer solutions with ion strength corrected by KCl were used for pH adjustments of the samples. For measurements at strong acidic or alkaline conditions, diluted solutions of HCl or NaOH were used. Ultra pure water (0.055 μS/cm) from ELGA system was used for the solution preparation.
## Procedures.
Coulometric experiments were performed with three different electrolytes and different constant potentials: 0.01 M HCl (E = −0.8 V ), 0.001 M NaOH (E = −1.6 V ), phosphate buffer, and pH 7.0 (E = −1.2 V ). During electrolysis (in the interval from 0 to 60 min), 10 mL samples were withdrawn, passed through a preconditioned Zorbax C18 SPE cartridge (200 mg, Agilent, Palo Alto, USA) and dried with a vacuum water pump. Retained compounds were eluted using 1 mL of methanol and directly analyzed by GC/MS.
Liquid-liquid extraction of quinoxalinones from spiked water samples (8-20 μg of I per 100 mL water) was performed using a 250 mL separatory funnel. A volume of 100 mL of ethylacetate was added to 50 mL of spiked water sample in the funnel and shaken for 5 min. The upper analyte-containing organic layer was removed, transferred into a dryer and evaporated to dryness under reduced pressure at 70 - C. The sample was then reconstituted in 1 mL of methanol, diluted in 10 mL of the background electrolyte (1 mM NaOH) and analyzed using DPV. The standard addition method was used for the quantification. Although some earlier studies reported these compounds also as hydroxo derivatives [bib_ref] Dissociation Constants of Organic Bases, Perin [/bib_ref] , many properties (reactivity, IR-spectra) indicate that the keto form prevails [bib_ref] Chemistry of 1,2-dihydroquinoxaline-2-ones, Fryšová [/bib_ref] [bib_ref] Thermochemical and theoretical studies of 2-hydroxyquinoxaline, 2,3-dihydroxyquinoxaline, and 2-hydroxy-3-methylquinoxaline, Ribeiro Da Suva [/bib_ref]. Hydrogen bond is likely to play a significant role in the reduction process, stabilizing the planar molecule.
Quinoxalin-2(1H)-one derivatives are reduced within the whole acidic range. In acidic solutions, diffusion-controlled electrode process is manifested by a dual wave , curves (1a) and (1b). Both waves are approaching each other as acidity decreases, whereas in the alkaline range (for III in neutral) they merge into one single DC-wave or DPV-peak , curves (2a) and (2b), resp.). With increasing pH, a potential shift towards more negative values occurs, indicating that protons are involved in the electrode process.
DP voltammetric curves of II in 0.5 M HCl (1b) and in Britton-Robinson buffer pH 11.5 (2b).
Depolarizer concentration is 0.1 mM, methanol content 10% (v/v).
To determine the dissociation constant by spectrophotometry, modified Henderson-Hasselbalch equation was used:
[formula] pK a = pH − log A − A 1 A 2 − A ,( 1 ) [/formula]
where A is absorbance at given pH value, A 1 and A 2 are absorbances of solutions containing more than 99 % undissociated and dissociated forms, respectively. Dissociation constants of derivatives I (pK a = 9.85) and II (pK a = 10.04) were established in 10% (v/v) methanol. The dissociation constant of the derivative I was found to be in agreement with the reported value (pK a = 9.90) [bib_ref] Dissociation Constants of Organic Bases, Perin [/bib_ref]. The estimation of Compound
[formula] /[nA] pH ≈ 1 pH= 7.0 pH = 9.2 m-nitrobenzoic acid −689.0 - −650.8 benzil −344.7 −330.1 - 3-methyl-quinoxalin-2(1H)-one −344.1 −336.8 −338.6 [/formula]
dissociation constant of the derivative III by spectral method was not successful. The pK a values found were verified on the basis of the E 12 dependency of the total DC-tast wave on pH. For analytes I and II, dissociation constants both of the oxidized forms and the two dissociation constants of the reduced forms were determined by the intercepts of four linear segments of the functional dependency ). The found dissociation constants of the oxidized forms of I and II are in good agreement with the spectrophotometric method. In case of III, additional protolytic equilibrium is produced as a result of the protophilic property of the amino group. Therefore, two dissociation constants of the oxidized form, and three dissociation constants of the reduced form were established from the intercepts of
[formula] E 1/2 = f (pH) de- pendence. [/formula]
The curve break ) corresponds with pK R2 . However, it is situated close to pK Ox1 , and thus its position is difficult to identify with certainty due to low experimental point count. An overview of dissociation constants obtained polarographically is given in.
To evaluate independently the expected two-electron reduction of the analytes studied, the following methods were used.
(1) A comparison of cathodic DC-tast waves of analytes with two-electron wave of benzil or with four-electron wave of m-nitrobenzoic acid, respectively was performed. subsequent GC/MS analysis. The products with m/z increased by 2 Da were found in electrolyzed solution [fig_ref] Figure 5: Reconstructed chromatograms for the selected m/z values [/fig_ref] which corresponds to the two-electron reduction. They were identified as respective 3,4dihydroquinoxalin-2(1H)-ones (see [fig_ref] Figure 6: Proposed scheme of electrochemical reduction of 3-methylquinoxalin-2 [/fig_ref].
The amino derivative III did not yield sufficiently volatile products, and was therefore not analyzed using this method. Nevertheless, its similar behavior at mercury electrode indicates that the two-electron reduction can also be expected.
Based on the experimental results and considering the stabilizing effects of tautomery and mesomery, following mechanism of electrode reduction process can be concluded for the I and II derivatives [fig_ref] Figure 6: Proposed scheme of electrochemical reduction of 3-methylquinoxalin-2 [/fig_ref].
Rather more complex mechanism can be expected for III where amino group is also involved in the protolytic equilibria. However, deprotonization of the NH 3 + group of the reduced form, which is probably allied to that of oxidized form, is not obvious in the E 1/2 (DC-tast) = f (pH) dependency. Based on these observations, supposed reaction scheme for III (excluding tautomery and mesomery) can be deduced [fig_ref] Figure 7: Proposed scheme of electrochemical reduction of 7-amino-3-methylquinoxalin-2 [/fig_ref].
As the appropriate slopes of linear segments in the E 1/2 = f (pH) dependency rather differ from the theoretical values for the reversible diffusion controlled processes, it can be assumed that electrode reduction of I-III does not exhibit a purely reversible character. In case of irreversible reduction, [bib_ref] Electrochemical reduction of 6-substituted purines correlation with structural and energetic characteristics, Janik [/bib_ref] :
[formula] E 1/2 = f (pH) dependency is defined by (2)E 1/2 = E f − p 0.0592 αz pH,(2) [/formula]
where p is number of protons, α is a charge transfer coefficient, E f is potential of E 1/2 = f (pH) at pH = 0 as read off from the graph. The product of parameters αz [fig_ref] Table 3: Values of product αz and number of protons p calculated from DC... [/fig_ref] was determined from DC-curves:
[formula] (a) E 1/4 − E 1/2 = 0.0517 αz , or (b) E 1/2 − E = 0.0592 αz log 2x(3x − 1) 5(1 − x) , x = I E I lim ,(3) [/formula]
where I E is the current obtained at a potential E and I lim denotes the limiting current. Individual linear slopes of E 1/2 = f (pH) correspond to those calculated using (4) [bib_ref] Electrochemical reduction of 6-substituted purines correlation with structural and energetic characteristics, Janik [/bib_ref] :
[formula] dE 1/2 dpH = −0.0592 p αz ,( 4 ) [/formula]
confirming the supposed mechanism of the overall reduction of I-III at mercury dropping electrode. In acidic media, the total two-electron reduction is divided into two one-electron steps exhibiting separate waves at low pH values . Considering the polarization of curve shape and taking into account the behavioral analogies of related quinoid compounds [bib_ref] Polarographisches Verhalten einiger Isorosindonderivate, Kotouček [/bib_ref] [bib_ref] Controlled-potential electrolysis of polarographically observed phenazine and its mono Noxide, Nakaya [/bib_ref] , it is obvious that the intermediate reduction product is semiquinone radical.
Consecutive reduction via semiquinone (Sem) can be described as follows:
Ox + e ⇐⇒ Sem, The equilibrium of all three forms:
[formula] Sem + e ⇐⇒ Red.(5)Ox + Red ⇐⇒ 2Sem(6) [/formula]
is characterized by a semiquinone formation constant K:
[formula] K = [Sem] 2 [Ox][Red] .(7) [/formula]
Semiquinone formation can also be characterized by the de pendency of formal half-wave potentials of both partial reactions (E 1 and E 2 ). Both potentials will be situated symmetri-cally around the mean inflection point (i.e., ≈ E 1/2 ). For the equilibrium is valid:
[formula] E = E 1 + RT F ln [Ox] [Sem] = E 1/2 + RT 2F ln [Ox] [Red] = E 2 + RT F ln [Sem] [Red] ,(8)E 1 = E 1/2 + RT 2F ,( 9 ) [/formula]
and similarly,
[formula] E 2 = E 1/2 − RT 2F ln K.(10) [/formula]
The Scientific World Journal For the difference of formal potentials is valid
[formula] E 1 − E 2 = RT F ln K.(11) [/formula]
If K = 1, all tree potentials are identical (E 1 = E 2 = E 1/2 ). If K > 1, potentials decrease in the direction
[formula] E 1 > E 1/2 > E 2 . [/formula]
The value of the constant K was calculated from potentials read in 1/4, 1/2, and 3/4 of the DC-tast wave using [bib_ref] Contribution to polarography of semiquinone forming systems, Stránský [/bib_ref] :
[formula] K = 10 (Ei·2F)/(2·3RT) − 3 2 10 (E i ·2F)/(2·3RT) , where E i = E 1 4 − E 1 2 = E 1 2 − E 3 4 .(12) [/formula]
The shape of the corresponding voltammetric curve is influenced by the value of the semiquinone formation constant (from 0 to ∞). In alkaline media, K values of the studied quinoxalinones are <16 (e.g., for I, pH 9.4, K = 8.42), and the curve is flatter. Individual reduction steps are not separated from each other. As acidity increases, K values grow significantly (e.g., for I, pH 2.9, K = 1262). As a result, the original two-electron single-wave signal splits into two individual waves. The effect becomes even more obvious with increasing K values. For compounds of types I and II this phenomenon can be observed in solutions of pH < 7.5 only. As for semiquinone formation, an initial electron incorporation into the position 4 is supposed by Pflegel and coauthors [bib_ref] Polarography of 3-alkylquinoxalones. 7. Polarography of heterocyclic compounds, Pflegel [/bib_ref]. However, such a mechanism seems unlikely with respect to distribution of the electron density in the molecule. Moreover, a positive inductive effect of methyl group in position 3 together with the increased basicity of the N4-atom indicates that in a strongly acidic solution (pH < 2), protonation of the N-atom occurs at first. The gap in position 3 is then filled with an electron, forming a semiquinone free radical. Its reduction proceeds further by a reception of another electron and two protons. At pH 2.0-2.7 (semiquinone form prevailing), the reception of the electron and two protons is accomplished already in the first step [fig_ref] Figure 8: Dependencies of half-wave potentials of consecutive [/fig_ref]. Within the range 2.7-7.6 (pK R1 < pH< pK R2 ), the reduction process involves the reception of one electron and one proton in both steps producing a semiquinone. Two separate waves are approaching each other as pH increases, merging gradually into a single wave. At higher reactant concentrations, a possible semiquinone dimerization must also be taken into account [bib_ref] Polarography of 3-alkylquinoxalones. 7. Polarography of heterocyclic compounds, Pflegel [/bib_ref] , making the whole mechanism even more complex.
The reduction of the amino derivative III follows a similar pattern as that of I and II except for the semiquinone formation proceeding only in the pH range of prevailing protonized form, that is, pH < 5 [fig_ref] Figure 8: Dependencies of half-wave potentials of consecutive [/fig_ref]. At higher pH, the deprotonized amino group possessing a conjugated free electron pair accelerates the reduction process by its inductive effect to such an extent that the resulting semiquinones are not apparent in the form of separated peaks in the signal curve. Based on experimental data, a reduction scheme for III involving a formation of semiquinone can be deduced [fig_ref] Figure 1: Chemical structures of 3-methylquinoxalin-2 [/fig_ref].
## Adsorption.
Adsorption is likely to affect the electrode processes by lowering the reaction reversibility. The effect was reported for I in pH range 0-4.5 as a prewave occurring in the more positive potential range characterizing adsorption of the reduced form [bib_ref] Polarography of 3-alkylquinoxalones. 7. Polarography of heterocyclic compounds, Pflegel [/bib_ref].
Cyclic voltammograms [fig_ref] Figure 1: Chemical structures of 3-methylquinoxalin-2 [/fig_ref] demonstrate the rather poor electrode process reversibility of quinoxalinones.
The semiquinone formed during the first reduction can be reoxidized in the reversed scan. However, the difference in cathodic and anodic peak potentials is significantly higher than the theoretical value of 59 mV (as expected for reversible one-electron process), and the difference increases with
## Medium
3-methyl-quinoxalin-2-one 3,6,7-trimethyl-quinoxalin-2-one 7-amino-3-methyl-quinoxalin-2-one 1st peak 2nd peak 1st peak 2nd peak 1st peak 2nd peak 0 the increasing scan rate (v). Dependencies log I p = f (log v) are linear within the whole pH range with slopes different for both the first and the second cathodic peak [fig_ref] Table 4: Slopes of the regression lines calculated for log-log dependencies of cathodic CV-peak... [/fig_ref].
The comparison of the line slopes with the analogous data for both purely diffuse (Randles-Sevcik equation) and tensametric currents indicates that adsorption occurs in acidic and neutral milieu, especially for I and II during the first reduction step (slopes 0.6-0.8). The adsorption of III in acidic medium is less evident. At neutral pH, with prevailing electroneutral oxidized form, the adsorption becomes more significant.
During the second step of reduction and in alkaline solutions, the adsorption takes no effect for any of the analytes. Under such conditions, with the slope values smaller than 0.5 characterizing purely diffuse process, other factors (kinetic and catalytic) influencing the electrode processes cannot be excluded.
The influence of adsorption on electrochemical reduction was confirmed also by elimination voltammetry with linear scan (EVLS) [bib_ref] Theory of current elimination in linear scan voltammetry, Dračka [/bib_ref] [bib_ref] Electrochemical elimination methods, Trnková [/bib_ref]. The elimination function which eliminates simultaneously charged and kinetic currents and conserves diffusion current [bib_ref] Theory of current elimination in linear scan voltammetry, Dračka [/bib_ref] : f (I) = 17.485 I − 11.675 I 1/2 − 5.8284 I 2 [bib_ref] Electrochemical reduction of 6-substituted purines correlation with structural and energetic characteristics, Janik [/bib_ref] was used. Symbol I denotes the reference voltammetric current measured at reference scan rate, I 1/2 and I 2 are currents measured at half and double scan rates, respectively. The theoretical elimination curve f (I) for an adsorbed electroactive substance has a characteristic peak-counterpeak form [bib_ref] Application of elimination voltammetry to the resolution of adenine and cytosine signals..., Trnková [/bib_ref]. As can be seen from [fig_ref] Figure 1: Chemical structures of 3-methylquinoxalin-2 [/fig_ref] , the elimination function f (I) calculated for the species I in 0.01 M HCl exhibits a peak-counterpeak shape at potential of the first cathodic peak. Similar shape of the elimination function was observed also with the substance II in acidic and neutral media confirming the influence of adsorption on the first step of electrode process. The theoretical ratio:
[formula] I p/ I p + Icp ,(14) [/formula]
where I p and I cp are the heights of peak and counterpeak of the elimination curve, respectively, has the value of 0.4097 for a totally adsorbed species reducing without a preceding chemical reaction [bib_ref] Identification of current nature by elimination voltammetry with linear scan, Trnková [/bib_ref]. In case of species I and II the ratio The derivative III revealed the elimination curve with peak-counterpeak shape in neutral solutions [fig_ref] Figure 1: Chemical structures of 3-methylquinoxalin-2 [/fig_ref] where the uncharged form of molecule prevails. The ratiohas value of 0.60 at pH 6 and I ref = 40 mV/s. The elimination curves indicated no influence of adsorption for both the strong acidic media with prevailing protonized state of the III and the alkaline media with anionic form of quinoxalinone. In conclusion, the elimination voltammograms confirmed that the predominantly uncharged oxidized form of quinoxalinones adsorbs at the surface of mercury electrode.
## Determination of quinoxaline-2(1h)-one derivatives.
Alkaline media (0.001 M NaOH) are generally suitable for determination of I and II derivatives by the DPV method. A single peak in DP-voltammograms at E p = -1.042 V was obtained. Calibration curves were linear in the concentration range 10-0.5 μM [fig_ref] Table 5: Regression parameters of calibration curves y = ax + b [/fig_ref]. Concentration dependencies exhibit nonlinearity [fig_ref] Figure 1: Chemical structures of 3-methylquinoxalin-2 [/fig_ref] in acidic and neutral media due to adsorption complicating the reaction mechanism. However, linear dependency for I and II in 0.1 M-HCl for low concentrations was obtained (c < 2 μM). Neutral solution has proven to be suitable for the amino derivative III, for which linear calibration curves in the concentration range 0.1-1.0 μM using DPV and DPP were obtained. Calibration curves become nonlinear at higher concentrations. Main parameters of the calibration curves and the reached limits of detection (LODs) are summarized in [fig_ref] Table 5: Regression parameters of calibration curves y = ax + b [/fig_ref].
Determination of low concentrations of I-III in model samples was performed at the same conditions as the corresponding calibrations. The resulting data are summarized in [fig_ref] Table 6: Determination of derivatives of I-III, method of standard additions [/fig_ref].
Because of their biologically activity, the need for analytical determination of quinoxalinones in body fluids can be expected in the future. As a model experiment of their isolation from aqueous media for subsequent voltammetric determination, a solid phase as well as liquid-liquid extractions were performed.
The solid phase extraction (SPE) was carried out using C18 SPE-cartridges containing 200 or 500 mg of sorbent. The cartridges were preconditioned using methanol and redistilled water. Samples of water (100 mL) spiked with 8-20 μg of analyte were adjusted using buffer to final pH = 7.0 and supplied onto SPE cartridges through Teflon tubings (3-5 mL/min). Analyte elution from the drained SPE-cartridge was accomplished with 2 mL of methanol. The eluate was then dried by nitrogen stream at ambient temperature, redissolved in 1 mL of methanol, diluted to 10 mL with the background electrolyte (0.001 M-NaOH for I and II, neutral buffer for III) and subjected to DP-voltammetric analysis using the standard addition method. Recovery ranged between 56 -62% for II and and 44 -50% for III indicating the C18 stationary phase is not very suitable for the preconcentration of these compounds from water samples. Due to the low recovery, the described SPE procedure cannot be recommended for analytical purposes. Seven various solvents were tested for liquid-liquid extraction: hexane, benzene, dichloromethane, trichloromethane, carbon tetrachloride, diethyl ether, and ethyl acetate. For separation procedure, see Experimental. Ethyl acetate-water sample (2 : 1 by volume) and extraction time 5 min proved to be the most suitable preconcentration procedure. Distribution ratio for the species I was 8.5, extraction recovery achieved 94.5% at 20 - C.
# Conclusions
The study of electrochemical behavior of three quinoxaline-2-one derivatives at mercury dropping electrode has proven the pyrazine ring to be the electroactive center undergoing a two-electron reduction. The electrode process is pH-dependent. The protonization of nitrogen in position 4 precedes the electron acceptance forming a semiquinone radical which is relatively stable in acidic solutions. Its further reduction is manifested by separated current signals. A positive mesomeric effect of the nonprotonized amino group in position 7 of the derivative III accelerates the semiquinone reduction yielding a single peak. The final products of the electrochemical reduction are the corresponding 3,4-dihydroquinoxalin-2(1H)-ones. The electrode process is affected by the adsorption of the depolarizers on the mercury surface and is most pronounced when the quinoxalinone molecule is uncharged. The adsorption causes decreased reversibility of the overall electrode reaction and nonlinearity of concentration dependences. The suggested reaction mechanism was verified by the means of DC-polarography, coulometry with subsequent GC/MS analysis, and EVLS. The understanding of the mechanism was applied in developing of analytical method for the determination of the studied compounds.
[fig] Figure 1: Chemical structures of 3-methylquinoxalin-2(1H)-one (I), 3,6,7-trimethylquinoxalin-2(1H)-one (II), and a 7-amino-3-methylquinoxalin-2(1H)-one (III). [/fig]
[fig] 2. 4, Figure 2, Figure 2, Figure 3: Results and Discussion 2.4.1. Mechanism of Electrode Reaction. Electron deficiency in a molecule is essential for its electrochemical reduction. For quinoxaline derivatives, the electron deficiency is set by their quinoid structure. An influence of keto-enol-tautomery should be considered for the compounds of interest (Keto-enol-tautomery of quinoxalin-2(1H)-one derivatives. DC-tast polarographic curves of 3,6,7-trimethylquinoxalin-2(1H)-one (II) in 0.5 M HCl (1a), in Britton-Robinson buffer pH 11.5 (2a) and benzil curve of in 0.5 M HCl (3). [/fig]
[fig] 2, Figure 4: Potentiostatic coulometry at mercury pool cathode was performed of I and II (c = 0.Dependencies of half-wave potentials of DC-waves for I (a) and III (b) on pH in HCl and Britton-Robinson solutions. The numbers indicate slopes (mV/pH) of corresponding linear segments. [/fig]
[fig] Figure 5: Reconstructed chromatograms for the selected m/z values. The data were obtained by GC/MS analysis of I (0.1 mM) electrolyzed at constant potential −0.8 V in acidic solution (0.01 M HCl in water with 10% (v/v) of methanol). [/fig]
[fig] Figure 6: Proposed scheme of electrochemical reduction of 3-methylquinoxalin-2(1H)-one (I). [/fig]
[fig] Figure 7: Proposed scheme of electrochemical reduction of 7-amino-3-methylquinoxalin-2(1H)-one (III). [/fig]
[fig] Figure 8: Dependencies of half-wave potentials of consecutive (red and blue symbols) and overall (black symbol) DCwaves for I (a) and III (b) on pH in HCl and Britton-Robinson solutions. The numbers indicate slopes (mV/pH) of particular linear segments for consecutive processes. [/fig]
[fig] 14: was 0.76 and 0.55, respectively, in 0.01 M HCl at I ref = 40 mV/s and increased with increasing pH of solution. This implies the growing influence of kinetics of the preceding chemical reaction (most likely protonation of the oxidized quinoxalinone form). In alkaline solutions where the anionic form of both substances prevails, the courses of the elimination function provided no evidence of adsorption. [/fig]
[table] Table 3: Values of product αz and number of protons p calculated from DC curves for compounds I, II, and III. [/table]
[table] Table 4: Slopes of the regression lines calculated for log-log dependencies of cathodic CV-peak current [nA] on scan rate [mV/s]. [/table]
[table] Table 5: Regression parameters of calibration curves y = ax + b (significance level α = 0.05, number of calibration points n = 10). E P versus SCE, r-correlation coefficient, LOD = 3s b /a. [/table]
[table] Table 6: Determination of derivatives of I-III, method of standard additions (number of measurements n = 4). [/table]
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A systematic review on diagnostic test accuracy of magnetic resonance neurography versus clinical neurosensory assessment for post-traumatic trigeminal neuropathy in patients reporting neurosensory disturbance
Objectives:To perform a systematic review of published studies on diagnostic accuracy of magnetic resonance neurography (MRN) vs clinical neurosensory testing (NST) for posttraumatic trigeminal neuropathy (PTTN) in patients reporting neurosensory disturbances (NSD). methods: Human studies except case reports, reviews, systematic reviews and meta-analyses were included. PubMed, Embase, Web of Science and Cochrane Library were consulted. Risk of bias assessment was conducted using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Predetermined data extraction parameters were noted and summarized. Results: 8 studies met eligibility criteria of which 7 were retrospective, representing 444 subjects. Most studies were at high risk of bias with low applicability concerns. Populations and objectives were divergent with a large variation in timing (3 days-17 years post injury) and parameters (multiple coil designs, fat suppression techniques, additional contrast agent) of MRI acquisition. T 2 weighted 3 T imaging with short echo times (2.2-100 ms) and fat suppression was applied in seven studies, techniques varied. Determination of sensitivity and specificity could not be performed due to the methodological variation between studies and lacking comparative data between index and reference tests. Based on limited data, PTTN correlated reasonably well between clinical assessment, intraoperative findings and MRN abnormalities (k = 0.57). Increased signal intensity correlated with persistency of neurosensory disturbances in one study. Intra-(ICC 0.914-0.927) and interobserver (k = 0.70-0.891) MRN variability was considered good to excellent. One retrospective study showed substantial impact of MRN on clinical decision making in one-third of patients. conclusion: Currently, there is insufficient scientific knowledge to support or refute the use of MRN. Based on limited data, MRN seems promising and reliable in detection and grading of PTTN. Methodological issues underline the importance for prospective blinded studies with standardization of signal intensity calculation and rigorous reporting of MRI acquisition parameters. A systematic review on diagnostic accuracy of magnetic resonance neurography in posttraumatic trigeminal neuropathy Van der Cruyssen et al neurography versus clinical neurosensory assessment for post-traumatic trigeminal neuropathy in patients reporting neurosensory disturbance. Dentomaxillofac Radiol 2020; 50: 20200103.
## Neuropathic pain
# Background
The peripheral trigeminal nerves are a daily concern for anyone operating in the head and neck area.There is a risk of damage to these branches in numerous dentoalveolar and oral or maxillofacial surgeries such as wisdom tooth extraction, endodontic treatments, placement of implants and administration of local anesthesia.Once damage occurs, there is usually a neurosensory disturbance which can be superimposed with neuropathic pain and phenomena such as allodynia and hyperalgesia. Diagnosing these post-traumatic trigeminal neuropathies (PTTN) and predicting prognosis in the early post-traumatic period is not straightforward.Currently, diagnosis is mainly based on patient-reported neurosensory disturbances (NSD) and qualitative or quantitative psychophysical neurosensory tests (NST), which have their own methodological problems.Electrophysiological tests are available but are difficult to apply in the trigeminal distribution.Additionally, they cannot precisely depict the localization and extent of trauma, which is important if surgical management is considered.
From a clinical but also medicolegal point of view, it is important to be able to make a distinction in severity between nerve damage, localization and sensory profiles.Many patients experience spontaneous recovery, but in select cases with severe nerve damage, a microsurgical release or repair may be appropriate. It is generally agreed that a faster intervention leads to better neurosensory recovery.The current standard in diagnosing pathology of the peripheral sensory nervous system is quantitative sensory testing (QST). It was introduced by the German Research Network on Neuropathic Pain in 2006 and is already strongly substantiated in its value, being that it can clarify if a neurosensory deficit is present or not.However, for the time being, it remains unclear how these tests evolve in the transition from the acute to the chronic phases of trigeminal nerve damage and if they can predict prognosis and treatment outcomes in PTTN.Magnetic resonance neurography (MRN) is an MRI technique in which dedicated sequences are used to enhance the visualization of the peripheral nervous system and its pathology.It has the potential to visualize and quantify nerve injuries and the associated severity of damage.Evidence has already been demonstrated for plexus lesions and in neuromusculoskeletal imaging, but to the best of our knowledge no aggregate analysis of literature is known for the diagnostic accuracy and value in post-traumatic trigeminal nerve lesions.Therefore, the main objective of this study was to conduct a systematic review of diagnostic test accuracy (DTA) of MRN vs clinical neurosensory testing or patient-reported NSD in patients with PTTN. Secondary objectives were to identify currently used MRN sequences, their parameters and performance as well as how they correlate with nerve injury severity. Finally, we looked for any impact on clinical decisionmaking when adding MRN to the diagnostic work-up.
# Methods
## Systematic search
The PICO question included (P) patients suffering from PTTN resulting in NSD within the trigeminal distribution who (I) underwent MRI in (C) comparison with clinical (neurological) examination or patient-reported NSD and (O) to assess techniques reported, its diagnostic accuracy, performance and correlation with nerve injury severity. The current systematic review was registered in the International Prospective Register of Ongoing Systematic Reviews (PROSPERO; https:// www. crd. york. ac. uk/ PROSPERO/ display_ record. php? RecordID= 117971; number: CRD42018117971) and was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis-Diagnostic Test Accuracy (DTA) guidelines (see Appendix). The abstract was written using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis-DTA for Abstracts checklist. An experienced librarian was consulted before starting the study to co-create the search method. A systematic search was conducted in PubMed, Embase, Web of Science, and Cochrane Library in October 2019 and updated in February 2020. The search query is illustrated inand consisted of two concepts: "MRI" and "PTTN". These concepts were combined using the AND operator. Reference lists of included studies also were screened.
## Selection criteria
The search was limited to original research articles without restrictions on language or publication date.
Inclusion criteria included cohort studies, observational case-control, cross-sectional, randomized controlled trials (RCTs) and case series. In general, studies were included if the investigated patients were diagnosed with PTTN on the basis of sensory tests or Exclusion criteria included animal trials, case reports, reviews, systematic reviews and meta-analyses.
## Screening and selection of records
The first author (FVDC) executed the literature search and exported all references to Rayyan QCRI after deduplication.Two researchers (FVDC and FP) independently screened titles and abstracts according to inclusion and exclusion criteria. Disagreements were resolved in a consensus meeting with a third researcher (TMC). The first author screened the reference lists for additional articles that did not appear in the systematic search. Both researchers again independently determined which articles should be retained and consensus was reached in a second consensus meeting with the three researchers. Web of Science "Magnetic resonance imag*" OR "MRI" OR "nuclear magnetic resonance imag*" OR "arterial spin label*" OR "diffusion tensor imag*" OR "diffusion weighted imag*" OR "dynamic contrast-enhanced magnetic resonance imag*" OR "functional magnetic resonance imag*" OR "multiparametric resonance imag*" OR "perfusion weighted imag*" OR "neurography" OR "NMR" OR "MR tomography" OR "NMR tomography" OR "MRI-scan" OR "functional MRI" OR 'functional magnetic resonance imag*" OR "diffusion MRI" OR "diffusion weighted MRI" OR "nuclear magnetic resonance imag*" OR "fMRI" "Trigeminal nerve injury" OR "Trigeminal nerve injur*" OR "fifth cranial nerve injur*" OR "traumatic fifth nerve palsies" OR traumatic trigeminal neuropath*" OR "injury cranial nerve V" OR "traumatic fifth nerve palsy" OR "trauma trigeminal nerve" OR cranial nerve V injury" OR "fifth nerve trauma" OR "trigeminal nerve contusion" OR "trigeminal nerve transection" OR "trigeminal nerve avulsion" OR "inferior alveolar nerve" OR "lingual nerve" or "mandibular nerve"
Cochrane library # 1: [mh "magnetic resonance imaging"] # 2: ((magnetic NEXT resonance NEXT imag*) OR MRI):ti,ab,kw # 3: (nuclear NEXT magnetic NEXT resonance NEXT imag*):ti,ab,kw # 4: (arterial NEXT spin NEXT label*):ti,ab,kw # 5: (diffusion NEXT tensor NEXT imag*):ti,ab,kw # 6: (diffusion NEXT weighted NEXT imag*):ti,ab,kw # 7: (dynamic NEXT contrast NEXT enhanced NEXT magnetic NEXT resonance NEXT imag*):ti,ab,kw # 8: (functional NEXT magnetic NEXT resonance NEXT imag*):ti,ab,kw # 9: (multiparametric NEXT resonance NEXT imag*):ti,ab,kw # 10: (perfusion NEXT weighted NEXT imag*):ti,ab,kw
## Risk of bias assessment
The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool was used to assess the risk of bias and applicability concerns.Four levels were tested, including patient selection, index test, reference standard and flow and timing. A total score was plotted and indicates if included studies were at high, low or unclear risk of bias or applicability concern. FVDC and FP both independently assessed the included studies according to the QUADAS-2 manual. Discrepancies were discussed in a meeting with a third researcher aiding (TMC) in reaching a consensus. Resulting scores were plotted on a stacked bar chart.
## Recorded variables, data collection and analysis
Predetermined variables were extracted from the selected articles when possible and included: type of study, use of a reporting guideline, number of patients, age and gender, inclusion criteria, review questions, timing of MRI acquisition, investigated nerve branch, number of nerves observed, reference test, MRI device, coil type, sequence and sequence settings, use of post-processing techniques, use of contrast, evaluator level, blinding of evaluators, number of readings, type of analysis, formulas used for calculations, measurement areas and region of interests, intra-and interobserver variability, nerve caliber and relative signal intensity, correlation of MRN with NST, clinical and surgical findings, impact on clinical management and the author's conclusions. The first author extracted the data and correctness was verified by the second author.
# Results
## Study selection
The search yielded 483 articles, and 1 additional article was retrieved by reference list screening. After deduplication, 298 articles remained. These were screened based on title and abstract, after which 41 articles remained for full-text analysis. Eight articles were retained for the systematic review. Overview of the selection procedure is shown in.
## Study characteristics
Most included studies were retrospective (7/8) and 5 of these were case series, representing 444 subjects in total.Two studies applied a case-control designand one study a prospective cohort design.None of the articles mentioned the use of a reporting guideline. Using the QUADAS-2 tool, most studies were at high risk of bias but with low applicability concerns (Table 2,. The inclusion criteria and studyspecific research questions turned out to be divergent. There was a large variation in timing of the MRI acquisition (3 days-17 years). All studies assessed the inferior alveolar nerve (IAN) and some additionally included lingual nerve injuries (4/8). The reference test mostly consisted of a clinical (neurological) evaluation. Four studies added intraoperative findings as a reference test.In three studies it was unclear which reference test was applied.Due to the low methodological quality with widely varying methods, a DTA-analysis nor a meta-analysis could be performed. Consequently, after consultation with all authors, it was decided to provide a broad overview of the study and MRN characteristics, the evaluation methods used, their results and the conclusions drawn by the authors of the selected articles.
# Synthesis of results
## Characteristics of included studies & mri parameters:
An overview of all MRN parameters is given inDessouky et al. The impact of MRN on clinical decision-making was reported in one study by Cox et al. They stated that 29% did not have a change in clinical management and in 35% of cases MRN had substantial impact on their management, meaning a change in treatment.
# Discussion
MRN appears promising in the detection and grading of post-traumatic trigeminal lesions and correlates with clinical and surgical findings as well as neurosensory testing. However, there is a large heterogeneity in the reported studies with high risk of bias. None of the studies reported the use of a guideline or framework such as the STARD guideline.to allow adequate evaluation of the peripheral nervous system.Different methods have been described to achieve this and were observed in the selected studies of this review.Future studies should identify which of these sequences render the best suppression and thus nerve selective imaging of the peripheral trigeminal branches.
Post-processing was performed in all studies in which multiplanar reformatting was applied along the course of the nerve. Given the tortuous course of the trigeminal nerve, this would allow for a more holistic assessment. An isotropic voxel size is preferable to further assess its course in three dimensions, improving resolution and possibly reducing artifacts.This requires a thin slice thickness to adequately visualize these fine nerve branches, which are often less than 2 mm in diameter.Image interpretation and reporting was diverse with considerable methodological concerns. The outcomes that were assessed ranged from qualitative anatomic considerations towards quantitative RSI calculations. SI calculations require a methodological approach to allow standardization, especially if pulsed sequences are used.Since the RSI value seems of prognostic importance as illustrated by Cox et al, determining a standard approach and cutoff values is important for future research into DTA of MRN.In the included studies no cutoff values for relative signal intensity were defined; however the study by Dessouky et al did report sensitivity and specificity for MRN compared to clinical neurosensory testing and surgical findings, suggesting they determined cut-off values.They reported moderate to good correlation of MRN with injury severity, which was measured using NST or was surgically observed. Additionally, we need to consider that the region of interest where RSI values are measured would depend on the etiology of the PTTN and differ depending on the patient inclusion criteria, further complicating future comparison of studies. Therefore, mapping of the whole nerve trajectory could be a methodological approach to consider in future DTA studies.Finally, the use of MRN and its impact on clinical decision-making was demonstrated in one retrospective study by Cox et al.They illustrated a substantial impact in about one-third of patients, meaning a change in treatment. Although this concerns a small number of patients, it immediately raises the question whether this also has had an impact on outcomes and quality of life. Additionally, future studies should add a cost-benefit analysis of adding MRN to the diagnostic work-up. Limitations of this review are the small number of articles obtained, which were of low quality with different methodologies and results. No randomized controlled trials could be identified. Because of these arguments, DTA could not be determined.
In conclusion, there is insufficient scientific base to support or refute the use of MRN in the diagnosis and grading of PTTN. MRN seems promising in improving PTTN diagnostics and steering treatment decision.
## References
Dentomaxillofac Radiol, 50, 20200103
A systematic review on diagnostic accuracy of magnetic resonance neurography in posttraumatic trigeminal neuropathy Van der Cruyssen et al
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A Survey of Rain Attenuation Prediction Models for Terrestrial Links—Current Research Challenges and State-of-the-Art
# Introduction
The rapidly growing demands in high bandwidth, data rate, and availability requirements are pushing to deploy millimeter-wave frequency in wireless networks that can meet these requirements. The reason for such interests is the lower frequency bands' crowding and increasing demand for high data rates and bandwidth to accommodate ever-increasing customer services. However, the higher frequencies are considerably affected by rainfall. Rain is a natural process that attenuates the propagating signal at microwave and millimeter-wave frequencies. Therefore, it is necessary to mitigate rain attenuation to ensure the quality of microwave and millimeter-wave links. To this end, dynamic attenuation mitigation methods are implemented alongside attenuation prediction models that can predict the projected attenuation of the links. Therefore, multiple studies have been conducted on this issue worldwide. Studies on rain attenuation are used in geographically distributed locations to analyze and develop a rain attenuation model applicable over a wide frequency range, particularly radio frequencies over approximately 30 GHz for 5G and beyond network applications. However, to develop such a rain attenuation model, it is first necessary to determine the factors that affect attenuation. There is evidence that in addition to the rainfall, the frequency, path length (distance between the receiving and transmitting antennas), temperature, wind direction and velocity, pressure, and humidity can affect attenuation. Among others, rainfall intensity, frequency of operation, and link distance are significant parameters that determine rain attenuation. Various rain attenuation prediction models have mapped the correlation between rainfall intensity, path length, and frequencies with rain attenuation. An increment in rainfall rate increases the chance of interfering probability with radio waves [bib_ref] Real measurement study for rain rate and rain attenuation conducted over 26..., Shayea [/bib_ref]. In some studies, attenuation because of rain was reported at even lower frequencies, such as 5 GHz [bib_ref] Radar-derived path reduction factors for terrestrial systems, Goddard [/bib_ref] and 7 GHz [bib_ref] Improvement of a rain attenuation prediction method for terrestrial microwave links, Moupfouma [/bib_ref]. Many rain attenuation models have been proposed in the literature, and researchers have attempted to improve existing models to fit with local climatic conditions [bib_ref] Statistical Analysis of Rain at Millimeter Waves in Tropical Area, Al-Saman [/bib_ref] [bib_ref] Evaluation of Ka-Band Rain Attenuation for Satellite Communication in Tropical Regions Through..., Kalaivaanan [/bib_ref] [bib_ref] Rain attenuation at millimeter wave and low-THz frequencies, Norouzian [/bib_ref] [bib_ref] Investigation of the unified rain attenuation prediction method with data from tropical..., Abdulrahman [/bib_ref] [bib_ref] Rain attenuation statistics over millimeter wave bands in South Korea, Shrestha [/bib_ref] [bib_ref] Assessment of ITU-R predictions for Ku-Band rain attenuation in Malaysia, Khairolanuar [/bib_ref] [bib_ref] Application of the SC EXCELL model for rain attenuation prediction in tropical..., Lam [/bib_ref] [bib_ref] The Effects of Rain on Terrestrial Links at K, Ka and E-Bands..., Diba [/bib_ref]. The accurate estimation of attenuation because of rain in a specific radio link is essential for planning the link budget, maintaining the link quality, and designing the system. It was shown that rain attenuation could reduce the throughput of a link compared to sunny weather conditions [bib_ref] Availability and Fade Margin Calculations for 5G Microwave and Millimeter-Wave Anyhaul Links, Hilt [/bib_ref]. By deploying an appropriate rain attenuation model, even in the rain, a terrestrial link's throughput can be kept unchanged compared to a case without deploying any FMT and with the condition that other parts are usually working. FMT might be attained in several ways, such as power control, modulation techniques, adaptive waveform, and diversity techniques. If we do not consider power control of FMT owing to rain attenuation, then it is not possible to avoid overestimating or underestimating a transmission system's power. In fact, in every frequency band, the radio frequency (RF) engineer must follow the allowable power transmission requirements according to the spectrum management regulatory organization's rule. Thus overestimated the power of the transmitted signal strength may create interference into another frequency spectrum that is in use in the neighboring device if the engineers do not adhere to such specifications. On the other hand, underestimated power in the transmitted signal may further be attenuated by rain attenuation if the effect of rain is not mitigated either by changing modulation technique, adaptive waveform, or diversity control. Thus rain attenuation model plays a significant role in the FMT operation in a transmission system. As said earlier, higher rate data transmission demand leads to implement and use 5G and beyond wireless networks where millimeter-wave frequency band is a powerful candidate. However, the millimeter-wave coverage is short, leading to a higher number of terrestrial links in the 5G and beyond network. Thus, for the 5G and beyond the network's proper operation, accurate rain attenuation model deployment is substantial for terrestrial link. In this regard, lots of terrestrial rain attenuation models are proposed. However, a survey paper that contains an in-depth analysis of the models is not available, to the best of found knowledge. We have tried to fill this gap through this study. This paper exhibits parameters affecting rain attenuation, classification of recognized models, microwave and millimeter-wave frequency bands applicability to terrestrial links, efficient path length determination techniques, and enhancements or shortcomings of the models. Besides, a critical review of 18 well-known rain attenuation prediction models is assessed, classified, evaluated, compared, and summarized in this study. It is mentionable that previously we studied thoroughly brand-new learning-based rain attenuation models in [bib_ref] Learning-Assisted Rain Attenuation Prediction Models, Samad [/bib_ref]. That paper substantially differs from the current study as the main concentration of that article was learning-based rain attenuation models, including terrestrial and slant links. In the remainder of this study, the term model refers to the rain attenuation model and ITU-R (International Telecommunication Union-Radio-communication sector) database or DBSG3 (Study Group 3 databanks) will be used interchangeably.
# Contributions
This article thoroughly analyzes the main features, weaknesses, and unique characteristics of well-known and new models. In this article, the main points are:
- Section 2 includes extensive coverage on predicting the accurate rain rate for the ungauged area, techniques to generate rain rate corresponding attenuation time series [fig_ref] Table 1: Estimation techniques of rain attenuation time series [/fig_ref] , highly spatial resolution rain rate estimation techniques , and effective path length through correction techniques .
- To the best of our knowledge, there is no survey paper regarding the prediction of the rain attenuation of terrestrial links. We classified the most well-known and updated models in this study, which are presented in Section 3. -
We developed a brief overview of each of the selected models. The quantitative and qualitative features of various models are tabulated in [fig_ref] Table 6: Properties of rain attenuation models [/fig_ref] , respectively. -
We observed an inherent improvement in each model, criticizing the model by finding the drawbacks and unique features mentioned in [fig_ref] Table 8: Cont. [/fig_ref]. -
The comprehensive research concerns are summarized in Section 6. -
We have tabulated recent research works outcomes with short-ranged links at , and 120 GHz frequency where well-known ITU-R model predicts inaccurately .
## Preliminaries
This section discusses rain attenuation factors, data collection, available databases, experimental studies, and database sources to check the validity of the model.
## Rain attenuation factors
It is crucial to find a justification and insightful analysis to determine the variables that influence rain attenuation. Although rain is a crucial factor influencing rain attenuation, the link distance, frequency, and polarization play a significant role in the determination of rain attenuation. A brief review of more parameters for rain attenuation is presented here. In the literature, various researchers have found different rain attenuation factors for either terrestrial or slant links. In this regard, we compiled 17 parameters that can impact rain attenuation for microwave links using artificial or ML-based techniques [bib_ref] Learning-Assisted Rain Attenuation Prediction Models, Samad [/bib_ref].
## Rainfall rate data collection procedures
The rain rate is an essential parameter for determining rain attenuation. In this section, different data collection techniques for rain attenuation are discussed.
## Available databases
A newly devised model should check the efficiency for its validity the confirmation. In most cases, the model developer uses the ITU-R DBSG3 rain attenuation database. In some cases, weather databases European Center for Medium-Range Weather Forecasts (ECMWF) or ECMWF re-analysis-15 (ERA-15) were also used as secondary sources of determining the rainfall rate. These secondary databases lack rain attenuation information on terrestrial and earth-space links for tropical regions. Consequently, most of the models developed in tropical countries are needed to create facilities to prepare the rain attenuation databases.
## Experimental setup
A simple method of determining the rain rate is to set up an experiment to deploy measuring equipment such as the use of a disdrometer, weather station, and rain gauges that measure the rain rate at lower integration times ( 1 min intervals), which can be saved in a personal computer with the help of a dedicated data logger [bib_ref] Real measurement study for rain rate and rain attenuation conducted over 26..., Shayea [/bib_ref] [bib_ref] Statistical Analysis of Rain at Millimeter Waves in Tropical Area, Al-Saman [/bib_ref] [bib_ref] A study on the rain attenuation prediction model for ubiquitous computing environments..., Choi [/bib_ref] [bib_ref] Rain fade slope model for terrestrial microwave links, Chebil [/bib_ref]. In some cases, the radar information of the rain cell was used to measure the rain rate. The problem with radar-based techniques is that massive investments are required to collect rain rate information if radar systems have not been deployed for other purposes [bib_ref] Data and theory for a new model of the horizontal structure of..., Capsoni [/bib_ref] [bib_ref] Method for prediction of attenuation on earth-space links based on radar measurements..., Leitao [/bib_ref].
## Rain rate data generation: synthetic technique and logged data
The rain rate time series in a specific area is essential because it is used to calculate the attenuation in a fixed radio transceiver infrastructure [bib_ref] Time series rainfall spike modelling from Markov chains and queueing theory approach..., Diba [/bib_ref]. The general procedure for collecting the rain rate time series is to collect the data by employing an experimental setup. Thus, the general approach is time-consuming because a minimum of one year of data should be collected over a particular area. Cost is also associated with this process.
In addition to this experimental technique, a synthetic method can be used to calculate the time series using a mathematical approach. [fig_ref] Table 1: Estimation techniques of rain attenuation time series [/fig_ref] , summarizes the various types of synthetic time series assessment techniques.
## Rain rate prediction from spatial interpolation techniques
To accurately determine the rain attenuation, it is necessary to consider the spatial distribution of the rainfall intensity. The rain rate cannot be measured everywhere using the rain rate collector, which significantly reduces the accuracy of the experimental setup. However, an intense spatial resolution rain rate is required for accurate estimation. There exist some synthetic techniques by which the undetermined rain rate can be estimated to solve the problem at a particular location.
The inverse distance weighting (IDW) technique as per Equation (1) can be used to determine the rainfall rate at ungauged locations [bib_ref] Rain Attenuation Measurements and Analysis at 73 GHz E-Band Link in Tropical..., Al-Samman [/bib_ref] [bib_ref] Rainfall rate and attenuation performance analysis at microwave and millimeter bands for..., Diba [/bib_ref] :
[formula] R p = N ∑ i=1 w i R i ,(1) [/formula]
where N is the number of rain gauges. The rain value w i depends on the location of d i in the estimated position p is given by Equation (1), and w i is given by Equation (2):
[formula] w i = d −2 i N ∑ i=1 d −2 i .(2) [/formula]
The average rainfall rate was then determined from these estimated values, along with the rain gauge readings used in this analysis. Using Equation (1) the rain rate can be predicted up to 10-30 km. Unfortunately, the rainfall data available in the weather database ERA-40 provided by the ECMWF suffer from a low spatial resolution 1.125 - × 1.125 - latitude per longitude grid.
The spatial-temporal rainfall distribution mechanisms based on the top-to-bottom data analysis approaches are surveyed in. This survey compared most techniques that predict high-resolution space-time rainfall using remote sensing, conventional spatial interpolation, atmospheric re-analysis of rainfall, and multi-source blending techniques, and discussed issues in integrating various merging algorithms. In the article, it was shown that the maximum spatial resolution is available by the Global Satellite Mapping of Precipitation Near Real-Time (GSMaP-NRT) dataset with a resolution of up to 0.01 - with an update of once per hour, which is clearly higher than the ECMWF database. presents an analysis of different high-resolution spatial rainfall estimation techniques.
Another technique for generating the rain rate is applying the local rain data to the MultiEXCELL model [bib_ref] MultiEXCELL: A new rain field model for propagation applications, Luini [/bib_ref]. This model was used into generate synthetic rain rates. Transmitting and detecting specific differential phase-shifted signals through a dual-band radar system has been experimented with in. As a result of this experiment, the authors noticed the scattering effects in the detected signals that arise due to the radar signals' differential reflection. A corrector factor should be used for the reflected and differently reflected signals in order to eliminate the scattering effects. The statistical uncertainties of rainfall are then calculated by considering the propagation of the power-law relations.
R(Z h , Z dr , K dp ) = 9.6046Z 0.072 h Z −0.017 dr K 0.824 dp .
(3)
## Ref.
Estimation Techniques [bib_ref] Time series prediction of rain attenuation from rain rate measurement using synthetic..., Das [/bib_ref] The proposed technique generates rain attenuation time series using storm speeds from 1 to 12 m/s in a two-layered rain structure model. Also, temperature, altitude, and height are used as per the geographic location.
[27]
[formula] A(t) = a 0 ·e √ 2d AG /βa ·W(t)+d AG ·va /βa ·t 1+d AG ·a 0 t 0 e √ 2d AG /βa ·W(s)+d AG ·va /βa ·s ds [/formula]
where a 0 : 0-0.5 dB, W(t): Wiener process, β a , v a : gamma distribution parameters, d AG : Dynamic parameter β of the Maseng-Bakken model. [bib_ref] Worst Month Tropospheric Attenuation Variability Analysis: ITU Model vs. Rain Gauge Data..., Liu [/bib_ref] It proposed an enhanced technique to generate rain attenuation time series where precise rain rates are not available at global scale using ITU-R model. The technique uses mean and standard deviation of rain rate either from NOAAand ITU-R modeland the output of Gaussian noise through a low-pass filter (LPF: k/p + β, cut-off frequency f c : 0.2 MHz) into a non-linear memoryless device, where A offset is the calibration factor, A offset : exp(m + σQ −1 (P 0 /100)) and Q: zero-mean, unit variance Gaussian probability density function.
[formula] [31] A(x 0 ) = k A L A 0 R α A (x 0 + ∆x 0 , ξ)dξ + k B L B L A R α B (x 0 , ξ)dξ [/formula]
where L A and L B are the radio path lengths, ∆x 0 is the shift due to the presence of layer B, x 0 = v · t, and v is the average storm speed (typically 10 m/s).
[32]
[formula] A(t 0 ) = 1 cos θ d 0 +S A d 0 k A R(l) α A dl + d 0 +S A +S B d 0 +S A k B 3.134 α B R(l) α B dl , where θ: link elevation angle, (α A , k A ), (α B , k B ): [/formula]
power-law coefficients that converts the rain rate into specific attenuation for layers A and B, respectively, and R: rain rate along the link. [bib_ref] Rain attenuation time series synthesizer based on copula functions, Kourogiorgas [/bib_ref] A copula is a multivariate distribution function expressed by marginally uniform random unit interval variables and it can avoid dependence index like in log-normal distribution. The procedure is: ρ = sin πτ 2 → zero mean Gaussian random variables correlated matrix→normal CDF→desired random variable→inverse CDF of the desired distribution. [bib_ref] Rain attenuation time series synthesizer based on the gamma distribution, Andrade [/bib_ref] The procedure is:
[formula] R G = e −β·|τ| → [β EMB , β gamma ] → H(z) = √ 1−e −2βTs 1−z −1 e −βTs , [/formula]
where T s : sampling time, and β EMB and β gamma are 12.3d −0.95 × 10 −4 and 6.9d −0.6 × 10 −4 , respectively. [bib_ref] A rain attenuation time-series synthesizer based on a dirac and lognormal distribution, Boulanger [/bib_ref] The procedure is:
[formula] a k = 1 N ∑ N−1 j=0 M j e − i2π N kj = M j → a k = h k (c G ) × e k → M j = −1 (a k ), [h k = 0.5], where M(t): [/formula]
Gaussian process, and −1 are direct and inverse Fourier transforms, respectively.
[36]
Compute the stochastic differential equation: [bib_ref] Radar-derived path reduction factors for terrestrial systems, Goddard [/bib_ref] , where µ and γ are found by fitting to experimental first order statistics of rain attenuation, β a and S a are the parameters of the diffusion coefficient of the M-B model. [bib_ref] Advanced time series synthesizer for simulation of joint rain attenuation conditions, Nebuloni [/bib_ref] Compute:
[formula] dA(t) = µ 4 d a µ 2 λ A(t) − A 2 (t) + µ 2 .dt + d a µ 3 λ A(t)dW(t), where d a = 2β a S 2 a λ µP(t i ) = 1 − P 0,i → z i = T z (r i ) → find M z (d) → Gaussian PDF → ρ j (τ) → H i (z), [/formula]
where P 0,i is the possibility of rain in the ith station, r i represents a nonlinear transformation T z , and ρ j is the temporal autocorrelation function of rain attenuation for ith link. . Highly spatial resolution rainfall estimation models.
## Ref.
Technique or Resolution [bib_ref] Statistical Analysis of Rain at Millimeter Waves in Tropical Area, Al-Saman [/bib_ref] Analyzed millimeter-wave and showed that the ITU-R predicted rainfall rate of region P is up to 0.01% of time (agrees → 99.99% of time and disagrees → 0.01% of time).This multi-source blending technique to estimate high-resolution space-time rainfall scales to develop and merge remote sensing, conventional spatial interpolation, atmospheric re-analysis of rainfall, and multi-source blending techniques.
[38] It presented gauged-based data re-analysis at a resolution of 0.5 - × 0.5 - .
[In GSMaP-NRT, it analyzed the satellite, microwave-infrared, and near real time weather dataset to compare better predictability presented resolution about 0.01 - × 0.01 - .
[40] ECMWF: 1.125 - × 1.125 - [bib_ref] The impact of spatial-temporal averaging on the dynamic-statistical properties of rain fields, Yang [/bib_ref] It proposed the spatial and the temporal correlation functions to determine rainfall rate. . Techniques to calculate effective path length (EPL) or path length coefficient factor (PCF).
## Ref. epl or pcf parameter settings remarks
[2] r = 1/ 1 + 0.03(100P) βl m Method: Practical measurement; Frequency band: 7-38 GHz; Path length: 58 km; and rain rates were collected over 1-min time interval.
The correction factor depends on β; link length; and p% of rain The correction factor only depends on the A rad,d (t), and γ R (t).
[formula] [3] r rad (t) = A rad, d (t)/γ R d[42] r = 1 1+ L 2636 R(P)−6.2 [/formula]
Method: Practical measurement Rain rate: 5-min point at 11 GHz frequency; 42.5 km long radio link with R > 10 mm/h
The correction factor depends on the radio link length and rain rate.
[43] r = 1.08L −0.5108 (7 GHz for 0.01% of the time)
Method: Practical virtual link; Link length: 1-10 km; Time exceedance: 0.01%; Frequency 7 GHz
The reduction function depends only on the total path length. Estimation: Exponential curve fitting [bib_ref] Prediction of rain attenuation in terrestrial links using full rainfall rate distribution, Mello [/bib_ref] d e f f = The correction factor depends only on the rain rate exceedance of p% of the time.
Estimation: exponential curve fitting The correction factor depends only on the slant path length and the rain cell diameter.
[formula] [45] r(R 0.01 , L) = L × ( −R 0.01 1+ζ(L)×R 0.01 ) ITU-R database[49] r = A/(kR α TX L slant ) [/formula]
Method: MultiEXCELL rain simulation. Calculation: rain attenuation is calculated via the numerical approach. Rain field size: 1 km × 1 km to 250 km × 250 km
The correction factor depends on calculated attenuation, specific attenuation conversion coefficients, 'measured' rain rate at the transmitter end, and the LOS link length.
[formula] [50] r = 1 0.477L 0.633 R 0.073α 0.01% f 0.123 − 10.579 1 − e −0.024L [/formula]
(1) Can be used worldwide; (2) frequency band: 5-100 GHz;
(3) Maximum path length is 60 km
The correction factor depends on the frequency (GHz), specific attenuation coefficient (α), and link length (L). . Cont.
## Ref. epl or pcf parameter settings remarks
[formula] [51] r = N e cos θ h R −h S tan θ R < R 0 N e cos θ ( 1 0.056+0.012R ) R ≥ R 0 [/formula]
The number of effective cells (N e ) is calculated after analyzing ITU-R DBSG3 database.
To define the rain cell, it needs to know the cell (R 0 ) boundary rain rate.
[formula] [52] r = 1 1.77d 0.77 R −0.05 0.01 f ≤ 40 GHz 1 0.477d 0.633 R 0.073 0.01 f 0.123 2 f > 40 GHz [/formula]
It was based on the measured attenuation of smaller than 1 km terrestrial link and frequency 26/38 GHz.
It concluded that the distance factor is inconsistent for a link length smaller than 1 km.
## Ref. technique or resolution remarks
[formula] [53] E = 1 nCountry 415 ∑ i=1 W i log R i DBSG3 R i S−B [/formula]
This test was used to re-analysis based rain rate and the rain rate provided by the ITU-R DBSG3 database.
[54]
[formula] E R0.01 = E 2 ψ + E 2 φ + ∆R 0.01 2 where E 2 ψ = ∂R 0.01 ∂ψ 2 σ 2 ψ and E 2 φ = ∂R 0.01 ∂φ 2 σ 2 φ . [/formula]
The model was developed and verified using DBGS3 along with CHIRPS rainfall (ψ), and TPW (φ) in the ERA-Interim Reanalysis database. Authors have not compared with measured data and the precise calculation of rain rate R 0.01 showed lower accuracy (uncertainty is about 14%).
The wet-antenna effect has relation with the bias value of the signal in the receiver section. However, an appropriate bias compensation technique has not yet been developed.
A rain-rate-retrieval algorithm was designed using radar reflectivity derived from the rain rate in [bib_ref] Rain-rate estimation algorithm using signal attenuation of Ka-band cloud radar, Oh [/bib_ref]. Based on the Doppler velocity, the derived radar reflectivity was classified as low-and high-rain cases. This model paved the way for blending reflectivity and attenuation to predict the rain rate. However, beyond reflectivity and attenuation, other factors, such as seasonal variation and rain type, were not considered.
The minimum observed attenuation and the maximum observed attenuation were calculated through a commercial microwave link (CML) within a fixed interval [bib_ref] Empirical study of the quantization induced bias in commercial microwave links' min/max..., Ostrometzky [/bib_ref]. Using these minima and maxima, the observed attenuation value averaged rain-intensity can be calculated asR
[formula] i = b max(A r_ max i − B, 0) αL ,(4) [/formula]
where B (in dB) is the induced bias value because of to the mixture of the transformation of the min/max with the quantizer, the negative values of (A r_ max i − B) are counted as zeroes when they exist,ã = a · [ln(K) + 0.57722] b , and the a and b parameters refer to the power-law relationship of specific coefficients and K is the number of instantaneous samples per interval from which the maximum attenuation is extracted.
Since 2000, numerical weather prediction (NWP) has become popular in predicting rainfall and has drawn interest from the meteorological forecasting industries, researchers, and other stakeholders. However, owing to decreased portability and implementation coverage in remote locations, NWP-based techniques are not a potential technique for remote area application. Therefore, the prediction of learning supported rain diminution is standard because the problem of the NWP technique can be solved. In [bib_ref] Deep learning-based effective fine-grained weather forecasting model, Hewage [/bib_ref] [bib_ref] A neural network-based local rainfall prediction system using meteorological data on the..., Kashiwao [/bib_ref] [bib_ref] An extensive evaluation of seven machine learning methods for rainfall prediction in..., Cramer [/bib_ref] [bib_ref] Improved rainfall prediction using combined pre-processing methods and feed-forward neural networks, Tran Anh [/bib_ref] [bib_ref] Rainfall Monitoring Based on Machine Learning by Earth-Space Link in the Ku..., Xian [/bib_ref] ML-based rainfall prediction techniques were presented. [fig_ref] Table 4: Error estimation techniques for rain rate prediction [/fig_ref] lists some of the error estimation techniques for rain rate prediction.
## Distance correction
The rain attenuation (A) was calculated by multiplying the specific attenuation and the distance between the transmitting and receiving antennas.
[formula] A = γL e .(5) [/formula]
Assuming the effective path length L e to be 1 km, in Equation (5) the specific attenuation and the link attenuation are equal. Equation (5) is true if the rain and cloud are uniformly distributed over the entire path between the transmitting and receiving antennas. However, if the distance between the transmitting and receiving antennas is not 1 km:
[formula] L e = A γ .(6) [/formula]
Owing to the non-uniform distribution of rain, the values of the specific and link attenuation (for 1 km length) are different, which defines a term called the effective path length. This implies that the effectual and actual distance varies for non-uniform rain distributions and links. The effectual distance is usually calculated based on the rainfall distribution [bib_ref] Real measurement study for rain rate and rain attenuation conducted over 26..., Shayea [/bib_ref]. Many models calculate the effective path length using a correction factor, referred to as the path adjustment factor. In terrestrial links, all the link lengths remain within a single rain cell for a short link or many cells for a long link. A brief discussion on the parameters that affect either the effective path length or path length adjustment factor is presented in the next section. In most cases, the accuracy of the model discussed above was calculated using the measured rain attenuation data, which was then compared to the attenuation derived through the attenuation formula. In some cases, the root means square (RMS) and standard deviation (STD) values were calculated to validate the model. contains all of the most critical effective path length or distance correction factors proposed in the literature. The distance correction factor is more crucial for the E-band, which is a probable frequency band for developing 5G and beyond wireless communication networks, as discussed in Section 6.4.
## Frequency and polarization
The specific attenuation can be determined from the rainfall rate, frequency and polarization using the following power-law relation [bib_ref] The aR b relation in the calculation of rain attenuation, Olsen [/bib_ref] [bib_ref] Statistics of terrestrial millimeter-wave rainfall attenuation, De Bettencourt [/bib_ref].
[formula] A sp (dB/km) = xR y 0.01 ,(7) [/formula]
where R 0.01 is the rain rate, and x and y are regression coefficients that depend on several factors such as: polarization, carrier frequency, temperature, and rain drop size distribution [bib_ref] The aR b relation in the calculation of rain attenuation, Olsen [/bib_ref]. The values of x and y can be determined experimentally as empirical values. ITU-R P. 838-3provides the prediction values for x and y for 1-100 GHz frequency bands at horizontal and vertical polarizations. In this section, various parameters of rain attenuation, rain rate data collection procedure, available public domain databases, time-series generation techniques, percentage of time exceedance of rain (Equation (7)), specific attenuation coefficient determination procedure, and the procedure of distance correction factor have been discussed. All the data collected or modified through these techniques can be used by the rain attenuation models, which will be discussed in the next section.
## Rain attenuation models: terrestrial links
Existing terrestrial models can be classified into five categories based on the formulation of the rain attenuation model. These include the empirical, physical, statistical, fade slope, and optimization-based models.
## -
Empirical model: The model is based on experimental data observations rather than input-output relationships that can be mathematically described. The model is then classified as an empirical category.
- Physical model: The physical model is based on some of the similarities between the rain attenuation model's formulation and the physical structure of rain events. - Statistical model: This approach is based on statistical weather and infrastructural data analysis, and the final model is built as a result of regression analysis in most cases. - Fade slope model: In the fade slope model, the slope of attenuation from the rain attenuation versus time data was developed with a particular experimental setup. Later, these data were used to predict rain attenuation. - Optimization-based model: In this type of model, the input parameters of some of the other factors that affect the rain attenuation are developed through optimization (e.g., minimum error value) process. [fig_ref] Figure 1: Taxonomy of terrestrial rain attenuation models [/fig_ref] , represents a taxonomy of the well-known and recently developed rain attenuation models used in this study.
## Empirical models
## Moupfouma model
This model [bib_ref] Electromagnetic waves attenuation due to rain: A prediction model for terrestrial or..., Moupfouma [/bib_ref] uses the rain rate exceeded by 0.01 percent of the time and the calculation of the proportion of time-correlated with the excess of any given interest attenuation.
[formula] γ R 0.01 = kR α 0.01 (8) A 0.01 = γ R 0.01 × L eq ,(9) [/formula]
where L eq is the equivalent path length for which the rain propagation is assumed to be uniform.
## Budalal model
In this model [bib_ref] Modification of Distance Factor in Rain Attenuation Prediction for Short Range Millimetre-wave..., Budalal [/bib_ref] according to the 300 m link's attenuation analysis with frequencies of 26 and 38 GHz, the authors found attenuation inconsistency provided by the latest ITU-R model. They then investigate the specific attenuation (γ th ) as per ITU-R P.838-3and found an inconsistency between the effective specific attenuation (γ e f f ) can be defined as Equations (10) and (11):
[formula] I f γ = 1 1.77d 0.77 R −0.05 0.01 , for f ≤ 40 GHz, d < 1 km(10)I f γ = 1 0.477d 0.633 R 0.073 0.01 f 0.123 2 , for f > 40 GHz, d < 1 km.(11) [/formula]
It is inferred that the model can be used for short-range outdoor links with frequencies higher than 25 GHz in 5G networks.
## Perić model
This model is also referred to as a dynamic model [bib_ref] Dynamic rain attenuation model for millimeter wave network analysis, Perić [/bib_ref]. It depends on the cumulative distribution function of the rain intensity of the area of interest, the number of rain events in which the rain intensity threshold is exceeded, the rain advection vector intensity, and the rain advection vector azimuth. The model considers the spatial distribution within a 10 km radius around an antenna and is suitable for small geographical areas, up to 10 km × 10 km. Furthermore, it has not been tested in a real-world network environment.
## Garcia model
It is one of the modified version [bib_ref] Modified Lin's empirical formula for calculating rain attenuation on a terrestrial path, Garcia-Lopez [/bib_ref] of Lin model [bib_ref] 11-GHz radio: Nationwide long-term rain rate statistics and empirical calculation of 11-GHz..., Lin [/bib_ref] , assuming that the path length reduction coefficient changes with the path length and rainfall rate. The developed model was tested with in Paris, Stockholm, Dijon (France), and Kjeller (Norway), with variations in frequency and path length. The model is best suited for temperate European regions.
[formula] A = kR α 1−min L 1 0.5 + [L(3R 1−min − 3.9L + 245)/2636 ] [/formula]
, for R > 10 mm/h , L > 5 km. [bib_ref] The Effects of Rain on Terrestrial Links at K, Ka and E-Bands..., Diba [/bib_ref] This model improves the limitation of the 5-min rain rate requirement of the original Lin's model. This model's drawback is that it was only tested at 11 GHz and not at higher frequencies. Furthermore, the model did not consider spatial rain distribution variations. Another limitation of this model is that it only applies to terrestrial links.
## Da silva/unified model
This model [bib_ref] Improved unified method for the prediction of rain attenuation in terrestrial and..., Da Silva Mello [/bib_ref] uses the full rainfall rate distribution with multiple nonlinear regressions from the rain attenuation database. It is primarily developed for terrestrial links and can be later extended to slant links. For a terrestrial link,
[formula] A p = k (R e f f T (R p , d)) α . d 1 + d/d 0 (R p ) ,(13) [/formula]
where R e f f T is the approximate effective rain rate for terrestrial links and the and the cell diameter d 0 is given by Equations (14) and (15) respectively.
[formula] R e f f T = 1.74R 0.786+0.197/d (14) d 0 = 125R −0.33(15) [/formula]
R e f f (R p , L s , θ) = 1.74R 0.786+0.197/L s cos θ · cos θ + 120 L s 2.88 R −0.186 sin θ .
For the terrestrial case L s = d, the second term in the brackets vanishes as θ = 0 - , and the expression is reduced to the terrestrial case prediction method. With the correct consistency for terrestrial and slant paths, the model exhibits good performance; however, the error has not been compared with real attenuation data.
## Mello model
According to this model [bib_ref] Unified method for the prediction of rain attenuation in satellite and terrestrial..., Mello [/bib_ref] the cumulative probability distribution of rain attenuation for terrestrial link can be determined by the Equation (17):
[formula] A p = k 1.763R 0.753+0.197/L s cos θ α d 1 + d 119R −0.244 .(17) [/formula]
## Abdulrahman model
According to this model [bib_ref] Rain attenuation predictions on terrestrial radio links: Differential equations approach, Abdulrahman [/bib_ref] the rain attenuation is given by the Equation (18):
[formula] A%p = µ[S(R%p)](18) [/formula]
where
[formula] S(R%p) = βR α−1 %p (19) β = k[α + b(1 − r%p)]d eff (20) µ = R%p α + b(1 − r%p) .(21) [/formula]
## Crane model
This model [bib_ref] Prediction of attenuation by rain, Crane [/bib_ref] establishes rain distribution from a global perspective and the USA's precise rain distribution maps. From these maps, the rain rate distribution can be calculated.
If the path length D > 22.5 km, then the rain rate should be modified:
[formula] R P = R P D 0 D(22) [/formula]
where D 0 = 22.5 km
[formula] A R p , D = kR α p e uαd −1 uα − b α e cαd cα + b α e cαD cα , d D 22.5 km A R p , D = kR α p e uαD −1 uα , 0 < D d(23) [/formula]
where the constants are given by Equation (24)
[formula] u = ln[be cd ] d , d in km b = 2.3R −0.17 p , R p in mm/h c = 0.026 − 0.03 ln R p d = 3.8 − 0.6 ln R p .(24) [/formula]
## Physical models
## Crane two-component (t-c) model
This model [bib_ref] A two-component rain model for the prediction of attenuation statistics, Crane [/bib_ref] is based on different integration techniques for heavy and light rainfall regions. The author proposed two versions of the T-C models: the first is a simple T-C model and was published in 1982. The model consisted of several steps. (1) Determining the propagation path for the global climate. (2) Finding a mathematical relation between the projected path length in the rain cell and debris region. (3) Fixing the expected amount of attenuation. (4) Deriving the required rain rate to produce rain attenuation and calculating the probability that the specified attenuation is fixed in step (3).
[formula] P(a > A) = P c (a + D c /W c )e −R /R c + P D (1 + D D /W n D )η ln R n − ln R D σ D .(25) [/formula]
The model was primarily developed for Western Europe and the USA, and has difficulty in determining rainfall parameters, such as the probabilities of occurrence and mean rainfall, for weak and strong rain cells. Sometimes these weak and strong rain cells are referred to as debris and cell, respectively. The model was verified for both the satellite and terrestrial links.
## Ghiani model
This modelis based on a PCF-correction-based model for terrestrial links. It can be modeled by simulation with Equation (26) and analyzed with Equation (27):
[formula] A = L γ R (l)dl = L kR(l) α dl.(26) [/formula]
(1) Calculate
[formula] A = kR TX α LPF.(27) [/formula]
(2) Calculating the PCF: PCF = A/kR TX α L for the number of rain maps generated by the MultiEXCELL model. This results in the following expression:
[formula] PF av = a( f , L)e −b( f ,L)R + c( f , L),(28) [/formula]
where the symbols a, b, and c are taken from the regression coefficients. These three coefficients depend on the values of frequency and path length.
(3) Because the effect of the frequency is negligible
[formula] A(P, L) = kR(P) α L a(L)e −b(L)R + c(L) ,(29) [/formula]
where the constants are given by the set of equations in (30),
[formula] a = −0.8743e −0.1111R + 0.9061 b = −0.0931e −0.0183R + 0.1002 c = −0.6613e −0.178R + 0.3965.(30) [/formula]
This model's drawback is that the RMS of the prediction error against the ITU-R database did not exhibit better performance compared to the ITU-R and Brazilian models. Thus, a better terrestrial link rain database from DBSG3 or Comité Consultatif International des Radiocommunications (CCIR) was required for examination before its final application.
## Excell/capsoni model
The parameters of this statistical model [bib_ref] A comprehensive meteorologically oriented methodology for the prediction of wave propagation parameters..., Capsoni [/bib_ref] of the horizontal rain structure can be determined based on the local statistical distribution of the point rainfall intensity. The model was validated using the COST 205, 1985 database. This model consists of several rain cell structures, collectively refereed to as kernels. In such a rain cell, the rainfall rate at a distance l from the center is given by:
[formula] R = R peak e −l/l 0 .(31) [/formula]
Probability of attenuation equation:
[formula] P(A) = ∞ R E E.[0.5ln 2 (R peak /R E ) + r ln(R peak /R E )]. [−P(R p ) ]d(ln R peak )(32) [/formula]
where r = 1/4πl 0 . Rain distribution can be calculated as:
[formula] P(R) = P 0 ln n ( R * R ).(33) [/formula]
Here, P(R) = 0 indicates that the probability of rain is zero, which will be true at the rain cell boundary. A simplified version of the model with the point rain intensity at point (x,y) can be defined as:
[formula] R(x, y) = R M e − ( x lx ) 2 + y ly 2(34) [/formula]
along a cell radius:
[formula] R(x, y) = R M e − √ x 2 +y 2 l 0 .(35) [/formula]
In the sense of the rain attenuation model, this model does not provide attenuation. However, it facilitates the generation of a synthetic rain rate from which attenuation can be predicted using a suitable prediction model. There are critics that the exponential rain peak is not present [bib_ref] HYCELL-A new hybrid model of the rain horizontal distribution for propagation studies:..., Féral [/bib_ref] in nature, and the model does not differentiate between stratiform and convective rain.
## Statistical models
## Itu-r model
This model [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref] is primarily based on a distance factor that relies on the rain rate R 0.01 , frequency, link length, and power-law relationship coefficients of the specific attenuation α (furthermore, it is a function of frequency and polarization). The attenuation and the distance factors can be calculated as:
[formula] A 0.01 = kR 0.01 α dr (36) r = 1 0.477d 0.633 R 0.073α 0.01 f 0.123 − 10.579(1 − e −0.024d ) .(37) [/formula]
The attenuation, A p , which exceeded for a percentage of time p other than 0.01%, was determined by the simplification of the attenuation A 0.01 . This model, validated in Malaysia, showed good agreement with the measured attenuation [bib_ref] Rain attenuation statistics over 5G millimetre wave links in Malaysia, Ghanim [/bib_ref].
## Singh model
This model [bib_ref] Proposed Model for Radio Wave Attenuation due to Rain (RWAR), Singh [/bib_ref] provides an easy calculation mechanism compared to the ITU-R model. The specific attenuation follows the ITU-R model for the frequency band of 1-100 GHz. After calculating the specific attenuation, the curve fitting technique using the MATLAB software cubic polynomial Equation (38) is approximated for the specific attenuation.
[formula] A(dB/km ) = c 3 f 3 + c 2 f 2 + c 1 f + c 0 ,(38) [/formula]
where the coefficients c 3 , c 2 , c 1 , c 0 of Equation [bib_ref] A gauge-based analysis of daily precipitation over East Asia, Xie [/bib_ref] for the horizontal polarization are given by:
[formula] c 3 h = 1.422 × 10 −9 x 2 + 2.03 × 10 −7 x − 1.21 c 2 h = 1.963 × 10 −7 x 2 + 8.618 × 10 −7 x + 0.0019 c 1 h = 2.114 × 10 −6 x 2 + 0.01x − 0.036 c 0 h = 3.10 × 10 −5 x 2 − 0.040x − 0.031(39) [/formula]
and for the vertical polarization:
A similar approach-based technique was proposed in [bib_ref] Prediction of Rain Attenuation and Impact of Rain in Wave Propagation at..., Kestwal [/bib_ref]. However, it was considered the original power-law relationship rather than the simplified polynomial form in that proposal. The second difference is that the constants k, α referring to the Equation [bib_ref] 11-GHz radio: Nationwide long-term rain rate statistics and empirical calculation of 11-GHz..., Lin [/bib_ref] depends only on frequency and either vertical or horizontal polarization.
[formula] A(dB/km) = kR α (41) a h = 4.21 × 10 −5 f 2.42 , for 2.9 GHz ≤ f ≤ 54 GHz a v = 4.09 × 10 −20 f 0.069 , for 54 GHz ≤ f ≤ 180 GHz b h = 1.41 f −0.0779 , for 8.5 GHz ≤ f ≤ 25 GHz b v = 2063 f −0.272 , for 25 GHz ≤ f ≤ 164 GHz.(42) [/formula]
## Fade slope models
## Andrade model
In the Andrade model [bib_ref] Short-term rain attenuation predictor for terrestrial links in tropical area, Andrade [/bib_ref] the variance of the fade slope is proportional to the attenuation as per Equation (43):
[formula] f ( f s |A) = 1.38 √ k · A 1 + f s 2 k·A 6.7 .(43) [/formula]
The predictor can estimate the next attenuation level A(t i + t p ) from the current attenuation value A(t i ) and fade slope:
[formula] A t i + t p = A(t i ) + f s t p ,(44) [/formula]
where t p is the prediction time, it can be considered that t p = 10, which corresponds to the minimum prediction time, that is, the sampling time of the experimental data.
## Chebil model
In the Chebil model [bib_ref] Rain fade slope model for terrestrial microwave links, Chebil [/bib_ref] the variance of the fade slope is proportional to the attenuation as per Equation (45):
[formula] p(ξ | A) = 1 σ ξ √ 2π exp 0.5 ξ σ ξ 2 ,(45) [/formula]
where the σ ξ is given by Equation (46) σ ξ = 0.00012A 3 − 0.003A 2 + 0.027A − 0.0016.
## Optimization-based models
## Develi model
This modelis based on the Differential evolution approach (DEA) optimization technique and experimentally tested at 97 GHz on terrestrial link in the United Kingdom (UK). The steps of the DEA attenuation model are as follows:
(1) The rate of rainfall and percentage of the time exceedance is related to the rain attenuation by equation:
[formula] z(t) = K ∑ k=0 a k x k (t) + N ∑ n=1 b n y n (t),(47) [/formula]
where a k , b n (k = 0, 1, ..., K, n = 1, 2, ..., N) are the model parameters. K + N is the total number of input variables in the model.
(2) The mean absolute error is:
[formula] E = 1 M M ∑ k=1 |m k (t) − z k (t)|,(48) [/formula]
which can be alternatively represented as:
[formula] E = 1 M M ∑ k=1 |m k (t) − f (x k (t), y k (t), a 0 , ..., a K , b 1 , ..., b N )|.(49) [/formula]
The mean absolute error given by this equation is treated as the cost function and used to obtain the optimized error by applying the DEA algorithm.
(3) Mutation:
[formula] ζ M,i = ζ n,opt + P mut (ζ n,p 1 − ζ n,p 2 ), for i = p 1 and i = p 2 ,(50) [/formula]
where n is the generation index, Pmut is the mutation variable, p 1 , p 2 and i are three arbitrarily chosen individual indexes, and the M and opt refer to the gene pool and the optimal entity in the population, respectively.
## Livieratos model
This model [bib_ref] Rain Attenuation Along Terrestrial Millimeter Wave Links: A New Prediction Method Based..., Livieratos [/bib_ref] was developed using a DBSG3 database-based on a supervised machine-learning (SML) technique. In this rain attenuation model, the SML technique was blended with a Gaussian process (GP). A rain attenuation algorithm must be trained in a particular area of interest to measure the different interdependencies of the parameters for detecting rain attenuation in a specific region, weather, or carrier frequency.
## Pinto model
This model [bib_ref] Improved ITU Model for Rainfall Attenuation Prediction of in Terrestrial Links, Pinto-Mangones [/bib_ref] is based on the actual distance correction mechanism through the distance correction factor (r) along with the effective rainfall rate distribution (R eff ). It uses the quasi-Newton method in addition to particle swarm optimization (PSO); minimizing the root mean square error (RMSE) is the objective function in both cases.
[formula] A p = k a 1 R p (a 2 +a 3 /d ) α d · 1 a 4 d a 5 R p a 6 f a 7 + a 8 (a − e a 9 d ) .(51) [/formula]
The a i (i = 1, 2, . . . , 9) coefficients can be calculated using quasi-Newton multiple nonlinear regression (QNMRN) and the Gaussian RMSE (GRMSE) algorithm. These coefficients were further fine-tuned using the PSO technique. The model performance has not been compared with the recently developed model, except for ITU-R P.530-17 [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref]. Thus, there is a need for further verification before application, except for the temperate climate and Malaysia rainfall database areas.
## Comparative study of the models
In the previous section, we have highlighted the working principles of 18 rain attenuation models. In this section, a comparative study of these models is presented. [fig_ref] Table 5: Related input parameters/functions of terrestrial rain attenuation models [/fig_ref] presents different input parameters and functional values as inputs to predict the selected models' attenuation. The link range, supported frequency bands, important regional parameters, supported link types, and remarks about whether the model is spatially friendly are presented in [fig_ref] Table 6: Properties of rain attenuation models [/fig_ref]. [fig_ref] Table 5: Related input parameters/functions of terrestrial rain attenuation models [/fig_ref] shows that most terrestrial models have implemented the effective path length technique. The main reason for using path reduction for the long terrestrial link is that several rain cells affect the terrestrial link during rain. As discussed in Section 2.3, the necessity to correct the distance between the transmitter and receiver to estimate the rain attenuation and a few other techniques of determining the effective path are tabulated in . The suitability of these well-known models to the fitting application area in the climatic region and whether a standard dataset validates these models are tabulated in [fig_ref] Table 7: Terrestrial rain attenuation models' few performance metrics [/fig_ref]. Finally, in [fig_ref] Table 8: Cont. [/fig_ref] , all the significant contributions, special features, and limitations of the models are presented.
## Model evaluation techniques
After developing the rain attenuation model, it should be tested to validate its applicability. There are a few well-known techniques specified by the ITU-R guidelines [bib_ref] Acquisition, Presentation and Analysis of Data in Studies of Radiowave Propagation, Document..., Itu-R Recommendations [/bib_ref] , to test a model's performance. A different version of the implementation of this guideline is available in the literature. The Equation [bib_ref] Modification of Distance Factor in Rain Attenuation Prediction for Short Range Millimetre-wave..., Budalal [/bib_ref] was used to measure the performance of newly developed model [bib_ref] Enhancement of the Synthetic Storm Technique for the Prediction of Rain Attenuation..., Luini [/bib_ref] [bib_ref] A new rain attenuation prediction model for the earth-space links, Lu [/bib_ref]. The details of the symbols description in Equation [bib_ref] Modification of Distance Factor in Rain Attenuation Prediction for Short Range Millimetre-wave..., Budalal [/bib_ref] are given in [bib_ref] A new rain attenuation prediction model for the earth-space links, Lu [/bib_ref]. The logarithm value of measured attenuation divided by the predicted attenuation was used as the model evaluation criteria in [bib_ref] Prediction of attenuation by rain, Crane [/bib_ref]. The coefficient of determination or an input-to-output correlation function to determine the performance of the model [bib_ref] Improved ITU Model for Rainfall Attenuation Prediction of in Terrestrial Links, Pinto-Mangones [/bib_ref]. The Equation [bib_ref] Development of a new global model for estimating one-minute rainfall rate, Singh [/bib_ref] represents the coefficient of the determination function. The coefficient of determination, denoted by R, is the quotient of the explained variation to the total variation (total sum of squares (TSS)) in a model of simple or multiple linear regression. The RMSE, and the RMS function (Equation (57)) is also used to validate the performance of the rain attenuation model [bib_ref] Electromagnetic waves attenuation due to rain: A prediction model for terrestrial or..., Moupfouma [/bib_ref] [bib_ref] Improved ITU Model for Rainfall Attenuation Prediction of in Terrestrial Links, Pinto-Mangones [/bib_ref]. The goodness-of-fit function, which in general, measures how well do the observed data correspond to the fitted (assumed) model Equation (55) was also used in [bib_ref] Rain attenuation statistics over millimeter wave bands in South Korea, Shrestha [/bib_ref]. This model is also referred to as the Pearson goodness-of-fit statistic function as per given in Equation [bib_ref] Empirical study of the quantization induced bias in commercial microwave links' min/max..., Ostrometzky [/bib_ref]. Furthermore, some researchers used X 2 (Equation (56)) to find the accuracy of the model [bib_ref] A comprehensive meteorologically oriented methodology for the prediction of wave propagation parameters..., Capsoni [/bib_ref].
[formula] V i = 100 · A m,i 10 0.2 · ln A p,i A m,i , 0 < A m,i < 10 100 · ln A p,i A m,i , A m,i ≥ 10(52)V i = ln( A measured A model-specified )(53)R 2 = Explained variation Total variation (54) (P) T = A %p, p − A %p, m A %p, m × 100[%](55)X 2 = ∑ j (O j − E j ) 2 E j(56)D ei = µ 2 ei + σ 2 ei ,(57) [/formula]
where V i is the test variable, A %p, m is the predicted attenuation, A m,i is the measured attenuation, A m (dB) measured attenuation, and A p (dB) predicted attenuation. The definition of E i and σ 2 ei are given by the equation the equations below. The validation results in Malaysia showed least RMSE compared to ITU-R P.530-17 [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref] , and Crane model [bib_ref] Prediction of attenuation by rain, Crane [/bib_ref].
[formula] E i = A p i − A m i A m i × 100(i = 1 to N)(58)σ 2 ei = 1 N N ∑ i=1 e 2 i − (µ ei ) 2 .(59) [/formula]
Validated with ITU-R, and Malaysian database Yes Yes [fig_ref] Table 8: Cont. [/fig_ref]. Terrestrial rain attenuation models' constraints, major contribution, drawbacks, and special feature (if there exists).
## Ref. constraints contribution drawbacks
Special Feature (If Any) [bib_ref] Electromagnetic waves attenuation due to rain: A prediction model for terrestrial or..., Moupfouma [/bib_ref] To predict the attenuation, it needs a special rain rate R 001 (mm/h) that exceeded for 0.01% of time It proposes effective path length with new functional parameter ζ(L).
It substantially overestimates the measured link attenuation at higher rain rates.
It is suitable for the prediction of the cumulative attenuation.
[52]
The actual reasoning of addressing the high prediction error at a short-link through distance modification factor is not justified.
It gives a solution for ITU-R model [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref] for short-link.
The case f ≤ 40 GHz, d < 1 km has not been verified.
It was verified through different short-links experimental databases around the globe.
[66]
The model can predict rain limited to 10 km × 10 km, and it has not been tested on a real network environment.
It provides a mechanism to simulate and measure radio link's throughput. It mathematically calculates the rainfall intensity in the center and in the outer region of the rain structure.
A single rain structure is limited in size.
It facilitates to simulate dynamics behavior of rain owing to link capacity changes by rain attenuation and traffic re-routing.
[67] Maximum frequency support is 11 GHz.
It proposes a path reduction coefficient as a function of path length and rain rates.
The path reduction coefficient depends on more than 5 km distance while [bib_ref] Electromagnetic waves attenuation due to rain: A prediction model for terrestrial or..., Moupfouma [/bib_ref] model describes this limit as 7 km.
It proposes to use 1-min rainfall rate using the original Lin's model [bib_ref] 11-GHz radio: Nationwide long-term rain rate statistics and empirical calculation of 11-GHz..., Lin [/bib_ref].
[68] It has not been tested with real measured attenuation database It calculates effective path length in a common technique both for terrestrial and slant links, although the vertical aspects differ from the horizontal structure of rain cell.
It is not verified with a real measured attenuation database.
It has tried to unify the path length correction factor.
[69] It shows good performance for the low percentage of rain rate It introduced the effective rainfall rate concept.
The tropical climatic case was not tested.
It is applicable for both terrestrial and slant links. [bib_ref] Rain attenuation predictions on terrestrial radio links: Differential equations approach, Abdulrahman [/bib_ref] It is exclusively applicable for the tropical region.
It defined the rate of change of attenuation concerning rain rate.
It needs smaller time percentages in the range 0.005 ≤ %p ≤ 0.001 as the input parameter.
ITU-R model [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref] still showed less mean error compared to it.
[71]
It can predicted maximum 30 dB attenuation owing to rain.
It uses rain's geophysical statistics and rain structure to predict the attenuation of terrestrial and slant links.
It comparatively complicated procedure to calculate attenuation.
It is considered as one of the critical models in practice. [bib_ref] A two-component rain model for the prediction of attenuation statistics, Crane [/bib_ref] It is difficult to determine the probabilities of occurrence and mean rainfalls at the center and at the boundary of a rain cell.
It includes the non-uniform heavy and light rain region concept in the signals propagation path.
It is computationally complex: it needs almost ten equations to solve.
It includes the joint statistics required for space diversity system design.It needs an additional terrestrial link rain database before final deployment.
It showed to fit spatial rain behavior through matching synthetic rain map and electromagnetic wave.
It showed good accuracy compared to the ITU-R model, but the performance did not exceed the Brazilian and ITU-R models.
The spatial variability of precipitation along terrestrial links is achieved through the synthetic rain cell simulation technique. [bib_ref] A comprehensive meteorologically oriented methodology for the prediction of wave propagation parameters..., Capsoni [/bib_ref] It considers attenuation to be zero below 5 mm/h, especially beyond 20 GHz, which is not justified well.
It integrates rain attenuation, site diversity gain interference by scattering factors.
It needs deployment location's rain height, which may not be available accurately.
It gives site diversity gain and interference by rain scattering. [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref] It is not verified well in heavy rainy tropical regions.
It uses horizontal reduction and vertical adjustment factors to predict attenuation.
The path length reduction is inappropriate for a short-range link [bib_ref] Measurement of rain induced attenuation over a line of sight link operating..., Sharma [/bib_ref].
It uses 2 parameters called L 1 and L 2 of the connection between antennas and RX entrance points (dB).
[77]
The cubic polynomial coefficients were not validated using a separate testing dataset.
It facilities to compute tedious task of computing the k and α in ITU-R model.
The applicable climate regions are not defined well. [bib_ref] Short-term rain attenuation predictor for terrestrial links in tropical area, Andrade [/bib_ref] A detailed weather condition is not mentioned in the experimental campaign dataset.
It is one of the most pioneer work that contributes fade slope model for the terrestrial link.
To remove other noise in the fade slope model, a pre-processing stage is normally used; however, no prepossessing was used in it.
It can predict attenuation before 10-s. [bib_ref] Rain fade slope model for terrestrial microwave links, Chebil [/bib_ref] It is a major contribution for fade slope model for terrestrial link.
It did not produce a good fit for an attenuation level of 1 dB.
It showed good performance in the goodness-of-fit test.
[80]
For different climatic zones, it needs to determine the different coefficient of rain and percentage of the time.
It showed excellent agreement with the measured values of rain attenuation.
It needs to calculate the coefficients (Taylor's series fashioned) of rain and % of the time.
The DE optimization algorithm was used as an optimization tool.
[81]
The climatic regions or frequency zones can be facilitated, employing the proper training data set that can convey the experimental information, but it may be difficult to attain.
It is one of the pioneer ML-based rain attenuation models; it does not have geographical limitations.
The tropical behavior of attenuation is not tested yet using this model.
It must train the algorithm with the unique data set in rare climatic conditions.
[82]
The performance was not compared with most latest ITU-R model [bib_ref] Recommendation P.530-16: Propagation Data and Prediction Methods Required for the Design of..., Itu-R Recommendations [/bib_ref].
It attempted to overcame the main limitation of the original ITU-R model (single value of the rainfall rate cumulative distribution).
The performance was compared with a suspended version ITU-R model.
The parameter adjustment factor was corrected by QNMNR (quasi-Newton multiple nonlinear regression) followed by particle swarm optimization (PSO) technique. . ITU-R model is ineffective for short distance.
## Current research scope and challenges
## Use of learning techniques
In recent technological developments, artificial intelligence (AI) has played an essential role in finding optimized and practical solutions to techniques. Many AI sub-domain techniques and applications have been reported in [bib_ref] The Effects of Rain on Terrestrial Links at K, Ka and E-Bands..., Diba [/bib_ref] [bib_ref] Prediction of rainfall using artificial neural networks for synoptic station of Mashhad:..., Khalili [/bib_ref]. The field of AI has been vast and has existed for many decades. AI can be described as a set that includes both ML and DL. Some significant fields include weather and climate, security, science, policy, mining, medical science, marketing, manufacturing, management, insurance, finance, environmental, engineering, energy, education, and agriculture. Similarly, the AI technique can predict the amount of attenuation and fading margin for a satellite or terrestrial communication radio link. In [bib_ref] Application of the deep learning for the prediction of rainfall in Southern..., Yen [/bib_ref] , an AI-based model was developed to predict rain in southern Taiwan by applying the AI technique with data obtained from another region in Taiwan. The prediction was accurate, with minimum error. In the future, this prediction can be used to predict rain attenuation instead of rain rate. Different models consider a few of the available sets of parameters for natural path length correction, contributing to some errors. An effective AI-based path length reduction technique can be applied to obtain an optimum path length; using such a model, and it may be possible to obtain the most accurate path length correction factor.
## Need to access rain data regularly
Because of climate change, the world's weather conditions, and hence rain event behaviors, are changing [bib_ref] The changing character of precipitation, Trenberth [/bib_ref] [bib_ref] Changes in precipitation with climate change, Trenberth [/bib_ref] [bib_ref] Impact of climate change on rainfall over Mumbai using Distributionbased Scaling of..., Rana [/bib_ref]. On account of this, the model will require periodic testing against measured and estimated attenuation to ensure its radio link availability at the desired level. If there is an intolerable difference between the measured and estimated attenuation, the model parameters will require modification. However, if the model estimates attenuation by analyzing a rain database, it may be necessary to update the database with a recent rain rate.
## Adoption of enhanced synthetic storm technique (esst)
An enhanced synthetic storm mechanism is proposed in [bib_ref] Enhancement of the Synthetic Storm Technique for the Prediction of Rain Attenuation..., Luini [/bib_ref]. The ESST receives an input report regarding storm velocity and rain height. These parameters can be extracted from other sources with the discriminating procedure of stratiform and convective rain events. Furthermore, depending on the type of event, the model can produce various rain heights. [fig_ref] Table 1: Estimation techniques of rain attenuation time series [/fig_ref] presents SST-based few other techniques for rain attenuation time series.
The model can be applied to the earth-space link at 30-300 GHz to calculate the time series attenuation. The experimental results show that the ESST closely aligns with the predicted results. Many models reported in this paper are based on the SST technique, for example, the physical-mathematical models, the global synthetic storm technique (GSST) model, and the artificial neural network-based model. It is expected that the implementation of the ESST technique may yield more accurate predicted values of rain attenuation.
## Rain attenuation research for 5g and beyond network
The effects of rain on the short-distance E-band (probable frequency band that can be used in 5G and beyond networks) is highly unpredictable by existing models. The preliminary outcome in [bib_ref] The Impact of Rain on Short E-Band Radio Links for 5G Mobile..., Luini [/bib_ref] shows that the existing general use of the ITU-R model does not fit well to predict rain attenuation for short-ranges <2 km path length.
A continuous 43-month long campaigned experimental data for link length d = 2.3 km on attenuation of 72.56 GHz at Budapest with the Telenor Hungary network is reported in [bib_ref] Frigyes, I. E-band terrestrial radio-propagation and availability aspects, Csurgai-Horváth [/bib_ref]. In this work, the authors showed that the ITU-R model predicted higher attenuation than the measured attenuation, especially in the 33-45 dB attenuation range. They proposed a formula to predict attenuation using the following equation:
[formula] p A = a · exp(−L e b · A),(60) [/formula]
where a = 0.2991, b = 0.1281, and L e is the effective path length. Article [bib_ref] Modification of Distance Factor in Rain Attenuation Prediction for Short Range Millimetre-wave..., Budalal [/bib_ref] shows experimented results on the terrestrial link at a frequency of 26 GHz and 38 GHz, where the link distance was 300 m between the transmitter and receiver at UTM, Malaysia. The link availability of the 26 GHz and 38 GHz experimental systems was 98.6% and 99.5%, respectively. The visualization of the collected data shows that the wellused ITU-R model [bib_ref] Prediction Methods Required for the Design of Terrestrial Line-of-Sight Systems, Document ITU-R..., Itu-R Recommendations [/bib_ref] does not fit well for distance d < 1 km [bib_ref] Rain attenuation statistics over millimeter wave bands in South Korea, Shrestha [/bib_ref] [bib_ref] Modification of Distance Factor in Rain Attenuation Prediction for Short Range Millimetre-wave..., Budalal [/bib_ref] [bib_ref] The Impact of Rain on Short E-Band Radio Links for 5G Mobile..., Luini [/bib_ref] [bib_ref] Rain statistics investigation and rain attenuation modeling for millimeter wave short-range fixed..., Huang [/bib_ref] [bib_ref] The measurements of rain attenuation for terrestrial link at millimeter wave, Kim [/bib_ref] [bib_ref] Terrestrial link rain attenuation measurements at 84 GHz, Hong [/bib_ref] [bib_ref] Effect of rain attenuation for a 10-Gb/s 120-GHz-band millimeter-wave wireless link, Hirata [/bib_ref]. presents some of recent measured attenuation studies that found the drawbacks of the ITU-R model for short-distance links at and 120 GHz frequencies.
# Conclusions
This study has conducted the most recent and well-known comprehensive survey of the rain attenuation prediction models for the terrestrial link. The existing models have been classified as physical, statistical, empirical, optimization-based, or fade slope models based on the model development and formulation basis. They have been reviewed concerning innovative concepts, input parameters, advantages, and disadvantages. A careful comparison of several terrestrial link's rain attenuation model has been introduced and reviewed. According to this survey, no sole prediction model can be regarded as a comprehensive model to satisfy all specifications for diverse infrastructure setup related parameters, geographic locations, or even climatic variations over time. The open research challenges have been addressed to research further the prediction model for the 5G and beyond network's densely formed microwave and millimeter-wave links. It should be remembered that the rain attenuation prediction model for millimeter-wave applications can also be more effectively developed as most of the currently developed model predictions are yet inaccurate. We believe that this survey will inspire researchers to develop an accurate terrestrial link's rain attenuation prediction model either at the regional or global level. The comparative study can help people who work in terrestrial link design, link budget planning, and radio wave propagation management areas.
# Acknowledgments:
The writers would like to thank the editor and anonymous reviewers for their useful comments for improving the quality of this paper.
## Conflicts of interest:
The authors declare no conflict of interest.
## Abbreviations
The following abbreviations are used in this manuscript: for d < 1 km K dp Specific differential phase L A , L B Radio path lengths in two layer structure of rain M z (d)
Correlation of a Gaussian random process N e
## Number of effective cells p(r)
Probability as a function of rainfall rate R e f f T Effective rain rate for terrestrial links S(R%p)
Slope of attenuation with respect to rain rate T s Sampling time W(t)
Wiener process W i Weight for the ith site x(t) Percentage of the time x 0 Distance given by advacation velocity of rain (SST model:
[formula] x 0 = v · t) y(t) [/formula]
Rate of rainfall z(t)
Rain attenuation Z dr Differential reflectivity Z h Radar reflectivity
[fig] Figure 1: Taxonomy of terrestrial rain attenuation models. [/fig]
[fig] c 3 v: = −5.520 × 10 −12 x 3 + 3.36 × 10 −9 x 2 − 1.21 × 10 −7 x − 6.10 × 10 −6 c 2 v = 8.10 × 10 −9 x 3 − 4.552 × 10 −7 x 2 − 3.03 × 10 −5 x + 0.001 c 1 v = −5.71 × 10 −9 x 3 + 6 × 10 −7 x 2 + 8.707 × 10 −3 x − 0.018 c 0 v = −1.073 × 10 −7 x 3 + 1.068 × 10 −4 x 2 − 0.0598x + 0.0442. [/fig]
[fig] Author: Contributions: M.A.S. anticipated, reviewed the related literature, evaluated, and outlined the rain attenuation models of terrestrial microwave links. D.-Y.C. played a major role in coordinating research. M.A.S. conscripted the paper and subsequently modified and justified it by F.D.D. and D.-Y.C. All authors have read and agreed to the published version of the manuscript. Funding: The BrainKorea21Four Program supported this research through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (4299990114316). Additionally, this research was also partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019R1F1A1058128). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig]
[table] Table 1: Estimation techniques of rain attenuation time series. [/table]
[table] Table 4: Error estimation techniques for rain rate prediction [/table]
[table] Table 5: Related input parameters/functions of terrestrial rain attenuation models. [/table]
[table] Table 6: Properties of rain attenuation models. GHz corresponding rain attenuation with vertical polarization is mentioned, ♣, ♠ : Recent study showed that ITU-R model overestimates attenuation within short-link about within 2 km, -: unavailable. [/table]
[table] Table 7: Terrestrial rain attenuation models' few performance metrics. [/table]
[table] Table 8: Cont. [/table]
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Advances in infection control
Several initiatives took place in recent years in relation to nosocomial infection control in order to increase patient safety. Some of these initiatives will be commented in this brief review.RESUMOVárias iniciativas aconteceram nos últimos anos em relação ao controle das infecções no ambiente hospitalar para aumentar a segurança do paciente. Algumas dessas iniciativas são comentadas nesta breve revisão.
# Introduction
Nosocomial infections such as ventilator-associated pneumonia and central venous catheter-related bloodstream infection are important causes of morbidity and mortality. Implementation of prevention measures for these infections have showed zero infection rate in a number of intensive care units, [bib_ref] Successful prevention of ventilator-associated pneumonia in an intensive care setting, Marra [/bib_ref] as well as at different hospital units. [bib_ref] The Lowbury Lecture. The United States approach to strategies in the battle..., Jarvis [/bib_ref] Changes in health professional behavior, [bib_ref] Positive Deviance: a New Tool for Infection Prevention and Patient Safety, Marra [/bib_ref] who actively participate in reducing infection rates, have reduced morbidity, mortality, related costs and have also shown more safety for patients.
In addition, the increase of participation, the implementation of new technologies, such as antibacterial or antiseptic-impregnated invasive devices, the use of ultrasound resource for central venous catheter insertion, and chlorhexidine-impregnated dressing are welcoming resources for better practice not only for physicians but also to all multidisciplinary health team responsible for patients' care. [bib_ref] Eliminating Infections in the ICU: CLABSI, Latif [/bib_ref] Despite all innovations nosocomial infection, hand hygiene is still the most important procedure for preventing infections in hospitals.Health professionals often complain about the difficult for hand hygiene. The major complaints are related with handwashing problems, such as dry skin and injuries caused by soap or detergent, and other reasons. Professionals report that handwashing procedure is time-consuming and cause interruption in routine patient care tasks.Many studies have shown that frequent and repetitive handwashing (with soap and water, but in this case with chlorhexidine), in a number of American and European hospitals present compliance rates below 50%. [bib_ref] Compliance with handwashing in a teaching hospital, Pittet [/bib_ref] Currently, strategies to increase compliance with handwashing are focused on reduction of time needed for this task. International guidelines for infection prevention related with intravascular catheters emphasize hand hygiene.Daily there are a number of opportunities for non-compliance to hand hygiene in hospital units particularly because of high-complexity patients. However, because patients are hospitalized in private rooms, great difficulties exist to measure hand hygiene compliance.An alternative for conventional hand antisepsis is the use of alcohol-based hand rubs. Hand hygiene compliance rate increased when chlorhexidine was replaced by alcohol-based hand rubs. [bib_ref] No time for handwashing!? Handwashing versus alcoholic rub: can we afford 100%..., Voss [/bib_ref] Several studies reported that hand hygiene compliance is poor among health professionals. [bib_ref] Eliminating Infections in the ICU: CLABSI, Latif [/bib_ref] [bib_ref] No time for handwashing!? Handwashing versus alcoholic rub: can we afford 100%..., Voss [/bib_ref] [bib_ref] Compliance with handwashing in a teaching hospital, Pittet [/bib_ref] Recent studies have showed that include technologies such as electronic counters and video camera monitoring in order to give real-time feedback for professionals
## Advances in infection control
## Avanços no controle das infecções
Alexandre Rodrigues Marra 1 regarding hand hygiene have increased compliance. [bib_ref] Using high-technology to enforce low-technology safety measures: the use of third-party remote..., Armellino [/bib_ref] In our hospital, we use a radio frequency identification, which does not require wireless internet, named ZigBee (i-Healthsys, São Carlos, Brazil). This system enables to monitor handwashing, without the need of human observation. The system interacts with health professional by a light flashing. The use of this new technology increased hand hygiene compliance at our institution. [bib_ref] New technologies to monitor healthcare worker hand hygiene, Marra [/bib_ref] In addition, this technology may bring benefit to our patients and we believe this procedure should be applied in other hospitals.
A number of side effects may occur in hospitals, among them are infectious events that in the past were considered expected and preventable. Currently, these events are not accepted and many of them, such ventilator-associated pneumonia and central venous catheter-related bloodstream infection, are no longer covered by the American health system (Medcare and Medcaid). [bib_ref] The Lowbury Lecture. The United States approach to strategies in the battle..., Jarvis [/bib_ref] [bib_ref] Eliminating Infections in the ICU: CLABSI, Latif [/bib_ref] Unfortunately in Brazil this is not a reality yet, but, perhaps, in the near future it will be.
einstein. 2016;14(1):108-9
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Dependence of column ozone on future ODSs and GHGs in the variability of 500-ensemble members
State-of-the-art chemistry-climate models (CCMs) have indicated that a future decrease in ozonedepleting substances (ODSs) combined with an increase in greenhouse gases (GHGs) would increase the column ozone amount in most regions except the tropics and Antarctic. However, large Arctic ozone losses have occurred at a frequency of approximately once per decade since the 1990s (1997, 2011 and 2020), despite the ODS concentration peaking in the mid-1990s. To understand this, CCMs were used to conduct 24 experiments with ODS and GHG concentrations set based on predicted values for future years; each experiment consisted of 500-member ensembles. The 50 ensemble members with the lowest column ozone in the mid-and high latitudes of the Northern Hemisphere showed a clear ODS dependence associated with low temperatures and a strong westerly zonal mean zonal wind. Even with high GHG concentrations, several ensemble members showed extremely low spring column ozone in the Arctic when ODS concentration remained above the 1980-1985 level. Hence, ODS concentrations should be reduced to avoid large ozone losses in the presence of a stable Arctic polar vortex. The average of the lowest 50 members indicates that GHG increase towards the end of the twenty-first century will not cause worse Arctic ozone depletion.From its worst state in the 1990s, the ozone layer has been gradually recovering due to ozone-depleting substance (ODS)-reducing measures implemented internationally as a consequence of the Montreal Protocol and its amendments 1,2 . Ozone amounts and ozone recovery are affected by ODSs, such as chlorofluorocarbons 3-7 , and by greenhouse gases (GHGs)[8][9][10][11][12]. Moreover, in the high latitudes of the northern hemisphere (NH), ozone amounts in winter and spring are greatly influenced by interannual variations of the atmosphere 13,14 . Record lows in the total ozone during the Arctic spring were observed in 1997, 2011 and 2020 15,16 . A future projection using ODS values specified by the World Meteorological Organization (WMO) predicted that by the 2060s, total ozone may still episodically drop to 50-100 DU below the corresponding long-term ensemble mean 17 . A GHG increase may cause severe ozone loss in the Arctic in the future18,19. The large interannual variations in ozone amount are due to interannual variations in temperature and circulation, and hence are influenced by the large interannual variations of planetary waves 20,21 , which are partly influenced by the Quasi-Biennial Oscillation (QBO), the 11-year solar cycle and the El Niño Southern Oscillation (ENSO) 22-30 , but more predominantly by the intrinsic variability associated with wave-mean flow interaction 31,32 . Given the chaotic nature of the atmosphere, as wave propagation and dissipation are sensitive to zonal-wind and temperature fields 33 , a small difference in these fields may result in a large difference in the dynamical state of the future atmosphere 34 . Temperature and atmospheric circulation changes influence the ozone layer through chemical reactions and ozone transport, and ozone changes in turn influence temperature and circulation through radiation processes in the atmosphere32,35,36. The large interannual variations may make it difficult to understand the effects of ODSs and GHGs on long-term ozone variations 37 . In future, GHG concentrations will increase further 38-42 , while ODS concentrations will decrease owing to regulation 1,2,43 . Therefore, in order to predict the future behaviour of the ozone layer, it is essential to understand the dependence of ozone amounts on ODS and GHG concentrations in the context of interannual atmospheric variations. For this purpose, we performed 500-member ensemble simulations using chemistry-climate models (CCMs) constructed on the Model for Interdisciplinary Research on Climate (MIROC) versions OPEN www.nature.com/scientificreports/ 3.2 and 5. The CCMs were developed at Japan's National Institute for Environmental Studies (NIES) and the University of Tokyo. The dependences of column ozone, polar cap temperature, and zonal mean zonal wind on ODS and GHG concentrations were analysed.ResultsMIROC3.2 CCM.As stated in the Introduction, there is a need to examine ozone dependence on ODS and GHG concentrations in the NH mid-and high latitudes in the context of interannual atmospheric variations. However, in this study, interannual variations due to the QBO, 11-year solar cycle and ENSO (see 'Input Data for CCM run' in the Methods section) were ignored; instead, the focus was on those caused by the intrinsic variations of the wave-mean flow interaction and the chaotic nature of fluid dynamics. This CCM setup still produced large interannual variations (variability among the ensemble members) in ozone and other atmospheric parameters in the NH mid-and high latitudes.Twenty-four experiments were performed using pairings of ODS and GHG levels for selected years(Table 1). In addition, an experiment was performed using atmospheric data from the year 2000 (ODS-2000&GHG-2000 for comparison. Each experiment is a 510-year timeslice simulation that was run continuously, where each year is assumed to be an independent realization, but the first 10 years were excluded from the analysis. The ODS concentrations from past years were selected to simulate concentrations predicted to be achieved in the future under ODS regulation. For the ODS-1960&GHG-2040 pairing, minimal ozone destruction was predicted due to the low halogen concentrations; therefore, to save computing resources, no run was performed for this pairing. Instead, we assumed that ODS and GHG dependence for this pairing could be interpolated using the ODS-1960&GHG-2030 and ODS-1960&GHG-2050 ODS concentration levels were expressed using historical data from the WMO-A1 scenario. ODS concentrations are expected to decrease in the future, having peaked around 1995, due to ODS regulations. In the WMO-A1 scenario, equivalent effective stratospheric chlorine (EESC) in the polar regions is calculated by adding 65 times the number of bromine atoms to the number of chlorine atoms 44 ; under this scenario, ODS concentration in 1990 corresponded to that in 2006, that in 1985 corresponded to that in 2030, that in 1980 corresponded to that in 2044, and that in 1960 corresponded to no year of the current century. GHG (CO 2 , CH 4 , and N 2 O) concentration levels were also expressed by year, based on the RCP 6.0 scenario.Figure 1shows histograms of the minimum column ozone values between 45° and 90° N from March to May for the 24 runs performed using the MIROC3.2 CCM, with the same arrangement of panels as inTable 1. At mid-and high latitudes during the NH spring, the distribution of minimum column ozone values was relatively narrow for the smallest ODS value (that for 1960 from the WMO-A1 scenario). As the ODS concentration increased (from left to right panels), the peak values of the distribution decreased. As can be seen inFig. 1, the peak broadens and flattens, extending towards smaller minimum column ozone values. However, the largest minimum column ozone values remained relatively constant.It is interesting to note that even under high-GHG conditions, several ensemble members exhibited very small column ozone minima in the presence of high ODS concentrations, such as the ODS-1995&GHG-2095 pairing (top right panel inFig. 1). This suggests that extensive ozone depletion could occur in future high-GHG atmospheres if ODS concentrations remain high. For instance,Fig. 1shows that the ensemble members with spring column ozone minima less than 200 DU notably increased with ODS concentrations above the 1985 level and with all selected GHG concentrations. Note that although the spring column ozone minimum is a good measure of the strength of spring ozone depletion in a region over a given time period, it may not comprehensively represent ozone depletion throughout the region and period, as it is a localized and instantaneous quantity. Thus, we also calculated the area from 45° to 90° N wherein the column ozone values were less than 220 DU from March to May(Supplementary Fig. S1) and the time-integrated value (Supplementary Fig. S2). The results showed that ensemble members with large ozone loss areas appeared when ODS concentrations were above those in 1985, which was consistent with the results of the analysis of the spring column ozone minimum. Hence, the spring column ozone minimum is an appropriate measure for detecting ensemble members showing extreme ozone depletion associated with changes in ODS concentration.With increasing GHG concentrations (from bottom to top panels inFig. 1), the peak of the distribution (maximum bin count) shifted to higher column ozone minima. This can be explained by an increase in ozone transport [fig_ref] Table 1: the top row shows the GHG-2095 experiments, the bottom row the GHG-2000... [/fig_ref]. Reference years for the ODS and GHG concentrations used for CCM runs with 500-member ensembles. Each ○ indicates a pairing used for a CCM run; -indicates a pairing not used for a run. Years in parentheses are the years after 2000 with the same EESC levels as those before 2000 in the top row based on the WMO-A1 scenario. The GHG concentration levels for the years in the left column are based on the RCP 6.0 scenario.
## Scientific reports
| (2023) 13:320 | https://doi.org/10.1038/s41598-023-27635-y www.nature.com/scientificreports/ to high latitudes due to the strengthening of the meridional circulation when GHGs are more abundant [bib_ref] Removal of chlorofluorocarbons by increased mass exchange between the stratosphere and troposphere..., Butchart [/bib_ref] [bib_ref] A simulation of the separate climate effects of middle Atmospheric and Tropospheric..., Sigmond [/bib_ref] [bib_ref] Changes in the strength of the Brewer-Dobson circulation in a simple AGCM, Eichelberger [/bib_ref] [bib_ref] Acceleration of the Brewer-Dobson circulation due to increases in greenhouse gases, Garcia [/bib_ref] [bib_ref] Chemistry-climate model simulations of twenty-first century stratospheric climate and circulation changes, Butchart [/bib_ref] [bib_ref] The Brewer-Dobson circulation, Butchart [/bib_ref]. The distribution of ozone loss areas (ozone values less than 220 DU) showed a trend consistent with that of the spring column ozone minima, with the area decreasing as GHGs increased [fig_ref] Figure 2: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref].
In the mid-and high latitudes during the Southern Hemisphere (SH) spring, the distributions were much narrower than those for the NH spring [fig_ref] Figure 2: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. Increasing ODS concentrations (from left to right panels) induced a shift in the peak value towards lower column ozone minima and a narrower distribution. The narrowing of the column ozone minimum distribution implies less variability in that quantity among the ensemble members. The narrowing is considered to be a result from a nearly complete PSC-driven ozone destruction in the lower stratosphere, where most of the column ozone exists, at a grid and time indicating the column ozone minimum. Thus the column ozone minimum becomes less sensitive to meteorological variability as ODSs increase. The narrowing also may be associated with increased stability of the Antarctic polar vortex, with a stronger zonal mean zonal wind and with lower temperatures inside the vortex. However, narrowing of the distribution was not evident with regard to the area less than 220 DU as ODSs increase [fig_ref] Figure 3: MIROC3 [/fig_ref].
In contrast to the NH results, an increase in GHG concentration had little impact on the width and amplitude of the distribution of SH column ozone minima (from bottom to top panels in [fig_ref] Figure 2: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. The ozone loss area distribution also showed little dependence on the GHG concentration [fig_ref] Figure 3: MIROC3 [/fig_ref]. The NH mid-and high-latitude histograms [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref] suggest that the ODS and GHG dependence of the spring column ozone minimum differed between the 500-member ensemble mean and the means of the members with the most extreme column ozone values. [fig_ref] Figure 3: MIROC3 [/fig_ref] shows the dependence on ODS and GHG concentrations of the ensemble-mean spring column ozone minima at mid-and high latitudes of both hemispheres, as predicted by the MIROC3.2 CCM. The 500-member ensemble mean is represented in the middle panels, the mean of the 50 members with the largest column ozone minimum (upper 50) is shown in the left panels, and the mean of the 50 members with the smallest column ozone minimum (lower 50) is shown in the right panels. The upper and lower 50 members represent extreme cases and statistically once-in-a-decade events, respectively. This grouping is based on the fact that Arctic springs with extremely low total ozone and chemical ozone losses tend to occur approximately once a decade In the NH mid-and high latitudes (upper panels), the mean spring column ozone minimum of the 500-member ensemble depended on both GHG levels (expressed on the vertical axis as CO 2 concentrations) and ODS concentrations (expressed on the horizontal axis as EESC at 45°-90° N/S and 50 hPa during spring). The column ozone minima decreased as the ODS concentration increased, but increased with increasing GHG concentrations. For the upper 50 members, a very small dependence on ODS and GHG concentrations was evident. The average for the lower 50 members indicated clear ODS dependence. The difference between the means of the upper 50 and lower 50 members in the NH was likely caused by the large interannual variability of the Arctic polar vortex among the ensemble members. In the NH, catalytic destruction of ozone by ODSs appeared to occur at a significant level for ensemble members with a stable, colder polar vortex, whereas it did not develop under an unstable polar vortex. To confirm this, we examined the ODS and GHG dependence of the March values for the Arctic polar cap temperature at 63-90° N and 50 hPa and for the zonal mean zonal wind (polar night jet) at 60-70° N and 50 hPa, as shown in [fig_ref] Figure 4: MIROC3 [/fig_ref]. The lower 50 ensemble members showed a clear ODS dependence. With increasing ODS concentrations, the anomalies became larger for both the polar cap temperature (colder) and polar night jet (stronger). This implies that for the lower 50 members, the Arctic polar vortex became stronger when the ODS concentration increased. The 500-member ensemble showed weak ODS dependence for the polar cap temperature and the polar night jet. For the upper 50 members, the polar cap temperature and polar night jet strength showed an ODS dependence trend opposite to that for the lower 50. For all three sets of members, the polar cap temperature increased and the polar night jet strength decreased with increasing GHG concentration.
However, the aforementioned results regarding ODS and GHG dependence of the polar night jet and polar cap temperature apply only to the lower stratosphere in the given latitudes, thus providing only a partial picture of the ODS and GHG dependence over the entire meridional section. The future decrease in ODS from the 1995 level to the 1960 level increases the polar cap temperature not only at 50 hPa but also in the entire polar stratosphere, as well as weakening the polar night jet in the stratosphere and mesosphere. The GHG increase from the 2000 level to the 2095 level also weakens the polar night jet by a smaller magnitude, but the temperature increase in the polar region was limited to the lower stratosphere (see Supplementary Figs. S4 and S6). Both the decreasing ODS and increasing GHG were associated with statistically significant differences (significance level 95% or more) among the lower 50 members with regard to polar cap temperature (63-90° N, 50 hPa) and polar night jet strength (60-70° N, 50 hPa) for March.
In the SH mid-and high latitudes, the column ozone minimum depended only on the ODS concentration for the means of the upper 50 members, the 500-member ensemble or the lower 50 members, as indicated by the www.nature.com/scientificreports/ almost vertical contours in the lower panels of [fig_ref] Figure 3: MIROC3 [/fig_ref]. No obvious GHG dependence was observed for all three sets of members; similarly, the polar cap temperature and polar night jet strength in the lower stratosphere were ODS-dependent and almost GHG-independent for all three sets of members [fig_ref] Figure 5: MIROC3 [/fig_ref]. The difference in ODS and GHG dependence between the NH and SH was linked to the difference in stability between the polar vortices in each hemisphere, which results in a difference in wave activity. The Antarctic polar vortex is more stable and tighter than the Arctic polar vortex, and the transport of chemical constituents and heat is less effective in the Antarctic than in the Arctic. Thus, ozone in the Antarctic is more vulnerable to chemical destruction resulting from ODS increases.
## Miroc5 ccm.
The results of the MIROC5 CCM were essentially similar to those of MIROC3.2 with regard to the ODS and GHG dependence of the spring column ozone minimum at mid-and high latitudes. However, there were several differences, including that MIROC5 consistently produced narrower distributions of the spring column ozone minimum in the NH than MIROC3.2, except for the four ODS-1960 pairings (see Supplementary . This is because spring total ozone in the Arctic was higher with MIROC5 than with MIROC3.2 [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. Furthermore, unlike with MIROC3.2, larger ODS amounts in the SH did not lead to narrower distributions with MIROC5 . Column ozone minima in both the NH and SH showed weaker ODS dependence with MIROC5 compared with MIROC3.2 [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. GHG dependence in the NH was weaker with MIROC5 than with MIROC3.2. In the SH, GHG dependence of total ozone was barely evident for both MIROC5 and MIROC3.2.
With regard to the ODS dependence of the Arctic polar cap temperature (63-90° N, 50 hPa) and zonal mean zonal wind (westerly, 60-70° N, 50 hPa) in March, results for the lower 50 ensemble members were similar to those of MIROC3.2. The higher the ODS concentration, the lower the polar cap temperature and the stronger the polar night jet. The upper 50 members did not show consistent ODS dependence between the two models (see [fig_ref] Figure 4: MIROC3 [/fig_ref] and [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref].
For the lower 50 ensemble members, the GHG dependence of polar cap temperature in the NH spring was weaker than that predicted by MIROC3.2, and that of polar night jet strength was almost the same as, or slightly weaker than, that predicted by MIROC3.2 (right panels in [fig_ref] Figure 4: MIROC3 [/fig_ref] and [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref] , indicating that increased GHG led to a slightly weaker polar night jet and nearly unchanged polar cap temperature. This was also evident from the meridional distribution of the changes in zonal mean zonal wind (westerly) and zonal mean temperature associated with the GHG increase between 2000 and 2095 (middle panels of Supplementary ). According to MIROC5, the GHG-associated differences among the lower 50 members with regard to Arctic temperature and wind at 50 hPa fell below the statistical significance level of 95%. The upper 50 members did not show consistent GHG dependence between the two models. For the SH spring, MIROC5 indicated weaker ODS dependence for polar cap temperature and polar night jet strength than MIROC3.2 [fig_ref] Figure 5: MIROC3 [/fig_ref] and [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. GHG dependence of temperature and wind was not evident in the SH, except for polar night jet strength among the 500-member ensemble and the lower 50 (see [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref].
# Discussion
The results of the 500-member ensemble simulations using the MIROC3.2 and MIROC5 CCMs showed some common features regarding the ODS and GHG dependence of the spring column ozone minimum. The lower 50 ensemble members showed a clear ODS dependence. This implies that given the large interannual variations in ozone amounts in the NH mid-and high latitudes, severe Arctic ozone depletion could occur in a future year with a stable polar vortex if ODS concentrations remain high, even accounting for future increases in GHG concentration. In addition, both models found that an increase in GHG was associated with a small increase in the mean column ozone minimum for the upper 50 members, the lower 50 members and the 500-member ensemble in the NH.
According to the MIROC3.2 run using ODS and GHG values for the year 2000, the zonal mean column ozone values for the Arctic winter/spring had a minimum around March [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. This was due to the smaller magnitude of downward motion at high latitudes in winter/spring in MIROC3.2 than in MIROC5 [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref]. This implies that the Arctic polar vortex is more stable in the MIROC3.2 CCM than in the MIROC5 CCM, which leads to slightly different impacts on the ODS-and GHG-dependent variations in the spring column ozone minima. It is not easy to conclude whether the 500-member ensemble mean result from MIROC3.2 regarding the March minimum around 80-90° N is realistic in comparison with the 8-year average of Total Ozone Mapping Spectrometer (TOMS) observations, as TOMS observations were not available for polar winter. The MIROC3.2 results for ODS and GHG dependence may indicate possible conditions in the case of an Arctic polar vortex that is slightly more stable than the current polar vortex.
The fact that climate change has some effect on the Arctic severe ozone loss, in addition to the increased halogen loading in the atmosphere, has been discussed [bib_ref] Arctic ozone loss and climate change, Rex [/bib_ref] [bib_ref] Climate change favours large seasonal loss of Arctic ozone, Von Der Gathen [/bib_ref] [bib_ref] Increased polar stratospheric ozone losses and delayed eventual recovery owing to increasing..., Shindell [/bib_ref] [bib_ref] Future Arctic temperature and ozone: The role of stratospheric composition changes, Langematz [/bib_ref]. In terms of the effect of future global warming on an extreme ozone-destruction event in the Arctic, the lower 50 ensemble members from MIROC3.2 CCM and MIROC5 CCM indicate an increase in the column minimum ozone and temperature as GHG concentrations www.nature.com/scientificreports/ increase towards the end of the twenty-first century. This ozone and temperature dependence in the NH mid-and high latitude lower stratosphere on GHGs is different from the dependence reported by the local maxima of PSC formation potential (PFP LM ) based on CMIP6 GCM output [bib_ref] Climate change favours large seasonal loss of Arctic ozone, Von Der Gathen [/bib_ref] , which shows a higher PFP LM (lower temperature) towards the end of the twenty-first century in SSP5-8.5 and SSP3-7.0 scenarios, but similar to the result from the EMAC CCM on the point that cold winters do not increase towards the end of the twenty-first century [bib_ref] Future Arctic temperature and ozone: The role of stratospheric composition changes, Langematz [/bib_ref].
The EMAC CCM result also shows that the temperature change is different between early winter and late winter/early spring. Thus, the dependences of ozone amount and temperature on GHG concentration in the NH mid-and high latitude lower stratosphere in the future are highly uncertain and model dependent, because the lower stratosphere is located near the boundary of radiative warming in the troposphere and radiative cooling in the stratosphere as GHGs increase, and because dynamical heating due to enhancement of the meridional circulation could occur in the mid-and high latitude lower stratosphere. The GHG response is a combined effect of these processes and, hence, complex. These processes include many model-dependent factors for simulation, such as the horizontal and vertical resolutions, the radiation parameters, and the gravity wave parameterization. The difference in GHG scenarios between our CCM (RCP-6.0) and other studies may cause some differences in the results. Furthermore, the temperature dependence in [fig_ref] Figure 4: MIROC3 [/fig_ref]. However, these are very rare cases (one or a few in 500 ensemble members) and ODS concentrations at the end of the twenty-first century will not be so high under future ODS regulations.
In conclusion, the average of the 50 ensemble members with the lowest column ozone in the mid-and high latitudes of the Northern Hemisphere from the two CCMs suggests that ODS concentrations should be reduced to avoid large ozone losses not only in the Antarctic but also in the mid-and high latitudes of the NH in the case of a stable Arctic polar vortex. It is unlikely on a once-in-a decade basis that GHG increase towards the end of the twenty-first century will cause a worse Arctic ozone-depletion event.
# Methods
The multi-ensemble simulations were initiated by setting the ODS and GHG surface concentrations to globally uniform, temporally constant values. A continuous 510-year calculation was then performed at these fixed ODS and GHG levels; for example, one of the runs consisted of a 510-year continuous calculation using ODS concentrations for 1995 under the WMO-A1 scenario and GHG concentrations for 2000 under the RCP 6.0 scenario. This run is presented in the bottom right corner of [fig_ref] Table 1: the top row shows the GHG-2095 experiments, the bottom row the GHG-2000... [/fig_ref]. The first 10 years were excluded from the analysis as preliminary outputs obtained before the ozone amount in the CCM reached a steady seasonal cycle at the specified ODS and GHG concentrations. The last 500 years were analysed as an ensemble of 500 members.
The MIROC3.2 and MIROC5 CCMs are based on versions 3.2 and 5 of the MIROC atmospheric general circulation model (AGCM), respectively, incorporating a common stratospheric chemistry module that was developed at NIES and the University of Tokyo. MIROC (Model for Interdisciplinary Research on Climate) was developed by the University of Tokyo, NIES and the Japan Agency for Marine-Earth Science and Technology [bib_ref] Studies on the climate system and mass transport be a climate model, Numaguti [/bib_ref] [bib_ref] Improved climate simulation by MIROC5: Mean states, variability, and climate sensitivity, Watanabe [/bib_ref]. The spatial resolution of the CCMs is a T42 spectral truncation (2.8° by 2.8°) in the horizontal direction, and the models have 34 vertical levels of hybrid sigma-pressure vertical coordinates from the surface to 0.01 hPa (approximately 80 km). The MIROC3.2 CCM performs simulations as recommended by the international Chemistry-Climate Model Initiative (CCMI) and its predecessor, Chemistry-Climate Model Validation 2 (CCMVal2) in order to project the future ozone layer and analyse the relationship between ozone changes and climate change. The MIROC5 CCM is a newly developed CCM intended to be coupled with an ocean model to study interactions between atmospheric composition and climate. However, in this study, as the MIROC3.2 CCM was not coupled with an ocean model, the MIROC5 CCM was also not coupled so as not to interfere with comparison of the results. Sea surface temperature (SST) and sea ice distributions were derived from external data, as described in the next section.
The MIROC3.2 and MIROC5 CCMs use a common stratospheric chemistry module with 42 photolysis reactions, 142 gas-phase chemical reactions and 13 heterogeneous reactions for multiple aerosol types [bib_ref] A nudged chemistry-climate model simulation of chemical constituent distribution at northern high-latitude..., Akiyoshi [/bib_ref] [bib_ref] Chlorine partitioning near the polar vortex edge observed with ground-based FTIR and..., Nakajima [/bib_ref]. Three types of polar stratospheric clouds (PSCs) were incorporated: a H 2 SO 4 -HNO 3 -H 2 O supercooled ternary solution (STS), nitric acid trihydrate (NAT) and ice (ICE). The reaction probabilities for STS were calculated using the scheme used for Arctic ozone loss simulation 67 , and those for NAT and ICE were obtained from JPL-2010. These PSCs were assumed to have a single radius without size distribution. In this study, particle number density was assumed to be 10 particles/cm 3 for STS, 1 particle/cm 3 for NAT and 0.01 particles/cm 3 for ICE. The value of the particle number density for STS was based on stratospheric aerosol observations [bib_ref] Stratospheric aerosols: Observation and theory, Turco [/bib_ref] and was derived as a number density parameter of the log-normal distribution (σ = 1.8). Those for NAT and ICE were based on the parameters used for the simulations [bib_ref] The influence of the OH + NO2 + M reaction on the..., Ruhnke [/bib_ref] [bib_ref] Denitrification through PSC formation: A 1-D model incorporating temperature oscillations, Drdla [/bib_ref]. Assuming a spherical configuration, PSC radius was calculated from the particle number density and condensation volume, with the latter calculated from the saturation vapour pressures of H 2 SO 4 , HNO [bib_ref] Stratospheric sink for chlorofluoromethanes: Chlorine atom-catalysed destruction of ozone, Molina [/bib_ref] and H 2 O. Then, the sedimentation velocities of the PSCs were calculated based on the radius as a function of pressure and temperature, using the Stokes terminal settling velocity with the Cunningham correction factor.
The photolysis rates of the chemical constituents were calculated online from the radiation flux in the CCMs, with 32 spectral bins for each CCM. The radiation fluxes and ozone concentrations in the models were consistent with each other, indicating an interaction between these parameters. concentration was assumed to be uniform not only at the surface but also throughout the model atmosphere, with the same mixing ratio as that at the surface. The surface concentrations of the other ODSs and GHGs were assumed to be horizontally uniform, as they were assumed to be transported to the troposphere, stratosphere, and mesosphere of the model, and degraded by photochemical reactions and reactions with free radicals. Assuming a horizontally uniform concentration of the ODSs and GHGs at the surface should not produce any serious errors in examining long-term, planetary-scale changes in ozone amount, as approximately 90% of column ozone exists in the stratosphere, and the actual ODS and GHG distributions at the surface are levelled out at the upper troposphere/lower stratosphere because of the large-scale transport and photochemical reactions in the troposphere. The CCMs used monthly data for SST and sea ice from the Coupled Model Intercomparison Project Phase 5 (CMIP-5) simulations performed with the MIROC5 coupled atmosphere-ocean general circulation model. The monthly SST and sea ice data were averaged over 10 years. For example, the data average for 1995-2004 was used for the five GHG-2000 experiments , and the data average for 2025-2034 was used for the five GHG-2030 experiments. Therefore, the ENSO signals in the 10-year averaged data were removed. The solar flux data were based on the data for CCMI Phase 1 simulations, which were supplied by WCRP/SPARC SOLARIS-HEPPA 72 ; however, in this study, the data average for 1960-2000 was used. Hence, the effects of interannual solar flux variations, such as the 11-year solar cycle, were removed. Furthermore, the MIROC3.2 and MIROC5 CCMs do not generate QBO internally. Thus, the simulations in this study did not include the effects of ENSO, solar flux and QBO.
Output data from CCM runs. The CCMs output two-dimensional data (longitude/latitude) for column ozone and three-dimensional data for the ozone mixing ratio, temperature, zonal wind, meridional wind, air density and some other meteorological quantities and chemical constituents. In this study, we examined only the column ozone, temperature, zonal wind, total reactive chlorine concentration (Cly) and total reactive bromine concentration (Bry).
Analysis of the spring column ozone minimum. The spring column ozone minimum in the NH midand high latitudes was determined by searching for the minimum value for each ensemble member in the region between 45° and 90° N from March to May. The amount of column ozone in the Arctic polar vortex is often low because of the catalytic destruction of ozone during these months. As the Arctic polar vortex shows large temporal and spatial variability, the region for analysis included not only the polar regions, but also the northern part of the mid-latitudes. In the SH, the spring minimum values for each ensemble member were determined in the region between 45° and 90° S from September to November, corresponding to the ozone hole months. A histogram of the spring ozone minimums of the 500-member ensembles was created for each experiment [fig_ref] Table 1: the top row shows the GHG-2095 experiments, the bottom row the GHG-2000... [/fig_ref]. The horizontal axes of Figs. 3 and 4 represent average EESC at 50 hPa for the NH (45-90° N, March-May) and SH (45-90° S, September-November). The EESC was calculated by adding 65 times the reactive bromine concentration to the concentration of reactive chlorine.
Analysis of the area with column ozone of less than 220 DU. Because the column ozone bias of the MIROC3.2-CCM and MIROC5-CCM, based on TOMS observations, is not large globally (see [fig_ref] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90°... [/fig_ref] , we used a threshold of 220 DU, the ozone value indicating an ozone hole, to determine the ozone loss area for both models. The grids that indicated column ozone amounts of less than 220 DU were taken from daily column ozone data for the area between 45° and 90° N from March to May and the area between 45° and 90° S from September to November, and the daily values of the areas of the low-ozone grids were integrated with respect to the day for the three months of spring. Then, the ensemble distribution of the values was calculated, with units of 10 8 km 2 ·day (see Supplementary Figs. S2 and S3). Polar cap temperature and zonal mean zonal wind at 50 hPa. The Arctic polar cap temperature and zonal mean zonal wind at 50 hPa [fig_ref] Figure 4: MIROC3 [/fig_ref] were calculated by averaging the daily temperature data in the polar cap region (63-90° N; model grids of 9 points on latitude by 128 points on longitude) and the daily zonal wind data in the zonal wind maximum region (60-70° N; grids of 5 points on latitude by 128 points on longitude), respectively, during March. Similarly, the Antarctic polar cap temperature and zonal mean zonal wind at 50 hPa [fig_ref] Figure 5: MIROC3 [/fig_ref] were calculated by averaging the daily temperature data in the polar cap region (63°-90° S; model grids of 9 points on latitude by 128 points on longitude) and the daily zonal wind data in the zonal wind maximum region (55-65° S; grids of 5 points on latitude by 128 points on longitude), respectively, during October.
## Data availability
The model output used in this study are available from https:// doi. org/ 10. 17595/ 20221 109. 001.
[fig] Figure 1: Histogram of the spring column ozone minimum values between 45° and 90° N from March to May for the 24 MIROC3.2 CCM runs with 500-member ensembles. Results are shown by panels for all runs using different ODS and GHG concentrations. The panels are arranged in the same order as in [/fig]
[fig] Figure 2: Histogram of the spring column ozone minimum values between 45° and 90° S from September to November for the 24 MIROC3.2 CCM runs with 500-member ensembles. Results are shown by panels for all runs using different ODS and GHG concentrations. The panels are arranged in the same order as inTable 1: the top row shows the GHG-2095 experiments, the bottom row the GHG-2000 experiments, the left column the ODS-1960 experiments, and the right column the ODS-1995 experiments. The spring column ozone minimum values are depicted on the horizontal axis at intervals of 20 DU. The bin size for the column ozone minima on the horizontal axis is 2 DU, with the bin of the smallest value at 30-32 DU and the largest at 248-250 DU. The bin count (between 0 and 100) is indicated on the vertical axis. Column ozone values (average with standard deviation, maximum and minimum) for the 500-member ensembles of each experiment are represented in the upper parts of the panels. [/fig]
[fig] Figure 3: MIROC3.2 CCM results for the lower 50 ensemble members, full 500 members and upper 50 members regarding dependence of spring column ozone minimum values in the mid-and high latitudes on ODS and GHG concentrations. The upper panels show results for the area between 45° and 90° N from March to May (NH spring), and the lower panels show results for the area between 45° and 90° S from September to November (SH spring). Column ozone minimum values are averaged over the 50 ensemble members with the largest column ozone minima (upper 50, left), the full ensemble (500 members, middle) and the 50 members with the smallest column ozone minima (lower 50, right). The horizontal axis shows the mean spring EESC concentrations of the ODSs in ppbv for the NH (45-90° N, 50 hPa, March-May) and SH (45-90° S, 50 hPa, September-November). The vertical axis denotes the CO 2 concentration in ppmv. Black circles denote the EESC and CO 2 concentrations used in the 24 CCM runs. Colour levels represent column ozone minimum values in DU. The column ozone minimum values obtained from the 24 runs are interpolated and extrapolated in the (EESC, CO 2 ) space. Most of the extrapolated regions are masked with black. Scientific Reports | (2023) 13:320 | https://doi.org/10.1038/s41598-023-27635-y [/fig]
[fig] Figure 4: MIROC3.2 CCM results for the lower 50 ensemble members, full 500 members and upper 50 members regarding dependence of the polar cap temperature and polar night jet strength in the mid-and high latitudes of the NH (50 hPa, March) on ODS and GHG concentrations. The polar cap temperatures and polar night jet strength were calculated using the March mean values for daily temperature (63-90° N, 50 hPa) and daily zonal mean zonal wind speed (60-70° N, 50 hPa). Colour levels represent the temperature (in K) and zonal mean zonal wind speed (in m/s). The values of all experiments are interpolated and extrapolated in the (EESC, CO 2 ) space. Most of the extrapolated regions are masked with black. [/fig]
[fig] Figure 5: MIROC3.2 CCM results for the lower 50 ensemble members, full 500 members and upper 50 members regarding dependence of the polar cap temperature and polar night jet strength in the mid-and high latitudes of the SH (50 hPa, October) on ODS and GHG concentrations. The polar cap temperatures and polar night jet strength were calculated using the October mean values for daily temperature (63-90° S, 50 hPa) and daily zonal mean zonal wind speed (55-65° S, 50 hPa). Scientific Reports | (2023) 13:320 | https://doi.org/10.1038/s41598-023-27635-y [/fig]
[table] Table 1: the top row shows the GHG-2095 experiments, the bottom row the GHG-2000 experiments, the left column the ODS-1960 experiments, and the right column the ODS-1995 experiments. The spring column ozone minimum values are depicted on the horizontal axis at intervals of 20 DU. The size of each bin for the column ozone minima on the horizontal axis is 2 DU, with the bin of the smallest value at 90-92 DU and that of the largest value at 308-310 DU. The bin count (between 0 and 100) is indicated on the vertical axis. Column ozone values (average with standard deviation, maximum and minimum) for the 500-member ensembles of each experiment are represented in the upper parts of the panels. [/table]
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Polymeric Packaging Applications for Seafood Products: Packaging-Deterioration Relevance, Technology and Trends
Citation: Laorenza, Y.; Chonhenchob, V.; Bumbudsanpharoke, N.; Jittanit, W.; Sae-tan, S.; Rachtanapun, C.; Chanput, W.P.; Charoensiddhi, S.; Srisa, A.; Promhuad, K.; et al.
# Introduction
In 2021, world fishery market trade was forecast to increase by 12% in value and 3.7% in volume, with production projected to increase by 4 million tons from 2020 to 2022 . Fishery product prices, especially shrimp, remained stable until July 2021 and then increased from August 2021 due to high freight costs of US$ 0.70-0.80 per kg for products exported from Asia to North America and Europe. Processed seafood is popular as ready-to-eat meals or snacks equipped with detailed serving instructions that normally involve reheating using a microwave oven [bib_ref] Microwave pasteurization for ready-to-eat meals, Tang [/bib_ref]. Consumption of frozen packaged foods had predicted a CAGR of 5.98% from 2021 to 2028, while average year-on-year growth of 15.35% in 2020 was attributed to consumer panic buying behavior during the COVID-19 outbreak. Cold chain distribution requires high maintenance of both containers and packaging systems to maintain product quality. Seafood is a rich source of nutrients with specific aromas and tastes, while increasing global seafood production reflects consumer demand for packaged seafood products. lactic acid bacteria also contribute to fish deterioration. Photobacterium phosphoreum together with H 2 S spoilage bacteria trigger the production of trimethylamine during post-harvest metabolism. Production of hypoxanthine from the fish body during storage is triggered by Enterobacteriaceae, Pseudomonas, Shewanella putrafaciens, and Photobacterium phosphoreum.
The effect of microbial growth on changes in other fish quality parameters was reported by [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref]. Microbial growth was linearly correlated with melanosis and trimethylamine production of Pacific white shrimp stored under MAP [fig_ref] Figure 1: Interrelation of total viable count on changes in [/fig_ref]. This finding indicated that microbial growth strongly stimulated melanosis formation in shrimp following the mechanism shown in [fig_ref] Figure 2: Melanosis formation through polyphenol oxidation and its prevention via microbial inhibition and... [/fig_ref] , while the peptidoglycan binding protein (Gram positive bacteria) and β-1,3-glucan binding protein (Gram negative bacteria) contributed to pro-polyphenol oxidase activation that then induced melanosis formation [bib_ref] Melanosis in crustaceans: A review, Gonçalves [/bib_ref]. Trimethylamine dramatically increased after the total viable count (TVC) reached 6.8 log cfu/g. These results suggested that reduction of trimethylamine and melanosis was dependent on the microbial inhibition ability of CO 2 . Increasing TVC also inversely reduced the firmness. The microbials consumed nutrients such as lipids or proteins, causing proteolytic denaturation and resulting in a loss of firmness.
onaceae, and lactic acid bacteria also contribute to fish deterioration. Photob phoreum together with H2S spoilage bacteria trigger the production of trimeth ing post-harvest metabolism. Production of hypoxanthine from the fish bod age is triggered by Enterobacteriaceae, Pseudomonas, Shewanella putrafaciens, a rium phosphoreum.
The effect of microbial growth on changes in other fish quality param ported by [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref]. Microbial growth was linearly co melanosis and trimethylamine production of Pacific white shrimp stored [fig_ref] Figure 1: Interrelation of total viable count on changes in [/fig_ref]. This finding indicated that microbial growth strongly stimula formation in shrimp following the mechanism shown in [fig_ref] Figure 2: Melanosis formation through polyphenol oxidation and its prevention via microbial inhibition and... [/fig_ref] , while the p binding protein (Gram positive bacteria) and β-1,3-glucan binding protein tive bacteria) contributed to pro-polyphenol oxidase activation that then ind sis formation [bib_ref] Melanosis in crustaceans: A review, Gonçalves [/bib_ref]. Trimethylamine dramatically increased after the total (TVC) reached 6.8 log cfu/g. These results suggested that reduction of trimet melanosis was dependent on the microbial inhibition ability of CO2. Increa inversely reduced the firmness. The microbials consumed nutrients such as teins, causing proteolytic denaturation and resulting in a loss of firmness. Several studies have investigated microbial inhibition in seafood products using active packaging. [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref] [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref] found that chitosan film containing Ziziphora clinopodioides essential oil and pomegranate peel extract effectively retarded TVC and growth of Pseudomonas spp., Pseudomonas flourescens, Shewanella putrefaciens, Enterobacteriaceae, lactic acid bacteria, and Listeria monocytogenes in shrimp compared to the control and gelatin film, while incorporation of combined nanoparticles (ZnO, SiO, and CuO) into gelatin and polyvinyl alcohol film had antimicrobial activity equal to the positive control against S. aureus, L. monocytogenes, E. coli, P. fluorescens, V. parahaemolyticus, and A. caviae. Microbial activity in seafood products can be controlled via active packaging containing antimicrobial agents that delay post-harvest metabolism and extend the shelf-life.
## Chemical deterioration
Deterioration in seafood is mainly caused by lipid oxidation in high-fat content pelagic fish (mackerel, sardinella, and herring). Oxidation occurs as a result of the reaction between oxygen molecules and the double bond of the unsaturated fatty acid chain. Fish contain high amounts of mono or polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as major sources of nutrition benefits that are highly susceptible to oxidation. Packaging containing antioxidant agents, such as carvacrol essential oil [bib_ref] A novel composite edible film fabricated by incorporating W/O/W emulsion into a..., Yuan [/bib_ref] , gallic acid, and clove essential oil, loaded polymer reduced lipid oxidation of salmon fillets. [bib_ref] Corn starch/polyvinyl alcohol based films incorporated with curcumin-loaded Pickering emulsion for application..., Liu [/bib_ref] reported that MAP combined with εpolylysine, chitosan, and sodium alginate coatings delayed myofibril oxidation by inhibiting carbonyl groups in pufferfish.
Lipid oxidation in fish occurs enzymatically or non-enzymatically. Enzymatic lipid oxidation results from lipolysis by lipases, while non-enzymatic oxidation normally arises in hematin compounds such as hemoglobin, myoglobin, and cytochrome [bib_ref] Fish spoilage mechanisms and preservation techniques, Ghaly [/bib_ref]. Myoglobin is present in red meat such as tuna as an oxygen-binding protein similar to hemoglobin. High myoglobin content contributes to the reddish-brown color of the flesh. Undesirable discoloration in red meat is related to lipid oxidation associated with the binding between heme and myofibrillar protein and results in greater discoloration [bib_ref] Post harvest discoloration of dark-fleshed fish muscle: A review, Chaijan [/bib_ref]. The red color in tuna can be preserved by using carbon monoxide or nitric oxide that react with myoglobin (MB), resulting in MB-Fe +11 -CO and MB-Fe +11 -NO bonding, respectively. Therefore, Several studies have investigated microbial inhibition in seafood products using active packaging. [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref] [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref] found that chitosan film containing Ziziphora clinopodioides essential oil and pomegranate peel extract effectively retarded TVC and growth of Pseudomonas spp., Pseudomonas flourescens, Shewanella putrefaciens, Enterobacteriaceae, lactic acid bacteria, and Listeria monocytogenes in shrimp compared to the control and gelatin film, while incorporation of combined nanoparticles (ZnO, SiO, and CuO) into gelatin and polyvinyl alcohol film had antimicrobial activity equal to the positive control against S. aureus, L. monocytogenes, E. coli, P. fluorescens, V. parahaemolyticus, and A. caviae. Microbial activity in seafood products can be controlled via active packaging containing antimicrobial agents that delay post-harvest metabolism and extend the shelf-life.
## Chemical deterioration
Deterioration in seafood is mainly caused by lipid oxidation in high-fat content pelagic fish (mackerel, sardinella, and herring). Oxidation occurs as a result of the reaction between oxygen molecules and the double bond of the unsaturated fatty acid chain. Fish contain high amounts of mono or polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as major sources of nutrition benefits that are highly susceptible to oxidation. Packaging containing antioxidant agents, such as carvacrol essential oil [bib_ref] A novel composite edible film fabricated by incorporating W/O/W emulsion into a..., Yuan [/bib_ref] , gallic acid, and clove essential oil, loaded polymer reduced lipid oxidation of salmon fillets. [bib_ref] Corn starch/polyvinyl alcohol based films incorporated with curcumin-loaded Pickering emulsion for application..., Liu [/bib_ref] reported that MAP combined with ε-polylysine, chitosan, and sodium alginate coatings delayed myofibril oxidation by inhibiting carbonyl groups in pufferfish.
Lipid oxidation in fish occurs enzymatically or non-enzymatically. Enzymatic lipid oxidation results from lipolysis by lipases, while non-enzymatic oxidation normally arises in hematin compounds such as hemoglobin, myoglobin, and cytochrome [bib_ref] Fish spoilage mechanisms and preservation techniques, Ghaly [/bib_ref]. Myoglobin is present in red meat such as tuna as an oxygen-binding protein similar to hemoglobin. High myoglobin content contributes to the reddish-brown color of the flesh. Undesirable discoloration in red meat is related to lipid oxidation associated with the binding between heme and myofibrillar protein and results in greater discoloration [bib_ref] Post harvest discoloration of dark-fleshed fish muscle: A review, Chaijan [/bib_ref]. The red color in tuna can be preserved by using carbon monoxide or nitric oxide that react with myoglobin (MB), resulting in MB-Fe +11 -CO and MB-Fe +11 -NO bonding, respectively. Therefore, chemical deterioration depends on the chemical structure of the fish meat, while preservation is accomplished using an antioxidant agent or high CO 2 atmosphere.
## Biochemical deterioration
Post-mortem biochemical changes in fish result from autolysis by the action of proteolytic enzymes. [bib_ref] Fish spoilage mechanisms and preservation techniques, Ghaly [/bib_ref] [bib_ref] Fish spoilage mechanisms and preservation techniques, Ghaly [/bib_ref] explained that proteolytic enzymes are mostly present in the muscle and viscera of fish in the early rigor stages and contribute to postmortem degradation. Proteolytic enzymes break down protein, resulting in free amino acids and peptides during autolysis that lead to fish degradation, with production of the biogenic amines: histamine, tyramine, tryptamine, putrescine, and cadaverine [bib_ref] Biogenic amines in seafood: A review, Biji [/bib_ref] , followed by acidity, increased pH, and accumulation of basic nitrogen compounds such as trimethylamine (TMA) and total volatile-based nitrogen (TVB-N). The degradation of adenosine triphosphate (ATP) is also responsible for the biochemical post-mortem degradation of seafood products to form adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine, and hypoxanthine, determined as K value. A higher K value indicates a lower freshness level. [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref] reported that MAP with a high concentration of CO 2 (above 60%) retained a low TMA value. A combination of MAP and ε-polylysine, chitosan and sodium alginate coatings maintained Ca 2+ -ATPase activity by retaining the protein structure (α and β-sheet) of pufferfish, while TVB-N and TMA of salted dried Atlantic mackerel were reduced by vacuum packaging combined with an iron-based oxygen absorber [bib_ref] The Effect of Active and Vacuum Packaging on the Quality of Turkish..., Erkan [/bib_ref]. Biochemical deterioration mostly relates to the amine content in fish meat, and this can be delayed by MAP or active packaging.
## Melanosis
Melanosis occurs when a cell cannot regulate pigment production and appears as a black spot. This is a serious issue in crustacean products considering their high commercial value. Melanosis is harmless to human health but significantly decreases sensory market value and acceptance by the consumer. Goncalves and Oliveira (2016) [bib_ref] Melanosis in crustaceans: A review, Gonçalves [/bib_ref] categorized five stages of melanosis formation: (1) Gram positive and negative bacteria and fungi trigger the alignment of serine proteinase, (2) pro polyphenoloxidase then activates to become polyphenoloxidase (PPO), (3) PPO acts as a catalyst to convert phenol into colorless quinone, (4) oxidization of quinone results in melanin, and (5) melanin is responsible for melanosis formation in the crustacean carapace. Melanosis formation is accelerated by microorganisms, oxygen exposure, and high temperatures. Melanosis in crustaceans can be measured by PPO extraction and identification using SDS polyacrylamide gel electrophoresis (SDS-PAGE), color measurement, particularly whiteness [bib_ref] Inhibitory effect of a quercetin-based soaking formulation and modified atmospheric packaging (MAP)..., Qian [/bib_ref] or L value [bib_ref] Quality assessment of shrimps preserved with orange leaf essential oil incorporated gelatin, Alparslan [/bib_ref] , image analysis [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref] [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] , or sensory assessment.
Packaging systems integrated with active compounds are now widely developed to preserve crustaceans, particularly shrimp, by inhibiting melanosis formation on the carapace. MAP with a high CO 2 concentration to prevent melanosis in shrimp [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref] [bib_ref] Inhibitory effect of a quercetin-based soaking formulation and modified atmospheric packaging (MAP)..., Qian [/bib_ref] , while Laorenza and Harnkarnsujarit (2021) [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] developed a PBAT/PLA active film containing carvacrol, citral, and α-terpineol essential oils to prevent melanosis. [bib_ref] Quality assessment of shrimps preserved with orange leaf essential oil incorporated gelatin, Alparslan [/bib_ref] [bib_ref] Quality assessment of shrimps preserved with orange leaf essential oil incorporated gelatin, Alparslan [/bib_ref] also reported that edible coatings prepared from gelatin containing orange leaf oil retained a high L* value, indicating that melanosis was prevented. Application of packaging technology prevented melanosis in shrimp by minimizing oxidation and growth of melanosis-stimulating microbes.
## Polymeric packaging for seafoods
## Conventional polymer-based packaging
Conventional polymer-based packaging, including polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polyvinyl alcohol (PVOH), and polyamide (PA), still dominates the packaging industry with excellent mechanical, barrier, and thermal properties compared to plastic from other sources. Seafood is usually produced as frozen products, with a temperature during freezing of less than −40 - C and a frozen storage temperature −18 - C. Frozen seafood packaging must withstand low temperatures without cracking. Most frozen products become hard with sharp edges under freezing conditions due to their transition into an amorphous glassy state, causing impingement through flexible films and loss of package integrity. Glass transition temperature (Tg) plays a key role in the choice of packaging material properties. Polymers with higher Tg than freezing and storage temperatures are unsuitable for use with frozen products. Polyethylene has low glass transition temperature at up to −100 - C compared to other petroleum-based polymers, suggesting that it would be a suitable packaging material for products stored at extremely low temperatures. Petroleum-based packaging for seafood products is usually in a flexible (pouch, bag, or pocket) or rigid (tray or box) form. Flexible packaging is made from PP, PE, or nylon due to the high elongation at break, while rigid packaging is normally made from PET or PS due to poor elongation at break.
The development of conventional polymer-based packaging for seafood products includes functional polymeric packaging and MAP that delays microbial growth. [bib_ref] Characterization and preservation performance of active polyethylene films containing rosemary and cinnamon..., Dong [/bib_ref] [bib_ref] Characterization and preservation performance of active polyethylene films containing rosemary and cinnamon..., Dong [/bib_ref] found that the incorporation of rosemary and cinnamon essential oils into polyethylene reduced the degradation temperature and barrier properties of polypropylene film because the hydroxyl groups reacted with oxygen or water molecules and retarded shrimp deterioration. Nylon has excellent barrier properties and is mostly used as vacuum packaging in processed seafood products. Vacuum packaging with very low oxygen permeability maintained seafood quality by removing undesirable gases to minimize post-harvest metabolism [bib_ref] Role of vacuum packaging in increasing shelf-life in fish processing technology, Kumar [/bib_ref] , while incorporation of anthocyanin originating from plants with polyvinyl alcohol could monitor the freshness of shrimp.
Rigid and semi-rigid packaging such as PET-based trays play an important role in fishery product marketing, particularly in retail or convenience store fresh produce displays. Reduction of water-holding capacity in muscle structures causes high-drip loss of seafood products. This adversely affects appearance and consumer satisfaction. Liquid absorption pads are commonly used in the meat industry but are not popular in seafood packaging. The water absorbance pad is placed in direct contact with the fresh product and the water is still in contact with the product. Many patents of water-absorbent discs based on PLA modified material provided indirect contact water absorption between the fresh product and the absorption pad. They designed an outer dish body and a depository dish body in an overlapped mode containing a water accumulation cavity between them. The water absorption sponge is placed inside the water accumulation cavity. The water absorption sponge has a lower size than the height of the accumulation cavity so that the system allows for the absorption of the water in irreversible contact with the product. Rigid packaging is also used to reheat food, and PET, PP, PE, and PS are suitable for microwave heating. A tray can also be used as a seafood MAP to maintain quality and mitigate oxidation and microbial growth.
Thermal insulation is also important for seafood packaging. Seafood is highly prone to microbial and enzymatic degradation, and temperature control is important to maintain product quality. Polystyrene-based rigid packaging such as a tray or box is usually used as a thermal insulator in seafood product delivery to maintain low temperatures. Several types of box insulation packaging have been commercially produced and registered as patents such as insulator bags made from glass wool and non-woven fabric, rectangular parallelepiped-based insulator boxes with sodium polyacrylate, intermediate foam fish boxes from olefin, Styrofoam boxes with intermediate lids, and insulator boxes with inflatable bags and wood pulp-based absorbance as wave-shaped convex strips [bib_ref] Corrugated Carton Suitable for Transportation and Packaging of Fish Tank. China Patent..., Shang [/bib_ref]. Recently, conventional packaging still dominates in the packaging industry due to its excellent properties, processability, and lower price compared to bio-degradable polymers. However, end-use disposal of conventional polymer-based packaging is a serious problem; they are non-biodegradable due to their complex chemical structure of stable carbon-carbon linkages and are not broken down by microorganisms. Blending with biodegradable materials with high compatibility will improve the biodegradability without interfering with the properties.
## Bio-based packaging
The majority of bio-degradable packaging originates from natural biopolymers and synthetic degradable polymers such as polysaccharide-based materials, which are environmentally friendly in terms of disposal and end life. Starch is one of the primary materials used in bio-degradable packaging, which is a renewable source derived from plants. Starch is dominated by a hydroxyl group, resulting in a rigid network [bib_ref] Plasticized hydroxypropyl cassava starch blended PBAT for improved clarity blown films: Morphology..., Wadaugsorn [/bib_ref] and can be molded according to desirable shapes. Thus, it is suitable for rigid packaging such as a tray, which is now dominated by a conventional polymer. The tray is commonly used to protect food from damage caused by undesirable environmental conditions such as shock, vibration, pressure, and deformation.
Polysaccharide-based trays or foam were developed to replace Styrofoam as an environmentally friendly packaging. A patent regarding a starch-based foam tray with at least two slots in the tray body combined with two fasteners, on which the tray body can be stacked and mutually form-embedded through the tray's grooves and fasteners. This tray contains plurality holes, can accommodate loads up to 80 kg, and can be used as a fish bulk container. A patent was also registered for biodegradable resin foam sheets prepared from cellulose acetate as the main component with a starch and talc or eggshell powderbased modifier. This had high water-resistant properties as cup traysfor high water product packaging.patented a biodegradable tray composited from starch, bamboo pulp, succinic acid, fiber, paper pulp fiber powder, calcium alginate fiber, milk protein fiber, polyethylene resin, and a toughening agent (polyvinyl alcohol or acrylate rubber), which automatically degrades after use.
Antimicrobial trays made of high-amylose corn starch and cinnamyl aldehyde were patented by. They developed the trays by ultrasonic waves without added surfactant to prolong product shelf-life due to the slow release of cinnamyl aldehyde.patented a food packaging tray equipped with a sealed compartment using a gasket and cold-seal adhesive with no hot-sealing process. This packaging is to carry the reheated food from the store to keep the food safe and hygienic. They created the easy reseal system by reactivating the cold seal adhesive between the adhesive surface and another surface contacted together. Therefore, the tray plays an important role in seafood packaging, particularly in the selling and distribution, to keep products safe and hygienic for the consumer. Recently, starch-based rigid packaging has been widely developed and commercialized for tableware, packaging, cushioning, and insulation. The application of starch in this field still has limitations due to low water resistance since seafood products have high water content. Future research can be focused on improving the water resistance of starch by chemical modification, lamination, or blending with other materials that have great water resistance.
## Innovative polymeric packaging enhancing and monitoring seafood quality and freshness
## Active polymer technology for seafoods
Active packaging was described by Labuza in 1987 [bib_ref] Applications of 'Active Packaging' for improvement of shelf-life and nutritional quality of..., Labuza [/bib_ref] as packaging, packaging materials, or packaging solutions that can interact with food and have a specific function beyond protecting food from the outside environment. The definition of active packaging was then expanded as technology advanced to face new problems. Nowadays, active packaging is defined as a packaging material that releases or treats substances from or into food or the environment. Active packaging methods can be divided into oxygen scavengers [bib_ref] Pyrogallol loaded thermoplastic cassava starch based films as bio-based oxygen scavengers, Promsorn [/bib_ref] , carbon dioxide generating systems, ethylene scavengers, flavor and odor absorbers, antioxidants, and antimicrobials [bib_ref] Active packaging in food industry: A review, Prasad [/bib_ref]. The technology of inserting the active compound into the packaging can be divided by coating [bib_ref] Enhancement of agricultural produce quality and storability using citral-based edible coatings; the..., Arnon-Rips [/bib_ref] , incorporating [bib_ref] Characterization and preservation performance of active polyethylene films containing rosemary and cinnamon..., Dong [/bib_ref] , surface modification [bib_ref] Review of surface treatment methods for polyamide films for potential application as..., Tyuftin [/bib_ref] , and adding directly into the packaging (i.e., essential oil into MAP) [bib_ref] The combined efficacy of carvacrol and modified atmosphere packaging on the survival..., Nair [/bib_ref] , which are able to modify and improve product quality, achieving desirable purposes. The active agents are mostly applied in a polymer and effectively preserve the seafood quality [fig_ref] Table 1: Previous recent research on active packaging for seafood products [/fig_ref] , including essential oils [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref] [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] [bib_ref] Antifungal films from trans-cinnamaldehyde incorporated poly(lactic acid) and poly(butylene adipate-co-terephthalate) for bread..., Srisa [/bib_ref] , plant phenolic compounds, or nano minerals [bib_ref] Polyvinyl alcohol/gelatin nanocomposite containing ZnO, TiO 2 or ZnO/TiO 2 nanoparticles doped..., Azizi-Lalabadi [/bib_ref]. Films showed a yellowish color and improvement in water tolerance, elasticity, and antioxidant activity (56-85% of ABTS inhibition).
Fillets packed in active film had a pleasant smell and flavor, an increase in golden color, and higher stiffness than fillets packed in control film.Chitosan or lysozyme PLA Grass carp fillet
The film provides an antimicrobial agent to form an amine bond to inhibit E. coli and S. aureus.
Active film prolonged the fillet up to 3 days. PLA/chitosan was more effective in inhibiting bacterial growth than PLA/lysozyme.Essential oil (Carvacrol, citral and α-terpineol)
## Pla/pbat pacific white shrimp
Essential oil modified barrier properties and microstructure affected by polymer-essential oil interacted via hydrogen bonding and carbonyl groups.
Shrimp deterioration was prevented by active film. Citral and carvacrol more effectively stabilized protein conformation in muscle tissues, retained drip loss and adhesion between the cephalothorax and abdomen.
[37]
Green tea ground waste Potato starch, gelatin, carboxymethyl cellulose (CMC)
## Salmon
The film had high water vapor permeability (WVP) but limited germination due to a low pH. The DPPH radical scavenging of the tray containing tea waste was 80.75%.
The active tray + film provided potential inhibition against biogenic amine accumulation, 19% lower spoilage bacteria of salmon than the control after 6 days of storage. [bib_ref] Shelf-life extension of salmon using active total biodegradable packaging with tea ground..., Jamroz [/bib_ref] The incorporation of essential oils in the polymer takes advantage of the volatility of antimicrobial agents that migrate from the packaging to the food inside. [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] found that incorporation of carvacrol, citral, and α-terpineol essential oils released from the PBAT/PLA matrix into the headspace. The release behavior of these essential oils was related to shrimp quality with lower microbial growth, lipid oxidation/thiobarbituric acid reactive substance (TBARS), and melanosis formation, while films containing essential oils delayed glycogen metabolism revealed in FTIR absorbance at wavenumbers 2800 to 3040 cm −1 , which also agreed well with the protein degradation and head loss retardation. Shrimp packaged in films containing carvacrol, citral, and α-terpineol essential oils are shown in [fig_ref] Figure 3: Shrimp packaged in PBAT/PLA film containing 6% α-terpineol, carvacrol, and citral during... [/fig_ref].
sorbers, antioxidants, and antimicrobials [bib_ref] Active packaging in food industry: A review, Prasad [/bib_ref]. The technology of inserting the active compound into the packaging can be divided by coating [bib_ref] Enhancement of agricultural produce quality and storability using citral-based edible coatings; the..., Arnon-Rips [/bib_ref] , incorporating [bib_ref] Characterization and preservation performance of active polyethylene films containing rosemary and cinnamon..., Dong [/bib_ref] , surface modification [bib_ref] Review of surface treatment methods for polyamide films for potential application as..., Tyuftin [/bib_ref] , and adding directly into the packaging (i.e., essential oil into MAP) [bib_ref] The combined efficacy of carvacrol and modified atmosphere packaging on the survival..., Nair [/bib_ref] , which are able to modify and improve product quality, achieving desirable purposes. The active agents are mostly applied in a polymer and effectively preserve the seafood quality [fig_ref] Table 1: Previous recent research on active packaging for seafood products [/fig_ref] , including essential oils [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref] [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] [bib_ref] Antifungal films from trans-cinnamaldehyde incorporated poly(lactic acid) and poly(butylene adipate-co-terephthalate) for bread..., Srisa [/bib_ref] , plant phenolic compounds, or nano minerals [bib_ref] Polyvinyl alcohol/gelatin nanocomposite containing ZnO, TiO 2 or ZnO/TiO 2 nanoparticles doped..., Azizi-Lalabadi [/bib_ref].
The incorporation of essential oils in the polymer takes advantage of the volatility of antimicrobial agents that migrate from the packaging to the food inside. [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] [bib_ref] citral and alpha-terpineol essential oil incorporated biodegradable films for functional active packaging..., Laorenza [/bib_ref] found that incorporation of carvacrol, citral, and α-terpineol essential oils released from the PBAT/PLA matrix into the headspace. The release behavior of these essential oils was related to shrimp quality with lower microbial growth, lipid oxidation/thiobarbituric acid reactive substance (TBARS), and melanosis formation, while films containing essential oils delayed glycogen metabolism revealed in FTIR absorbance at wavenumbers 2800 to 3040 cm −1 , which also agreed well with the protein degradation and head loss retardation. Shrimp packaged in films containing carvacrol, citral, and αterpineol essential oils are shown in [fig_ref] Figure 3: Shrimp packaged in PBAT/PLA film containing 6% α-terpineol, carvacrol, and citral during... [/fig_ref]. Ziziphora clinopodioides essential oil and pomegranate peel extract in chitosan and gelatin film showed a synergistic effect, and were effective in inhibiting microbial growth in shrimp [bib_ref] Application of chitosan and gelatin based active packaging films for peeled shrimp..., Mohebi [/bib_ref]. Essential oils were also applied in bilayer packaging to control the release direction of volatile compounds. [bib_ref] Release of cinnamon essential oil from polysaccharide bilayer films and its use..., Arancibia [/bib_ref] [bib_ref] Release of cinnamon essential oil from polysaccharide bilayer films and its use..., Arancibia [/bib_ref] developed bilayer agar and alginate film containing cinnamon essential oil for chilled shrimp packaging. They found that cinnamon essential oil in agar was effective in inhibiting microbial growth due to essential oil interaction differences between agar and alginate. [bib_ref] Characterization and preservation performance of active polyethylene films containing rosemary and cinnamon..., Dong [/bib_ref] [bib_ref] Characterization and preservation performance of active polyethylene films containing rosemary and cinnamon..., Dong [/bib_ref] also developed bilayer active packaging from low density polyethylene (LDPE) containing rosemary and cinnamon essential oils as an inside layer. They found that films containing combined rosemary and cinnamon essential oils were more effective in reducing TVB-N, TBARS, and microbial count. Essential oils containing strong antimicrobial and antioxidant activity were released from the packaging and brought into contact with the food to preserve quality.
Plant extracts with antioxidant properties incorporated into polymers as seafood packaging were also reported [fig_ref] Figure 4: Active packaging with various active agents [/fig_ref]. loaded potato peel extract onto starch film. The 2,2 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) inhibition of the film was measured at 56-85% within 7 days and increased the golden color of smoked sea bream fillet. Grapefruit seed extract incorporated with sodium alginate retarded the microbiological limit of shrimp for up to 4 days longer than the control, with lower TVB-N, pH values, and off-flavors due to dispersion of nanoparticles from the packaging to the shrimp [bib_ref] Enhancing safety and quality of shrimp by nanoparticles of sodium alginate-based edible..., Baek [/bib_ref]. [bib_ref] Influence of chitosan-gelatin edible coating incorporated with longkong pericarp extract on refrigerated..., Nagarajan [/bib_ref] developed an active coating prepared from gelatin and chitosan incorporated by longkong pericarp extract. They found that the active coating effectively inhibited melanosis and polyphenol oxidase activity of black tiger shrimp, while lipid (TBARS, peroxide value, and anisidine value) and protein oxidation (loss of sulfhydryl group) were inhibited. These results indicate that the incorporation of plant extracts rich in antioxidants into polymers has the potential to preserve the quality of shrimp. However, further investigation is required regarding the stability of plant extracts, particularly thermal and oxidative degradation. onto starch film. The 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) inhibition of the film was measured at 56-85% within 7 days and increased the golden color of smoked sea bream fillet. Grapefruit seed extract incorporated with sodium alginate retarded the microbiological limit of shrimp for up to 4 days longer than the control, with lower TVB-N, pH values, and off-flavors due to dispersion of nanoparticles from the packaging to the shrimp [bib_ref] Enhancing safety and quality of shrimp by nanoparticles of sodium alginate-based edible..., Baek [/bib_ref]. [bib_ref] Influence of chitosan-gelatin edible coating incorporated with longkong pericarp extract on refrigerated..., Nagarajan [/bib_ref] developed an active coating prepared from gelatin and chitosan incorporated by longkong pericarp extract. They found that the active coating effectively inhibited melanosis and polyphenol oxidase activity of black tiger shrimp, while lipid (TBARS, peroxide value, and anisidine value) and protein oxidation (loss of sulfhydryl group) were inhibited. These results indicate that the incorporation of plant extracts rich in antioxidants into polymers has the potential to preserve the quality of shrimp. However, further investigation is required regarding the stability of plant extracts, particularly thermal and oxidative degradation. Nanoparticles were also reported as a potent antimicrobial agent incorporated with polymers [fig_ref] Figure 4: Active packaging with various active agents [/fig_ref]. With their small molecule size, large surface area, and reactivity via the hydroxyl group, nanoparticles are easy to insert into a polymer [bib_ref] Antimicrobial packaging efficiency of ZnO-SiO 2 nanocomposites infused into PVA/CS film for..., Al-Tayyar [/bib_ref]. Silicon dioxide (SiO2) [bib_ref] Characterisations of cassava starch and poly(butylene adipate-co-terephthalate) blown film with silicon dioxide..., Phothisarattana [/bib_ref] , zinc oxide (ZnO) [bib_ref] Blown film extrusion of PBAT/TPS/ZnO nanocomposites for shelf-life extension of meat packaging, Phothisarattana [/bib_ref] , and titanium dioxide (TiO2) [bib_ref] Butylene Adipate-Co-Terephthalate) and Thermoplastic Starch-Blended TiO 2 Nanocomposite Blown Films as Functional..., Phothisarattana [/bib_ref] are common nanoparticles used in food applications. Al-Tayyar et al. (2020) [bib_ref] Antimicrobial packaging efficiency of ZnO-SiO 2 nanocomposites infused into PVA/CS film for..., Al-Tayyar [/bib_ref] reviewed the active functions of nanoparticles. They found that the semiconductive properties of nanoparticles were able to generate reactive oxygen species (ROS) and that Zn 2+ antimicrobial ions were provided by nanoparticles in polar media. The nanoparticles interacted with carboxyl and amine groups on the bacterial membrane surface [bib_ref] Chapter 15-Microbially synthesized nanoparticles as next generation antimicrobials: Scope and applications, Busi [/bib_ref]. Another possibility was oxidative damage of the bacterial cell surface membrane by hydrogen peroxide by nanoparticles. prepared active films from gelatin and polyvinyl alcohol containing SiO, ZnO, TiO, and copper oxide (CuO). They found that films with these nanoparticles significantly reduced the total viable Shewanella putrefaciens, Enterobacteriaceae, and Pseudomonas spp. counted in shrimp and L. monocytogenes, S. aureus, and E. coli inoculated from shrimp. Moreover, polyvinyl alcohol and gelatin film containing ZnO and TiO2 extended the shelf-life of shrimp by up to 6 days longer than the control [bib_ref] Polyvinyl alcohol/gelatin nanocomposite containing ZnO, TiO 2 or ZnO/TiO 2 nanoparticles doped..., Azizi-Lalabadi [/bib_ref].
Several patents have been registered regarding active packaging, including beta-cyclodextrin and essential oil inclusion compounded in polyvinyl alcohol. They found no mold in the film after 18 days of incubation under 90% RH at 28 °C. A patent on multilayer active packaging was registered by Roberto et al. (2014) [bib_ref] Material for Packaging Fresh Food of Animal Origin Inhibiting the Development of..., Roberta [/bib_ref]. They developed a Nanoparticles were also reported as a potent antimicrobial agent incorporated with polymers [fig_ref] Figure 4: Active packaging with various active agents [/fig_ref]. With their small molecule size, large surface area, and reactivity via the hydroxyl group, nanoparticles are easy to insert into a polymer [bib_ref] Antimicrobial packaging efficiency of ZnO-SiO 2 nanocomposites infused into PVA/CS film for..., Al-Tayyar [/bib_ref]. Silicon dioxide (SiO 2 ) [bib_ref] Characterisations of cassava starch and poly(butylene adipate-co-terephthalate) blown film with silicon dioxide..., Phothisarattana [/bib_ref] , zinc oxide (ZnO) [bib_ref] Blown film extrusion of PBAT/TPS/ZnO nanocomposites for shelf-life extension of meat packaging, Phothisarattana [/bib_ref] , and titanium dioxide (TiO 2 ) [bib_ref] Butylene Adipate-Co-Terephthalate) and Thermoplastic Starch-Blended TiO 2 Nanocomposite Blown Films as Functional..., Phothisarattana [/bib_ref] are common nanoparticles used in food applications. Al-Tayyar et al. (2020) [bib_ref] Antimicrobial packaging efficiency of ZnO-SiO 2 nanocomposites infused into PVA/CS film for..., Al-Tayyar [/bib_ref] reviewed the active functions of nanoparticles. They found that the semiconductive properties of nanoparticles were able to generate reactive oxygen species (ROS) and that Zn 2+ antimicrobial ions were provided by nanoparticles in polar media. The nanoparticles interacted with carboxyl and amine groups on the bacterial membrane surface [bib_ref] Chapter 15-Microbially synthesized nanoparticles as next generation antimicrobials: Scope and applications, Busi [/bib_ref]. Another possibility was oxidative damage of the bacterial cell surface membrane by hydrogen peroxide by nanoparticles. prepared active films from gelatin and polyvinyl alcohol containing SiO, ZnO, TiO, and copper oxide (CuO). They found that films with these nanoparticles significantly reduced the total viable Shewanella putrefaciens, Enterobacteriaceae, and Pseudomonas spp. counted in shrimp and L. monocytogenes, S. aureus, and E. coli inoculated from shrimp. Moreover, polyvinyl alcohol and gelatin film containing ZnO and TiO 2 extended the shelf-life of shrimp by up to 6 days longer than the control [bib_ref] Polyvinyl alcohol/gelatin nanocomposite containing ZnO, TiO 2 or ZnO/TiO 2 nanoparticles doped..., Azizi-Lalabadi [/bib_ref].
Several patents have been registered regarding active packaging, including betacyclodextrin and essential oil inclusion compounded in polyvinyl alcohol. They found no mold in the film after 18 days of incubation under 90% RH at 28 - C. A patent on multilayer active packaging was registered by Roberto et al. (2014) [bib_ref] Material for Packaging Fresh Food of Animal Origin Inhibiting the Development of..., Roberta [/bib_ref]. They developed a multilayer film consisting of paper (outer layer), polyethylene, or biodegradable plastic materials, an optional third metallic layer with an adhesive layer in the interposition, and an inner layer containing Rosmarinus officinalis, Citrus limon, or Vitis vinivera in contact with the packaged food. Results showed that the biogenic amine of fish samples was inhibited by up to 45%, 36%, and 39% after 2, 4, and 7 days of incubation, respectively.developed another patent in active packaging as a dual function O 2 absorbing and CO 2 emission pouch for fishery products. Sodium bicarbonate, citric acid, and iron powder effectively absorbed O 2 with less than 1% concentration remaining within 24 h, while CO 2 remained high (80%) within 48 h. They explained that O 2 absorption was based on the principle of iron oxidation, whereas CO 2 emission came from the reaction between bicarbonate compounds and acids from citric acid releasing carbon dioxide along with the formation of other compounds. Furthermore, a patent for active amine scavenging for fish packaging was registered by [bib_ref] Active Amine Scavenging Film for Fresh Fish Packaging, Karlheinz [/bib_ref] [bib_ref] Active Amine Scavenging Film for Fresh Fish Packaging, Karlheinz [/bib_ref] , consisting of at least one layer of ethylene with an unsaturated carboxylic acid group neutralized by a metal ion capable of adsorbing an undesirous amine from the headspace of the package. Active packaging is designed to have an active compound/system with the purpose of preventing deterioration factors and extending the shelf-life of food products. Active packaging has started to be commercialized; however, the production volume is still low due to safety and regulation. The low stability of active agents also becomes a major factor in causing the short shelf-life of active packaging. Future research focuses on protecting the active agent from damage such as oxidative and undesirable release amounts by microencapsulation as control release, lamination, or multilayer consisting of the high barrier layer.
## Polymeric sensors for seafood
Intelligent packaging is defined as a system equipped with tools that are sensitive to environmental changes and continue to inform the users about the changes, while also providing information related to the function and properties of the packaged foods [bib_ref] Active Packaging of Food, Day [/bib_ref]. Intelligent packaging facilitates decision-making, warning of potential risks, food safety, and freshness [bib_ref] Monitoring the quality of perishable foods: Opportunities for intelligent packaging, Heising [/bib_ref]. Intelligent packaging can be divided into time-temperature indicators or gas indicators, biological sensors, humidity indicators, barcoding techniques, and radio frequency identification systems [bib_ref] A critical review on intelligent and active packaging in the food industry:..., Firouz [/bib_ref]. Food deteriorates during storage due to microbial activity, producing metabolites such as volatile amines and organic acids, which react and are then detected by the sensor-equipped intelligent packaging system that makes significant visible changes as a signal to assess the freshness level of the product [bib_ref] Active and intelligent biodegradable packaging films using food and food waste-derived bioactive..., Bhargava [/bib_ref]. Bioactive compounds extracted from plants have been widely investigated as freshness indicators in intelligent packaging. [bib_ref] Active and intelligent biodegradable packaging films using food and food waste-derived bioactive..., Bhargava [/bib_ref] [bib_ref] Active and intelligent biodegradable packaging films using food and food waste-derived bioactive..., Bhargava [/bib_ref] compiled freshness indicators such as anthocyanin, curcumin, betalains, chlorophyll, carotenoids, tannins, quercetins, and brazilin, while chemical-based sensors incorporated with polymers were developed, including alizarin, pelargonidin [bib_ref] Intelligent colorimetric indicator film based on bacterial cellulose and pelargonidin dye to..., Liu [/bib_ref] , cyanidin-3-glucoside [bib_ref] Preparation and characterization of intelligent packaging film for visual inspection of tilapia..., Shi [/bib_ref] , and rhodamine B [bib_ref] On-package ratiometric fluorescent sensing label based on AIE polymers for real-time and..., Liu [/bib_ref].
Biological/bioactive sensors are the most common intelligent packaging applied to seafood products, with metabolism outcomes such as ammonia, volatile nitrogenous compounds, and acidity (pH value) (Table 2 and [fig_ref] Figure 5: Mechanism of color change in pH−sensitive anthocyanins to monitor shrimp freshness [/fig_ref]. [bib_ref] A pH-intelligent response fish packaging film: Konjac glucomannan/carboxymethyl cellulose/blackcurrant anthocyanin antibacterial composite..., You [/bib_ref] explained that anthocyanin loses the cations on the original oxygen atoms in C-ring and decreased the original salt ion concentration, resulting in a yellow color, and gradually turned blue in response to an increase in pH. Curcumin is susceptible to humidity changes and is suitable for use as a humidity indicator. Higher humidity promoted contact between ammonia and water molecules, resulting in ammonium cations and hydroxide, leading to a more alkaline environment and accelerating red color formation. Metal-based colorimetric indicators such as gold, silver, or copper nanoparticles were also reported to be able to detect meat spoilage. Metal colorimetric indicators are suitable as volatile sulfuric compound detectors due to the high affinity between bonded metallic cations and sulfide anions. However, metal-based indicators, particularly Cu, were reported to have high stability in pH changes or no changes were found in variant pH values [bib_ref] A colorimetric indicator based on copper nanoparticles for volatile sulfur compounds to..., Teymouri [/bib_ref]. They also found that copper nanoparticles changed from dark yellow to red and finally black in response to an increase in hydrogen sulfide concentration in the fish. Fish spoilage results from the reaction of the volatile sulfur gas hydrogen sulfide with metals.
Common derivatives of anthocyanin include delphinidin, peonidin, pelargonidin, cyanidin, petunidin, and malvidin [bib_ref] Intelligent colorimetric indicator film based on bacterial cellulose and pelargonidin dye to..., Liu [/bib_ref]. Pelargonidin was applied in intelligent packaging of tilapia fillets by [bib_ref] Intelligent colorimetric indicator film based on bacterial cellulose and pelargonidin dye to..., Liu [/bib_ref]. They found that the orange-red color originating from the flavylium cation was reduced due to the formation of quinone, resulting in carbinol pseudobase formation. Absorbance increased with increasing pH from 7 to 10 as a result of the redshift phenomenon that commonly occurs in anthocyanin. Anthocyanin as cyanidin-3-glucoside was also applied in intelligent packaging for tilapia fillets. The red color at pH < 4 originated from the flavonoid cation and then turned colorless in the pH range of 5-6 due to the conversion of the flavonoid cation to carbinol pseudobase and chalcone, while cyanidin shifted to quinonoidal anhydrobase with a purple/blue color at pH 6-8 [bib_ref] Preparation and characterization of intelligent packaging film for visual inspection of tilapia..., Shi [/bib_ref].
Dye-based colorimetric indicators have been used as potential sensors. Alizarin contains a phenolic hydroxyl group that can easily deproteinize in neutral pH, resulting in color changes from yellow to red, while the second deproteination of the phenolic hydroxyl group occurs in alkaline pH [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref] , resulting in a purplish red color. [bib_ref] On-package ratiometric fluorescent sensing label based on AIE polymers for real-time and..., Liu [/bib_ref] found that the AIE-stimuli-responsive polymer tetraphenylethylene/polymethacrylic acid (TPE/PMMA) with rhodamine B acted as an acidity-dependent indicator that changed polymer conformation by ionization of the carboxylic acid group, thereby exhibiting a change in fluorescence intensity. This indicator was more sensitive in response to amines (trimethylamine and dimethylamine) than ammonia in salmon. Intelligent packaging based on poly and polystyrene sulfonate as flexible ammonia gas sensors for fish meat by coating them onto metal electrodes was patented by. The response value increased with increasing ammonia up to 94%, with humidity reaching 85%. Intelligent packaging now monitors food consumption safety using sensors that can detect various deterioration factors. The natural-based biosensor has a high potential as a dual-function biosensor as well as an antimicrobial agent to monitor the freshness and preserve the quality of seafood products. Color change from pale-blue to yellow-green in response to shrimp spoilage.
[112]
Malva sylvestris anthocyanins PLA, polyethylene glycol (PEG), and calcium bentonite (CB) Shrimp, fish roe, meat and chicken fillet pH-sensitive Color change from light red (pH 2) to green (pH 11) and more sensitive to shrimp and fish roe rather than chicken and meat correlated to TVB-N value.
[113]
Anthocyanin-rich purple potato extract 2,2 6,6tetramethylpiperidine-1-oxyradical, oxidized bacterial cellulose and thymol shrimp
Volatile ammonia detector
Color changed to dark purple in response to shrimp spoilage after 32 h.
[114]
Curcumin nano capsules Soy protein isolate and cellulose nanocrystals shrimp pH-sensitive, ammonia detector, anti-radical scavenging
The yellow (pH 3-7) color becomes reddish-brown (pH 8-11) in response to TVB-N changes in shrimp during storage.
[103]
Curcumin Corn starch, polyvinyl alcohol
## Pangasius bocourti
(catfish) pH-sensitive
Color changed from yellow to orange in the range acidic (pH 3) to neutral (pH 7), and turned to red at pH 8-10 in response to TVB-N changes. [bib_ref] Corn starch/polyvinyl alcohol based films incorporated with curcumin-loaded Pickering emulsion for application..., Liu [/bib_ref] Copper nanoparticles Salmon trout Volatile sulfur compound
The color of white, yellow and brown as a colorimetric indicator related to fresh, semi-fresh, and spoiled salmon, respectively.
[104]
Alizarin Gelatin and lavender essential oil shrimp pH-sensitive and ammonia detector, antimicrobial activity
The color changed from yellow to red-brown in response to increasing TVB-N in shrimp after 3 days of storage.
[98]
Donor-π-acceptor (D-π-A) Cellulose Fish Amine detector
The color changed from red to yellow in response to putrid fish, while the emission changed to bright cyan. [bib_ref] A dual-channel indicator of fish spoilage based on a D-π-A luminogen serving..., Duan [/bib_ref] Pelargonidin Bacterial cellulose Tilapia fillet pH-sensitive
The color change from red to colorless in response to the TVB-N value and sensory changes of tilapia fillets.
[99]
Cyanicin-3-glucoside Bacterial cellulose Tilapia fillets pH-sensitive
Color changed from red to green in pH range 3-10. During application, rose-red fresh tilapia turned to purple (acceptable) and lavender (spoilage). Color change from pink (fresh) to dark blue (spoilage) was linearly correlated with TVB-N, indicating that the sensing label was feasible and non-destructive for quantitative TVB-N.. The response value increased with increasing ammonia up to 94%, with humidity reaching 85%. Intelligent packaging now monitors food consumption safety using sensors that can detect various deterioration factors. The natural-based biosensor has a high potential as a dual-function biosensor as well as an antimicrobial agent to monitor the freshness and preserve the quality of seafood products.
## Other combined technologies with polymeric packaging to preserve seafood quality and freshness
## Modified atmosphere packaging for seafood
Modified atmosphere packaging (MAP) controls the gaseous atmosphere surrounding the food inside its packaging using specific polymeric packaging materials with appropriate levels and gas barriers to maintain gas transfer from and to the environment for food preservation [fig_ref] Figure 6: Application of various gases in modified atmosphere packaging with active absorbance in... [/fig_ref] [bib_ref] Modified atmosphere packaging and other active packaging systems for food, beverages and..., Embleni [/bib_ref]. MAP is a non-thermal technology with multiple advantages for extending seafood shelf-life [bib_ref] Inhibitory effect of a quercetin-based soaking formulation and modified atmospheric packaging (MAP)..., Qian [/bib_ref]. Lipid oxidation, protein degradation, microbial growth, and melanosis can be prevented by providing poor O 2 , rich CO 2 [bib_ref] Controlled atmosphere as coadjuvant to chilled storage for prevention of melanosis in..., Martinez-Alvarez [/bib_ref] , and argon (Ar). Gas concentration inside the MAP changes during storage due to gas-food interaction. Oxygen decreased due to consumption by microorganisms, while CO 2 decreased due to muscle absorption by seafood or meat [bib_ref] Inhibition of melanosis and microbial growth in Pacific white shrimp (Litopenaeus vannamei)..., Kimbuathong [/bib_ref] [bib_ref] Inhibitory effect of a quercetin-based soaking formulation and modified atmospheric packaging (MAP)..., Qian [/bib_ref]. A reduction of CO 2 also occurred due to gas transfer from the packaging into the environment. They also found that higher O 2 concentrations showed greater CO 2 losses. However, the phenomenon of gas absorption activity requires further detailed investigation. and horse mackerel. This finding indicated that hydrogen incorporation gave advantages to biogenic amine inhibition due to the presence of antioxidant properties. Moreover, hydrogen gas is a permitted food additive with the code of E949. A patent about the application of MAP on fisheries products was registered by Jing et al. (2020)for gas concentrations of 40-60% CO2, 0-10% O2, and 30-40 N2. They found that a gas composition of 60:5:35 of CO2:O2:N2 with a ratio between gas volume and puffer fish of 3:1 was optimal for packaging in terms of puffer fish quality. Packaging systems with low O2 and various concentrations of CO2, N2, Ar or H effectively preserved seafood quality and prevented lipid oxidation and biogenic amine production. Seafood products have different nutritional values, especially protein and lipid profiles, required specific gas concentration, gas permeability of packaging, and environmental conditions, to optimize MAP efficacy on seafood products. Modelling of MAP system for handling and storage which in turn influence shelf life of foods has been previously reviewed [bib_ref] Modelling approaches for designing and evaluating the performance of modified atmosphere packaging..., Belay [/bib_ref]. Future research needs to be focused on investigating, calculating, or modeling the appropriate MAP for seafood products with a specific characteristic. CO2:O2:N2 ratio 1) Increased CO2 to 60-80% effectively reduced microbial growth, melanosis, and lipid oxidation. O2 de- CO 2 is the mainstay gas used in MAP due to its ability to maintain product quality, particularly limited microbial growth. However, CO 2 is generally highly soluble in meat, particularly in muscle or fatty tissue, and is influenced by pH, lipid type, and lipid content. [bib_ref] Effect of salt on CO2 solubility in salmon (Salmo salar L.) stored..., Abel [/bib_ref] applied NaCl in salmon to reduce CO 2 solubility in MAP. They found that increasing NaCl concentration effectively reduced CO 2 solubility in salmon meat, due to changes in water fraction affected by increase in electrolyte concentration, leading to salting-out phenomena and resulting in inbound and unfree water content no longer available for CO 2 uptake.
The combination of MAP with preservation methods, such as plant extract, cinnamic acid [bib_ref] Inhibitory effect of a quercetin-based soaking formulation and modified atmospheric packaging (MAP)..., Qian [/bib_ref] , nisin, nitric oxide, ozonated or chlorinated water, and ε-polylysine [fig_ref] Table 3: Previous research on modified atmosphere packaging for seafood products [/fig_ref] , was reported to be more effective in retarding seafood deterioration and significantly delayed post-mortem metabolism rate such as ATPase activity, sarcoplasmic and myofibril degradation, lipid oxidation, biogenic amine and microbial growth.
The incorporation of hydrogen gas into MAP, called reducing atmosphere packaging (RAP), was investigated by [bib_ref] The effects of hydrogen incorporation in modified atmosphere packaging on the formation..., Sezer [/bib_ref]. The use of RAP with 4% hydrogen significantly inhibited the formation of biogenic amines, namely heterocyclic, aromatic, and aliphatic di-amines (histamine, tyramine, putrescine, cadaverine) in rainbow trout and horse mackerel. This finding indicated that hydrogen incorporation gave advantages to biogenic amine inhibition due to the presence of antioxidant properties. Moreover, hydrogen gas is a permitted food additive with the code of E949. A patent about the application of MAP on fisheries products was registered byfor gas concentrations of 40-60% CO 2 , 0-10% O 2 , and 30-40 N 2 . They found that a gas composition of 60:5:35 of CO 2 :O 2 :N 2 with a ratio between gas volume and puffer fish of 3:1 was optimal for packaging in terms of puffer fish quality. Packaging systems with low O 2 and various concentrations of CO 2 , N 2 , Ar or H effectively preserved seafood quality and prevented lipid oxidation and biogenic amine production. Seafood products have different nutritional values, especially protein and lipid profiles, required specific gas concentration, gas permeability of packaging, and environmental conditions, to optimize MAP efficacy on seafood products. Modelling of MAP system for handling and storage which in turn influence shelf life of foods has been previously reviewed [bib_ref] Modelling approaches for designing and evaluating the performance of modified atmosphere packaging..., Belay [/bib_ref]. Future research needs to be focused on investigating, calculating, or modeling the appropriate MAP for seafood products with a specific characteristic. ε-polylysine, chitosan and sodium alginate coatings and MAP delayed myofibril oxidation, preserved Ca 2+ -ATPase activity, α-helix and β-sheet contents, and stabilized tertiary structure during cold storage.
## Thermal insulation packaging
Maintaining the freshness of seafood products is important, particularly during handling and distribution. The cold chain system is the main stage for the fresh seafood product supply chain until it reaches the consumer safely. Low temperatures are mostly obtained by ice (flake ice, tube ice, block ice, crushed ice). However, packaging equipped with thermal insulation is necessary to stop the ice from melting during storage and distribution. Expanded polystyrene, polyurethane foam, and expanded polyurethane foam are common thermal insulators used for cold chain distribution. They contain many pores filled with air and have very poor thermal conduction properties that inhibit the flow of heat energy [fig_ref] Figure 7: Insulator box containing numerous pores, giving low thermal conductivity to maintain the... [/fig_ref]. [bib_ref] The Effect of Cool Box Insulator Type on the Temperature Characteristics and..., Sormin [/bib_ref] [bib_ref] The Effect of Cool Box Insulator Type on the Temperature Characteristics and..., Sormin [/bib_ref] reported that Styrofoam had better cool temperature maintenance, resulting in a faster cooling rate of fish (0.13 - C/min) compared to the cool box insulator "ela sago" (0.045 - C/min). Patents for insulator boxes for fisheries' products are listed in [fig_ref] Table 4: Cont [/fig_ref].
Nowadays, environmentally friendly thermal insulators have attracted research attention to replace conventional material-based products in cool chain systems. [bib_ref] The Development of Thermal Insulation System on Fish Cargo Hold by Utilizing..., Ariany [/bib_ref] [bib_ref] The Development of Thermal Insulation System on Fish Cargo Hold by Utilizing..., Ariany [/bib_ref] found that cellulose-based insulators with low thermal conductivity could be applied to fishery products to reduce the magnitude of heat flux on the fish to 61.31% of the maximum heat flux, although the system required further improvements, particularly the thickness. Thermal insulation-equipped packaging prepared from feathers had low thermal conductivity compared with expanded polystyrene. The feathers were extremely lightweight and environmentally friendly, with good mechanical properties and high thermal insulation due to the hollow shaft structure. Cardboard-based thermal insulators consisting of double E-fluted corrugated board sandwiches between PE laminated and metalized E-fluted cardboard had similar thermal insulation properties to commercial expanded polystyrene; however, the complex structure of cardboard-based insulator thermal insulators requires further investigation for foldability and manufacturability performance [bib_ref] Thermal insulation requirements and new cardboard packaging for chilled seafood exports, Navaranjan [/bib_ref]. Rigid packaging that can maintain low temperatures is required in the seafood supply chain to preserve product quality and prevent loss. Natural-based thermal insulators have started to be investigated to replace conventional polymers. Future research needs to investigate the durability of the natural-based insulator to meet the requirement for an insulator box for distribution and storage. It could be conducted by simulation with heat transfer and distribution, shock, and vibration taken into consideration. larly the thickness. Thermal insulation-equipped packaging prepared from feathers had low thermal conductivity compared with expanded polystyrene. The feathers were extremely lightweight and environmentally friendly, with good mechanical properties and high thermal insulation due to the hollow shaft structure. Cardboard-based thermal insulators consisting of double E-fluted corrugated board sandwiches between PE laminated and metalized E-fluted cardboard had similar thermal insulation properties to commercial expanded polystyrene; however, the complex structure of cardboard-based insulator thermal insulators requires further investigation for foldability and manufacturability performance [bib_ref] Thermal insulation requirements and new cardboard packaging for chilled seafood exports, Navaranjan [/bib_ref]. Rigid packaging that can maintain low temperatures is required in the seafood supply chain to preserve product quality and prevent loss. Natural-based thermal insulators have started to be investigated to replace conventional polymers. Future research needs to investigate the durability of the natural-based insulator to meet the requirement for an insulator box for distribution and storage. It could be conducted by simulation with heat transfer and distribution, shock, and vibration taken into consideration. [fig_ref] Table 4: Cont [/fig_ref]. Patents related to thermal insulators and box packaging for fishery products.
## Patent title invention details application patent source
Containing bag for fresh fish A bag contained waterproof material at the outer most portion, equipped with outer heat insulating material made from glass wool and nonwoven fabric. The cooling system, prepared from nitrogen gas sealed in the hollow layer reached cooling temperature of −1 - C to −5 - C. The mouth of the bag was equipped with fastener material (Velcro fastener or waterproof fastener).
Transportation and storage bag for high-economical commodity, particularly tuna and camellia in cold insulation state.
JP1995243741Portable cool box
The component of the cooling box was hollow with a rectangular parallelepiped case body, a lid, cold insulator, and support mechanism. The insulator material contained highly water-absorbing polymer sodium polyacrylate sealed in a hollow plastic case to maintain high water absorbency by forming a gel structure.
Portable cool box for retaining the freshness of fish or other objects which can be easily used in fishing or camping.
JP2013085550Intermediate dish of foamed fish box
The overlapped box containing two outer boxes and an upper layer made from cold resistant olefin sheet-based raw material. The depth of the main container was adjusted by calculating the amount of ice required. An edge over the upper layer created an overlapped insulator box.
Container for fresh fish at processing site or for transportation.
JP2018135150Non-direct contact cooling system using cold air for fresh products. KR1020180112388Corrugated carton suitable for transportation and packaging of fish tank A corrugated box for transporting fish tanks equipped with an inner and outer groove on the foam base. The inner groove is equipped with a rubber sleeve and the fish tank is placed on the outside of the rubber sleeve on the outer groove. Between the fish tank and rubber sleeve, rubber supporting feet are attached with a soft rubber cushion, EPE pads protect the fish tank and are reusable.
Corrugated paper box for fish tank transportation and packaging.
CN209427201 [bib_ref] Corrugated Carton Suitable for Transportation and Packaging of Fish Tank. China Patent..., Shang [/bib_ref] Circular fish tank buffering packaging box formed by one piece of paper A fish box formed by one piece of paper packaging as the main body, with linings and a handle, comprising a bottom plate, four end plates, a top plate, four lining plates and a bottom plate. The handle comprises two triangular top plates. Prevents fish transportation damage with reduced amounts of foam, reduced production cost, and material saving.
Paper box for fish transportation to minimize damage. CN209720191Packaging box for refrigerating fresh fish meat A box with an inside structure, including an inflatable bag with an inflatable interface on the top, foam plastic board, absorbent paper made from wood pulp layer and a non-woven layer on both sides, and a wave-shaped convex strip in the inner wall. A sealing gasket is attached between the box cover and box body, with a support pad placed in the bottom of the box.
Packaging box for refrigerating fresh fish meat. CN215246117
## Conclusions and future perspective
This paper highlighted seafood's post-harvest deterioration and reviewed recent advances in polymeric packaging technology. Novel technology regarding polymeric active packaging, intelligent packaging, MAP, as well as rigid insulation packaging for seafood products has been developed and patented. Furthermore, the removal of the smell of seafood products related to their chemical compounds has also become a challenge in terms of packaging technology. New active compounds have been successfully used to preserve product quality, such as essential oils, nanoparticles, and plant extracts, while intelligent bioactive sensors, such as anthocyanin and metal and dye-based sensors, have been used to monitor the freshness of seafood. Biodegradable insulator packaging for seafood is at the novel development stage. From these recent results, biodegradable and sustainable polymers will be increasingly investigated for packaging applications in the future. Functionalized materials as well as improve the stability and packaging performance of biodegradable materials for seafood packaging applications will be explored by global researchers. Chemical and physical modification of polymers, lamination, coating, and blending with other sustainable materials are the technologies to improve water resistance and performance for seafood packaging. Further research is required to scale-up the polymeric-based packaging to industrial market production to meet the needs of the fishery market and promote a circular economy in the post-pandemic situation.
# Data availability statement:
The data presented in this study are available on request from the corresponding author.
[fig] Figure 1: Interrelation of total viable count on changes in (A) melanosis, (B) firmn methylamine content of shrimp under a modified atmosphere during 12 h of storage duced with permission from Kimbuathong et al., 2019 [10]). [/fig]
[fig] Figure 2: Melanosis formation through polyphenol oxidation and its prevention via microbial inhibition and O2 removal using packaging technology. [/fig]
[fig] Figure 3: Shrimp packaged in PBAT/PLA film containing 6% α-terpineol, carvacrol, and citral during 12 days of storage at 4 °C. [/fig]
[fig] Figure 4: Active packaging with various active agents (essential oils, plant extracts, and nanoparticles) and its application for seafood products. [/fig]
[fig] Figure 5: Mechanism of color change in pH−sensitive anthocyanins to monitor shrimp freshness (adapted from Zhao et al. 2022[106] and You et al. 2000[102]). [/fig]
[fig] Polymers 2022 ,: 14, x FOR PEER REVIEW 17 of 27 [/fig]
[fig] Figure 6: Application of various gases in modified atmosphere packaging with active absorbance in salmon product. [/fig]
[fig] Figure 7: Insulator box containing numerous pores, giving low thermal conductivity to maintain the cold temperature of seafood products. [/fig]
[fig] Author: Contributions: Conceptualization, Y.L. and N.H.; methodology, Y.L. and N.H.; formal analysis, Y.L. and N.H.; investigation, Y.L. and N.H.; writing-original draft preparation, Y.L. and N.H.; writing-review and editing, Y.L., V.C., N.B., W.J., S.S.-t., C.R., W.P.C., S.C., A.S., K.P., P.W. and N.H.; supervision, N.H.; funding acquisition, N.H. All authors have read and agreed to the published version of the manuscript. Funding: Kasetsart University Research and Development Institute (KURDI) FF(KU) 25.64. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. [/fig]
[table] Table 1: Previous recent research on active packaging for seafood products. [/table]
[table] Table 2: Related research on intelligent packaging for seafood products. [/table]
[table] Table 3: Previous research on modified atmosphere packaging for seafood products. [/table]
[table] Table 4: Cont. Cooling packing box A Styrofoam-based insulator box equipped with an intermediate lid attached to the upper end of the main body. The intermediate lid had storage space for frozen food products in the middle, with holes to allow cold air to pass through the storage space in the intermediate lid. [/table]
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Effect of percutaneous assisted approach on functional rehabilitation for total hip replacement compared to anterolateral approach: study protocol for a randomized controlled trial
Background:The anterolateral approach is a commonly used technique for total hip replacement. It requires the detachment of a large part of the gluteus medius muscle. However, it is known that this muscle has a great impact on hip stability. Using the percutaneous assisted approach the damage to the gluteus medius can be limited. The purpose of this study is to compare the effect of the percutaneous assisted approach with the anterolateral approach on postoperative functional outcome. Methods/Design: This study uses a prospective, randomized, parallel-group design with blinded assessment and unblinded treatment to compare the percutaneous assisted approach with the anterolateral approach in total hip replacement surgery. The postoperative results of patients operated on using the percutaneous assisted approach will be compared with those of patients operated on using the anterolateral approach. Prior to surgery patients will undergo baseline measurements. These will consist of gluteus medius measurements (surface-electromyography, strength measurements of abductors and quadriceps and the Trendelenburg test), questionnaires (Oxford Hip Score and 36-item Short Form Health Survey) and functional measures (the Timed Get-Up-and-Go test, Five times Sit-to-Stand test and Six-Minute Walk test). These measurements will be repeated four and 12 weeks after surgery. After surgery both groups will receive usual care.Discussion:The gluteus medius is the main stabilizer of the hip joint. Therefore, we assume that functional outcome and gluteus medius function of patients after the percutaneous assisted approach will be better than after the anterolateral approach.
# Background
Total hip replacement (THR) is one of the most common orthopedic operations. Based on implant sales figures in the Benelux region of Europe (Belgium, The Netherlands and Luxembourg), the number of THR surgeries performed in 2001 has been estimated at 40,000 (1.52 THR per 1,000 inhabitants per year) [bib_ref] European hip arthroplasty: a survey in university hospitals, Scheerlinck [/bib_ref]. Every THR has an estimated cost of €9000 on averageand the length of hospital stay and postoperative care are contributing factors to these costs. Improvements in health-related quality of life (HRQoL) after surgery are reported. However, significant differences between patients and age-and sex-matched controls are noted. An example of this is the scores of the 36-item Short Form Health Survey (SF-36) subscales physical functioning (PF) (P = 0.01), role physical (RP) (P = 0.05) and vitality (P = 0.05) [bib_ref] Patient relevant outcome 7 years after total hip replacement for OA -a..., Nilsdotter [/bib_ref]. These lower scores suggest that although patients demonstrate full recovery on psychosocial and pain scales, a deficit in physical functioning remains. This lack of functional recovery has also been found in a review by Vissers et al. stating that physical functioning in patients (measured by self-perceived function, clinical measures and actual daily activity) had only recovered to about 80% of that of controls [bib_ref] Recovery of physical functioning after total hip arthroplasty: systematic review and meta-analysis..., Vissers [/bib_ref]. Furthermore, Rasch et al. found persisting muscle atrophy two years after surgery in the acting muscles around the hip [bib_ref] Persisting muscle atrophy two years after replacement of the hip, Rasch [/bib_ref]. This was caused by loss of muscle volume as well as infiltration of fat tissue.
Postoperative outcome can be influenced by the applied surgical procedure. Two approaches are used to perform THR, with the posterior approach being most commonly used in the USA. Leg lengthening and an increased dislocation rate are the downsides [bib_ref] Effect of surgical approach for total hip replacement on hip function using..., Edmunds [/bib_ref] [bib_ref] A clinical comparison of the anterolateral and posterolateral approaches to the hip, Ritter [/bib_ref] [bib_ref] A comparison of the anterolateral, transtrochanteric, and posterior surgical approaches in primary..., Vicar [/bib_ref]. A popular technique in Europe is the anterolateral approach (AA). By using this, a large part of the gluteus medius muscle is released to obtain good access to the acetabulum. There are little or no dislocations reported [bib_ref] A comparison of the posterolateral and anterolateral approaches to total hip arthroplasty, Roberts [/bib_ref] , but limping during the first three months is a wellknown problem [bib_ref] Effect of surgical approach for total hip replacement on hip function using..., Edmunds [/bib_ref] [bib_ref] A clinical comparison of the anterolateral and posterolateral approaches to the hip, Ritter [/bib_ref].
Muscles are major contributors to hip contact force, with gravitational and centrifugal forces combined contributing less than 5%. One of the most important muscles for this is the gluteus medius [bib_ref] Contributions of individual muscles to hip joint contact force in normal walking, Correa [/bib_ref]. It is responsible for stabilizing the femoral neck in the acetabulum during the different stages of gait [bib_ref] A: The functional anatomy of hip abductors, Al-Hayani [/bib_ref] [bib_ref] The functional anatomy of tensor fasciae latae and gluteus medius and minimus, Gottschalk [/bib_ref]. This makes it a very important muscle for normal functioning and gait. The gluteus medius is often already weakened before surgery in patients with osteoarthritis of the hip [bib_ref] Comparison of gluteus medius muscle activity during functional tasks in individuals with..., Dwyer [/bib_ref]. Since the AA detaches approximately two thirds of the gluteus medius, it affects normal function. Consequently, it can be assumed that interventions sparing the gluteus medius are probably beneficial for patients' postoperative functional outcome.
Damage to the gluteus muscle can be limited using the percutaneous assisted approach (PAA). In this technique, a second small incision (1 cm) at the anterior border of the femur is made. A canula is placed underneath the muscle and used to pass the reamers in the direction of the acetabulum [fig_ref] Figure 1: Percutaneous assisted approach, side view [/fig_ref]. There is no need to enlarge the skin incision or to release more muscle insertion to achieve good working access to the acetabulum. There are two advantages of this procedure: sparing of the gluteus medius muscle and safe access to the acetabulum to obtain perfect positioning of the implants.
Interventions sparing the gluteus medius are probably beneficial for patients' postoperative functional outcome. The purpose of this study is to evaluate the effect of the PAA on postoperative functional outcome compared to the AA.
# Methods/design
## Study design
This study uses a prospective, randomized, parallel-group design with blinded assessment and unblinded treatment to compare the PAA with the AA in THR surgery.
# Ethical approval
Full ethical approval has been given by the ethics committee of the Antwerp University Hospital (UZA) and Hospital Network Antwerp (ZNA) (approval number: B300201318915).
## Patient selection
Patients will be recruited at the orthopedics department of the ZNA Middelheim hospital in Antwerp, Belgium. Patients with a hip complaint for which THR is indicated are screened for eligibility by a specialized surgeon (PM). Eligible patients will be between 50 and 80-years-old and suffer from unilateral hip arthritis or avascular necrosis in need of THR. Comorbidities affecting functional outcome will be used as exclusion criteria. These consist of symptomatic lumbar pathology or those in need of surgery or intervention on the ipsilateral knee and/or ankle or foot. Additionally, neurological disorders, according to the International Classification of Diseases, such as Parkinson's disease and previous cardiovascular accidents (CVA) are excluded from this study.
Eligible patients who provide informed consent will be sent to the Antwerp University Hospital (UZA) where an appointment for preoperative (baseline) measurements will be made. These consist of gluteus medius measures, HRQoL-questionnaires and functional measures. Patients will be given an information sheet and a written informed consent will be obtained prior to baseline measurements.
## Gluteus medius measures
Surface-electromyography of gluteus medius muscle Patients will perform a single leg stance (SLS) and a maximal voluntary isometric contraction (MVIC) of the gluteus medius during surface-electromyography (SEMG) measurement. Both have shown excellent reliability (SLS intraclass correlation coefficient (ICC) = 0.93; MVIC ICC = 0.98) [bib_ref] Reliability and interpretation of single leg stance and maximum voluntary isometric contraction..., Norcross [/bib_ref]. Electrodes will be positioned 2.5 cm posterior of the middle of the line between the top of the iliac crest and the greater trochanter. This is more posterior than the method of Hermens et al. [bib_ref] Development of recommendations for SEMG sensors and sensor placement procedures, Hermens [/bib_ref]. This decision is based on a cadaver-study by Van Leemput et al. (Van Leemput W, Collier E, Saeys N, Stassijns G: Measuring tensor fasciae latae and gluteus medius with surface EMG: a cadaver-based study. Unpublished). They demonstrated that the distance from the needle tract to the central point of the muscle was significantly less when the electrodes were placed more posterior (P <0.0001). Therefore we prefer this method.
## Quadriceps and abductor muscle strength
Quadriceps and abductor strength will be measured with the microFET 2 hand-held dynamometer (Biometrics Motion nv, Almere, The Netherlands). Measurements with this device are highly reliable, with excellent intrarater reliability (ICC 0.921 to 0.929) and valid, with high correlations with the Timed Get-Up-and-Go test (TGUG) (r = −0.710 to −0.862) and gait speed (r = 0.728 to 0.825) [bib_ref] Reliability and validity of three strength measures obtained from community-dwelling elderly persons, Schaubert [/bib_ref].
For quadriceps force the patient is seated with the knees bent 90°. Resistance will be administered on the ventral side of the lower leg, just proximal of the ankle joint. Abductor force will be measured in a supine position. Resistance will be administered on the lateral side of the upper leg, just proximal of the knee joint. Patients will be asked for a MVIC. The tests will be repeated three times. The mean value of the three measurements will be recorded.
## Trendelenburg test
This test measures abductor force in a functional way. The patient is asked to raise one leg (sound side) and raise the non-stance side of the pelvis as high as possible for 30 seconds. The response is classified as followed [bib_ref] The significance of the Trendelenburg test, Hardcastle [/bib_ref] :
1. Normal: The pelvis on the non-stance side can be lifted maximally for 30 seconds. 2. The pelvis on the non-stance side can be lifted but not maximally 3. The pelvis is elevated but not maintained for 30 seconds 4. No elevating of the pelvis on the non-stance side 5. Drooping of the pelvis 6. Non-valid response: due to hip pain or uncooperative patient
Test-retest reliability coefficients (κ) are found to be greater than 0.75 [bib_ref] Low back pain: clinimetric properties of the Trendelenburg test, active straight leg..., Roussel [/bib_ref].
## Hrqol-questionnaires
All patients will be required to complete the Oxford Hip Score (OHS) and the 36-item Short Form Health Survey (SF-36) questionnaires. The OHS is a disease-specific questionnaire that consists of 12 questions for the evaluation of pain and hip function in relation to various activities. Each question contains five quantifiable answers, leading to a total score that can range from 12 (least problems) to 60 (most problems). For our study population we will use the Dutch version. This version is believed to have excellent test-retest reliability (ICC = 0.97) and a very high correlation with the visual analogue scale score of the hip (r ≥ 0.7) [bib_ref] Oxford Heup Score": the translation and validation of a questionnaire into Dutch..., Gosens [/bib_ref]. Changes in the OHS are also closely related to the patients' satisfaction with their surgery [bib_ref] The value of short and simple measures to assess outcomes for patients..., Fitzpatrick [/bib_ref]. Furthermore, it is very sensitive to change in the first postoperative year [bib_ref] Oxford Heup Score": the translation and validation of a questionnaire into Dutch..., Gosens [/bib_ref].
The SF-36 is a generic questionnaire that contains 36 items measuring health on eight different dimensions. These dimensions cover functional status, wellbeing and overall evaluation of health. It draws attention to broader problems of physical functioning than the OHS [bib_ref] Comparison of measures to assess outcomes in total hip replacement surgery, Dawson [/bib_ref]. The SF-36 is not very sensitive to individual changes, but is effective for measuring group changes, so it becomes a good instrument for scientific research [bib_ref] Magnitude and meaningfulness of change in SF-36 scores in four types of..., Busija [/bib_ref]. Internal-consistency reliability of the Dutch version meets the 0.70 level (Cronbach's alpha) recommended for group comparisons on all scales (range: 0.78 to 0.92). The PF and Bodily Pain (BP) scales even met or exceeded the 0.90 level recommended for individual comparisons [bib_ref] Translation, validation, and norming of the Dutch language version of the SF-36..., Aaronson [/bib_ref].
## Functional measures
All patients will have functional measurements recorded using the TGUG test, the Five Times Sit-to-Stand test (5tSTS) and the Six-Minute Walk (6MWT) test. In the TGUG test, the participant is asked to stand up and walk 3 m, turn around and walk back to the chair and to sit down. The score recorded is the time used to complete the test expressed in seconds [bib_ref] Up & Go": a test of basic functional mobility for frail elderly..., Podsiadlo [/bib_ref]. Yeung et al. investigated the psychometric properties of the TGUG test in inpatients on an orthopedic rehabilitation ward [bib_ref] The timed up and go test for use on an inpatient orthopaedic..., Yeung [/bib_ref]. They found a high ICC (0.80). The standard error of measurement was 10.2 seconds. Change in TGUG scores correlated with the changes in pain (r = 0.21; P <0.01) and function (r = −0.23; P <0.01).
The 5tSTS test is an easily feasible test where the patient has to stand up and sit back down five times as quickly as possible is a good predictor of falling. A worse score (a longer time needed to complete the test) on the 5tSTS implies a greater chance of falling [bib_ref] The comparative ability of eight functional mobility tests for predicting falls in..., Tiedemann [/bib_ref]. Furthermore it predicts problems with daily activities [bib_ref] Bohannon RW: Test-retest reliability of the five-repetition sit-to-stand test: a systematic review..., Zhang [/bib_ref]. The test-retest reliability is good to high (ICC 0.64 to .096) in most populations and settings and validity is scored good (compared to 1 Repetition Maximum (1RM) leg press, Pearson correlation coefficient r = 0.68, P <0.05) [bib_ref] Reliability and validity of physical fitness field tests for adults aged 55..., Ritchie [/bib_ref]. The standard error of measurement (6.3% of mean 5tSTS) and the minimum detectable change (17.5% of mean 5tSTS) are low [bib_ref] The five-times-sit-to-stand-test (FTSST), the short version of the activities-specific balance confidence (ABC)..., Goldberg [/bib_ref].
The 6MWT requires a 30 m hallway, but no exercise equipment or advanced training for technicians. The test measures the distance a patient can quickly walk on a flat, hard surface in a time-period of six minutes (the six-minute walking distance (6MWD)). The test will be carried out according to the American Thoracic Society guidelines. This means there will be a standard instruction and demonstration prior to the test; the researcher is not allowed to walk with the patient. No verbal feedback is given. Every minute the remaining time is mentioned with a standard phrase of encouragement, spoken in a neutral tone. The timer is started when the patient starts walking. The test-retest reliability is excellent (ICC 0.95). The 6MWD is significantly greater for active than for inactive adults (P <0.0001). It moderately correlates with chair stands (r = 0.67), standing balance (r = 0.52), gait speed (r = −0.73) and self-reported physical functioning (r = 0.55) [bib_ref] Mobility-related function in older adults: assessment with a 6-minute walk test, Harada [/bib_ref].
## Intervention
All patients will get a standard THR (cementless hydroxyapatitecoated cup and a titanium plasma-sprayed stem (Wright Medical, Warshaw, USA)) with a ceramic-on-ceramic (third generation BIOLOX™ delta (Wright Medical, Warshaw, USA) couple. Preoperative leg length and offset are marked to reconstruct the preoperative leg length and to obtain the optimal offset.
In the AA group, a standard transgluteal approach is used and the most optimal component position is achieved. In the PAA group, a muscle sparing technique is used. This will not compromise the component position, but fewer release and detachment of the gluteus medius is necessary. Both groups will receive usual care (UC) after surgery. This includes standard physiotherapy care consisting of mobilizing and strengthening techniques. All patients will receive a booklet containing information about the surgery, weight-bearing capacity after the surgery and rehabilitation in general.
## Follow-up measurements
All measurements will be repeated at four weeks and three months post-surgery. Six weeks post-surgery patients will be seen by the orthopedic surgeon for clinical and radiographic control. These are routine measurements to assess the postoperative course.
## Randomization, blinding and treatment allocation
Patients will be allocated to one of two treatment groups by the surgeon at the preoperative consultation. Randomization will take place by means of blinded envelopes. In total 10 envelopes are provided: five that allocate the patient to the AA group and five that allocate the patient to the PAA group. This is to ensure that the same number of patients is randomized in one of the two treatment groups. Patients receive the same information from the surgeon regardless of their group allocation. A blinded researcher will perform baseline and follow-up measurements. To ensure blinded assessment, SEMG will be carried out by an independent researcher. This because blinding is not possible due to different locations of scar tissue.
## Power and sample size calculation
Starting with a pilot study, we will include five people in each group. On this basis we will do power and sample size calculations in order to commence with the randomized controlled trial.
# Analysis
The primary outcome measure will be the score on the TGUG. Secondary outcome measures will include SEMG, strength of quadriceps and gluteus medius, OHS and SF-36 questionnaire score, score on the 5tSTS, the Trendelenburg test score and the 6MWT score. Normality of data will be checked via the Kolmogorov-Smirnov test. Differences between both treatment groups at baseline and follow-up measurements will be analyzed using the chi-square statistical method for categorical variables. For continuous variables, comparisons between both groups' baselines will be made using an independent samples t-test. Comparisons between both groups at the follow-up measurements will be made using a twofactor repeated measures Analysis of Variance (ANOVA) (group × time). Baseline values will be included as the first follow-up measurements. Differences between successive measurements within a treatment group will be analyzed using a one-factor repeated measures ANOVA.
Significance value for all tests is set at P <0.05. The results of all subjects will be analyzed, regardless of their treatment adherence (intention-to-treat analysis).
# Discussion
The aim of this study is to assess the difference in functional outcome after THR through the PAA, compared to THR through the AA. The main difference between these two approaches is their impact on the gluteus medius. Whereas the AA detaches two thirds of the gluteus medius, the PAA only detaches a small part of the muscle. Since the gluteus medius is one of the most important muscles for the stability of the hip joint [bib_ref] Contributions of individual muscles to hip joint contact force in normal walking, Correa [/bib_ref] [bib_ref] A: The functional anatomy of hip abductors, Al-Hayani [/bib_ref] [bib_ref] The functional anatomy of tensor fasciae latae and gluteus medius and minimus, Gottschalk [/bib_ref] , we hypothesize that functional outcome is better or is achieved faster if THR surgery is performed through the PAA instead of the AA.
For this study we chose to have multiple outcome measures (SEMG, strength of gluteus medius and quadriceps, the Trendelenburg test score, the 6MWD and scores on the OHS, SF-36, TGUG and 5tSTS tests) to be certain we measure all possible effects. According to Vissers et al. three aspects should measure functional outcome: self-perceived function, functional tests in a laboratory setting and actual daily activity [bib_ref] Recovery of physical functioning after total hip arthroplasty: systematic review and meta-analysis..., Vissers [/bib_ref]. Both selfperceived function (measured by the OHS and SF-36 questionnaires) and functional tests (measured by the 6MWT and the TGUG, 5tSTS and tests) are use as outcome measures in our study. However, we chose not to assess actual daily activity at this stage. Rasch et al. reported remaining muscle atrophy up to two years after THR [bib_ref] Persisting muscle atrophy two years after replacement of the hip, Rasch [/bib_ref]. Therefore, we chose to include muscle strength measurements (measured by the SEMG, MicroFET and Trendelenburg tests). If this protocol takes too much effort for our patients it is possible that we could reduce it by removing the 6MWT and the Trendelenburg test.
This study has the following limitations. First, complete blinding cannot be achieved because of the SEMG measurements of the gluteus medius and the difference in location of scar tissue for both surgical approaches. Therefore, we chose to have an independent researcher carry out SEMG measurements and note the results, ensuring the other measures will be blinded. Second, we will only evaluate the short-term outcome. Because of the difference in approach we assume that the intervention group will already show a great deal of improvement in the short-term, while the control group will improve less. In later studies it will be necessary to investigate long-term success as well.
The following strengths can be mentioned. First, we conduct a single centre study, ensuring a standardized approach. Second, we use a wide range of outcome measures allowing us to achieve a total picture of the rehabilitation process. This is why patients are asked to complete both a generic (SF-36) and a disease-specific questionnaire (OHS). The disease-specific questionnaire is believed to better measure changes in function. The generic questionnaire is used to detect changes in psychosocial terms as well.
## Trial status
## Competing interests
Funding of this study is partially provided by Stöpler Belgium NV.
Authors' contributions CH drafted the manuscript. WDH has been involved in drafting the manuscript, revised it critically for important intellectual content and made substantial contributions to the conception and design of the study. UVD revised the manuscript critically for important intellectual content. PM has been involved in drafting the manuscript, more specifically the technical description of the surgical approaches and the background of the surgical approaches. GS revised the manuscript critically for important intellectual content and made substantial contributions to conception and design of the study. All authors have approved the final manuscript for publication.
[fig] Figure 1: Percutaneous assisted approach, side view. [/fig]
[fig] Figure 2: Percutaneous assisted approach, top view. [/fig]
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Retinitis Pigmentosa Treatment with Western Medicine and Traditional Chinese Medicine Therapies
Current management of retinitis pigmentosa (RP) includes an attempt at slowing down the degenerative process through therapies that use either Western or traditional Chinese medicine (TCM). Novel therapies in Western medicine (WM) include use of tailormade gene therapy, transplantation of stem cells, or neuroprotection treatment. TCM treatment includes two major approaches. These are orally applied herbal decoctions and acupuncture. In fact, all TCM treatments are based on the differentiation of a symptom-complex, which is the characteristic essence of TCM. Thus, diagnosed RP may be treated via the liver, the kidney, and the spleen. The principle behind these treatments is to invigorate the blood and brighten the eyes by toning up the liver and the kidney. Also treatments to cope with deficiencies in the two concepts that are unique and fundamental to TCM are considered: Qi or "vital energy" and Yin and Yang or the harmony of all the opposite elements and forces that make up existence. In particular, the Qi deficiency that results from blood stasis is addressed in these treatments. This paper also puts forward the existing problems and the prospect of the future development on integrating TCM with WM.
# Introduction
Retinitis pigmentosa (RP) is a group of inherited degenerative retinal diseases, involving progressive degeneration of the retina, typically starting in the midperiphery and advancing toward the macula and fovea [bib_ref] Retinitis pigmentosa, Hartong [/bib_ref]. It is associated with night blindness, progressive peripheral visual field loss followed by reduction in central vision, and abnormalities in the electroretinogram (ERG). The prevalence of RP is reported to be 1 in 3000-7000 individuals worldwide [bib_ref] Retinitis pigmentosa: genes and disease mechanisms, Ferrari [/bib_ref] , with more than 1.5 million patients who suffer from progressive visual deterioration with this disorder [bib_ref] Novel VCP modulators mitigate major pathologies of rd10, a mouse model of..., Ikeda [/bib_ref]. Most RP patients suffering from low-vision or blindness are often severely disabled or legally blind by the end of the second, third, or fourth decade of life. For that reason, it is important that our work focuses on therapy. Over the last few decades, several therapies have been devised for the treatment of RP with gene therapy, stem cell therapy [bib_ref] Current concepts in the treatment of retinitis pigmentosa, Musarella [/bib_ref] , neuroprotection therapy, and TCM treatment, included amongst them.
## Western medicine therapies
2.1. Stem Cell Therapy. Various types of stem cells have been isolated from a variety of tissues including preimplantation embryos, fetuses, birth-associated tissues, and adult organs. Based on the source, these cells can be broadly classified into embryo-derived stem cells (ESCs) and adult tissue-derived stem cells. And based on biochemical and genomic markers, these stem cells can be broadly classified into embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HPSCs), and induced pluripotent stem cells (IPSCs). Stem cell therapy is a novel approach for vision restitution in retinitis pigmentosa [bib_ref] Stem cell therapy: a novel approach for vision restoration in retinitis pigmentosa, Uy [/bib_ref]. Transplantation of stem cells that can be stimulated to become replacement photoreceptors and be supportive of outer retina cells can theoretically lead to treatments that restore visual function [bib_ref] Shaping the eye from embryonic stem cells: biological and medical implications, Colozza [/bib_ref].
In recent years, stem cell transplantation therapy in RP has made progress. The mechanisms of stem cells therapy for this disease include the following. (1) Cell replacement: transplanted stem cells can differentiate into retinal cells and integrate into the retina of patients, and the differentiated stem cells replace the apoptotic or injured retinal cells [bib_ref] Stem cells as a therapeutic tool for the blind: biology and future..., Singh [/bib_ref].
(2) Nutritional support: the function of transplanted cells is to release diffusible factors and nutritional substances which act as a local cell delivery system for trophic factors and can promote photoreceptor cell survival [bib_ref] Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in..., Inoue [/bib_ref] [bib_ref] Non-invasive stem cell therapy in a rat model for retinal degeneration and..., Wang [/bib_ref] [bib_ref] Mesenchymal stem cells enhance GABAergic transmission in co-cultured hippocampal neurons, Mauri [/bib_ref].
(3) Protection of the retinal blood vessels and cones: bone marrow derived stem cells contain endothelial precursors. Through significant upregulation of many antiapoptotic genes, these stem cells can rescue retinal blood vessels that would ordinarily degenerate completely [bib_ref] Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit..., Otani [/bib_ref] [bib_ref] Rescue of retinal degeneration by intravitreally injected adult bone marrowderived lineage-negative hematopoietic..., Otani [/bib_ref]. Researchers have observed that in humans, either the patient's or a normal person's bone marrow cells may provide potential cone neuroprotection to preserve central vision [bib_ref] Bone marrow-derived stem cells preserve cone vision in retinitis pigmentosa, Smith [/bib_ref].
(4) Promotion synaptic connections: many studies have shown donor cells taken from developing mouse retina at a time coincident with the peak of rod genesis can blend in with cells in a normal adult or a degenerating mouse retina and subsequently build synaptic connections with the remaining retinal cells and, thus, effectively improve visual function [bib_ref] Retinal cells integrate into the outer nuclear layer and differentiate into mature..., Bartsch [/bib_ref]. Despite the recent progress made by stem cell transplantation therapy as a treatment for retinal degeneration, many challenges remain. Firstly, the problem of low rate of stem cell survival and migration needs to be resolved. Secondly, despite the retina being physically isolated from the immune system, the immune response that is triggered when stem cells are transplanted into the subretinal space during therapy and hampers their survival cannot be ignored. Thirdly, there are some biosafety issues. For example, the formation of tumors by transplants cannot be ruled out and, therefore, the therapeutic suitability of the stem cells transplanted which include factors such as the age of the patient cannot be ruled out.
## Gene
Therapy. The mechanism in gene therapy is the transfer of a therapeutic gene by use of viral or nonviral vectors and requires genetic modification of the ocular cells to produce its therapeutic effect. There are two methods to replace or correct abnormal genes: (1) gene augmentation therapies, where a normal gene is inserted into the genome to replace nonviable or diseased genes using a carrier vector;
(2) gene silencing therapies, in which the expression of the mutated gene is inhibited by use of ribozyme or RNA interference [bib_ref] Therapeutic challenges to retinitis pigmentosa: from neuroprotection to gene therapy, Sahni [/bib_ref]. Successful augmentation therapies are dependent on efficient transduction of the target cell by Adeno-associated virus (AAV) and sustained expression of the recombinant virus at a sufficient level. To date, more than 30 patients have so far received the gene therapy, ranging in follow-up from 90 days to 1.5 years and no major side effects have been reported [bib_ref] Human RPE65 gene therapy for leber congenital amaurosis: persistence of early visual..., Cideciyan [/bib_ref] [bib_ref] Gene therapy for leber's congenital amaurosis is safe and effective through 1.5..., Simonelli [/bib_ref]. Two approaches have been proposed to silence the abnormal gene: ribozymes and RNAinterference (RNAi).
RNAinterference (RNAi) knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies [bib_ref] RNA interference gene therapy in dominant retinitis pigmentosa and conerod dystrophy mouse..., Jiang [/bib_ref]. Some studies showed that allelespecific or non-allele-specific knockdown of a dominant GCAP1 mutant can ameliorate photoreceptor dystrophy in dominant RP and cone-rod dystrophy mouse models caused by GCAP1 mutations [bib_ref] RNA interference gene therapy in dominant retinitis pigmentosa and conerod dystrophy mouse..., Jiang [/bib_ref]. However, it may be difficult to judge which model is most relevant to a specific condition in humans. Some of the mutations between humans and animals are not similar. The mutation and the phenotype in the animal model must be viewed with some degree of caution. It cannot therefore be assumed that these are truly representative of the disease that is occurring in the patient until the phenotypes are critically examined using the same criteria. Translational clinical research initiatives are finally offering hope to relatives and patients with RP, but the safety of these techniques has yet to be established in large animal and human experiments.
## Neuroprotection therapy.
Neuroprotection provides a sympathetic environment to prolong the viability of the photoreceptors by their effect on the secondary biochemical pathways. It is a therapeutic strategy which can be achieved either by delivering neurotrophic growth factors, on the one hand, or inhibiting proapoptotic pathways on the other. It can also be delivered by the implementation of viability factors such as the rod-derived cone viability factor for the treatment of retinal neurodegenerative disease that is independent of the etiology of the degeneration. A number of neurotrophic growth factors that slow photoreceptor death in animal models have been identified: basic fibroblastderived growth factor (bFGF), brain-derived neurotrophic factor (BDNF), cardiotrophin-1, nerve growth factor (NGF), fibroblast growth factor (FGF), and CNTF.
The final common pathway of all types of RP is photoreceptor cell death. Leonard et al. [bib_ref] XIAP protection of photoreceptors in animal models of retinitis pigmentosa, Leonard [/bib_ref] reported that the X-linked inhibitor of apoptosis protein (XIAP) was thought to be the most potent member to promote cell preservation. They used Adeno-associated virus (AAV) mediated delivery of XIAP to study its neuroprotective effect. Their results showed that XIAP treated eyes of homozygous albino transgenic rats had significantly preserved outer nuclear layers than their contralateral untreated counterparts. Recent evidence [bib_ref] Photoreceptor cell death mechanisms in inherited retinal degeneration, Sancho-Pelluz [/bib_ref] [bib_ref] Photoreceptor rescue and toxicity induced by different calpain inhibitors, Paquet-Durand [/bib_ref] has highlighted the importance of calpain activation for both photoreceptor cell death and survival. The authors of these studies have proposed the use of highly specific calpain inhibitors to prevent or delay RP. Recent evidence derived from studies on rod-derived cone viability factor (RdCVF) protein injections in a type of rhodopsin mutation, in the P23H rat, showed that this induced an increase in cone cell number. This suggests that RdCVF is a promising therapeutic option for saving rods [bib_ref] Functional cone rescue by RdCVF protein in a dominant model of retinitis..., Yang [/bib_ref]. Another new study [bib_ref] Novel VCP modulators mitigate major pathologies of rd10, a mouse model of..., Ikeda [/bib_ref] has shown that decreased cellular ATP levels may result in the pathology of this eye disease and perhaps also in RP and other similar neurodegenerative diseases. Therefore, neuroprotection may prevent or forestall the progression of such incurable eye diseases that ultimately lead to blindness. Ikeda et al. [bib_ref] Novel VCP modulators mitigate major pathologies of rd10, a mouse model of..., Ikeda [/bib_ref] described small compounds (Kyoto University Substances, KUSs) that were developed to inhibit the ATPase activity of valosin-containing protein (VCP), which is the most abundant soluble ATPase in the cell. The authors showed that KUSs, as well as exogenous ATP or ATP-producing compounds, suppressed endoplasmic reticulum stress and demonstrably protected various types of cultured cells including retinal ones from death. KUSs prevented photoreceptor cell death and preserved visual function in rd10, a mouse model of RP.
## Traditional chinese medicine (tcm) therapies
## Tcm pathogenesis of rp.
TCM therapy is based mainly on the practice of Chinese medicine and is constantly enriched and developed by practical experience. In ancient times, Chinese people discovered that certain foods reduced or eliminated certain diseases. This became the basis of Chinese herbal medicine. It is thought that ancient peoples discovered that fomenting a painful area of the body with the warmth of a fire or the use of warm leather or bark bags or hot stones or sand eliminated the discomfort caused by pain. While using stone as a production tool, people discovered that after a certain part of the body being stabbed, the pain in another part could be relieved, thereby creating treatment methods of using stone and bone needles, which was developed into acupuncture and therefore the formation of meridian. Retinitis pigmentosa (RP) belongs to the high-altitude wind sparrow's vision category in TCM. High-altitude wind sparrow's vision was described in the book Taiping-sengxian Prescriptions in 992 AD for the first time. This classical work recorded more than 16,800 prescriptions and was descended to later generations popularly. Treatment determination based on syndrome differentiation is an essential principle in TCM in understanding and treating disease. It is a specific research and treatment method of disease in TCM and also one of the essential characteristics of TCM. TCM believes that the congenital deficiency with debilitation of the life gate fire is the main reason that causes RP. Other pathogenic factors of RP in TCM include liver and kidney deficiency with essence and blood insufficiency, or spleen and stomach deficiency with Qi and blood insufficiency. Finally, these elements can result in blood stagnation and vessel insufficiency, leading to a loss of nourishment to the eyes, and these will whittle down the spirit light of the pupil, narrow of the visual field, and nocturnal blindness. In TCM, this condition is referred to as high-altitude wind internal obstruction or high-altitude wind sparrow's vision. In recent years, many TCM ophthalmologists conducted further study of the pathogenesis of this disease. For example, Pengproposed and demonstrated blood stasis in deficient pattern type of pathogenesis in patients with retinitis pigmentosa. They put forward that in the treatment of this disease, some additional Chinese medicines which can remove the blood stasis and promote the blood circulation need to be used on the basis of ameliorating deficient pattern and, by doing so, may achieve more satisfactory results [bib_ref] Blood stasis in deficient pattern type of pathogenesis in retinitis pigmentosa, Qinghua [/bib_ref]. This study gives some new insights on Chinese medicine treatment for RP. However, the main hurdle remaining in the traditional Chinese medicine (TCM) theory is that the success of these treatments depends on proper symptomcomplex differentiation, which may also include the stage of the disease, the patient's age, systemic and environmental factors, and so on.
Orally applied herbal decoctions and acupuncture treatment have a role in relieving symptoms and improving visual function in patients.
## Oral herbal decoctions for rp treatment.
All TCM treatments are differentiated on the symptom-complex seen. This is the essence of TCM therapy. High-altitude wind sparrow's vision can be treated from the liver, the kidney, or the spleen. TCM differentiates the pathogenesis of this disease into four different syndromes, including deficiency of the liver-Yin and kidney-Yin, deficiency of the spleen and Qi, insufficiency of the kidney-Yang, and deficiency of the Qi and blood stasis. Among these multifaceted symptom complexes, deficiency of the liver-Yin and kidney-Yin is the most common clinical pattern observed. Different syndromes are prescribed very different treatments. For example, deficiency of the liver-Yin and kidney-Yin is treated by modified Ming Mu Di Huang decoction, which nourishes and tones the liver and the kidney and leads to invigorating the blood and brightening the eyes. In this formula of modified Ming Mu Di Huang decoction, the Chinese herbal drugs, Shudihuang, Danggui, Wuweizi, and Gouqizi, work on supplementing liver and kidney. The Chinese herbal drugs, Danpi, Danshen, Yemingsha, and Chongweizi, can also clear heat and invigorate blood. Chinese herbal drugs Shan Zhu Yu and Shen Di Huang reinforce the kidney, which may improve eyesight by replenishing vital essence. The formula achieves the purpose of seeking Yang within Yin, bringing true Yang back to its origin and unblocking the vessels and collaterals. So the actions of this formula are nourishing and tonifying liver and kidney. And they can invigorate blood and brighten the eyes. A recent study [bib_ref] Bright eye to add and subtract the hope for retinal pigment kidney..., Luo [/bib_ref] in China reported that using TCM treatment on RP resulted in a satisfactory clinical curative affect, and it was worth publicizing. In this study, researchers observed 83 eyes of 42 patients whose TCM diagnoses were highaltitude wind sparrow's vision (deficiency of the liver-Yin and kidney-Yin). After treatment by modified Ming Mu Di Huang decoction, the visual acuity and visual fields in these cases had obvious improvement. Many studies demonstrate that the modified Ming Mu Di Huang decoction may protect apoptosis of photoreceptor cells in retinal degeneration. So it stabilizes the symptoms and delays the progression of RP. The blood moving medicinals include Dang Gui and Dan San that enhance the effect of nourishing and invigorating blood. And thus, they make an impact on the preserving the vision and prevent optic atrophy or blindness.
3.3. Acupuncture for RP Treatment. Acupuncture has been applied as a therapeutic medical technique in China since at least 2,000 years ago, when stone knives and other sharp instruments were used. The term itself is derived from Latin "acus" meaning needles and "punctura" meaning puncture. In this form of treatment, some diseases of the body can be treated by puncturing the points on the body surface to regulate the meridians, Zang-Fu organs, and the circulation of Qi and blood. Acupuncture treatment in RP cases often takes the acupoints such as BL1 (Jing Ming), EX-HN7 (Qiu Hou), GB20 (Feng Chi), LB18 (Gan Shu), LB20 (Pi Shu), ST36 (Zu San Li), and SP6 (San Yin Jiao). The therapist selects 1-2 local points along with two distal acupoints each time. The points should be made with the reinforcing method of needle, which is thought to invigorate the body's healthy Qi and to strengthen weakened physiological function. The filiform needle should be kept for 20-30 minutes in place insertion. For patients who are chronically Yang deficient, one should apply the moxibustion on the distal points or use both needling with moxa. Moxibustion is a therapy in which burning moxa is used to produce a heat stimulation to the human body. It affects the function of the meridians and points to treat or prevent diseases.
Acupuncture may improve the activities and speed of the rod and cone cells of retina, enhance the neural networks and biological activity of the retina cells, and improve the inner circulation, the metabolic activities of retinal epitheliumphotoreceptors complexes, and the damages to visual function. Ma et al. [bib_ref] Acupuncture dialectical retinitis pigmentosa 15 cases, Ma [/bib_ref] used acupuncture dialectical therapy in 15 cases of RP and observed visual acuity, visual field, ERG, and other indicators. The clinical study showed that after treatment, the vision acuity, the visual field distribution, and the ERG-b wave had been improved. The total effective rate was 86.7% and the differences were statistically significant.
However, one problem of acupuncture therapy is that there is no uniform standard and parameters on acupuncture point selection, needling techniques, depth and angle of piercing, gas-getting status, needle retention time, and stimulation methods.
## Common clinical patterns and clinical treatments in
TCM. Some common clinical patterns and clinical treatments in traditional Chinese medicine is summarized in [fig_ref] Table 1: Common clinical patterns and clinical treatments in TCM [/fig_ref].
## Challenges and outlook
There are many topics in clinical studies on RP. The current approaches against RP include the Chinese herbal medicine, acupuncture, moxibustion, gene therapy, neuroprotection therapy, neurotrophic growth factors, antiapoptotic agents, ribozyme therapy, RNAi, retinal transplantation, dietary supplementation, retinal prostheses, stem cell therapy, and Journal of Ophthalmology 5 so on. However, due to the complexity of RP pathogenesis, multiple risk factors, long cycle of RP prevention and control, and the overall prognosis of severe RP remains dismal. There are some obstacles: the success of these treatments depends on proper patient selection; how to successfully translate new therapies in the animal models of the disease; how to effective delivery of the therapy, both genetic material and other neurotrophic factors, and stem cell to the target tissue have been a formidable task. Worldwide researches with numerous samples are expected.
From the long-term perspective, delaying the occurrence and progression of RP and establishing an efficient and practical prevention and control system is the focus of the future RP research in the world. TCM may be able to play an important role in this. Possible future research direction of integrating TCM with western medicine may include (a) TCM treatment of RP by regulating stem cells and (b) TCM treatment of RP by regulating microglia.
In addition, for complicated life phenomenon, both metabonomics and pharmacometabonomics take an organic conception of the human body, which conforms to the way of thinking of traditional Chinese medicine (TCM). The application of metabonomics and pharmacometabonomics in the TCM treatment in RP can deepen the evaluation of the therapeutic effects of TCM in RP through the study of intrinsic quality of TCM syndrome and the treatment by differentiation of syndrome. The research about integration of TCM with modern biological science and technology in RP may provide a new space for the development of therapy against RP, and these treatments would fill an enormous therapeutic gap that we have right now.
[table] Table 1: Common clinical patterns and clinical treatments in TCM. [/table]
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A Rare Case of Systemic Cystic Angiomatosis Involving the Bones, Spleen, Liver, and Lungs
Systemic cystic angiomatosis (SCA) is a rare disorder, usually involving the visceral organs with incidental detection during its insidious course. On radiography, it can present as multiple cystic lesions. In rare instances, it can present as mixed lesions (lytic, sclerotic) as was the case with our patient. The disease has a better prognosis than most vascular neoplasms involving the bones. We present a rare case of this disease, involving multiple organs, and presenting with an insidious onset.
# Introduction
Systemic cystic angiomatosis (SCA) is a rare disorder with multisystemic vascular involvement of the skeletal system and other organ systems, like the spleen, liver, and lungs. Classically, in the bones, SCA represents a maldeveloped vascular and/or lymphatic system, characterised by multifocal bony cysts with a honeycombed or "bubble" appearance, without aggressive osteolysis with neither peripheral soft tissue involvement nor a periosteal reaction [bib_ref] Cystic angiomatosis: case report and review of the literature, Levey [/bib_ref]. Most of the patients present around puberty and men seem to be more frequently affected than women [bib_ref] Cystic angiomatosis of bone: a report of three cases and review of..., Boyle [/bib_ref]. Visceral involvement is seen in one-third of the cases [bib_ref] Cystic angiomatosis, a heterogeneous condition: four new cases and a literature review, Najm [/bib_ref]. Here, we describe a case of SCA in a middle-aged female presenting with involvement of abdominal visceral organs, lungs, and bones.
## Case presentation
A 36-year-old non-diabetic, non-hypertensive female patient, who was healthy a month back, presented to us in the outpatient department with pain in the epigastric region, and upper and lower limbs pain for three weeks. It was not associated with other symptoms. The patient received a blood transfusion six months back since she was diagnosed as anemic; nothing special in her history could be elicited. The abdominal examination revealed a non-tender abdomen with gross splenomegaly without guarding or rigidity. Preliminary blood investigations showed normal hemoglobin of 12.1 gm/dl, packed cell volume (PCV) of 36.9%, total leucocyte count (TLC) of 6300 per microliter, and platelet count of 200,000 per microliter. The coagulation profile showed a normal prothrombin time of 11 seconds, an activated partial thromboplastin time of 28 seconds, and an international normalized ratio (INR) of 1.0. Preliminary tumour markers with values of serum carcinoembryonic antigen (CEA) (0.98ng/ml), CA-125 (15.87U/ml), and CA 19-9 (5.37U/ml) were within normal range.
On imaging, the chest radiograph posteroanterior (PA) view showed multiple well-defined, radio-dense nodular lesions of varying sizes scattered in the bilateral lung fields . Abdominal ultrasound (US) images showed gross splenomegaly (18.5cm) with multiple well-defined heterogeneously hyperechoic lesions of varying sizes scattered throughout the splenic parenchyma, measuring up to approximately 5 x 5 cm in the upper pole, with no increased vascularity on Doppler evaluation . After the US, a contrast-enhanced CT scan of the thorax and abdomen was performed which showed multiple solid calcified and non-calcified, parenchymal nodules of varying sizes randomly distributed in the bilateral lung fields . After contrast administration, non-calcified nodules showed minimal post-contrast enhancement . Few of the nodules had adjacent ground glass opacity on lung window settings . There was gross splenomegaly (18.5 cm) with multiple well-defined hypo-dense lesions showing peripheral punctate calcifications which showed minimal enhancement on post-contrast imaging. A hypodense nodule was noted in the right lobe of the liver which showed homogenous post-contrast enhancement .
## Figure 1: chest radiograph postero-anterior (pa) view and axial ct chest images (plain)
Multiple, well-defined radio-dense nodular lesions (red arrow) of varying sizes scattered in bilateral lung fields on chest radiograph (1A). Axial CT chest plain images show multiple solid, calcified (green arrow), and non-calcified (white arrow), parenchymal nodules of varying sizes randomly distributed in bilateral lung fields (1B, 1C). After contrast administration, non-calcified nodules (white arrow) showed minimal post-contrast enhancement (1D). A few of the nodules (blue arrow) had adjacent ground glass opacity on the lung window setting (1E).
## Figure 2: abdominal us (plain) images and ct images showing splenic and hepatic lesions
Abdominal US (plain) images (2A, 2B, 2C) showing gross splenomegaly (red arrow), with multiple, well-defined, heterogeneously hyperechoic lesions of varying sizes scattered throughout the splenic parenchyma, with no increased vascularity on Doppler evaluation (green arrow). Plain (2D) and contrast-enhanced (2E) CT images show multiple minimally enhancing lesions in the spleen (blue arrow). Hypo-dense enhancing nodule is noted in the right lobe of the liver (yellow arrow).
On bone window CT images, multiple mixed (sclerotic, lytic), mildly expansile lesions were seen in the left scapula, multiple bilateral ribs, vertebral bodies, sacrum, pelvic bones, left humeral head and proximal femur. Lesions were predominantly intramedullary in location, reaching up to the cortex, however, there was no cortical break, periosteal reaction, and soft tissue component .
## Figure 3: ct images in bone window setting
Multiple mixed (sclerotic, lytic), mildly expansile lesions (red arrows) were seen in the left scapula (3A), bilateral ribs, and vertebral body (3B), sacrum and pelvic bones (3C).
Taking into account the above findings and considering the clinical history, age, gender and imaging, primary splenic angiosarcoma with lung, liver, and skeletal metastases was thought as first differential diagnosis; lymphomatous etiology with multiple spleen, liver, lung, and skeletal deposits was considered as second differential diagnosis. For final tissue diagnosis, patient underwent US-guided biopsy of the splenic lesion which showed multiple small vascular channels with thin fibrous walls, lined by endothelium with no evidence of atypia. Features were consistent with benign vascular neoplasm. CT-guided biopsy of the lung nodules was done for further evaluation which showed multiple small vascular channels with thin fibrous walls, lined by endothelium with focal areas of hemosiderin laden macrophages and no evidence of atypia or malignancy. The lesion was thyroid transcription factor 1 (TTF-1) negative, cluster of differentiation (CD)34 and CD31 positive, and D2-40 negative in the endothelial cells . Features were consistent with benign vascular neoplasm. After correlating with similar splenic lesions, angiomatosis was considered.
## Figure 5: histological sections of core needle biopsy of splenic nodule (a,b) and lung nodule (c,d) with immunohistochemistry findings of lung specimen (e-h)
Histological sections of core needle biopsy of the splenic nodule (5A, 5B) and lung nodule (5C, 5D) showing multiple small vascular channels with thin fibrous walls, lined by flattened endothelium with no evidence of atypia. Features were consistent with benign vascular neoplasm. Immunohistochemistry findings of the lung specimen showed that the lesion was thyroid transcription factor 1 (TTF-1) negative (5E), CD34 and CD31 positive (5F, 5G), and D2-40 negative (5H) in endothelial cells.
Our patient is presently on a monthly dose of zoledronic acid of 4 mg, and is being followed up with radiological assessment of splenic, lung, liver, and bone lesions biannually. The patient is asymptomatic at present.
# Discussion
This case depicts a rare disease in which a final diagnosis could only be reached after a biopsy; its pathophysiology remains poorly understood. It is a benign disease but follows a progressive course by involving multiple organs and can lead to catastrophic outcomes due to visceral and spinal involvement. Our patient presented in middle age, hence, a metastatic disease with multiorgan involvement was the first possibility kept as differential, and the main aim of multiple diagnostic tests was to rule out the same. A similar case reported by Kumar et al.considered metastatic disease due to multi-organ involvement; however, post the biopsy, turned out to be SCA.
In a systematic review by Najm et al. [bib_ref] Cystic angiomatosis, a heterogeneous condition: four new cases and a literature review, Najm [/bib_ref] , only 48 patients with SCA have been reported so far. The mean age of presentation was 22 years. Spinal involvement is seen in up to 50% of patients [bib_ref] Skeletal angiomatosis limited to the hand: radiographic and scintigraphic correlation, Toxey [/bib_ref]. The presentation of SCA is highly variable. Majority of the cases present with bony cysts most commonly affecting the femur and pelvic bones [bib_ref] Cystic angiomatosis of the skeletal system, Jacobs [/bib_ref]. Most commonly patients presented with joint or back pain, however, our patient presented with pain in the epigastric region and on evaluation was found to have gross splenomegaly. On reviewing the literature, the spleen is the most common extra skeletal organ which is affected by SCA and was involved in one-fourth of the cases [bib_ref] Gorham-Stout syndrome with chylothorax in a six-year-old boy, Deveci [/bib_ref].
On MRI and CT, splenic involvement usually presents as multiple slightly enhancing nodules. In our case, splenic lesions were multiple and of varying sizes with peripheral punctate calcifications, showing mild enhancement, while the coagulation profile of the patient was normal. On PET-CT, the lesions were nonspecific with no metabolic activity, and some showed moderately increased non-pathological metabolic activity [bib_ref] Imaging features of systemic cystic angiomatosis, Marraoui [/bib_ref]. Huang et al. [bib_ref] Diffuse skeletal hemangiomatosis mimicking metastastic disease on 18F-FDG PET/CT, Huang [/bib_ref] reported a case in which a 53-year-old man who presented with back pain and proximal right femur pain showed FDG avid lesions which were initially thought as neoplastic etiology but on histologic examination, led to a diagnosis of diffuse skeletal angiomatosis. In our case, the lesions showed no significant FDG uptake. Other sites include skin and soft tissues, the thymus, kidneys, mediastinum, and mesentery. Lung and pleural involvement are uncommon. The complications include bony pains, pathological fractures, deformities, splenic rupture, and spinal cord compression [bib_ref] Cystic angiomatosis, a heterogeneous condition: four new cases and a literature review, Najm [/bib_ref].
On imaging, the typical skeletal lesions of SCA appear as well-defined intramedullary cysts with preservation of cortical bone surrounded by a sclerotic rim without any periosteal reaction and few showing honeycomb appearance [bib_ref] Cystic angiomatosis: case report and review of the literature, Levey [/bib_ref]. In our case, multiple lytic sclerotic lesions involving both axial and appendicular skeleton were noted. Few of them showed expansion, however, no lesion showed cortical break, periosteal reaction, and soft tissue component. Due to the involvement of multiple bones, it mimicked bone metastases, lymphoma, multiple myeloma, Paget's disease, and histiocytosis. On histological examination, a single layer of flattened endothelial cell-lined vascular channels is typically observed [bib_ref] Cystic angiomatosis of bone: a report of three cases and review of..., Boyle [/bib_ref].
There is no confirmed treatment for SCA. However, spontaneous regression leading to cure has been reported [bib_ref] Cystic angiomatosis (hamartous haemolymphagiomatosis) of bone. A clinicopathological study of three cases, Schajowicz [/bib_ref]. Bisphosphonates, interferon, and radiotherapy are different treatment modalities mentioned in the literature [bib_ref] Systemic cystic angiomatosis mimicking metastatic cancer, Iranpour [/bib_ref] [bib_ref] Cystic angiomatosis, pleural effusion and multiple bone lesions mimicking a metastatic malignant..., Anjos [/bib_ref]. In high turnover states, by inhibiting osteoclastic activity, bisphosphonates decrease osteolysis and increase mineralization [bib_ref] Bortezomib and zoledronic acid on angiogenic and vasculogenic activities of bone marrow..., Moschetta [/bib_ref]. By inhibiting the proliferation of endothelial cells, they exert an anti-angiogenic effect [bib_ref] Cystic bone angiomatosis: a case report treated with aminobisphosphonates and review of..., Marcucci [/bib_ref]. The effect of radiation therapy is difficult to assess as the course of this disease is usually static [bib_ref] Skeletal angiomatosis in association with gastro-intestinal angiodysplasia and paraproteinemia: a case report, Clayer [/bib_ref] [bib_ref] Asymptomatic skeletal cystic angiomatosis may be managed conservatively with close observation, Aumann [/bib_ref].
# Conclusions
We would like to emphasize that radiological diagnosis of SCA is challenging and requires the help of pathological and laboratory investigations to reach a final diagnosis. However, we suggest that it should be kept as a differential diagnosis in patients having lesions involving multiple organs, with imaging features as described above, in most of the radiological modalities. Thus, a high index of suspicion with careful analysis of patient history and imaging features is required to establish an accurate diagnosis.
# Additional information disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
[fig] FIGURE 4: PET images showing lung, skeleton, and splenic lesions Bilateral lung fields (4A, 4B) showing multiple non-fluorodeoxyglucose (non-FDG) avid parenchymal nodules (green arrow) of varying sizes. Splenic lesions (red arrow) show minimal FDG uptake (4C, 4E, 4F). Skeletal lesions (yellow arrow) are also non-FDG avid (4E). [/fig]
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Capability to make well-founded decisions: an interview study of people with experience of sickness absence who have common mental disorders
Background: Sickness absence and rehabilitation processes can be challenging for an individual. At a time of generally reduced capacity, the individual must comprehend and navigate through several options. The aim of this study was to investigate the prerequisites for support, knowledge and information related to decision making experienced by people on sickness absence due to common mental disorders.Methods:A qualitative explorative approach was used. Face-to-face interviews took place with 11 sick-listed individuals with common mental disorders. Patients were recruited from different sources in the western part of Sweden, such as primary health care centres, patient organizations and via social media. Data analysis was performed using manifest content analysis, meaning that the analysis was kept close to the original text, and on a low level of interpretation and abstraction.Results:The analysis revealed three themes that described experiences of decision making during the sick leave and rehabilitation process: Ambiguous roles challenge possibilities for moving on; Uncertain knowledge base weakens selfmanagement; and Perceived barriers and enablers for ending sick leave.Conclusions:Our findings suggest that alternatives need to be found that address sickness absence and rehabilitation processes from a complex perspective. Collaboration between stakeholders as well as shared decision making should be considered when the time for return to work is discussed with sick-listed individuals. Other factors in the context of the individual must also be considered. Current knowledge on strategies to improve health/well-being while being in the sick leave process need to be elaborated, communicated and adapted to each individuals' unique situation, including clarifying rights, obligations and opportunities during the sick-leave process.
and social insurance institutions [bib_ref] Social insurance literacy: a scoping review on how to define and measure..., Stahl [/bib_ref]. Decisions must be made, some of which could be life changing or at least critical in relation to a continued work life. Thus, the ability to make well-founded decisions is key during this process. However, studies on the specific needs of patients for decision making during sickness absence and the rehabilitation processes are scarce. More research exist on experiences of the sick-leave process itself including factors that facilitate or complicates return to work. Some recurring factors include different kinds of coworker support [bib_ref] Meta-synthesis of qualitative research on return to work among employees with common..., Andersen [/bib_ref] [bib_ref] Common psychosocial factors predicting return to work after common mental disorders, cardiovascular..., Gragnano [/bib_ref] , a supportive employer-employee relationship [bib_ref] Common psychosocial factors predicting return to work after common mental disorders, cardiovascular..., Gragnano [/bib_ref] , accommodation of work and work strain [bib_ref] Common psychosocial factors predicting return to work after common mental disorders, cardiovascular..., Gragnano [/bib_ref] , help to maintain routines in life and work identity [bib_ref] Return-to-work support for employees with mental health problems: identifying and responding to..., Cameron [/bib_ref] , professional support and coordination between different actors/systems [bib_ref] Meta-synthesis of qualitative research on return to work among employees with common..., Andersen [/bib_ref] [bib_ref] Common psychosocial factors predicting return to work after common mental disorders, cardiovascular..., Gragnano [/bib_ref] , help to improve mental health and foster self-efficacy and balance in life [bib_ref] Common psychosocial factors predicting return to work after common mental disorders, cardiovascular..., Gragnano [/bib_ref] [bib_ref] Experiences within the process of sick leave, Hansen Falkdal [/bib_ref] , and ability to participate in one's own sick-leave/rehabilitation process [bib_ref] Experiences within the process of sick leave, Hansen Falkdal [/bib_ref].
One of the few studies with a focus on decision making [bib_ref] Experiences of factors contributing to womens ability to make informed decisions about..., Mårtensson [/bib_ref] , a qualitative study of women with experiences of sickness absence, identified "feeling capable and having belief in one's capacity" as important for making wellfounded decisions. The study participants also expressed a need "to be sure" and they described that, initially, they were active in information seeking but became gradually less active because they had difficulties finding the information they needed. Finally, the study identified "a supportive context, commitment, respect and shared responsibility" as important for enhancing decision making. This study put forward that making well-founded decisions was not only a cognitive, intellectual exercise but also a multifaceted process including emotional and social aspects.
This aligns with a variety of theoretical models that emphasize the importance of understanding the (non) actions of individuals' in their context. One such perspective, when discussing sustainable employability, suggested by Van der Klink et al. [bib_ref] Sustainable employability--definition, conceptualization, and implications: a perspective based on the capability approach, Van Der Klink [/bib_ref] is based on Amartya Sen's Capability Approach [bib_ref] Capability and well-being, Sen [/bib_ref] , which illustrates how the individual's capability to conceive, revise and exercise freedom (act in his or her own interest) is connected to how well their respective resources harmonize with surrounding institutions. Van der Klink et al. [bib_ref] Sustainable employability--definition, conceptualization, and implications: a perspective based on the capability approach, Van Der Klink [/bib_ref] further interpret the capability approach as follows: "Capabilities therefore represent a person's opportunity and ability to achieve valuable outcomes, taking into account relevant personal characteristics and external factors: being able and enabled" (p. 74). An individual's capability to make, for example, health promotive decisions is only partly determined by his or her collective resources (functionalities), because their usefulness is connected to how well they connect with the mandate, organization and performance of the societal institutions that govern the area of life in question. Thus, in a sickness absence and rehabilitation process, if understood from this perspective, it is important to focus not only on the individual's resources, values, health status and so on but also on the existence of reciprocal links between these and for example, the support structures of the social security system. This insight is of particular importance for people affected by common mental disorders (CMDs), because their health situation limits their ability to make the type of decisions that are needed for the use of societal services. Furthermore, trusting relationships with professionals is crucial to their experience of health care and social services, especially so when information about services is fragmented or inadequate or different stakeholders convey conflicting messages [bib_ref] Service users' experiences of participation in decision making in mental health services, Dahlqvist Jönsson [/bib_ref]. In addition, members of this group have experienced being excluded from decision making due to lack of knowledge and information and because it was felt that they were not capable of participating in a decision due to insufficient insight as a result of their illness [bib_ref] Service users' experiences of participation in decision making in mental health services, Dahlqvist Jönsson [/bib_ref].
Quality collaboration with, for example, health care staff is therefore an important building block, which the capability approach deems necessary, between an individual's resources and the institution. Self-confidence and peace of mind can also be understood as functionalities. When they are lacking, the possibility to express needs, ask for more information or for new suggestions in vocational rehabilitation is reduced. The individuals do not feel that they are capable of participating in a decision when they do not have the whole picture [bib_ref] Bringing Sen's capability approach to work and human resource practices, Subramanian [/bib_ref].
In Sweden, where this study was carried out, sickness insurance is universal and encompasses all citizens, and non-citizens with working permission. The first 7 days of sick-leave are self-certified while a medical certificate is needed from the 8th day. Sickness benefits are provided if the person has a reduction of work capacity with at least 25% due to disease, injury or medical symptoms. There is at present no limit for how long a person can be sick-listed. For persons with CMD the reduction of work capacity can be difficult to explain and physicians find it particurlary difficult to asses work capacity in CMD [bib_ref] Assessing work capacity -reviewing the what and how of physicians' clinical practice, Nordling [/bib_ref]. Another factor that has been raised as problematic in the Swedish system is collaboration between the Social Insurance Agency, the health care and the employer. Thus, the Swedish system puts high demands in particular on long-term sick-listed individuals to navigate rules and regulations and to communicate with several different professional representatives.
So far, few studies have explored the perceived needs of persons affected by CMDs during the sick-leave process [bib_ref] Experiences of factors contributing to womens ability to make informed decisions about..., Mårtensson [/bib_ref] [bib_ref] Critical factors for the returnto-work process among people with affective disorders: voices..., Porter [/bib_ref]. To strengthen their capability to use health care and social services effectively, we need to better understand how representatives of this group experience the conditions for making well-founded decisions during sickness absence and the rehabilitation process. This kind of knowledge can also be important for future interventions to prevent a prolonged rehabilitation process and shed light on possible interactions between important factors.
The aim of this study was to investigate the prerequisites for support, knowledge and information related to decision making experienced by people on sickness absence due to CMDs.
# Methods
## Design
A qualitative explorative approach was decided as the most appropriate method for the study. This enables individuals with CMDs to describe how they perceive their situation and how they make decisions during the rehabilitation process in their own words. Face-to-face interviews were considered the most suitable method for data collection, but potential participants were also offered focus group interviews. However, all participants chose individual interviews.
## Study participants and recruitment
Purposive sampling was used, and the inclusion criteria for participation in the study were that the participants should be between 18 and 65 years of age and have ongoing or recent experience of sickness absence from work or studies due to CMDs. People with severe depression or anxiety, or individuals who were suicidal were not invited, mostly for ethical reasons. We strived to capture as many different ideas and experiences as possible, therefore a variation in characteristics regarding age, sex, education, occupation, length of sickness absence and country of birth was desirable.
To recruit participants, we put flyers in the waiting rooms at primary health care centres and health care staff were asked to spread the word about the study when they met patients fulfilling the inclusion criteria. The health care staff were not reimbursed for their efforts. Flyers were also put up on pin boards at common venues (e.g. libraries, food stores) within Region Västra Götaland. We sent e-mails through a patient organization for people with mental health problems and an advertisement was posted on their webpage. Advertisements through official social media channels (Facebook and LinkedIn) and a webpage for the University of Gothenburg were also used.
## Interviews
The interviews were semi-structured, and a thematic interview guide was created by the research group, consisting of researchers with varied professional backgrounds and experience of insurance medicine. To capture relevant experiences for the aim of the study, the interview guide was constructed in line with the dimensions according to the Capabilities Approach. The thematic guide was piloted in one interview, and no suggestions for changes were made. The interview guide consisted of four domains:
- the period sick-listed (e.g. how the days at the beginning, the middle and the end went down and how health and the ability to be active varied within the process) - information regarding CMD and sickness absence - the sick-listing process (e.g. support or lack of support from stakeholders, friends and relatives, and what worked well and not so well within the process) - the path to recovery (regarding health and return to work), causes of CMD and sick-listing, and spontaneous ideas for web-based support.
The participants were interviewed at the University of Gothenburg (except for two telephone interviews). All interviews were conducted and audio recorded by CA (a registered occupational therapist and Doctor of Medicine) and RF (Master of Public Health) between June and September, 2017. One interview was transcribed from the interviewer's memory immediately after the interview due to technical problems with the audio recording. The interviews lasted between 42 and 94 minutes.
Recruitment was originally planned to continue until theoretical saturation was achieved. However, the recruitment of participants went slowly, so when 11 interviews were conducted and the information was found to be relevant and rich in content, it was decided that the data collection could cease.
Of the 11 interviewees, all participants lived in urban areas. The background of the participants varied as anticipated [fig_ref] Table 1: Sociodemographic description of the participants [/fig_ref]. They were employed within both public and private sectors and involved in both operative and administrative tasks (e.g. mailman, nurse, and engineer). At the time of recruitment, 10 of the participants were on sick leave, but some had been sick-listed partly, full time or a variation of this over time. One participant was no longer on sick leave but had started an educational course. In addition, the participants had different experiences of the sick leave process depending on the length of the sick leave spells.
# Analysis
A manifest content analysis inspired by Graneheim and Lundman [bib_ref] Qualitative content analysis in nursing research: concepts, procedures and measures to achieve..., Graneheim [/bib_ref] was performed, which means that the analysis was kept close to the original text (the transcribed interviews) and on a low level of interpretation and abstraction.
The interviews were transcribed verbatim by a professional agency and two of the authors (CA and RF) from the multidisciplinary team began the analysis. They each read the transcripts to gain a common understanding of the whole. Then, the interviews were read line by line to identify meaning units related to the aim of the study. The meaning units were compared and a high consistency was found among the chosen meaning units. Differences were discussed, and after a second review, the meaning units were decided and grouped according to the four domains in the interview guide.
In the next step, conducted by two of the authors (CA and AJ, a registered nurse and Doctor of Medicine), the meaning units were condensed and coded. The codes were regrouped according to their content, and each group with similar content constituted a theme. The themes were scrutinized to make sure that no data related to the aim were missed or excluded. Based on the themes, categories and subcategories were identified. The themes, categories and subcategories were then labelled.
One author, ME (a psychologist and PhD) analysed the meaning units connected to the domains in the interview guide. These were first merged into larger units and then merged into three overarching themes. The concordance between the results of the two analysis procedures was good, and the final results were agreed upon after a critical review by the entire research group.
## Ethics
Due to the sensitive nature of the topic, efforts were made to handle the interview situation with care. All participants were informed about the purpose of the study, that participation was voluntary and that it was possible to leave the interview at any time without justifying why. The participants signed an informed consent form before starting the interview. All participants completed the interview. The Regional Ethical Review Board in the University of Gothenburg, Sweden (Dnr 810-16), approved the study.
# Results
We identified three themes from the interview material that captured the participants' perceptions of the interaction between the person on sick leave and representatives of the institutions involved. These three themes describe three aspects of the sick-leave and rehabilitation process. The first theme, Ambiguous roles challenge the possibilities for moving on, focuses on the factors that the respondents described as relevant for moving forward in the process. It includes a duality related to perceptions of one's own responsibility and not having enough energy for participating in important decisions. The second theme, Uncertain knowledge base weakens self-management, describes the quality of information exchange as something necessary for good governance of the process. It concerns both the experience of information not being presented or clarified, and uncertainty about how one's own statements were received. The third theme, Perceived barriers and enablers for ending sick leave, includes expressed needs for external support as well as the use of personal characteristics to take a constructive part in the sick-leave process [fig_ref] Table 2: Overview of the results new doctors every time [/fig_ref]. Citations are given to illustrate the subcategories and ensure an empirical foundation [bib_ref] On quality of qualitative studies, Larsson [/bib_ref] , and labelled according to sex, age and length of sick leave.
## Ambiguous roles challenge possibilities for moving on
This theme embraces the respondent's perceptions about their own role in the sick-leave process. In addition, it captures how they viewed their relationship with representatives of, for example, the Social Insurance Agency, health care and the workplace. It includes a duality related to perceptions of one's own responsibility and not having enough knowledge or energy to participate in important decisions. In particular, the need for coordination was mentioned when respondents discussed the interaction between different stakeholders, especially when it did not develop satisfactorily, or in a way that made sense to the respondents.
## The influence of demands and responsibilities
The participants described that they took, and wanted to take, personal responsibility for making things happen during the sick-leave process, yet underlined that this often demanded more effort and energy than they felt they could spare. Such demands were sometimes experienced as being due to lack of understanding or knowledge from the professionals, The respondents put forward that they felt that they were expected to take wide-ranging responsibility for their own sick-leave process. This perceived responsibility to be active weighed heavy on them and was described as a source of worry over, for example, not being able to give the correct health information or suggest the most relevant intervention to the physician. A certain amount of energy and strength seems to be necessary, and even expected in the sick-leave process.
[formula] … [/formula]
The participants also indicate that lack of this functionality is not neutral in the sick-leave process, but something that consumes energy, and therefore, could hinder the process from moving forward.
## The significance and authority of the physician
The interaction with physicians was regarded as key to the process because many actions depended on the physician; for example, communication with the Social Insurance Agency when representing the patient and writing a correct and informative sickness certificate. However, worries were expressed about the capacity to convey problems in a way that truthfully reflected one's health situation. The encounter with the physician was sometimes perceived as an occasion where they had to display their collective strengths, be sharp, and do their best.
Because the doctor's appointment is an achievement, you kind of have an adrenaline rush during that meeting; so in that situation, it may be difficult for the doctor to see how bad you actually are. (F, 33, 15 months)
Lack of continuity of physicians (e.g. because of temporary positions and changes) was experienced as complicating and delaying the process, and led to concerns about not being fully understood. Seeing a new physician at each appointment made it necessary to tell the same story repeatedly, reducing the options for discussing plans for the future.
But especially in the beginning, when there were
## Years)
These concerns about not being fully understood led to feelings of uncertainty in relation to the next visit and to stressful feelings of having to formulate strategies for how to get a new certificate as an alternative to being forced to go back to work. This was further amplified when communications about the rules for being on sick leave and getting back to work were experienced as unconstructive.
In some situations, the physician's communication style was even perceived as threatening.
So after four months, you know, she was frantic, more or less. "Yes, but … You have to start working now". But I cannot work.
## Years)
The capacity to repeatedly, efficiently and convincingly portray problems and health status to the physician was described as a key functionality. In addition, the quality of the relationship was described as being potentially hampered by organizational limitations, such as frequent changes of staff and the extra explanatory burden it put on the individual.
## The impact of roles in the workplace
The importance of the workplace in managing the sickleave process was described in several ways related to roles that appear while being at work. Support from colleagues and managers contributed to their active participation in planning for return to work. Regular meetings with a manager induced a feeling of being a valuable employee and acceptance of special needs connected to the health situation; for example, a need for less social interaction was highlighted as important for decisions connected to work adjustment. In contrast, absence of strategies by management or their lack of knowledge about the ways to support and adjust for the employees return to work was described as a challenge. Examples include lack of planning, unclear organization or distribution of responsibilities related to the return to work. Such uncertainties led either to disappointment over promises not fulfilled or that the person on sick leave had to remind the employer about how to create conditions for return to work. One woman looked forward to starting return to work training; however, the conditions were not the best for her. She explained:
… Not at my usual workplace; it was at another place. And I crashed again, after … my six hours a week became way too much.
## .5 years)
Thus, when the workplace was experienced as a safe and well-known arena, with support from employer and staff, this became an enabling factor during the sick-leave process. However, when too much responsibility was placed on the person involved, or when there was a lack of interest and knowledge about how to support a sick-listed employee, the effect was the opposite.
## The need for coordination and continuity
The need for coordination of different interventions was expressed as central for planning to return to work. Insufficient information and contradictory messages from health care professionals caused confusion for the sick person. Interventions both within the health care system and between different stakeholders were not always synchronized, and/or planned for but not executed. Examples included care measures not given at the right time, or when there was a lack of consensus about regulations for sick leave, leading to insecurity, worries and anxiety. Another perceived problem was that several promises were made that were never fulfilled, as quoted below:
But I have not received any information on how to handle the stress. Because at first they thought that I should talk to a counsellor, but then it never happened. And I do not know why. The health centre … . they said that occupational health care will take over my care and then occupational health care said "We cannot take you over" … (F, 41, 2 months) This category also includes statements about being the most important coordinator or being completely left out of the planning process. Uncertainty about getting the best treatment and the possibility of taking an active part in the planning were also discussed.
You do not really dare to speak out. There are different people; it feels like there is no coordination, not even within their own house. They say they work in teams, but it does not feel like the team always has all the information.
## .5 years)
On the other hand, coordination and consensus regarding the application of rules and about suitable interventions led to a feeling of security and an overview of what to relate to during the sick-leave process.
But it feels like you should get more information but maybe just suggestions about getting help from a rehab coordinator. Because if you are paralyzed by anxiety and probably depression as well, you will not be able to talk to any of them [The Social Insurance Agency]. The participants expressed the need for synchronization between the individual's specific situation and the possibilities for support within the health care and social security systems. This could further be interpreted as need for bridging between the individual functionalities and the contextual capabilities.
## Uncertain knowledge base weakens self-management
Within the second theme, thoughts and experiences on information were discussed. Information comprised both the ability to make sense of the information that was given and that the best information concerning the individual situation was available. Lack of knowledge for the individual as well as caregivers and other stakeholders emerged. The individual experienced lack of knowledge about making decisions on possible interventions and self-care. On the other hand, the professionals did not appear to understand all the dimensions in the return to work process and measures needed to support this process.
## Understanding available information to make healthy decisions
The participants stated a desire to gain and search for more credible knowledge concerning health issues and matters related to the sick-leave process. Web sites were used as a source of knowledge, as well as well-informed friends or acquaintances. On the other hand, worries were expressed about what information could be trusted. Another hesitation concerned the participant's own ability to perceive and comprehend the information. In some situations, the strength to search for information was lacking.
## … i feel that i have missed something; this is it "how do you deal with anxiety?" … . i do not believe in cbt [cognitive behavioural therapy]. but, somehow cbt is done in sweden, because the national board of health and welfare says so. yes, but show me that it's good. i can read research reports. i do not have difficulty reading information … but i do not have the strength to search for it. (f, 44, 3.5 years)
Uncertainty due to unclear information about rights, obligations and opportunities was expressed. Not knowing what the next step is as the basis for decisions and who is responsible for these decisions and actions led to insecurity.
… It can take several years to come back. And I might have wanted to know that information from the beginning. First, being put on sick leave for three weeks does not mean you're healthy in three weeks. "We will probably put you on sick leave for much, much longer, but I want to get in touch with you at regular intervals. "
## .5 years)
Clearer and more transparent information was asked for, such as a schedule for actions and how to prioritize these. This was related both to information about the sick-leave process and about options for treatment. The informants also felt insecure about how their information about the situation was perceived and handled.
The capacity to become an active participator in your own process was discussed with a focus on information as a transformative tool, connecting the person with the surrounding context. When such information was not communicated, the possibilities for cooperation was hampered.
## Deficiency of knowledge
Representatives from the health care system, including physicians, do not explicitly inform about the options available for the person on sick leave. Instead, the person is asked about his or her preferences without knowing the alternatives or the effect on return to work.
## … they have asked me what help i need. it is very difficult when you do not know what there is to choose from. i do not know; this information does not appear anywhere. (f, 20, 3 months)
The individuals on sick leave needed information that they could rely on concerning the different options, but they also needed support or guidance in how to implement the chosen actions step-wise adjusted for themselves.
## One thing that i think has been my main problem is that you have been told that exercise is great. … mm, great. but what type of exercise? i could not work that out myself. i just want "now you have to do this". (f, 34, 3.5 years)
Another knowledge deficiency concerns paying attention to early signs of illness and risks for sick leave. Examples were given about trying to solve the situation while struggling to stay at work, leading to a total breakdown before seeking health care.
The great difficulty and lesson is not to ignore signals. And that's probably the hardest part. You notice that with people who "are" their working lives and so may not be on sick leave yet. But I can see quite strong signs or symptoms telling me that it is starting to be, you know, a risk zone.
## Years)
Not being able to understand and describe one's shortcomings led to an inability to start making necessary adjustments to the new circumstances, both in the workplace and outside work. The perception of one's own ability was sometimes overrated, both before and during sick leave.
## And limitations and … or not limitations, but values, have been incredibly strong. i have been doing
just as much at home, with the children, driving and going here and there. And all the projects we have to do in the house and so on. In hindsight, I can see that it was doomed to crash.
## .5 years)
A lack of knowledge also occurred in other important instances during the sick-leave process. Shortcomings were described, both in the health care system and the Social Security Agency, concerning assessment of work ability, especially when factors at work and in everyday life coincide in influencing the ability to work. Shortcomings also appeared within the employer in adapting the work situation to the individual's need for change and support.
## … that they get a really proper reminder about rehabilitation responsibilities, what is important to think about, what … how you have to try to create conditions for this person to get back to work. and i was never offered job rehabilitation in that situation. this was one thing … it was actually an important thing that i never understood. because i should not have worked. i should have been rehabilitated because i had no idea what i was or was not capable of. (f, 33, 15 months)
In this category, the need for new insights and new knowledge, from both lived experiences and professional expertise, became apparent. In other words, there is lack of knowledge in order to support the capabilities, both whitin the individual, as well as in the surrounding systems.
## Perceived barriers and enablers for ending sick leave
This theme comprises statements about the ability to adjust to the new situation. The statements focused on both external and internal factors that were described as having a positive or negative impact on the process and the ability to return to work.
## The importance of time
Time was an important factor for recovery. Time was described as important for regaining a sense of one's capacity, and too much pressure and demands was experienced as counterproductive. Furthermore, the sick leave had to include meaningful activities to have a positive impact on the recovery process. One woman explained how she needed time during her sick leave:
Time has been, again, a very important factor. To get … proper time to get back to who you are. So at first, it was very much about sleeping.
## .5 years)
She also highlighted:
## I feel that even if i'm not completely healthy, i do not know if i will be healthier if i go home. and i need something to do with my time that i feel is meaning-
## .5 years)
Time was also considered as important for learning new aspects about one self. This was exemplified as learning how to handle the impact of illness, how to set limits for commitments, and how to speak up for one's rights. To learn how thought patterns influence behaviour and everyday life was seen as part of the change towards recovery.
## Years)
Another example was the ability to make new plans, for example, change of workplace.
## But i have been too cowardly to resign from a permanent job. but now i feel it is not possible to work and i cannot sacrifice my health to work as i do … so this has taught me that you have to dare … (f, 41, 2 months)
The respondents spoke about time as an enabling factor for changing inner resources. Having enough time during sick leave was also important to actually fulfil commitments, such as carry out treatment programs.
## Managing everyday activities
To manage everyday activities was described as an important factor on the road to recovery. One positive aspect of managing everyday activities created structure in everyday life while not able to work. Maintaining everyday activities, such as having a family role and social contacts, or taking part in some work activities, created the feeling of being an able person and not just being sick.
It is very good to be out and work when you have depression, because you are forced to meet people. Even if you work only for a few hours, not full time, you meet people. So it's not really the best thing in my opinion to be on full time sick leave, because it's easy for you to just lie down and sleep and not get out.
## Months)
Allowing oneself to take part in activities that were enjoyable, such as skating, taking a walk in the woods or engaging in your personal interests, was experienced as health promoting. On the other hand, some had difficulties allowing themselves to have fun, because it led to a bad conscience and a feeling of slipping away from responsibilities such as return to work.
## I cannot do anything fun, because then i "play hooky". (f, 34, 3.5 years)
The participants also talked about how some everyday activities, including family relationships, were experienced as a strain when trying to focus on returning to work. This was exemplified as having the responsibility for children with special needs.
… Several of my children have special needs; this adds to the work involved in having children and taking care of a home, and than to be good enough about that … .
## Years)
Not being able to manage everyday activities could also be seen as an indicator of the individual's capacity to cope with self-care matters, and even less ability to work.
The Swedish Social Insurance Agency is forcing me to return to work as soon as possible, but I can not even cook for myself yet. These statements on the importance of everyday activities reflect the reciprocal relationship between inner capacities and contextual factors. External possibilities can lead to a stronger sense of self-efficacy, whereas the opposite can lead to stagnation in the recovery process.
## The importance of support and recognition
The participants emphasized the importance of support from partners, relatives and significant others during the recovery process. The support involved being helped with practical matters, such as self-care, housework and planning tasks, as well as emotional support and understanding of the circumstances. Encounters with people in similar situations increased the feeling of not being alone, which was beneficial in their situation.
The fact that you feel that you are not alone, you hear about other people's problems, and maybe hear how they managed, it allows me to handle my own problem, in a different way. … If you then have problems with resources, okay, then think that you should collect a few ideas instead of just from one person … . I met a very nice person at physiotherapy, and I now have contact with them and I can talk to them. We do not have the same diagnosis but we still understand each other's situation.
## .5 years)
One factor that emerged from the participants' experiences of professional support was the importance of being listened to and that someone believed in their story. This mutual respect was vital for achieving recovery. The participants related issues about how their situation and inabilities were perceived and described in the sickness certificate, and uncertainty about whether you had fulfilled the conditions according to the sick-leave rules. Both private and professional support could also consist of practical help and support with daily activities, as well as planning the recovery process in small steps and making individual adaptations to proposed measures.
## Personal characteristics
The participants' personal characteristics were described as both benefits and hindrances, exemplified by being stubborn, sustained, and having high demands. Inner traits such as stubbornness or sense of duty were described as supportive for achieving one's commitments at or out of work, or having the energy to engage in their own process.
## So i am stubborn as sin [laughs] and i place very
high demands on myself. I want to be good so that I will return to being a part of society.
## Years)
On the other hand, personal characteristics such as high demands, passivity or lack of a driving force could work in the opposite direction.
Yes, but it may be I have … it may be a negative self-image, or self-esteem, and as well as passivity or lack of drive or difficulty in starting … difficulties with initiation.
## Years)
Personal characteristics sometimes played an important role in the ability to adjust to a new situation. However, the impact of these qualities did not work in isolation, but appeared and were adjusted depending on the context.
# Discussion
The participants in this study perceived that information and knowledge are essential factors for decision making regarding health issues related to the rehabilitation process. The study also showed that a supportive workplace, as well as a person-centred approach from professionals, could prevent a prolonged rehabilitation process, and facilitate the person's own participation in planning for returning to work. However, the findings revealed three themes that highlight the complexity of how other important factors influence the process. Ideally, when roles between the person and professionals are clearly defined, adequate and relevant information is communicated, and the person has a supportive environment, the process can move forward. When obstacles arise in any one of the three themes, return to work can be delayed, and the lack of coordination can put high demands on the person.
## Findings in relation to previous studies
Previous studies have shown that the concept of health literacy can be important in understanding underlying factors for making well-grounded decisions [bib_ref] Experiences of factors contributing to womens ability to make informed decisions about..., Mårtensson [/bib_ref]. Just as with definitions on health literacy [bib_ref] Health literacy -a heterogeneous phenomenon: a literature review, Mårtensson [/bib_ref] , our findings show that this is not only a matter of obtaining, perceiving, analysing and valuing information. Making sense of information also relies on the context, for example, where and how this information is delivered and adapted to the actual situation of the sick-listed person. In our study, participants spoke of normally being a capable person but for the moment losing the ability to obtain and make sense of information. The context also matters in the sense that, in contrast to being in a familiar situation as a competent worker, being on sick leave is a completely new situation, where previous knowledge cannot be applied. Our findings also correspond to another aspect of health literacy, underlining the importance of the organization [bib_ref] The health literate health care organization 10 item questionnaire (HLHO-10): development and..., Kowalski [/bib_ref]. In this sense, the organization contributes to whether or not the person experiences enough support and information, for example, by how well the planned measures are adjusted for the person on sick leave. The question remains whether deficient knowledge on how to move forward in the process lies within the individual or the organization. According to our findings, this probably applies to both.
Our results could also be discussed in relation to the concept of empowerment, which has been described on an individual level, as well as on an organizational and community level [bib_ref] The role of empowerment and quality of life in depression severity among..., Johanson [/bib_ref]. Empowerment has been used to explain factors concerning the situation while being in the rehabilitation process [bib_ref] Does the quality of encounters affect return to work? Lay people describe..., Müssener [/bib_ref] , highlighting the importance of the individual level, where the interaction between the person and the professional has been suggested to be an important factor. This can lead to either positive or negative self-evaluation, with an impact on what is called psychological empowerment or disempowerment [bib_ref] Focus on health, motivation, and pride: a discussion of three theoretical perspectives..., Svensson [/bib_ref]. This could be exemplified in our study, whereby uncertainty about roles and contradictory information were expressed as leading to worries and insecurity about the next step. Empowerment is further defined as taking part in your own rehabilitation process, and also having a sense of control, as well as actual control over daily life. Such control is not only dependent on inner psychological factors but also on contextual or structural factors, such as having access to support and knowledge, and participation in meaningful activities [bib_ref] Patient empowerment: a critique of individualism and systematic review of patient perspectives, Agner [/bib_ref]. In our study, the participants expressed the importance of managing ordinary, everyday activities as a road to recovery, as well as support from important people around them.
Skoglund et al. [bib_ref] A bridge over troubled water? A qualitative study of primary care patients'..., Skoglund [/bib_ref] , in their study on patients on sick leave, found that understanding that things change, and change takes time, was important. This can be compared with our study where the need for time was found to have a role to play in recovery while learning about oneself and finding strategies to change the work situation. Filling the time with structure, meaningful occupations and support from others have also been described in other studies [bib_ref] A bridge over troubled water? A qualitative study of primary care patients'..., Skoglund [/bib_ref]. Also, the timing of return to work actions is an important factor highlighted by the participants in this study. That a mutual decision on this has been made, for example, between the health professionals and the patient, is crucial and coincides with what Corbiere et al. [bib_ref] Employee perceptions about factors influencing their return to work after a sick-leave..., Corbiere [/bib_ref] found in their study on decision making and return to work.
Achieving increased work capacity, and thereby shortening the sick-leave process, is a matter of interaction between finding strategies at work as well as in other everyday activities and has been highlighted in another study on interventions for return to work [bib_ref] Work and everyday activities: experiences from two interventions addressing people with common..., Jansson [/bib_ref]. The authors discuss that awareness of one's own capacity and self-reflection are promoted by breaking the traditional split between a focus on work life on the one hand and other aspects of everyday life on the other. Similar themes have been discussed in other studies proposing that rehabilitation efforts need to be individualized [bib_ref] Critical factors for the returnto-work process among people with affective disorders: voices..., Porter [/bib_ref] , taking both the individual level and social environment into account [bib_ref] The experience of return to work self-efficacy among people on sick leave, Lork [/bib_ref].
Such conclusions can be related to one of the categories in our study, deficiency of knowledge, where both participants and professionals had difficulties in interpreting and describing early signals, risk of sick leave, and the relevant interactive part of coping mechanisms in work as well as other everyday contexts. Taking the whole-life situation into account in the rehabilitation process could provide new insights into factors that promote more sustainable coping strategies [bib_ref] Work and everyday activities: experiences from two interventions addressing people with common..., Jansson [/bib_ref]. Other findings under the same category concerned non-transparent information from, for example, the health care system about the measures available, how to get access to such interventions, and the timing and extent for such proposals. Too much was left to the individual to decide, with the risk of not being able to make a decision, or choosing and doing too much. A better understanding of the different factors that contribute to the potential for increased return to work could be warranted [bib_ref] Womenʼs experiences of being in the sick leave process, Andersson [/bib_ref] [bib_ref] Factors related to the return to work potential in persons with severe..., Bejerholm [/bib_ref]. Engaged professionals need to provide just the right information and planning related to the individual situation, and this could also be discussed in relation to ethical considerations [bib_ref] Ethical perspectives in work disability prevention and return to work: toward a..., Ståhl [/bib_ref]. The authors propose that different ethical considerations and cultural differences between stakeholders in the rehabilitation process could help explaining the deficiencies in, for example, mutual planning for the best measures that will support the individual. Such an approach could help explain the lack of communication between stakeholders, highlighted in a study [bib_ref] Communication characteristics between clients and stakeholders within the Swedish sickness insurance system..., Karlsson [/bib_ref] on different types of debate and information exchange between stakeholders, leading to unnecessary uncertainty in making decisions, both among professionals and the person on sick leave.
Last, but not least, the findings in this study correspond to the capability approach applied to the sick leave situation, describing the complexity between work ability and health [bib_ref] Sustainable employability--definition, conceptualization, and implications: a perspective based on the capability approach, Van Der Klink [/bib_ref]. Several categories could be summarized as describing the interplay between capabilities and functionalities in both the individual and in the surrounding context. To be highlighted, two categories stand out as something not previously observed or elaborated. One is the need for a coordinative effort as a bridge between inner and outer capabilities. The other is the need for adding new knowledge in order to understand the interplay between capabilities even better.
## Strengths and weaknesses of the method
In general, trustworthiness can be suggested to be relatively high in the study. The variation among the participants in the study was acceptable in terms of age (20-55 years) as well as the length of sick leave (2 months to 10 years), leading to a variability in different exeperiences during sick leave. In terms of education, participants were relatively highly educated; most had a university education and others had upper secondary education. This might have affected the participants' work situation, and thus the ability to adapt the return to work process in a positive way, as well as the person's requirements for information during such a process. The problems described by the participants affected the result, however, it was difficult to establish in what way. The participants were mainly women, which is in line with the fact that there are more women than men on sick leave with CMDs, although a gender balance would have been desirable. All researchers participated in the analysis via a so-called peer check through intersubjective dialogue. In addition, age, sex and professional background varied among the researchers, which contributed to a variation in perspectives of the phenomena under study.
We do not believe that the participants were damaged in any way due to the study. The overall purpose of studying experiences of information and support in people with CMDs and sickness absence was to contribute to future improvements of the situation. In addition, before the interviewees shared their stories with the researchers, they were informed about their rights. Two interviewers with different experiences conducted the interviews. They were in continuous contact to maintain the same approach and quality, including indicating to the participants that their experiences were important and taken seriously. Furthermore, the participants were invited to contact the interviewers and other responsible researchers with any questions regarding the project or their own participation, but nobody took that opportunity.
One advantage in this study was that the participants shared their experiences in an open and honest way, which yielded rich data. The broad competences of the research group increased the possibilities for data to be handled in a skilful and ethical manner.
# Conclusions
Our findings suggest that alternatives need to be found that address sickness absence and rehabilitation processes from a complex perspective. Collaboration between stakeholders, and consideration of factors in the context of the individual need to be expanded to improve the situation for sick-listed people. Current knowledge of strategies to improve health/well-being while being in the sick leave process need to be elaborated, communicated and adapted to each individuals' unique situation, including clarifying rights, obligations and opportunities during the sick-leave process. One way of consolidating these needs could be through patient education with a focus on the complexity of being on sick leave, instead of coping strategies related to the specific diagnosis only. Such interventions could be accomplished both face to face and online.
Better understanding of knowledge on how to detect and describe early signs of activity limitations related to the capacity to work is needed. Further research on the effects of timing and interaction of different actions supporting the rehabilitation process could contribute to more substantiated decisions concerning sick leave.
Abbreviations CMD: Common mental disorder; SIA: Social Insurance Agency; CBT: Cognitive behavioural therapy.
[table] Table 1: Sociodemographic description of the participants (n = 11) [/table]
[table] Table 2: Overview of the results new doctors every time. It felt like you had to repeat yourself; you had to tell the same story over and over again; you had to get to know a new person. (F, 34, 3.5 [/table]
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lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli
Bacterial persisters are nongrowing cells highly tolerant to bactericidal antibiotics. However, this tolerance is reversible and not mediated by heritable genetic changes. Lon, an ATP-dependent protease, has repeatedly been shown to play a critical role in fluoroquinolone persistence in Escherichia coli. Although lon deletion (Dlon) is thought to eliminate persister cells via accumulation of the cell division inhibitor protein SulA, the exact mechanism underlying this phenomenon is not yet elucidated. Here, we show that Lon is an important regulatory protein for the resuscitation of the fluoroquinolone persisters in E. coli, and lon deletion impairs the ability of persister cells to form colonies during recovery through a sulAand ftsZ-dependent mechanism. Notably, this observed "viable but nonculturable" state of antibiotic-tolerant Dlon cells is transient, as environmental conditions, such as starvation, can restore their culturability. Our data further indicate that starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Dlon persisters, and Z-ring architecture is important for persister resuscitation in both wild-type and Dlon strains. Our in-depth image analysis clearly shows that the ratio of cell length to number of FtsZ rings for each intact ofloxacin-treated cell predicts the probability of resuscitation and, hence, can be used as a potential biomarker for persisters. IMPORTANCE The ATP-dependent Lon protease is one of the most studied bacterial proteases. Although deletion of lon has been frequently shown to reduce fluoroquinolone persistence, the proposed mechanisms underlying this phenomenon are highly controversial. Here, we have shown that lon deletion in Escherichia coli impairs the ability of persister cells to form colonies during recovery and that this reduction of persister levels in londeficient cells can be transient. We also found that altered Z-ring architecture is a key biomarker in both wild-type and lon-deficient persister cells transitioning to a normal cell state. Collectively, our findings highlight the importance of differentiating persister formation mechanisms from resuscitation mechanisms and underscore the critical role of the nonculturable cell state in antibiotic tolerance.
frequently shown to reduce fluoroquinolone persistence [bib_ref] Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of..., Theodore [/bib_ref] , suggesting that this protein may be an attractive target for small molecular inhibitors [bib_ref] Leveraging peptide substrate libraries to design inhibitors of bacterial lon protease, Babin [/bib_ref]. However, the proposed mechanisms underlying the lon-dependent persistence state are highly controversial [bib_ref] Persistence increases in the absence of the alarmone guanosine tetraphosphate by reducing..., Chowdhury [/bib_ref] [bib_ref] Commentary: what is the link between stringent response, endoribonuclease encoding type II..., Van Melderen [/bib_ref]. Lon was initially thought to induce persistence by degrading antitoxin molecules through a ppGpp/polyphosphate-dependent mechanism, but this model is no longer supported, as the reported observations were due to artifacts from a notorious laboratory contaminant. Another suggested mechanism for the role of Lon in bacterial persistence depends on the cell division inhibitor protein SulA and is based on the observation that deletion of sulA restores fluoroquinolone persister levels in lon-deficient strains [bib_ref] Highly contingent phenotypes of lon protease deficiency in Escherichia coli upon antibiotic..., Matange [/bib_ref]. In this model, accumulation of SulA in the absence of Lon should inhibit FtsZ-dependent ring formation in fluoroquinolonetreated persister cells [bib_ref] Highly contingent phenotypes of lon protease deficiency in Escherichia coli upon antibiotic..., Matange [/bib_ref] [bib_ref] Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring, Bi [/bib_ref] [bib_ref] Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia..., Mukherjee [/bib_ref] , thus impairing persister cell resuscitation and colony formation. Notably, this hypothesis, which remains unverified, may contradict the notion of persister cell dormancy. In other words, persister cells may not respond to fluoroquinolones or express SulA due to their dormant state. However, two independent groups analyzed the SOS response in ofloxacin-treated Escherichia coli cultures at single-cell resolution [bib_ref] Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in..., Goormaghtigh [/bib_ref] and found that antibiotic-induced DNA damage is similar in both persisters and antibiotic-sensitive cells. These findings demonstrate that persister cells can respond to external factors and indicate the presence of unique physiological activities in these cells that may be essential for their survival and resuscitation.
Studies of persisters are based on the premise that if a proposed mechanism is essential for the persister phenotype, genetically perturbing that mechanism should eliminate or reduce persister abundance. Such cells are quantified by persistence assays (e.g., clonogenic survival assays) in which culture samples are collected at various intervals during antibiotic treatment, washed, and plated on standard growth medium to enumerate surviving cells that can colonize in the absence of antibiotics [bib_ref] Definitions and guidelines for research on antibiotic persistence, Balaban [/bib_ref]. Unfortunately, this standard method does not distinguish persister formation mechanisms from resuscitation mechanisms. Thus, it is unclear from previous studies whether targeting Lon protease chemically or genetically eradicates persister cells or simply converts them to a viable but nonculturable (VBNC) state. To address this question, in the current study, we used vector constructs that allow fine-tuning of recombinant protein expression to verify that lon deletion (Dlon) impairs the resuscitation of ofloxacin (OFX) persisters by inhibiting FtsZ-dependent ring formation. We further showed that the reduction of persisters among lon-deficient cells can be transient based on environmental conditions, such as starvation. Indeed, starvation-induced SulA degradation or expression of Lon during the recovery period (i.e., after removal of antibiotics) restores the ability of nonculturable Dlon cells to form colonies by facilitating Zring formation, which represents a potential biomarker for Dlon persister cells transitioning to the normal cell state.
# Results
Lon is required for resuscitation of fluoroquinolone persisters. Fluoroquinolone antibiotics such as OFX inhibit DNA gyrase, leading to formation of double-stranded DNA breaks and induction of DNA repair mechanisms in persister cells [bib_ref] Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in..., Goormaghtigh [/bib_ref] [bib_ref] Induction of the SOS response by new 4-quinolones, Phillips [/bib_ref]. Consistent with previous studies [bib_ref] Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of..., Theodore [/bib_ref] [bib_ref] Highly contingent phenotypes of lon protease deficiency in Escherichia coli upon antibiotic..., Matange [/bib_ref] , we found that Dlon in E. coli MG1655 cells also significantly reduces levels of persisters compared to those found in a wild-type (WT) strain in cell cultures treated with OFX for 6 h [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref] in the supplemental material). While deletion of sulA from the WT strain (DsulA) did not affect OFX persister levels in E. coli, its deletion from the Dlon strain (DlonDsulA) restored persister levels of the lon-deficient cells [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref] , further verifying the reproducibility of previous studies [bib_ref] Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of..., Theodore [/bib_ref] [bib_ref] Highly contingent phenotypes of lon protease deficiency in Escherichia coli upon antibiotic..., Matange [/bib_ref]. Using a sulA reporter (pMSs201-P sulA -gfp) in which the sulA promoter (P sulA ) is fused to a gene encoding green fluorescent protein (GFP) [bib_ref] A comprehensive library of fluorescent transcriptional reporters for Escherichia coli, Zaslaver [/bib_ref] , we assessed sulA expression in both WT and Dlon cells after OFX treatment [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref]. Although we identified a subset of OFX-treated cells that could not express GFP, we detected a significant number of GFP-positive cells expressing sulA in both the WT and Dlon strains, containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells with increased filamentation observed in lon-deficient cells, an expected morphological feature mediated by SulA accumulation [bib_ref] SulA-dependent hypersensitivity to high pressure and hyperfilamentation after high-pressure treatment of Escherichia..., Aertsen [/bib_ref] [bib_ref] Role of sulA and sulB in filamentation by lon mutants of Escherichia..., Gottesman [/bib_ref] [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref].
To determine whether increased accumulation of SulA converts Dlon persisters to VBNC cells, we overexpressed Lon protease from a low-copy-number plasmid in lon-deficient cells during the recovery period on agar plates. This plasmid expression system was constructed using a cassette containing the isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible T5 promoter, lon, and the strong LacI q repressor, which was integrated into a pUA66 plasmid variant, thereby generating pUA66-lon. An empty vector (EV) without lon and a noninduced condition with pUA66-lon served as controls. Stationary-phase Dlon cells harboring either pUA66-lon or EV were diluted in fresh medium and treated with OFX for 6 h. Lon expression was not induced before or during treatment. OFX-treated cells were then collected, washed with sterile phosphate-buffered saline (PBS) solution to remove the antibiotic, and spotted onto Luria-Bertani (LB) agar plates containing different concentrations of IPTG to induce lon expression. We found that Lon overexpression during recovery rescues growth of OFX persisters in a concentration-dependent manner; this increase in colony-forming ability was not observed in the absence of IPTG or with the EV control [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref]. The same phenomenon was also observed in cell populations obtained from exponential-phase cultures [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref] to E). We verified that presence of the plasmid-based expression systems does not significantly alter the levels of persister cells observed in wild-type (WT) and Dlon strains . In addition, overexpressing Lon in OFX-treated WT cells during recovery did not significantly affect WT persistence . Next, to determine the role of Lon in E. coli persistence before and/or during OFX treatment, we added IPTG to cultures before and/or during treatment. Treated cells were then transferred to agar plates with or without IPTG for recovery [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref]. Although adding IPTG, and, thus, induction of Lon, at different stages did not impact WT persistence, Dlon persisters could only be rescued when IPTG was added in LB agar plates during the recovery period [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref]. These data further highlight the importance of Lon in OFX persister resuscitation.
SulA induction in the absence of OFX reduces culturability of the Dlon strain. Given that exposure to UV light induces both DNA damage and expression of SulA in both WT and Dlon strains [bib_ref] An inducible DNA replication-cell division coupling mechanism in E. coli, Huisman [/bib_ref] [bib_ref] Second site mutations in capR (lon) strains of Escherichia coli K-12 that..., Gayda [/bib_ref] , we next tested whether the fluoroquinolone-mediated phenomenon described above also occurs in cells exposed to UV in the absence of OFX. To this end, we diluted stationary-phase cells in fresh medium, exposed these diluted cells to UV light for various time intervals, and then plated the cells on solid medium to determine total CFU. Cells harboring the P sulA -gfp reporter were also subjected to UV exposure, cultured in liquid medium for 2 h, and examined microscopically to assess SulA expression. We found that similar to OFX treatment, UV exposure induced sulA expression in both WT and Dlon strains and increased filamentation of Dlon cells . Further, whereas our chosen time intervals of UV exposure did not affect WT cell viability or CFU levels , in response to this treatment, cells lacking Lon displayed significantly lower CFU than WT cells in a UV exposure time-dependent manner . Next, we tested whether Lon overexpression could rescue this colony formation deficiency. Following UV exposure, Dlon cells containing pUA66-lon or EV were plated on solid agar medium containing different concentrations of IPTG. As expected, we found that Lon expression eliminated the observed reduction in the culturability of Dlon cells in response to UV . We further detected a positive correlation between IPTG (lon inducer) and CFU levels to E), suggesting that recovery is dependent on Lon protein concentration, as well as on levels of accumulated SulA. were then treated with 5 mg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 (t = 0) and 6 h (t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates (n = 4). Scale bar, 25 mm. (C and D) E. coli cells (WT and Dlon) containing an inducible lon overexpression plasmid (pUA66-lon) or empty vector control (pUA66-EV) were treated with OFX (5 mg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl b-D-1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Dlon E. coli containing pUA66-lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using oneway analysis of variance (ANOVA) with Dunnett's post hoc test. **, P , 0.01; ***, P , 0.001; ****, P , 0.0001; ns, nonsignificant.
Both chemical (OFX) and environmental (UV) induction of SulA attenuated the colonyforming ability of lon-deficient cells [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref] , suggesting this phenomenon should be reproduced by plasmid-mediated SulA overexpression in the Dlon strain. To accomplish this, we generated a plasmid (pBAD-sulA) expressing sulA under the arabinose-inducible P BAD promoter and transferred this plasmid into both WT and Dlon strains harboring pUA66-lon or EV. We first noticed that leaky expression from pBAD-sulA inhibited growth of Dlon cells in liquid pretreatment cultures [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. Therefore, we supplemented both WT and Dlon pretreatment cultures with 0.25 mM IPTG to maintain Dlon cell growth. Both WT and Dlon strains harboring the plasmids were grown to stationary phase, washed to remove inducer, and then plated on solid medium containing arabinose (sulA inducer) and/or IPTG (lon inducer) at various concentrations to differentially express SulA and/or Lon during colony formation. Notably, although we did not fine-tune expression levels of these proteins on plates, we observed reduced CFU of WT cells and almost no CFU of the Dlon strain (under the limit of detection) at higher arabinose concentrations (.10 mM) [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. Further, whereas lower arabinose concentrations (,5 mM) did not affect the colony-forming ability of WT cells, colony formation of the Dlon strain was significantly compromised [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref] but reversed upon addition of IPTG [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref] , similar to what we observed for cells exposed to OFX and UV [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref].
Starvation rescues Dlon persisters. Persister cells are typically quantified using clonogenic survival assays in which antibiotic-treated cells are plated on agar medium and then incubated for at least 16 h. Therefore, we next tested whether a longer incubation period is Lon overexpression rescues the colony-forming ability of Dlon cells exposed to UV. (A) WT and Dlon E. coli containing pMSs201-P sulA -gfp were grown to stationary phase, diluted 100-fold in fresh LB medium, and exposed to UV for the indicated times. After exposure, cells were cultured in a shaker for 2 h, and phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images from a representative replicate are shown, but similar data were obtained for four biological replicates. n = 4. Scale bar, 25 mm. (B) WT and Dlon E. coli were exposed to UV as described in panel A and spotted onto LB agar plates to enumerate CFU. n = 4. (C to E) Cells containing the inducible lon overexpression plasmid (pUA66-lon) or empty vector (pUA66-EV) control were exposed to UV for the indicated times and transferred to agar plates supplemented with IPTG at indicated concentrations to enumerate CFU. n = 4. Statistical significance for pairwise comparisons was assessed using one-way ANOVA with Dunnett's post hoc test. ***, P , 0.001; ****, P , 0.0001; ns, nonsignificant.
Persistence State Mediated by Lon Protease ® necessary for Dlon colony formation [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. We found that, although a small number of both WT and Dlon colonies emerged at later time points and that existing colonies grew larger with longer incubation times, the 2-to 3-log difference in CFU between the WT and Dlon strains did not change with longer incubations [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. We made a similar observation for strains harboring pUA66-lon or EV that were plated on agar medium lacking IPTG coli with or without the inducible lon overexpression plasmid (pUA66-lon) or empty vector control (pUA66-EV) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with OFX for 6 h. After treatment, cells were washed, serially diluted, and spotted onto LB agar plates with or without IPTG (1 mM). Plates were incubated for 48 h, and images were captured at indicated time points. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. (B) WT and Dlon E. coli with or without pUA66-lon or pUA66-EV were treated with OFX for 6 h, transferred to sterile PBS solution with or without 1 mM IPTG, and incubated at 37°C in a shaker for 7 days. Samples were collected at the indicated time points and spotted onto agar plates to enumerate surviving cells. n = 4. Statistical significance for pairwise comparison was assessed using one-way ANOVA with Dunnett's post hoc test. *, P , 0.05; **, P , 0.01; ***, P , 0.001; ns, nonsignificant. [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. Thus, while IPTG-induced Lon during recovery on agar medium could rescue Dlon cells harboring pUA66-lon [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref] , it was not clear whether Dlon cells that could not form colonies in the absence of IPTG [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref] were truly alive. To further investigate the ability of Dlon cells to form colonies and assess the resilience of Dlon persisters, cells with or without expression vectors were transferred to PBS solution after OFX treatment [bib_ref] Timing of DNA damage responses impacts persistence to fluoroquinolones, Mok [/bib_ref] [bib_ref] Viable but non-culturable and persistence describe the same bacterial stress state, Kim [/bib_ref] , and their colony-forming ability was tested daily by plating samples on agar medium with or without IPTG for 7 days [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. We found that persister subpopulations from both WT and Dlon strains were alive and able to survive for at least the 7-day duration of the experiment. Surprisingly, Dlon cells, even those without any expression vectors, could be gradually resuscitated when incubated in PBS solution [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref].
Given that SulA can be targeted by proteases other than Lon [bib_ref] ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease..., Seong [/bib_ref] and that starvation may further enhance its intracellular degradation [bib_ref] Viable but non-culturable and persistence describe the same bacterial stress state, Kim [/bib_ref] [bib_ref] Role of protein degradation in the survival of carbon-starved Escherichia coli and..., Reeve [/bib_ref] [bib_ref] Protein degradation in Escherichia coli. II. Strain differences in the degradation of..., Nath [/bib_ref] , we measured SulA concentrations in OFX-treated Dlon cell populations (including dead, VBNC, and persister cells) at days t = 0 and t = 2 during incubation in PBS solution and confirmed a decrease in SulA levels over time. Because persister cells were scarce, we used high-cell-density cultures for persister cell enrichment, which were generated using a procedure (see Materials and Methods) that does not eliminate the observed phenotypic switch during starvation but increases the number of resuscitated persister cells . To test if protein degradation is a general characteristic of starved cells, we used WT and Dlon cells harboring pMSs201-P sulAgfp and monitored GFP levels in OFX-treated cells during starvation. Although the antibiotic induced GFP expression from the sulA promoter, cellular GFP started to decrease when the OFX-treated cells were starved in PBS solution . Notably, we identified a subset of cells that did not respond to OFX or express GFP, consistent with our data in [fig_ref] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells [/fig_ref] and B. These cells might have been dead residual cells and/or VBNC cells; their condition remains to be determined. To show that the observed GFP reduction is not solely attributed to leakage of the proteins through damaged membranes, we stained the cells with propidium iodide (PI), Starvation enhances cellular protein degradation. (A and B) Stationary-phase cells of the Dlon strain were diluted 10-fold in fresh LB medium and treated with OFX for 6 h. Treated cells were collected, washed, and transferred to sterile PBS solution and cultured at 37°C in shaker for 2 days. Western blotting was performed at indicated time points (t = 0 and 2 days) to measure SulA expression (19 kDa). CFU counts were obtained before and after PBS incubation. n = 3. (C) The WT and Dlon strains with pMSs201-P sulA -gfp were transferred to PBS solution after OFX treatment and cultured at 37°C with shaking for 7 days. Cells were analyzed with a flow cytometer for GFP measurements at indicated time points. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. (D) OFX-treated cells described in panel C were stained with propidium iodide (PI) and analyzed with a flow cytometer. Live cells and ethanol (70% [vol/vol])-treated cells (dead cells) served as negative and positive controls, respectively. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. Pairwise statistical significance was performed using two-tailed t test with unequal variance, where **, P , 0.01. Persistence State Mediated by Lon Protease ® which does not permeate cells with intact membranes. PI staining verified that a significant number of intact WT and Dlon cells still exhibited increased GFP degradation after starvation . Also, we observed an enrichment of intact cells at later time points of PBS incubation, as some dead cells were eventually lysed during PBS incubation . Altogether, our results imply that the resuscitation of Dlon cells under starvation conditions might result from increased SulA degradation.
PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures. Given that Dlon potentially converts persisters to VBNC cells, we employed flow cytometry and an IPTG-inducible gfp expression system (pUA66-gfp) to monitor persister cell resuscitation. After OFX treatment, the WT and Dlon strains harboring pUA66-gfp were transferred to fresh LB recovery medium (liquid) containing IPTG. We note that GFP was not induced in pretreatment cultures or during OFX treatment. When persister cells are resuscitated and then proliferate in recovery cultures, the cells should express GFP in the presence of IPTG and be detectable with flow cytometry [bib_ref] Flow-cytometry analysis reveals persister resuscitation characteristics, Mohiuddin [/bib_ref]. Interestingly, our analysis revealed that a large number of OFX-treated cells in both WT and Dlon recovery cultures started to express GFP [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] , subpopulations highlighted with green circles) while preserving their membrane integrity [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref]. Although the GFP-expressing WT cells in recovery cultures corresponded to ;3.28 6 0.53% of the initial cell population (before OFX treatment), the percentage of these cells was much higher in the Dlon recovery cultures (;7.31 6 1.46%). Given that these cells can express GFP and that only a small fraction of them (i.e., persisters) could exit the nongrowth state and proliferate upon their transition to fresh medium (WT persisters, 0.125 6 0.032% of the initial population; Dlon persisters, ;0.0015 6 0.001% of the initial population), we presume that these GFP-positive cells largely exhibit VBNC phenotypes. The proliferating subpopulation in the WT recovery culture became more noticeable upon flow cytometry at approximately 7 to 8 h after transfer to new medium [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] , subpopulation highlighted with a red circle), whereas this subpopulation was not detected in the Dlon recovery culture throughout the course of the study [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref]. However, when starved in PBS solution for 2 days, Dlon persisters could be resuscitated in recovery cultures similar to WT cells [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] , subpopulations highlighted with a red circle). Although we did not observe many GFP-expressing cells after starvation, the recovery cultures still contained a significant number of intact cells [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] whose size started to increase upon their transfer to fresh medium, which was verified by their increased forward scatter (FSC-H) signals [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] , subpopulations highlighted with orange circles). Although it is not clear whether these intact cells are truly VBNC cells (which is beyond the study's scope), our data further showed that incubation in PBS solution facilitated persister resuscitation in both WT and Dlon recovery cultures. Specifically, starved persister cells were resuscitated in less time (2 to 3 h) [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] than persisters from unstarved cell cultures [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref]. In addition, the resuscitating cells seemed to be elongated after starvation, and their size (i.e., FSC-H) considerably increased [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] , subpopulations highlighted with red circles). Altogether, these results verify the existence of a phenotypic switch between a nonculturable to a culturable cell state in OFX-treated Dlon cells that can be facilitated by starvation conditions.
Starvation facilitates Z-ring formation in OFX-treated Dlon cells. Although SulA accumulation in lon-deficient cells is expected to inhibit Z-ring formation [bib_ref] Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia..., Mukherjee [/bib_ref] , this process has not been investigated in Dlon persisters. Z-ring formation at the possible division site of a bacterium takes place when FtsZ, a filamentous tubulin-like protein, assembles into a ring shape [bib_ref] Bacterial cell division and the Z ring, Lutkenhaus [/bib_ref] [bib_ref] Z-ring force and cell shape during division in rod-like bacteria, Lan [/bib_ref] [bib_ref] A widely conserved bacterial cell division protein that promotes assembly of the..., Gueiros-Filho [/bib_ref]. To study this event in Dlon persisters, we generated and validated a lowcopy expression system (pUA66-ftsZ-gfp) in which ftsZ is fused with gfp (46) and controlled by the IPTG-inducible T5 promoter [bib_ref] A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction..., Buss [/bib_ref]. Here, using this FtsZ-GFP construct, we observed Zring formation in exponentially growing WT cells of various sizes and shapes, such as smaller cells with single rings and filamentous cells with randomly or orderly spaced multiple rings (data available upon request), as reported elsewhere [bib_ref] A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction..., Buss [/bib_ref] [bib_ref] Regrowth-delay body as a bacterial subcellular structure marking multidrug-tolerant persisters, Yu [/bib_ref] [bib_ref] Identification and characterization of a negative regulator of FtsZ ring formation in..., Levin [/bib_ref]. We also confirmed that FtsZ-GFP expression did not affect the observed 2-to 3-log difference between WT and Dlon persistence (data available upon request).
To investigate cellular Z-ring formation in OFX-treated cultures, cells harboring the FtsZ reporter were transferred to an LB agarose pad after OFX treatment and then monitored with fluorescence microscopy. OFX-treated WT cells showed highly heterogeneous cell sizes and morphologies, (e.g., smooth and rough cells) [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. FtsZ-GFP proteins were primarily aggregated in rough cells [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref] , which were potentially dead, as their membranes were highly damaged . FtsZ assemblies were generally found to be structurally heterogeneous in smooth, intact cells and included regularly spaced multiple Z-rings or linear, spiral, elliptical, and "8"-shaped structures that may have been transitioning to a ring shape [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. Similar to the WT strain, the heterogeneous FtsZ assemblies and cell morphologies were Persistence State Mediated by Lon Protease ® also observed in the DsulA and DlonDsulA strains [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref]. Conversely, Z-ring formation was rarely seen in Dlon cell populations, which were instead enriched with cells in which FtsZ was dispersed in smooth cells or aggregated in rough cells [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. However, when starved in PBS solution for 2 days, OFX-treated Dlon cultures formed cell subpopulations with structurally heterogeneous FtsZ assemblies, including randomly or orderly spaced multiple rings and linear or spiral filamentous structures [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. When we monitored OFX-treated WT, Dlon, DsulA, and DlonDsulA cells on LB agar pads with timelapse microscopy after starvation, we noted that healthy, elongated cells of all strains containing multiple highly organized Z-rings could be resuscitated within 2 to 4 h [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. These cells first exhibited an extensive elongation, consistent with our flow cytometry data [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref] , and then divided at the septal points (49) [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. Notably, we did not observe were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with OFX for 6 h. IPTG (1 mM) was added to cultures before and during treatment to express FtsZ-GFP. After treatment, cells were collected, washed with PBS solution to remove the antibiotic, and spread on an LB agarose (1%) pad. The pad was then monitored with a fluorescence microscope to collect phasecontrast and fluorescence images. The magnified FtsZ assemblies highlighted by numbers are provided in the right panel. (C) Dlon cells harboring pUA66-ftsZ-gfp were transferred to PBS solution after OFX treatment and cultured for 2 days at 37°C with shaking (250 rpm). Then, cells were collected and transferred to pads to monitor FtsZ assemblies. Images from a representative replicate are shown, but similar data were obtained for four biological replicates. n = 4. Scale bar, 25 mm.
persister cells in which FtsZ polymerization underwent a structural change from linear or spiral to ring-shaped assemblies [bib_ref] Z-ring force and cell shape during division in rod-like bacteria, Lan [/bib_ref] [bib_ref] Identification and characterization of a negative regulator of FtsZ ring formation in..., Levin [/bib_ref]. While shorter cells with fewer rings were rarely resuscitated, cells with aggregated proteins or dispersed GFP could not be resuscitated at all. Of note, protein aggregation was not due to the overexpression of FtsZ from the low-copynumber plasmids, we still observed aggregation phenotypes in OFX-treated cells without any expression vector in phase-contrast micrographs (data available upon request). Also, detection of resuscitating persisters in OFX-treated Dlon cells with microscopy was extremely difficult when the cells were not starved in PBS.
Z-ring architecture is a key biomarker of Dlon persisters. Although starvation facilitated persister resuscitation and Z-ring formation in the Dlon strain, persister cells still represented only a small fraction of the intact cell subpopulation in antibiotic-treated cultures. To determine whether the Z-ring is a suitable biomarker for Dlon persisters during their transition to a normal cell state, we monitored hundreds of intact but diverse (in cell shape, size, and Zring architecture) OFX-treated Dlon cells with time-lapse microscopy after PBS starvation. Cell lengths, as well as Z-ring structures and numbers, were determined using phase-contrast and fluorescence microscopy directly after cells were transferred to microscope pads. Resuscitated cells were identified using time-lapse microscopy. Our in-depth image analysis revealed that persister resuscitation in the Dlon strain strongly correlated with cell size and the number of Zrings [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref]. Specifically, cells with linear or spiral FtsZ assemblies could not be resuscitated [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref]. Moreover, when we calculated the ratio of cell length in microns (L) to the number of Z-rings (Z) for each cell analyzed, we found a correlation between persister resuscitation and calculated L/Z values [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref] , which was supported by binomial regression analysis [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref]. Cells with L/Z % 1, L . 5 mm, and Z . 5 were generally resuscitated [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref]. While L/Z ratios of nonresuscitated cells were often much greater than 1 [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref] , we observed some nonresuscitating phenotypes in cells with L/Z % 1. However, these cells were generally smaller (L , 5 mm) and had fewer Z-rings (Z , 5) than persister cells [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref]. Although we did not perform this labor-intensive analysis for the DsulA and DlonDsulA strains, we were able to report similar results for the WT strain [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref] , verifying the existence of a conserved relationship between persister resuscitation and Z-ring architecture in E. coli MG1655 under the conditions studied here.
The capability of persister cells to form highly organized multiple Z-rings [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref] and [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref] should be enhanced by starvation [fig_ref] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures [/fig_ref]. We have also noticed that sulA deletion induces thick and well-defined Z-ring structures in persister cells [fig_ref] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells [/fig_ref]. As the sulA promoter (P sulA ) is tightly regulated by a RecA-LexA-dependent global DNA damage response mechanism, we presume that persisters (unlike VBNC cells) can successfully repair OFX-induced DNA damage, thus reducing SulA expression during the recovery period; this mechanism may explain the observed Z-ring structures in these cells [fig_ref] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker [/fig_ref]. Although the DNA repair mechanism(s) in persisters was not the main focus of this study, we analyzed P sulA activity of OFXtreated WT cells (harboring pMSs201-P sulA -gfp) in recovery cultures with fluorescence microscopy and flow cytometry. While most resuscitating WT cells were initially SulA positive after OFX treatment, their sulA promoter activity decreased when they began to elongate and divide in fresh, antibiotic-free medium . We also identified a cell subpopulation that continued to express SulA while elongating; however, these cells did not divide throughout the course of the study , implying the presence of DNA damage. Our flow cytometry analysis also showed that a large number of resuscitating cells had reduced sulA promoter activity . Although we did not monitor P sulA activity of OFX-treated cells after PBS starvation, as the GFP variant from pMSs201-P sulA -gfp was mostly degraded , the lack of DNA damage response in daughter cells highlights the ability of persister cells to efficiently repair OFX-induced cellular damage, which is consistent with a previously published study.
Lon overexpression during recovery facilitates Z-ring formation in OFX-treated Dlon cells. Finally, to demonstrate if Lon overexpression during recovery (without starvation) facilitates Z-ring formation in lon-deficient cells, we generated a pBAD-ftsZ-gfp plasmid (expressing the FtsZ reporter under the control of an arabinose-inducible promoter) and introduced it to the Dlon strain harboring pUA66-lon. Because growth of the Dlon strain with both expression vectors was markedly decreased on microscope pads, we performed resuscitation experiments in liquid cultures. Lon expression was not induced in cultures before and during antibiotic treatment. After OFX exposure, cells were transferred to fresh liquid medium with or without IPTG (the lon inducer), and at designated time points, samples from the resuscitation cultures were collected and plated on solid medium (with or without IPTG). We found that Dlon cells started to resuscitate approximately 8 h after their transfer to fresh liquid medium with IPTG, as evidenced by increased CFU levels due to cell proliferation near the same time point . As expected, the resuscitation of Dlon persisters was impaired in the absence of IPTG , and similar CFU profiles were obtained for Dlon cells containing the vector control . We also investigated the collected samples with fluorescence microscopy and found that, despite their scarcity, persister cells began to form Z-rings after 8 h of culturing in the presence of IPTG and exhibited highly heterogeneous morphologies . FtsZ assemblies were also found to be structurally heterogeneous (e.g., linear and spiral structures) . Conversely, this phenomenon was rarely observed in Dlon cultures in the absence of IPTG; rather, these cultures were enriched with nonresuscitating cells in which FtsZ was typically dispersed or aggregated .
# Discussion
The Lon protease has been shown to degrade misfolded proteins, RNases, heat shock proteins, and transfer-messenger RNA (tmRNA)-associated proteins, as well as components of chromosomal and/or plasmid-based toxin/antitoxin systems [bib_ref] SulA-dependent hypersensitivity to high pressure and hyperfilamentation after high-pressure treatment of Escherichia..., Aertsen [/bib_ref] [bib_ref] Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its..., Higashitani [/bib_ref] [bib_ref] Regulation of cell division in Escherichia coli: SOS induction and cellular location..., Schoemaker [/bib_ref] [bib_ref] Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis, Torres-Cabassa [/bib_ref] [bib_ref] Structural inhibition and reactivation of Escherichia coli septation by elements of the..., Dopazo [/bib_ref] [bib_ref] The product of the lon (capR) gene in Escherichia coli is the..., Chung [/bib_ref] [bib_ref] Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated..., Laskowska [/bib_ref]. Although these known functions and previously published data suggest Lon is involved in persister formation, our results have identified a crucial role for Lon in the resuscitation and recovery of persister cells. The work in this area, including the current study, suggests that SulA-Lon acts as a toxin-antitoxin module during quinolone treatment (SulA as a toxin) [bib_ref] Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of..., Theodore [/bib_ref] [bib_ref] Highly contingent phenotypes of lon protease deficiency in Escherichia coli upon antibiotic..., Matange [/bib_ref]. However, sulA only appears to be relevant in the absence of lon, as both the DsulA and double mutant DlonDsulA strains showed persister levels similar to those of WT cells. Nevertheless, Lon may still be an attractive target for small molecular inhibitors [bib_ref] Leveraging peptide substrate libraries to design inhibitors of bacterial lon protease, Babin [/bib_ref] , as it is a critical protein for bacterial cell survival.
One way to differentiate persister formation mechanisms from those involved in persister resuscitation is to controllably express the genes of interest in their respective mutant strains before, during, and after antibiotic treatment. If a gene is essential for persister resuscitation, its expression in the mutant strain during recovery should be sufficient to restore the colony-forming ability of antibiotic-treated cells, as shown in this study, in which the inducible expression of Lon in Dlon cells during recovery from OFX treatment increased their culturability. However, culturability of Dlon persisters can also be restored when the cells are starved after OFX treatment. Interestingly, this phenomenon was also observed in WT cells, as we detected an increase (;10-fold) in persisters of WT cultures during starvation [fig_ref] FIG 3: Starvation in PBS solution rescues Dlon persisters [/fig_ref]. These results are consistent with those of previous studies [bib_ref] Timing of DNA damage responses impacts persistence to fluoroquinolones, Mok [/bib_ref] [bib_ref] Toxin induction or inhibition of transcription or translation posttreatment increases persistence to..., Lemma [/bib_ref] , which found that starvation, toxin induction, and chemical inhibition of transcription or translation following antibiotic treatment rescued E. coli persisters. Although these stressors delay growth-related processes which are thought to facilitate DNA repair and survival [bib_ref] Timing of DNA damage responses impacts persistence to fluoroquinolones, Mok [/bib_ref] [bib_ref] Toxin induction or inhibition of transcription or translation posttreatment increases persistence to..., Lemma [/bib_ref] , our data suggest that SulA degradation might also play a crucial role in persister rescue. However, these two proposed mechanisms are not mutually exclusive; in fact, they may occur in the cells simultaneously, as both are necessary for persister cell resuscitation.
We measured SulA concentrations in OFX-treated Dlon cells, which included the entire cell populations of dead, VBNC, and persister cells. Unfortunately, direct measurement of persister cell physiology is challenging due to the difficulty in isolating pure samples. Many technical challenges arise from their low abundance, transient nature and similarities to VBNC cells, which are more abundant than persister cells. Given that (i) starvation enhances intracellular protein degradation [bib_ref] Viable but non-culturable and persistence describe the same bacterial stress state, Kim [/bib_ref] [bib_ref] Role of protein degradation in the survival of carbon-starved Escherichia coli and..., Reeve [/bib_ref] [bib_ref] Protein degradation in Escherichia coli. II. Strain differences in the degradation of..., Nath [/bib_ref] , (ii) SulA is a fairly unstable protein [bib_ref] ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease..., Seong [/bib_ref] , and (iii) cellular GFP was completely degraded in the entire cell population during incubation in PBS , the resuscitation of Dlon cells under starvation conditions potentially results from SulA degradation. This finding is further supported by our aforementioned experiments that show chemical (OFX) and environmental (UV) induction of SulA and plasmid-mediated SulA overexpression attenuate the colony-forming ability of lon-deficient cells.
One key insight from this study relates to issues associated with VBNC cells, which are generally more abundant than persister cells, can be metabolically active, stain as living cells, and preserve their membrane integrity [bib_ref] Establishment of a method to rapidly assay bacterial persister metabolism, Orman [/bib_ref]. Further, although they also survive antibiotic treatments, their resuscitation in standard medium is typically not observed [bib_ref] Bridging the gap between viable but non-culturable and antibiotic persistent bacteria, Ayrapetyan [/bib_ref]. In general, the clinical importance of VBNC cells of pathogenic bacteria is recognized, as certain bacteria (e.g., Mycobacterium tuberculosis) are known to survive in the human body for years without growing or causing symptoms [bib_ref] Mechanisms of latency in Mycobacterium tuberculosis, Parrish [/bib_ref] [bib_ref] Persistent bacterial infections, antibiotic tolerance, and the oxidative stress response, Grant [/bib_ref]. These cells are fastidious under laboratory conditions and are most effectively detected and identified by PCR and immunological methods [bib_ref] Persistent bacterial infections, antibiotic tolerance, and the oxidative stress response, Grant [/bib_ref] [bib_ref] Immunologic diagnosis of tuberculosis: a review, Chan [/bib_ref]. Critically, although perturbation of certain mechanisms may reduce persister levels in a cell population, VBNC cells may not be eliminated, which poses an important health concern. In this study, our data show that environmental signals can trigger VBNC cell resuscitation. Therefore, measuring VBNC cell levels in cultures is essential to better understand the mechanism by which they can resume growth. We previously developed flow cytometry techniques for this purpose [bib_ref] Establishment of a method to rapidly assay bacterial persister metabolism, Orman [/bib_ref] [bib_ref] Identifying metabolic inhibitors to reduce bacterial persistence, Mohiuddin [/bib_ref] [bib_ref] Dormancy is not necessary or sufficient for bacterial persistence, Orman [/bib_ref] and showed here that resuscitation of VBNC Dlon cells occurs under starvation conditions.
Another important finding from our study helps shed light on the long-standing unsolved question of why only a small fraction of intact cells can be resuscitated after the removal of antibiotics. Although microscopy, which has been used extensively by many research groups to study rare persister cells [bib_ref] Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in..., Goormaghtigh [/bib_ref] [bib_ref] Enrichment of persisters enabled by a ß-lactaminduced filamentation method reveals their stochastic..., Windels [/bib_ref] [bib_ref] Bacterial persistence as a phenotypic switch, Balaban [/bib_ref] , can provide single-cell resolution, this approach cannot be reliably applied to distinguish persisters from VBNC cells without a persister-specific biomarker. This limitation is because both types of cells are in a growth-inhibited state during antibiotic treatment and can only be discriminated after persisters exit their persistence state and start to replicate following antibiotic removal. Resuscitation mechanisms may be regulated in a stochastic and threshold manner in persister cells during their transition to a normal cell state [bib_ref] Enrichment of persisters enabled by a ß-lactaminduced filamentation method reveals their stochastic..., Windels [/bib_ref] , but these mechanisms can only be elucidated if persister cells are detected and characterized during their transition and before they revert to normal, proliferating cells. Notably, our findings here suggest that Z-ring formation may be a key biomarker for distinguishing these cells.
Overall, the results of this study show that Lon plays a critical role in persister cell resuscitation through a mechanism dependent on SulA and FtsZ. We further demonstrate that the nonculturable state of antibiotic-tolerant Dlon cells is transient, as their colony-forming ability can be restored by starvation, which likely leads to SulA degradation. Finally, we show that starvation-induced SulA degradation or expression of Lon during recovery facilitates Zring formation in Dlon persisters, suggesting altered Z-ring architecture may be a biomarker for persister cells transitioning to a normal cell state.
# Materials and methods
Bacterial strains and plasmids. All experiments were conducted using E. coli MG1655 and its derivative strains. E. coli MG1655 and the pQE-80L and pUA66 plasmids were obtained from Mark P. Brynildsen at Princeton University, and pXY027 was a gift from Jie Xiao (plasmid no. 98915; Addgene, Watertown, MA, USA) [bib_ref] A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction..., Buss [/bib_ref]. The Dlon, DsulA, and DlonDsulA strains were generated using the Datsenko-Wanner method, as described previously [bib_ref] One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Datsenko [/bib_ref]. The pQE-80L plasmid has an isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible synthetic T5 promoter and a strong constitutive LacI q repressor. The pUA66-gfp plasmid variant was generated by transferring the T5-gfp-lacI q segment from pQE-80L-gfp into pUA66 that has kanamycin resistance gene (Kan r ). Removal of gfp from pUA66-gfp led to development of a modified pUA66 empty vector. The lon gene was then cloned into the modified pUA66 plasmid to generate the pUA66-lon expression system. The SulA reporter (pMSs201-P sulA -gfp) was generated in a previous study [bib_ref] A comprehensive library of fluorescent transcriptional reporters for Escherichia coli, Zaslaver [/bib_ref]. A pBAD plasmid variant containing the ampicillin resistance gene (Amp r ), an arabinose-inducible promoter (P BAD ), the araC gene encoding the P BAD inhibitor, and pBRR322ori were obtained from Thermo Fisher Scientific (catalog no. V44001; Waltham, MA, USA). The sulA gene was cloned into this pBAD plasmid to generate the pBAD-sulA expression system. To generate the pBAD-ftsZ-gfp expression system, a cassette containing the ftsZ gene fused with gfp, and a chloramphenicol resistance (Cm r ) gene was amplified from pXY027 and ligated to the P BAD -araC-pBR322ori cassette obtained from the pBAD plasmid. To generate the pAU66-ftsZ-gfp plasmid, the ftsZ-gfp DNA fragment, amplified from pXY027 plasmid, was cloned into the modified pUA66 empty vector. FtsZ-GFP expression systems were transferred to WT, Dlon, DsulA, and DlonDsulA strains that also contain the wild type ftsZ gene. All plasmids were generated using standard cloning methods (https://www.neb.com/tools-and-resources/feature-articles/foundations-of -molecular-cloning-past-present-and-future?__cf_chl_jschl_tk__=SNruurTMsKPXPp0X7YTcODunmJAHSq9MW9W NWyaasjM-1639784771-0-gaNycGzNCL0). Genetic modifications were verified by PCR and gene sequencing (Genewiz, South Plainfield, NJ, USA). A complete list of strains, plasmids, and oligonucleotides used in this study is presented in in the supplemental material.
Media, chemicals, and culture conditions. Unless stated otherwise, all chemicals, antibiotics, and enzymes used in these experiments were purchased from Thermo Fisher Scientific, VWR International (Radnor, PA, USA), Sigma-Aldrich (St. Louis, MO, USA), or New England Biolabs (Ipswich, MA, USA). All liquid media were prepared with ultrapure deionized (DI) water, having an electrical resistivity of 18.2 MX.cm. Luria-Bertani (LB) liquid medium was prepared by dissolving 10 g tryptone, 10 g sodium chloride, and 5 g yeast extract in 1 L ultrapure DI water. LB agar medium was prepared by dissolving 40 g premixed LB agar powder in 1 L ultrapure DI water. Phosphate-buffered saline (PBS, 1Â) solution was used to remove chemicals and antibiotics from bacterial cell cultures; this was prepared by mixing 10Â sterile PBS solution with autoclaved ultrapure DI water. Persister assays were performed with 5 mg/mL ofloxacin (OFX). The MIC of OFX for E. coli MG1655 was determined previously [bib_ref] Identifying metabolic inhibitors to reduce bacterial persistence, Mohiuddin [/bib_ref]. To select for plasmid maintenance, kanamycin (25 mg/mL), chloramphenicol (25 mg/mL), and ampicillin (50 mg/mL) were added to both liquid and solid cultures. To induce expression of Lon, FtsZ, and SulA, medium was supplemented with the inducer (IPTG and L-arabinose, depending on the plasmid used; see in the supplemental material for details) at the indicated concentrations. All chemicals and antibiotics added to bacterial cultures were dissolved in ultrapure DI water and sterilized by passage through 0.2-mm membrane filters. Solid and liquid media were sterilized by autoclaving. Overnight precultures and main cultures were prepared in test tubes containing 2-mL LB broth, and these were grown at 37°C with shaking (250 rpm) for 24 h. Overnight precultures were inoculated from cell stocks frozen in 25% glycerol at 280°C. Main cultures grown for 24 h were considered to be in stationary phase.
Cell growth and persister assays. To prepare main cultures, cells from overnight precultures were diluted 1:1,000 in 2-mL LB broth in 14-mL test tubes and cultured in a shaker for 24 h. Growth was assessed by measuring optical density at 600-nm wavelength (OD 600 ) using a Varioskan Lux multimode microplate reader (Thermo Fisher Scientific). Stationary-phase cells from the main cultures (t = 24 h) were diluted 100-fold in 2-mL LB in 14-mL test tubes and treated with OFX (5 mg/mL) for 6 h. At the indicated time points, 1 mL of culture was removed from the test tubes, collected into microcentrifuge tubes, and centrifuged at 13,000 rpm to pellet the surviving cells. After centrifugation, 900 mL of supernatant was removed from the tubes, and 900 mL PBS solution was added. This washing procedure was repeated at least twice to reduce the antibiotic concentration to sub-MIC levels. After the final centrifugation, 900 mL of supernatant were removed; cell pellets were resuspended in the remaining 100 mL of PBS solution, and concentrated cell suspensions were transferred to the wells of a round-bottom 96-well plate. Cell suspensions were then serially diluted (10-fold) in PBS solution, and 10 mL of the diluted cell suspensions were spotted onto agar plates to enumerate persister levels. When required, cells were spotted onto agar plates supplemented with IPTG (0.1 to 1 mM), L-arabinose (1 to 20 mM), kanamycin (25 mg/mL), chloramphenicol (25 mg/mL), or ampicillin (50 mg/ mL) at the indicated concentrations. To increase the limit of detection, the remaining 80 mL of the concentrated cell suspensions were plated. This serial dilution and plating procedure were also performed for cell cultures before antibiotic treatment to quantify the total number of cells in these cultures. To quantify persister cells in exponential-phase cultures, cells from an overnight culture were diluted (100-fold) in 25 mL LB in a 250-mL baffled flask, cultured until they reached an OD 600 of ;0.2, and then treated with OFX. At the indicated time points during treatment, cells were collected, washed, and plated to quantify CFU as described above. Plates were incubated at 37°C for at least 16 h to count CFU and then incubated up to an additional 48 h to see if new colonies emerged. Of note, we refer to overnight and main cultures as "pretreatment cultures" and OFX-treated cultures as "treatment cultures" in the manuscript. Solid and liquid cultures to which OFX-treated cells were transferred after removal of antibiotics are referred to as "recovery cultures." When indicated, the Lon expression was induced in pre-and treatment cultures with 1 mM IPTG.
UV exposure assay. WT and Dlon cells from stationary-phase cultures were diluted 1:100 in 1-mL LB, transferred to petri dishes, and exposed to UV light (UVP ChemStudio, catalog no. 849-97-0928-02; Analytik Jena, Jena, Germany) for different lengths of time (i.e., 0, 1, 2, and 4 min). After UV exposure, cells were collected, serially diluted in PBS solution, and then spotted onto agar plates (61 mM IPTG). Agar plates were incubated for at least 16 h at 37°C to enumerate CFU. Cells expressing SulA reporters were similarly exposed to UV; these cells were then transferred to test tubes and cultured for 2 h in a shaker prior to imaging analysis (described below).
SulA overexpression. WT and Dlon cells harboring the pBAD-sulA and pAU66-lon plasmids from stationary-phase cultures were serially diluted (10-fold) in PBS solution, and 10 mL of the diluted cell suspensions were spotted onto agar plates containing L-arabinose (the SulA inducer) at various concentrations (0, 1, 5, 10, and 20 mM) with/without 1 mM IPTG (the Lon inducer). Of note, pretreatment cultures of both WT and Dlon strains were supplemented with 0.25 mM IPTG for Lon expression to maintain the growth of Dlon cells, as the leaky expression of SulA from the pBAD-sulA plasmid impairs the growth of Dlon cells in liquid pretreatment cultures.
Starvation in PBS solution. Stationary-phase WT and Dlon cells were diluted 1:100 in 14-mL test tubes containing 2 mL LB and treated with OFX for 6 h. Treated cultures (2 mL) were collected, washed with PBS solution to remove antibiotics, resuspended in 2-mL sterile PBS solution, and cultured at 37°C with shaking (250 rpm) for 7 days. At the indicated time points (0, 1, 2, 3, 5, and 7 days), 100-mL cell samples were collected, serially diluted, and spotted onto agar plates (containing either 1-mM IPTG or no IPTG), as described above. Agar plates were incubated at 37°C for at least 16 h to enumerate CFU.
Western blotting for SulA protein detection in high-cell-density cultures. To quantify intracellular SulA protein levels, we used high-cell-density cultures. Briefly, stationary-phase cells were diluted 10-fold in 25 mL fresh LB medium in 250-mL flasks, treated with OFX for 6 h, and then washed with PBS solution to remove the antibiotic and resuspended in 25 mL PBS solution in flasks and cultured at 37°C with shaking (250 rpm). Cultures (including persister, VBNC, and dead cells) were collected into 50-mL falcon tubes on day 0 and day 2 during PBS starvation and centrifuged at 4,700 rpm for 15 min to sediment the cells, which were then frozen with dry ice and sent to RayBiotech (Peachtree Corners, GA, USA) for the SulA quantitation. Cells were also plated to quantify CFU. Cell lysis, protein quantitation, and detection were performed by RayBiotech scientists. Samples containing the same total protein concentration were analyzed by an automatic western machine, in which proteins are separated by size and immobilized in a capillary system. The target protein (SulA) was then chemiluminescently quantified using a primary antibody (rabbit anti-E. coli SulA polyclonal antibody; catalog no. MBS7004012; MyBioSource, San Diego, CA, USA) and a horseradish peroxidase-conjugated secondary antibody.
Monitoring GFP degradation. Stationary-phase cells of E. coli MG1655 WT and Dlon strains harboring the pMSs201-P sulA -gfp plasmid were diluted 100-fold in 2-mL LB in 14-mL test tubes and treated with OFX (5 mg/mL) for 6 h. After the treatment, the cells were washed with PBS solution to remove the antibiotic, and resuspended in PBS solution and cultured at 37°C with shaking (250 rpm). Cells at indicated time points during the PBS incubation were analyzed with a flow cytometer (NovoCyte flow cytometer; NovoCyte 3000RYB; ACEA Biosciences Inc., San Diego, CA). Cells were excited at 488 nm, and green fluorescence was detected with a 530/ 30 nm emission filter. Live cells with or without GFP expression systems were used as controls.
PI staining. OFX-treated cells were collected, washed with PBS solution, and resuspended in 0.85% NaCl solution to achieve a desired cell density for flow cytometry analysis (;10 6 cells/mL). After adding PI (20 mM), cells were incubated in dark at 37°C for 15 min. The cells were then analyzed with a flow cytometer. Unstained live cells were used to gate the cell population on the flow cytometry diagram using forward and side scatter parameters. PI-stained dead cells obtained from ethanol (70%) treatment were used as a positive control. PI-stained live cells were used as a negative control. Cells were excited at 561-nm wavelength, and red fluorescence was detected with a 615/20-nm bandpass filter. When necessary, PI-stained cells were also analyzed with fluorescence microscopy (see "Microscope imaging" section for details).
Monitoring persister cell resuscitation with flow cytometry. OFX-treated WT and Dlon cells harboring the pUA66-gfp plasmid were washed with PBS solution to remove the antibiotic and then resuspended in fresh LB medium containing 1 mM IPTG and cultured at 37°C with shaking (250 rpm). However, IPTG was not added in overnight and treatment cultures. At designated time points, cells were collected from the recovery culture and analyzed with a flow cytometer. Cells were excited at 488 nm, and green fluorescence was detected with a 530/30-nm emission filter.
Microscope imaging. (i) Z-ring imaging. Stationary-phase E. coli MG1655 WT, Dlon, DsulA, and DlonDsulA cells harboring the pUA66-ftsZ-gfp plasmid were diluted 1:100-fold in 2-mL LB broth in 14-mL test tubes and then treated with OFX for 6 h. IPTG (1 mM) was added to overnight and main cultures to induce ftsZgfp expression. Treated cells were then collected, washed to remove the antibiotic, transferred to 2 mL PBS solution in 14-mL test tubes, and cultured at 37°C with shaking (250 rpm). After starving the cells in PBS solution for 2 days, 1-mL cell suspensions were collected from PBS cultures, pelleted by centrifugation, and resuspended in 1 mL LB media. The resuspended cells were then transferred to LB agar pads, which were dried next to a Bunsen burner flame for 30 min. The agar pads were prepared by dissolving agarose (1%) in LB liquid medium followed by microwave sterilization. After cooling the agarose medium, kanamycin (25 mg/mL) and IPTG (1 mM) were added for plasmid retention and ftsZ-gfp expression. The LB agarose medium was then poured over a glass slide (25 by 75 mm), each side of which was enclosed by stacked slides to make the pad smooth and sufficiently thick [bib_ref] Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent..., Skinner [/bib_ref]. Once the agar pads containing cells were dried, glass coverslips (25 by 75 by 0.17 mm) were placed on the top of the cells. The slides with the coverslips were also taped at the corners to make them stable enough for the microscope imaging. Agar pads were maintained in an onstage incubator (catalog no. AMC1000; Thermo Fisher Scientific) in which temperature and humidity were controlled during resuscitation and microscope imaging. Both phase-contrast and fluorescence (GFP) images were obtained using a fluorescence microscope (Evos FL Auto 2; catalog no. AMAFD2000; Thermo Fisher Scientific) with a 100Â (oil) objective (Olympus, catalog no. AMEP4733; working distance, 0.3 mm) to determine cellular morphology and assess expression of the FtsZ-GFP fusion protein. An Evos GFP light cube (catalog no. AMEP4651) was used to acquire GFP images with 470/22-nm excitation and 510/42-nm emission wavelengths. To confirm that the pUA66-ftsZ-gfp plasmid can report the cellular ring structures, exponentially growing WT cells (harboring the reporter) that did not receive any treatment were monitored with microscopy as described above. The same microscopy procedure was also applied for the OFX-treated cells that were not starved.
(ii) Imaging cells harboring the pUA66-lon and pBAD-ftsZ-gfp plasmids. Stationary-phase WT or Dlon cells containing the pUA66-lon and pBAD-ftsZ-gfp plasmids were diluted 1:100 in 2 mL LB broth in 14-mL test tubes and then treated with OFX for 6 h. Arabinose (1 mM) was added to overnight and main cultures to induce ftsZ-gfp expression. OFX-treated cells were collected, washed with PBS solution, and resuspended in 2 mL LB, supplemented with 10 mM arabinose (61 mM IPTG) in 14-mL test tubes and grown in a shaker. At the indicated time points, one test tube was removed from the shaker; 1 mL of cell suspension was used for CFU enumeration, and the remaining 1 mL was used for the imaging process as described above.
(iii) SulA imaging. WT and Dlon cells containing the sulA reporter plasmid (pMSs201-P sulA -gfp) were collected after OFX treatment or UV exposure, serially diluted, spotted onto agarose pads, and imaged with microscopy, as described above. When necessary, cells treated with OFX or exposed to UV were transferred to 1 mL PBS solution in 5-mL test tubes and analyzed via flow cytometry to quantify GFPpositive (i.e., SulA-expressing) cells. Cells were excited at 470/20 nm, and green fluorescence was detected with a 510/42-nm emission filter. Cells carrying the EV were used as controls.
(iv) Image analysis. To capture a heterogenous cell population in each replicate, 10 different locations in each agarose pad, leading to a total number of cells ranging from 200 to 1,000, were selected and monitored with the use of the Evos FL Auto 2 imaging system. Cell morphology and FtsZ ring structures in microscope images were analyzed with ImageJ software [bib_ref] NIH Image to ImageJ: 25 years of image analysis, Schneider [/bib_ref]. Default ImageJ plugins and JavaScript (1.8.0) were used to process the images. The brightness of the phase-contrast images and the color of the fluorescent images were adjusted with "brightness/contrast," "color balance," and "sharpen" options. Given that dead, rough cells with aggregated proteins can be detected in both phase-contrast and fluorescent images, as shown in , we used phase-contrast images to investigate protein aggregation in the cells that do not have fluorescent protein expression systems. Cell lengths were measured manually using the segmented line tools in the ImageJ. The scale bars of raw images were used as a reference for the cell length measurements [bib_ref] NIH Image to ImageJ: 25 years of image analysis, Schneider [/bib_ref]. Similarly, the number of Z-rings within a bacterium was enumerated manually using the fluorescent images.
Statistical analysis. At least three independent biological replicates were performed for each experiment. A nonlinear logarithmic model was used to generate biphasic kill curves [bib_ref] Identifying metabolic inhibitors to reduce bacterial persistence, Mohiuddin [/bib_ref]. F statistics were used to compare kill curves and to assess statistical significance [bib_ref] Identifying metabolic inhibitors to reduce bacterial persistence, Mohiuddin [/bib_ref]. Pairwise comparisons were performed using oneway analysis of variance (ANOVA) with Dunnett's post hoc test or two-tailed t test with unequal variance. Each data point in the figures represents the mean value 6 standard deviation. For microscopy analyses, representative images from both phase-contrast and fluorescence microscopy are displayed in the figures. The threshold values for significance were set at *, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001; and ns, nonsignificant. GraphPad Prism was used to generate the figures and to perform the statistical analyses.
Binary logistic regression analysis from GraphPad Prism was used to determine whether the probability of resuscitation of a cell depends on the cell length (L), the number of Z-rings within the cell (Z) or the L/Z ratio. The outcomes (resuscitation status) are encoded as 1 (indicating a "success" in resuscitation) or 0 (indicating a "failure" in resuscitation). The form of the simple logistic model is expressed as follows:
log odds ð Þ¼ b 0 1b 1 X odds ¼ p 12p
where b 0 and b 1 are intercept and slope constants, respectively, and p is the probability of resuscitation of a cell. Wald test was used to determine if the slope of the simple logistic model (b 1 ) is significantly different from 0, which is equivalent to whether the odds ratio is 1.0 (i.e., P = 1.0).
# Supplemental material
Supplemental material is available online only.
[fig] FIG 1: Lon overexpression rescues colony formation in OFX-treated Dlon cells. (A and B) WT E. coli MG1655 (A) and Dlon E. coli MG1655 (B) [/fig]
[fig] FIG 3: Starvation in PBS solution rescues Dlon persisters. (A) WT and Dlon E. [/fig]
[fig] FIG 5: PBS starvation facilitates persister resuscitation in both WT and Dlon recovery cultures. (A) WT and Dlon containing an IPTG-inducible gfp expression plasmid (pUA66-gfp) were transferred to fresh LB recovery medium containing 1 mM IPTG after 6 h of OFX treatment. IPTG was not added to cultures before or during treatment. At designated time points during recovery, cells were collected and analyzed with a flow cytometer. (B) WT and Dlon cells containing pUA66-gfp were transferred to PBS solution after 6 h of OFX treatment and cultured at 37°C with shaking for 2 days. Cells were then collected and resuspended in fresh LB recovery medium and grown in the presence of 1 mM IPTG. At designated time points, cells were collected from the recovery culture and analyzed with a flow cytometer. (C and D) Cells described in panels A and B, respectively, were stained with PI at the indicated time point and analyzed with a flow cytometer. Live cells and ethanol (70% [vol/vol])-treated cells (dead cells) served as negative and positive controls, respectively. Proliferating cell fractions in the recovery populations are highlighted on the flow cytometry diagrams. A representative biological replicate is shown here. All replicates produced consistent results. n = 4. [/fig]
[fig] FIG 6: Starvation promotes Z-ring formation in OFX-treated Dlon cells. (A and B) WT (A) and the Dlon strain containing an IPTG-inducible FtsZ-GFP expression plasmid (pUA66-ftsZ-gfp) (B) [/fig]
[fig] FIG 7: The number and structural organization of Z-rings represent a key persister biomarker. WT and Dlon strains containing the IPTG-inducible FtsZ-GFP expression plasmid (pUA66-ftsZ-gfp) were transferred to PBS solution after 6 h of OFX treatment and cultured at 37°C with shaking. IPTG (1 mM) was added to cultures before and during OFX treatment to express FtsZ-GFP. Cells incubated in PBS solution for 2 days were then collected, pelleted by centrifugation, and resuspended in LB medium and were then spread on LB agarose pads containing 1 mM IPTG. The pads were monitored for 24 h with a fluorescence microscope with an onstage incubator. Cell lengths, as well as Z-ring structures and numbers, were determined for both resuscitating (persisters) and nonresuscitating (VBNC) cells to generate plots of the number of Z-rings versus cell length (A), the number of linear/spiral structures versus cell length (B), L/Z (cell length in mm/number of Z rings) ratios versus strain (C), L/Z versus cell length (D), and L/Z versus number of Z-rings (E). Pink color represents resuscitating cells, blue color represents nonresuscitating cells, and n represents the number of cells analyzed. [/fig]
[fig] FIG S1 ,: TIF file, 2.8 MB. FIG S2, TIF file, 2.7 MB. FIG S3, TIF file, 2.9 MB. FIG S4, TIF file, 2.6 MB. FIG S5, TIF file, 2.7 MB. FIG S6, TIF file, 2.8 MB. FIG S7, TIF file, 2.7 MB. FIG S8, TIF file, 2.9 MB. FIG S9, TIF file, 2.9 MB. TABLE S1, DOCX file, 0.02 MB. [/fig]
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Mechanisms of GOLPH3 Associated with the Progression of Gastric Cancer: A Preliminary Study
Study Design: To investigate the specific mechanisms by which Golgi phosphoprotein 3 (GOLPH3) affects the progression of gastric cancer and to explore its clinical significance.Methods: Immunohistochemical analysis was used to evaluate the correlations between GOLPH3, phosphorylated mTOR (p-mTOR), phosphorylated Akt (p-Akt), phosphorylated p70S6 (p-p70S6), phosphorylated 4E-BP1 (p-4E-BP1) and the clinicopathological features of gastric cancer. The mRNA expression levels of GOLPH3, mTOR, Akt, p70S6 and 4E-BP1 in gastric cancer, carcinoma-adjacent and paired normal tissue were analyzed using RT-PCR. Western blotting was used to determine the protein expression of GOLPH3, p-mTOR, p-Akt, p-p70S6 and p-4E-BP1 in tissues.Results: High expression protein levels of GOLPH3, p-AKT, p-mTOR, p70S6, p-4E-BP1 were positively associated with histological grade (p,0.05), depth of invasion (p,0.05), distant metastasis (p,0.05) and lymph node involvement (p,0.05). Compared with carcinoma-adjacent and paired normal tissues, the mRNA expression levels of GOLPH3, AKT, mTOR, p70S6 and 4EBP1 in gastric cancer tissues were significantly higher. The protein expression levels of GOLPH3, p-AKT, p-mTOR, p-p70S6 and p-4E-BP1 in gastric cancer tissues were also significantly higher than in carcinoma-adjacent and paired normal tissues. A strong positive correlation was observed between GOLPH3, p-mTOR, p-p70S6 and p-4EBP1 expression (r = 0.410, 0.303 and 0.276, respectively, p,0.05), but no significant correlation between the expression of GOLPH3 and p-Akt was observed.Conclusions:The GOPLH3 expression level is highly correlated with Akt/mTOR signaling in human gastric cancer samples. GOLPH3 combined with Akt/mTOR signaling activation may play an important role in the development, differentiation, invasion and metastasis of gastric cancer.
# Introduction
Although global statistics show that gastric cancer (GC) is the fourth most common cancer in men and the fifth most common cancer in women, the mortality from gastric cancer ranks second only to lung cancer. The global incidence of gastric cancer has been declining since World War II [bib_ref] Recent patterns in gastric cancer: a global overview, Bertuccio [/bib_ref]. However, in developing nations, including China, the morbidity and mortality of gastric cancer have remained high. Gastric cancer treatment has improved in recent years, but most patients are still diagnosed with advanced gastric cancer, frequently including invasion and distant metastasis, leading to an unsatisfactory treatment effect rate. Because gastric cancer presents a significant threat to human health and life, it is crucial to understand the pathogenesis of gastric cancer in the process of tumorigenesis, invasion and metastasis to guide early diagnosis and treatment.
It is commonly accepted that tumor formation involves the misregulation of numerous oncogenes, tumor suppressor genes and metastasis-related genes Golgi phosphoprotein 3 (GOLPH3), which is also known as GPP34, GMx33, or MIDAS, is a 34-kD membrane protein that was initially identified in the mouse Golgi apparatus by proteomic analysis [bib_ref] GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts..., Snyder [/bib_ref]. GOLPH3 belongs to the Golgi matrix protein family, which is highly conserved from yeast to humans [bib_ref] GMx33: A Novel Family of trans-Golgi Proteins Identified by, Wu [/bib_ref]. After GOLPH3 has been translationally modified, it is dynamically connected to the negative Golgi matrix, rapidly transported through the trans-Golgi network (TGN) to the cytoplasm, and distributed into plasma membrane vesicles and endocrine cells [bib_ref] GMx33: A Novel Family of trans-Golgi Proteins Identified by, Wu [/bib_ref]. Several studies have demonstrated that GOLPH3 is involved in anterograde and retrograde Golgi trafficking, receptor recycling, and glycosylation from the Golgi apparatus to the plasma membrane; GOLPH3 also plays a role in cytoskeletal interactions and maintenance of the Golgi structure [bib_ref] GMx33: A Novel Family of trans-Golgi Proteins Identified by, Wu [/bib_ref] [bib_ref] Proteomics characterization of abundant Golgi membrane proteins, Bell [/bib_ref] [bib_ref] MIDAS/GPP34, a nuclear gene product, regulates total mitochondrial mass in response to..., Nakashima-Kamimura [/bib_ref] [bib_ref] GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the Golgi to..., Dippold [/bib_ref]. Scott et al. first identified the oncogenic role of GOLPH3 by revealing its important role in tumor cell differentiation and proliferation and its presence in many solid cancers [bib_ref] GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer, Scott [/bib_ref]. Interestingly, some studies have suggested that GOLPH3 regulates cancer cells by enhancing mammalian target of rapamycin (mTOR) activity. mTOR is a serine/threonine protein kinase and a key integrator of phosphatidyl-inositol-3 kinase (RTK-PI3K) pathways known to regulate cell growth, proliferation, and survival in human cancer cells [bib_ref] GOLPH3 links the Golgi network to mTOR signaling and human cancer, Abraham [/bib_ref] [bib_ref] Signaling From the Golgi: Mechanisms and Models for Golgi Phosphoprotein 3-Mediated Oncogenesis, Scott [/bib_ref]. Many studies are currently investigating the relationship between the GOLPH3 and several solid cancers However, there is insufficient evidence to support the hypothesis that GOLPH3 expression is associated with human gastric cancer progression and prognosis, and the mechanistic basis by which GOLPH3 affects the tumorigenesis, invasion and metastasis of gastric cancer remains unclear. In this study, we investigated clinical significances of GOLPH3 and AKT/mTOR signaling in gastric cancer. Meanwhile, we discovered the relation of GOPLPH3 and Akt/mTOR signaling in gastric cancer to reveal the potential role of GOLPH3 in regulating mTORmediated gastric cancer tumorigenesis, invasion and metastasis.
# Materials and methods
# Ethical statement
All study procedures were reviewed and approved by the Institutional Ethics Review Board of the First Affiliated Hospital of Guangxi Medical University and conducted according to the principles expressed in the Declaration of Helsinki. Informed consent was exempted by the board due to the fresh specimens were handled and anonymized according to ethical and legal standards.
## Tissue samples and clinical data selection
The fresh tissue samples were collected from eighty patients with gastric cancer who underwent surgical treatment at the First Affiliated Hospital of Guangxi Medical University during the period from January 2012 to January 2013. Cancerous and carcinoma-adjacent tissues (3-cm distance from the cancer) and paired normal tissues were obtained from each patient, and the patient was diagnosed independently by two experienced pathologists according to the American Joint Committee on Cancer criteria [bib_ref] of the AJCC cancer staging manual: stomach, Washington [/bib_ref]. Complete original clinical data were available for patients diagnosed with gastric cancer. The group included 36 females and 44 males, with an average age of 55.4613.1 years (range 27-75 years). Additional patient characteristics are shown in [fig_ref] Table 3: Correlation between clinicopathological features and the p-P70S6 expression in gastric cancer specimen [/fig_ref]. Patients receiving chemotherapy or radiotherapy prior to surgery or who had a history of other tumors were excluded. In the following studies, a portion of the specimen taken during surgery was immediately snap-frozen in liquid nitrogen and subsequently stored at 280uC, and a portion was fixed at 10% buffered formalin for 24 h and embedded in paraffin.
## Immunohistochemistry
Paraffin-embedded specimens were sectioned into 3-mm-thick sections from the cancerous, carcinoma-adjacent, and paired normal tissue samples and mounted on glass slides. The sections were initially baked at 65uC for 2 h. Then, the sections were deparaffinized in 100% xylene and rehydrated in a descending ethanol series (100%, 90%, 80%, and 70% ethanol) and distilled water according to standard protocols. Thereafter, the sections were soaked in the citrate antigenic retrieval buffer, heated in an autoclave, and allowed to cool to room temperature. After the sections were washed in PBS three times for five minutes, 3% hydrogen peroxide was added to block endogenous peroxidase activity and non-specific antigen binding at room temperature for 20 min. After washing again with PBS, the sections were incubated with a specific antibody in a moist chamber overnight at 4uC. The following primary antibodies were used: GOLPH3 antibody at 1:100, p-mTOR antibody at 1:200, p-Akt antibody at 1:200, p-p70S6 antibody at 1:100, and p-4E-BP1 antibody at 1:200. For the negative control, the primary antibody was replaced with PBS, and the remaining steps were performed as previously described. The samples were then washed with PBS, and a polymer-reinforcing agent was added to the sections at room temperature for 20 min. After washing, the tissue sections were treated with HRP-tagged goat anti-rabbit IgG, biotinylated antirabbit immunoglobulin, and streptavidin peroxidase complex reagent for 30 min. The sections were then visualized in fresh 3-39 diaminobenzidine solution, hematoxylin counterstain, and 0.1% hydrochloric acid (to aid color separation). Finally, the slides were mounted in neutral gum and analyzed with a brightfield microscope.
The degree of immunostaining of each tissue section was assessed independently by two experienced pathologists who were blind to the patients' clinical data. The protein expression levels were evaluated using a semi-quantitative scoring system based on the total percentage of positively stained tumor cells and the staining intensity. The percentage of positively stained cells was scored according to the following criteria: 0 (#5% positive tumor cells stained), 1 (6-25% positive tumor cells stained), 2 (26-50% positive tumor cells stained), 3 ($51% positive tumor cells stained). The staining intensity was scored using a scale from 0 to 3 as follows: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow-brown), 3 (strong staining = brown). For the analysis, aggregate scores ,4 were defined as low expression, and scores $4 were defined as high expression.
## Reverse transcription polymerase chain reaction
Total RNA was extracted from fresh tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RT-PCR primers used in the study were designed and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. b-Actin was used as an internal reference. Information regarding the primer sequences used in the study is shown in [fig_ref] Table 4: Sequences of primers used for RT-PCR [/fig_ref]. The RT-PCR was performed using a two-step method. One microgram of total RNA was reverse-transcribed into cDNA at 37uC for 60 min and 85uC for 5 s. PCR was then performed according to the following conditions: heating to 95uC for 5 min; 40 cycles of denaturation for 30 s at 95uC, annealing at 60uC for 30 s, and extension for 30 s at 72uC; and final extension at 72uC for 7 min before the reaction was stored at 4uC. Seven micrograms of the final PCR product were loaded on a 2% agarose gel for electrophoresis and imaging analysis under ultraviolet light. The Quantity One software program (Bio-Rad, Hercules, CA, USA) was used to measure the bands and the b-actin gray value of the PCR products.
## Western blotting
The cancerous, carcinoma-adjacent and paired normal tissue samples were weighed and ground into small pieces in liquid nitrogen. Pre-chilled PBS was used to wash the specimens, which were then centrifuged at 5000 rpm at 4uC for 5 min. After . Correlation between clinicopathological features and the GOPLH3, p-mTOR expression in gastric cancer specimen. The associations between protein expression and clinicopathological variables were estimated using the chi-squared test. *Significant at the level of p,0.05. doi:10.1371/journal.pone.0107362.t001 . Correlation between clinicopathological features and the p-AKT1, p-4E-BP1 expression in gastric cancer specimen. The associations between protein expression and clinicopathological variables were estimated using the chi-squared test. *Significant at the level of p,0.05. doi:10.1371/journal.pone.0107362.t002
washing three times with PBS, pre-chilled phosphate lysis buffer and PMSF were added. The sample was sonicated at 180 W at 4uC for 5 min and centrifuged at 1,000 rpm at 4uC for 5 min; the supernatant was then immediately removed, and the protein concentration was quantified by the Bradford assay using a commercial kit purchased from Bio-Rad Laboratories. The supernatants were mixed with loading buffer and boiled at 100uC for 5 min to denature the proteins. Equal quantities of each sample were loaded into the wells of a 4-12% sodium dodecylsulfate-polyacrylamide gel, electrophoresed, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% BSA buffer for 1 h with shaking and then incubated at 4uC overnight with the individual primary antibodies diluted in TBST. The following primary antibodies were used: polyclonal rabbit anti-human GOLPH3 (1:1000, Abcam plc, Cambridge, UK), polyclonal rabbit anti-human p-p70S6 (Thr389)
## Statistical analyses
All of the statistical analyses were performed using the Statistical Package for the Social Sciences, version 17.0 (SPSS Inc., Chicago, IL, USA). The RT-PCR and Western blotting results are presented as the mean 6 SE. A one-way ANOVA followed by LSD multiple-range tests was used to analyze the differences between groups. The relationships between GOLPH3 and p-mTOR, p-Akt, p-4E-BP, and p-p70S6 expression levels were estimated using Pearson's correlation coefficient. The associations between protein expression and clinicopathological variables were analyzed using the chi-squared test. P,0.05 (two-tailed) was used as the level of statistical significance.
# Results
## Relationships between the clinicopathological variables and the protein expression levels by immunohistochemistry
Immunohistochemical detection showed that GOLPH3, p-Akt, and p-4E-BP1 were most common in the cytoplasm, and phosphorylated m-TOR (p-mTOR) was also present in the nucleus at a low level. However, p-p70S6 staining was observed only in the cytoplasm. GOLPH3 and phosphorylated Akt/mTOR pathway proteins were more highly expressed in the gastric cancer group than in the para-carcinoma tissue and paired normal mucosa groups [fig_ref] Figure 1: Representative GOLPH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 immunohistochemistry images in different tissues [/fig_ref] , . Furthermore, the relationships between the clinicopathological variables and protein expression were examined and are summarized in [fig_ref] Table 3: Correlation between clinicopathological features and the p-P70S6 expression in gastric cancer specimen [/fig_ref]. A statistical analysis revealed that the positive expression rate of GOLPH3 in the gastric cancer group strongly correlated with histological grade (p = 0.011), depth of invasion (p = 0.007), distant metastasis (p = 0.002), and lymph node involvement (p, 0.004), whereas it did not significantly correlate with age or gender (p = 0.801 and 0.833, respectively). Interestingly, the high expression levels of p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 were observed to be consistent with high GOLPH3 expression.
## Mrna expression of golph3 and mtor signaling pathway-related genes by rt-pcr
We extracted RNA from gastric cancer, para-carcinoma tissue, and matched normal tissue samples from eighty patients with gastric cancer. The expression levels of GOLPH3 and mTOR signaling pathway-related genes, including Akt, mTOR, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and p70 ribosomal protein S6 kinase (p70S6), were assessed in the three groups by RT-PCR. The expression levels of these genes in . The protein expression levels of GOLPH3, p-AKT1, p-mTOR, p-4E-BP1 and p-p70S6 in different tissues. gastric carcinoma were higher than in carcinoma-adjacent tissues and significantly higher than in paired normal gastric tissues. The mean density and expression distribution of GOPLH3, mTOR, Akt, 4E-BP1 and p70S6 are shown in [fig_ref] Figure 2: Representative images showed semi-quantitative RT-PCR for GOLPH3, Akt, mTOR, 4E-BP1 and p70S6... [/fig_ref]. A statistical analysis revealed that the expression levels of GOLPH3 and mTOR signaling pathway-related genes were significantly different in different gastric tissues (p,0.05).
## Protein expression of golph3 and mtor signaling pathway related proteins by western blotting
Western blotting analysis showed that GOLPH3, p-mTOR, p-Akt1, p-4EB-P1 and p-p70S6 were also expressed in the gastric cancer, carcinoma-adjacent, and paired normal gastric mucosa groups. Interestingly, a progressive increase was observed in the mean values of the aforementioned protein expression levels from the paired normal gastric mucosa group to the gastric cancer group [fig_ref] Figure 3: The GOPLH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 protein levels in the different... [/fig_ref] , and there was a significant difference between the groups (p,0.05).
## Correlations between the expression of golph3 and the signaling signaling pathway-related proteins
Pearson's correlation analysis revealed a strong correlation between GOLPH3 expression and that of the signaling pathway . A statistical analysis showed that the Pearson product moment correlation coefficients were all positive in the gastric cancer group. Interestingly, GOLPH3 expression was strongly associated with p-mTOR, p-4E-BP1 and p-p70S6 (r = 0.410, 0.203 and 0.128, respectively, p,0.05) expression, and a positive correlation between GOLPH3 and p-Akt was nearly significant (p = 0.054). Meanwhile, strong correlations were observed between the expression of p-4E-BP1 and the above-mentioned proteins with the exception of p-mTOR. Phosphorylated mTOR expression was also positively correlated with p-Akt expression (r = 0.521,
# Discussion
Although the incidence of gastric cancer has declined in recent years [bib_ref] Recent patterns in gastric cancer: a global overview, Bertuccio [/bib_ref] , it remains a serious threat to human health. Most gastric cancer patients are diagnosed in advanced stages and tend to present with tumor invasion and metastasis. Over the past decade, no great improvements have been achieved regarding the early diagnosis and treatment of gastric cancer. An understanding of the molecular biology underlying gastric cancer is still lacking. Since 2009, GOLPH3 has received considerable attention as an oncogene. This protein localizes to the cytoplasmic face of the trans-Golgi and has been closely associated with tumorigenicity [bib_ref] GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts..., Snyder [/bib_ref] [bib_ref] GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer, Scott [/bib_ref]. Large studies have found that GOLPH3 overexpression occurs in several human cancers, including epithelial ovarian carcinoma, renal cell carcinoma, glioblastoma multiforme, esophageal squamous cell carcinoma, and oral tongue cancer [bib_ref] High GOLPH3 expression is associated with a more aggressive behavior of epithelial..., Ma [/bib_ref] [bib_ref] GOLPH3 is a novel marker of poor prognosis and a potential therapeutic..., Xue [/bib_ref] [bib_ref] Overexpression of Golgi phosphoprotein-3 (GOLPH3) in glioblastoma multiforme is associated with worse..., Zhou [/bib_ref] [bib_ref] High expression of GOLPH3 in esophageal squamous cell carcinoma correlates with poor..., Wang [/bib_ref]. Evidence suggests that GOLPH3 is involved in tumorigenesis and correlates with poor prognosis. Furthermore, one study reported that GOLPH3 overexpression is associated with poor clinical outcome in gastric cancers [bib_ref] Overexpression of GOLPH3 is associated with poor clinical outcome in gastric cancer, Hu [/bib_ref]. However, the precise mechanisms underlying this relationship are unclear, and the exact molecular mechanism by which GOLPH3 is involved in gastric cancer tumorigenesis, invasion and metastasis is poorly understood. To investigate the potential molecular mechanism by which GOLPH3 is involved in gastric cancer, we first used immunohistochemistry to locate GOLPH3 expression in different gastric tissues. We found that GOLPH3 was primarily localized to the cytoplasm, and it was expressed in gastric cancer, para-carcinoma and paired normal gastric tissues. Interestingly, the rates of high GOLPH3 expression were 75.00% (60 out of 80) in gastric cancer tissues, 43.75% (35 out of 80) in carcinoma-adjacent tissues and 12.50% (10 out of 80) in normal tissues, respectively . Moreover, GOLPH3 over-expression was observed in gastric cancer tissue and was highly related to gastric cancer invasion and metastasis. Our results suggested that high GOLPH3 expression was significantly related to histological grade (p = 0.015), depth of invasion (p,0.001), lymph node metastasis (p,0.001), and distant metastasis (p = 0.006) in gastric cancer tissues . In addition, Western blotting and RT-PCR were performed to evaluate GOLPH3 expression at the protein and mRNA levels. Interestingly, the results were similar to those observed in the immunohistochemistry analysis. The above findings indicate that increased GOLPH3 expression is closely associated with the incidence of gastric cancer, and GOLPH3 may be involved in the gastric cancer invasion and metastasis.
Recently, an increasing number of signal transduction pathways have been reported to be involved in tumor invasion and metastasis. Mammalian target of rapamycin (mTOR), which is a serine/threonine kinase downstream of the phosphatidylinositol 3kinase (PI3K)/Akt signaling pathway and includes mTORC1 (mTOR/RAPTOR) and mTORC2 (mTOR/RICTOR), regulates protein synthesis, cell proliferation, cell metabolism and differentiation [bib_ref] Defining the role of mTOR in cancer, Guertin [/bib_ref] [bib_ref] Molecular mechanisms of liver regeneration and protection for treatment of liver dysfunction..., Fujiyoshi [/bib_ref]. Activated mTORC1 sequentially directly activates downstream targets, including p70S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and mTORC2 has been shown to play a critical role in Akt phosphorylation at Ser473, resulting in the full activation of Akt [bib_ref] Upstream and downstream of mTOR, Hay [/bib_ref] [bib_ref] TOR, a central controller of cell growth, Schmelzle [/bib_ref]. Although Akt directly activates mTOR/RAPTOR, it may also directly or indirectly activate the p70S6 kinase/4EBP1 pathway this pathway by phosphorylating the tumor suppressor TSC2 [bib_ref] mTOR? RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1..., Hresko [/bib_ref]. The Akt/mTOR pathway has been shown to be frequently activated in various malignant tumors, including breast cancer, urothelial carcinoma, ovarian cancer, pancreatic neuroendocrine tumors, human medulloblastoma, melanoma, endometrial cancer and hepatocellular carcinoma; therefore, this pathway represents a possible therapeutic target in many cancers [bib_ref] Prognostic and predictive value of p-Akt, EGFR, and p-mTOR in early breast..., Lazaridis [/bib_ref] [bib_ref] Aivation of the PI3K/Akt/mTOR pathway correlates with tumour progression and reduced survival..., Sun [/bib_ref] [bib_ref] Activation of mTOR signaling pathway associated with adverse prognostic factors of epithelial..., No [/bib_ref] [bib_ref] Mammalian target of rapamycin signaling activation patterns in pancreatic neuroendocrine tumors, Komori [/bib_ref] [bib_ref] mTOR is activated in the majority of malignant melanomas, Karbowniczek [/bib_ref] [bib_ref] The PI3K/AKT/mTOR pathway as a therapeutic target in endometrial cancer, Slomovitz [/bib_ref] [bib_ref] Pivotal role of mTOR signaling in hepatocellular carcinoma, Villanueva [/bib_ref]. In our study, RT-PCR and Western blotting revealed that the mRNA and protein expression levels of p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 in the gastric cancer group were significantly higher than in the para-carcinoma tissues and paired normal gastric tissue groups [fig_ref] Figure 2: Representative images showed semi-quantitative RT-PCR for GOLPH3, Akt, mTOR, 4E-BP1 and p70S6... [/fig_ref]. To determine whether the expression levels of p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 were associated with critical clinicopathological characteristics, we performed further analyses. In this study, 81.25% of the 80 patients with gastric cancer had high p-mTOR expression, 77.50% had high p-4E-BP expression, 76.25% had high p-p70S6 expression, and only 75.00% had high p-Akt expression . Surprisingly, an immunohistochemical analysis revealed that the expression levels of these protein expressions were significantly associated with the depth of invasion, lymph node and distant metastasis, and histological grade but were not associated with gender or age [fig_ref] Table 3: Correlation between clinicopathological features and the p-P70S6 expression in gastric cancer specimen [/fig_ref]. These results suggest that the Akt/mTOR pathway is activated in gastric cancer and may play an important role in gastric cancer tumorigenesis, invasion and metastasis.
Recently, Scott et al. revealed that GOLPH3 can promote cell transformation and tumor growth by constitutively activating the mTOR signaling pathway in melanoma cells and that GOLPH3 stimulates the mTOR signaling pathway via mTORC1 and mTORC2 complexes, thereby promoting tumor cell growth and proliferation [bib_ref] GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer, Scott [/bib_ref]. However, it is unclear whether this phenomenon also occurs in gastric cancer tissues. To our knowledge, our study is the first to explore the pathogenesis of GOLPH3 promoted tumorigenesis, invasion and metastasis of gastric cancer. Our data show a significant positive relationship between the expression of GOLPH3 and p-mTOR, p-4E-BP1, and p-p70S6 (r = 0.410, 0.203 and 0.128, respectively, p,0.05). However, the correlation between the expression of GOLPH3 and p-Akt was not significant (r = 0.258, p.0.05) . In particular, significantly higher GOLPH3 expression was accompanied by high expression of p-mTOR, p-4E-BP1, and p-p70S6 in gastric cancer tissues, and this relationship was significantly associated with the depth of invasion, histological grade, and lymph node and distant metastasis. Our results revealed various relationships between GOLPH3 and Akt/ mTOR signaling proteins, and the positive association between GOLPH3 and p-Akt was nearly significant. The specific causes of . Correlations between the expressions of GOLPH3, p-AKT1, p-mTOR, p-70S6, and p-4E-BP1. this relationship between GOLPH3 and p-Akt are unclear and should be further investigated. We speculate that the sample size may have been too small. In addition, GOLPH3 may primarily activate the Akt/mTOR signaling pathway via the activated mTORC1 complex rather than the mTORC2 complex, resulting in p70S6 and 4E-BP1 phosphorylation, thereby affecting gastric cancer. These results indicate that GOLPH3 may be involved in gastric cancer tumorigenesis, invasion and metastasis via abnormal activation of the Akt/mTOR signaling pathway.
In conclusion, this study detected the high expression levels of GOLPH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 in the gastric cancer group strongly which correlated with histological grade, depth of invasion, distant metastasis, and lymph node involvement by clinicopathological variables analysis and immunohistochemistry. Furthermore, the expression of GOPLH3 is highly correlated with the activation of Akt/mTOR signaling pathway in human gastric cancer samples. Based on these results, we speculate that GOLPH3 might be affected progression and metastasis of gastric cancer through the activation of Akt/mTOR signaling pathway. Moreover, we propose that the inhibition of the expression of genes in the GOLPH3 and mTOR signaling pathway may represent a novel target for gastric cancer therapy. Finally, the invasion and migration mechanisms of GOLPH3 in gastric cancer still need further investigation.
# Author contributions
[fig] Figure 1: Representative GOLPH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 immunohistochemistry images in different tissues (brown granules, original magnification6400). A: The gastric cancer group B: The carcinoma-adjacent tissue group C: The paired normal tissue group. Representative tissue sections from the gastric cancer group (n = 80), the carcinoma-adjacent tissue group (n = 80) and the paired normal tissue group (n = 80 [/fig]
[fig] Figure 2: Representative images showed semi-quantitative RT-PCR for GOLPH3, Akt, mTOR, 4E-BP1 and p70S6 transcription. cancer: The gastric cancer group (n = 80); paracancerous: The paracancerous tissue group (n = 80); normal: The paired normal gastric mucosa group (n = 80). A, B, C, D and E: The mRNA levels of GOPLH3, Akt, mTOR, 4E-BP1, p70S6 in the different measured by RT-PCR, respectively. F, G, H, I and J: Densitometric quantification of GOLPH3, Akt, mTOR, 4E-BP1 and p70S6 mRNA expression in different tissues, respectively. The mRNA expression was significantly different with each other and peaked at cancer. The data are presented as the mean 6 the SE. *p,0.05 versus cancer, # p,0.05 versus paracancerous. doi:10.1371/journal.pone.0107362.g002 p = 0.027). Phosphorylated p70S6 expression was also correlated with the expression of p-Akt (r = 0.274, p,0.001) and p-mTOR (r = 0.451, p = 0.007). [/fig]
[fig] Figure 3: The GOPLH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 protein levels in the different tissues. A: The protein levels of GOPLH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 in the different measured by western blotting. B, C, D, E and F: The GOPLH3, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6 protein levels in the different tissues, respectively. cancer: The gastric cancer group (n = 80); paracancerous: The paracancerous tissue group (n = 80); normal: The paired normal gastric mucosa group (n = 80). The data are presented as the mean 6 the SE. *p,0.05 versus cancer, # p,0.05 versus paracancerous. doi:10.1371/journal.pone.0107362.g003 [/fig]
[table] Table 3: Correlation between clinicopathological features and the p-P70S6 expression in gastric cancer specimen.The associations between protein expression and clinicopathological variables were estimated using the chi-squared test. *Significant at the level of p,0.05. doi:10.1371/journal.pone.0107362.t003 [/table]
[table] Table 4: Sequences of primers used for RT-PCR. [/table]
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Recurrent Adult Sacrococcygeal Teratoma Developing Adenocarcinoma: A Case Report and Review of Literatures
Sacrococcygeal teratomas (SCT) are most commonly seen in infants and children but are rare in adults. Most adult SCT are benign and mature with a minority of tumors having immature components or overt malignancy. Here, we report a 65-yearold female with a SCT developing adenocarcinoma. The patient was diagnosed with benign sacrococcygeal cystic teratoma on her initial hospital visit and was treated with surgical resection. She was followed up postoperatively and was noted to have a markedly elevated CA 19-9 level 13 months after the surgery. Radiological and clinical examination revealed thickening of the perirectal soft tissues, located near the inferior portion of her previous incision site. Histological evaluation of the lesion showed invasive, moderately differentiated adenocarcinoma. Immunohistochemical staining results were suggestive, but not diagnostic, of anal gland adenocarcinoma. This case report expands the knowledge regarding an adenocarcinoma arising from a previously benign, adult SCT.
# Introduction
Teratomas are germ cell tumors, composed of cells derived from one or more germ cell layers. Teratomas of the sacrococcygeal region are most commonly seen in neonates with an approximate prevalence of 1/27,000 live births, predominantly affecting females. However, they are extremely rare in adults. Most of the sacrococcygeal teratomas (SCT) are benign and mature; malignant transformation within adult SCT is exceedingly rare with only several case reports documented . Most of the adult SCT are located within the pelvis and may cause compressive symptoms such as lower back pain, bowel/urinary dysfunctions, and venous engorgement of the lower limbs. The diagnosis depends on radiological imaging and histopathologic analysis. Early and complete surgical resection relieves the patient's symp-toms and normally leads to a favorable prognosis. However, there is an approximately 15% recurrent rate; risk factors for recurrence include incomplete resection, immature component, and frank malignancy.
## Case description
The patient is a 65-year-old female who initially presented to our hospital in 2019, complaining of pelvic pressure and anorectal pain for 2 years. She also had difficulty with urination and altered bowel movements. Abdominal and pelvic computed tomography (CT) scan showed a 17 cm cystic retrorectal mass. Pelvic magnetic resonance imaging (MRI) revealed a 17:3 × 11:4 × 7:7 cm retrorectal cystic mass with solid mural nodule. Her preoperative CEA level was 3.7 ng/mL (normal range, <5 ng/mL), and the CA- was elevated at 266 U/mL (normal range, <37 U/mL). The patient received surgical resection via Kraske approach with resection of the involved coccyx. At surgery, she was found to have a large cystic neoplasm encompassing the entire space above the levators and extending superiorly to the pelvic floor and posteriorly to the coccyx. The tumor was densely adherent to the surrounding tissues as well as the wall of the rectum and contained brownish fluid. At pathologic examination, the cyst wall was rough and uneven with a wall thickness ranging from 0.2 to 0.8 cm. Microscopic examination of the tumor showed squamous epithelium (ectoderm), mature bone, smooth muscle (mesoderm), respiratory epithelium, pancreatic tissue, and enteric epithelium (endoderm)-2(f)). Noticeably, the tumor also demonstrated foci of proliferating glandular structures with interspersed vasculature ); cellular atypia was appreciated at high magnification . Immunostains showed the atypical glandular epithelium to be positive for CK7 and CDX2 (Figures 3(c) and 3(d)) and focally positive for CK20, suggestive of enteric/colonic differentiation. Despite a high Ki67 index ), the histology of the tumor lacked definite evidence of malignancy and the p53 was wild-type ). Overall, the above evaluations confirm the diagnosis of benign mature SCT. The patient was followed up postoperatively and was noted to have a markedly elevated CA 19-9 level (397.8 U/mL) 13 months after the prior surgery. She also had rectal and low back pain which prompted surveillance with a CT scan of the abdomen and pelvis showing thickening of the perirectal soft tissues. This was confirmed on a follow-up MRI of the abdomen and pelvis. On physical exam, she was noted to have a subcentimeter palpable mobile, hard, rubbery nodule in right inner subcutaneous fat tissues of the left buttock. PET/CT (not shown) demonstrated intensely FDG avid nodularity in the perianal region as well as along the peritoneal reflection involving the pelvic floor, pelvic sidewalls extending superiorly up to the presacral region which favors local recurrence. She was subsequently brought to the operating room and the perirectal soft tissue mass, which was near the inferior portion of her previous Kraske incision in the right buttock, was removed. The lesion was sent for frozen section, which consisted of yellow tan fibro-fatty tissue measuring 2:3 × 1:5 × 1:5 cm; the diagnosis was that of recurrent sacrococcygeal teratoma cannot exclude underlying malignancy. Permanent sections of the lesion revealed invasive, moderately differentiated adenocarcinoma, arising from the preexisting sacrococcygeal teratoma. The tumor cell islands displayed an infiltrative pattern at low magnification), with marked structural and cellular atypia appreciated at high magnification. In comparison to the patient's previous excision , these findings showed a similar histology but with a greater degree of nuclear atypia and definite invasive architecture. The malignant glands showed a high proliferative index on Ki-67), while p53 was still wild-type. Additional immunostains of the invasive glands were reactive for CK7, AE1/ AE3, Ber-EP4, and CK19, while nonreactive for CK20, CDX2, GCDFP-15, TTF-1, PAX8, PAX2, GATA3, WT-1, and vimentin. This immunophenotype was nonspecific but suggestive of anal gland adenocarcinoma. She was scheduled for chemotherapy with FOLFOX (Leucovorin, Oxaliplatin followed by 5FU) given every 14 days × 12 cycles.
# Discussion
Teratomas are composed of cells derived from one or more germ cell layers (ectoderm, mesoderm, and endoderm), and they are classified as a subgroup of germ cell tumors. Different theories exist regarding the origin of the SCT, including that they are derived from the totipotential cells in Hensen's node of the primitive knot. The majority of teratomas are located in the ovaries or testicles (gonadal teratomas); however, they can also develop in midline structures such as mediastinum, retroperitoneal space, presacral and sacrococcygeal areas, and brain/spine.
SCT are classified according to the Altman classification system/American Academy of Pediatrics Surgical Section: (2) Type II: the tumor is predominantly external but has a significant intrapelvic extension.
(3) Type III: the tumor is visible externally, but is predominantly located in the pelvic area with extension into the abdomen.
(4) Type IV: the tumor has no external presentation and is located in the pelvic bone.
In neonates and children, type I tumors have the lowest risk of malignancy and type IV tumors have the highest risk of malignancy.
Most neonatal SCT are externally visible, while most adult SCT present as a pelvic mass and often cause compressive symptoms such as lower back pain, bowel/urinary dysfunctions, and venous engorgement of the lower limbs. Sometimes, SCT can present as an anal fissure when associated with infection. Interestingly, this patient was found to have anal fissure in 2018 during perianal exam, which could be the early presentation of her SCT. Macroscopically, SCT can be cystic, solid, or a mixture of solid and cystic components. Histopathologically, SCT are classified into three categories: mature, immature, and malignant. Mature teratomas are benign and contain fully differentiated somatic tissues such as the epithelium, muscles, and bones. Immature teratomas consist of at least foci of embryonal structures or incompletely differentiated tissue components; primitive neuroectodermal structures are often present. Teratomas containing any malignant elements are considered to be malignant. In adults, there are only a few case reports noting malignant transformation from sacrococcygeal/presacral teratomas : two cases being mucinous adenocarcinoma, one case being adenocarcinoma of gastrointestinal origin, and four cases being nonspecific adenocarcinoma. Cytogenetic aberrations (amplifications of 8q and 12p) have been reported to be associated with malignant transformation to an adenocarcinoma. Elevated serum 3 Case Reports in Pathology tumor markers (CEA, CA19-9, AFP, and HCG) are suggestive of malignant transformation and could be useful to monitor postoperative recurrence .
The differential diagnosis of SCT in adults includes tailgut cyst, chordoma, meningocele, pilonidal cysts, rectal duplication or anal gland cysts, osteomyelitis of sacrum, giant cell tumor of sacrum, perirectal abscess, fistula, granu-loma, and tuberculosis. Our patient had a cystic lesion with histology showing components from all three germ cell layers, leading to a diagnosis of benign mature sacrococcygeal cystic teratoma. Noticeably, foci of atypical glandular structures were also identified in the teratoma, with a high Ki67 proliferative index. She was monitored after initial surgery and at 13 months, her CA 19-9 level was : Proliferating glandular structures with interspersed vasculature (a); cellular atypia was appreciated at 40X magnification (b). Immunostains of the atypical glandular epithelium were positive for CK7 (c) and CDX2 (d). The Ki67 proliferative index was high (e). The p53 was wild-type (f). Case Reports in Pathology markedly elevated. Radiological imaging favored local recurrence, and excisional biopsy was performed. Histology revealed invasive, moderately differentiated adenocarcinoma. Immunostains in the invasive glands were reactive for CK7, AE1/AE3, and Ber-EP4 and nonreactive for CK20, CDX-2, and GCDFP15. This immunophenotype was suggestive but not diagnostic of anal gland adenocarcinoma. The atypical glandular structures observed in the previously resected tumor did not meet the criteria of adenocarcinoma; however, these atypical features may act as a a histological indication of the tumor's ability to recur and even transform into adenocarcinoma. Early and complete surgical excision of SCT with coccygectomy is the mainstay of treatment. The coccyx may contain the nidus of totipotential cells; the risk of tumor recurrence is reported to be 30-40% without concomitant excision of the coccyx. Unlike gonadal teratomas, SCT are often unencapsulated, making it difficult to achieve a complete resection. The overall recurrence rate for mature teratomas is about 10%, and that for immature teratomas is about 20%. Complications of surgery include massive bleeding, bowel/urinary dysfunction, and dysesthesia. For malignant cases, additional treatment with chemotherapy and/or radiotherapy is required. Given the rarity of the entity, the standard treatment regimen has not been well established. Benign SCT have excellent prognosis with complete surgical excision; however, the prognosis of malignant SCT is poor.
[formula] HE, 20X (a) HE, 40X (b) CK7 (c) CDX2 (d) Ki67 (e) p53 (f) [/formula]
# Conclusion
Adult SCT are rare and often recognized following symptoms caused by compressive symptoms, including lower back pain, bowel/urinary dysfunctions, and venous engorgement of the lower limbs. Most cases are mature and benign; however, rarely malignant transformation can occur. Atypical structures with a high Ki67 proliferative index, if present, may help to alert that tumor recurrence or transformation into adenocarcinoma is possible. The diagnosis relies on radiological imaging (CT or MRI), serum markers, and histopathologic analysis. Complete surgical excision with coccygectomy is the mainstay treatment for benign cases (both mature and immature SCT) and often leads to a favorable prognosis. Malignant SCT require surgical excision with adjuvant therapy including chemotherapy and/or radiotherapy. Postoperative follow-up of the patients is crucial to detect recurrence.
## Conflicts of interest
The authors declare that they have no conflicts of interest.
## Case reports in pathology
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The Visual Attention and Psychological Responses from Older Customers to Wellness Service Pictures of Hotels
# Introduction
The health market segmentation of the hotel industry is gradually expanding [bib_ref] Segmentation and the senior traveler: Implications for today's and tomorrow's aging consume, Faranda [/bib_ref] , and the main consumer group of this market is older consumers (age 65 and older). Compared with young people, older consumers feel that they have limited time [bib_ref] The effect of ageing and time horizon perspective on consumers' response to..., Micu [/bib_ref] and expect to suffer losses in the future [bib_ref] Developmental expectations for the self and most other people: Age grading in..., Heckhausen [/bib_ref]. Therefore, they are concerned more with goals related to avoiding loss, such as maintaining body functions and delaying aging. However, hotel researchers still do not understand the impact of health service marketing advertisements provided by hotels on the visual and psychological responses of older consumers who are actively seeking methods to avoid losses.
To obtain a gradual expansion of the wellness market, hotels have started to focus on wellness service images when designing marketing images. Wellness service is defined as the service packages that provide appropriate professional knowledge and individual care to preserve or promote customers' health [bib_ref] Wellness tourism: Market analysis of a special health tourism segment and implications..., Mueller [/bib_ref]. In the hotel field, natural and built servicescapes represent the two ends of the environment design spectrum [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. Natural servicescapes refer to the use of the natural environment (e.g., mountains, rivers, tropical rain forests, and oceans) to build servicescapes in which consumers can enjoy physical activities [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. Natural servicescapes can shorten the distance and facilitate the interaction between individuals and natural environments [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] The nature of nature in nature-based tourism, Fredman [/bib_ref] , allowing consumers to enjoy a relaxing experience. Hotels can use the natural environment as a background for physical activity and encourage consumers to engage in physical activities in the natural environment. Built servicescapes refer to the use of indoor space (e.g., sound, visual design, functional equipment, and atmosphere) to create a better exercise experience [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] The impact of fitness center servicescape on individual behavior: The mediating role..., Ong [/bib_ref]. Hotels can influence consumer behavior by designing visual images of the physical environment, providing functional equipment, and creating relaxing spaces. Built servicescapes provide attractive designs, pleasant environmental conditions, sufficient activity space, and proper facility functioning to ensure that customers can perform physical activities satisfactorily [bib_ref] The impact of fitness center servicescape on individual behavior: The mediating role..., Ong [/bib_ref]. To design an effective wellness service image and promote the development of the wellness service market, this research aims to confirm the influence of marketing images based on natural or built servicescapes on the visual attention and psychological responses of older customers in terms of their well-being and willingness to pay.
In order for hotels to design tourist wellness service pictures that can effectively attract older customers, it is necessary to explore how to design the characteristics of the pictures from their perspective. Attention restoration theory argues that natural environments can easily attract the attention of individuals and stimulate them to engage in physical activity in such an environment [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. The interactive relationship between individuals and natural environments can improve the emotional states of individuals, which in turn leads to a number of restorative outcomes [bib_ref] Nature and health, Hartig [/bib_ref]. According to previous findings on the attention restoration theory in the fields of marketing [bib_ref] Validating the search, experience, and credence product classification framework, Girard [/bib_ref] [bib_ref] Exploring restorative potential of biophilic servicescapes, Purani [/bib_ref] , tourism [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] and environmental psychology [bib_ref] Nature and health, Hartig [/bib_ref] [bib_ref] The restorative benefits of nature: Toward an integrative framework, Kaplan [/bib_ref] [bib_ref] Restorative qualities of indoor and outdoor exercise settings as predictors of exercise..., Hug [/bib_ref] , hotel marketing managers can consider designing images that have visual clues related to recovery from mental fatigue, stress reduction, or improved emotional states, thereby improving marketing effectiveness [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref].
To accurately investigate the influence of image features on older consumers' visual and psychological responses, an eye tracking technique was adopted in the current study to record individual eye movement trajectories and analyze visual attention [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] Individual differences in selective attention to information graphics in televised sports, Cummins [/bib_ref]. Previous researchers have used this method to conduct choice experiments, where it was found that this technology makes it possible to obtain real-time eye movement information that expands the scope of information obtained from analyzing individual preferences [bib_ref] Eye tracking methodology; Theory and practice, Duchowski [/bib_ref] [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref]. For example, [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] found that images of natural servicescapes may attract more visual attention and arouse higher restorative perceptions among potential customers than built servicescapes.
The purposes of this study were thus to explore the potential divergent effects of hotel wellness service pictures with natural or built clues on visual attention, perceived well-being, and willingness to pay in older consumers. The findings will not only provide insight into the academic development of attention restoration theory in the tourism and hospitality field, but will also contribute to the ability of hotel marketing managers to develop effective tourism service advertisements.
## Wellness service-yoga
Many countries are beginning to face the challenge of an aging population. The ageing of the population has gradually increased the age of consumption in the hospitality industry, which in turn has created a new market segment. Compared to other age groups, the elderly population is a group of people with higher amounts of liquid capital and more travel experience [bib_ref] Essential customer service factors and the segmentation of older visitors within wellness..., Chen [/bib_ref]. Older adults are motivated to engage in tourism because it contributes to keeping them healthy and relaxed [bib_ref] Multi-dimensions of the perceived benefits in a medical hotel and their roles..., Han [/bib_ref] [bib_ref] Investigating customer loyalty formation for wellness spa: Individualism vs. collectivism, Han [/bib_ref].
Because well-being is a serious concern of older consumers, wellness services are the most attractive leisure option for the rapidly ageing population. Wellness services allow tourist hotels in the competitive tourism market to increase their operating income by enriching the choices of tourism products [bib_ref] Place-focused physical activity research, human agency, and social justice in public health:..., Blacksher [/bib_ref]. As a result, many hotels have started to use wellness as a part of their business strategies. The wellness service market differs from that of the traditional hotel market. Up until the present, research on wellness services has been rare, leading to a lack of understanding on the part of hotel researchers and managers of this potential market segment.
Yoga is suitable as a physical activity for older adults and yields both physical and psychological benefits. Many studies have also confirmed that yoga can effectively improve emotions in older adults (e.g., reducing depression and stress and improving mood) as well as cognitive functions (e.g., attention, memory, and resiliency) [bib_ref] Place-focused physical activity research, human agency, and social justice in public health:..., Blacksher [/bib_ref]. Thus, previous research in the field of well-being tourism has reported that older tourists are the target market for yoga tourism marketing. For example, Ali-Knight and Ensor (2017)explored yoga tourist profiles and found that seniors prefer this type of physical activity.
## Older consumer responses after observing wellness service pictures
Tourists always expect to recover from mental fatigue and reduce stress during a vacation [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] , so they pay special attention to wellness services that meet their needs. If the marketing messages provided by the hotel meets consumers' expectations for recovering from mental fatigue and relieving stress, this will affect their preferences, behavioral intentions, and choice decisions. Attention restoration theory is a cognitive theory that focuses on recovery from mental fatigue [bib_ref] The restorative benefits of nature: Toward an integrative framework, Kaplan [/bib_ref]. Spending time in an environment with a recovery potential has been regarded as a means of restoring life energy [bib_ref] The restorative benefits of nature: Toward an integrative framework, Kaplan [/bib_ref] [bib_ref] Restorative effects of natural environment experiences, Hartig [/bib_ref]. In the field of marketing and tourism research, such theories are considered to be an effective tool that can be used to understand and design marketing images and servicescapes that convey a restorative quality and influence behavioral decisions [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref].
According to the attention restoration theory, there are three key responses of older consumers after observing the wellness service images: visual attention [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] Restorative qualities of indoor and outdoor exercise settings as predictors of exercise..., Hug [/bib_ref] [bib_ref] Are we doing enough for visual research in tourism? The past, present,..., Park [/bib_ref] , perception [bib_ref] Landscape and well-being: A scoping study on the health-promoting impact of outdoor..., Abraham [/bib_ref] [bib_ref] Mental health benefits of neighbourhood green space are stronger among physically active..., Astell-Burt [/bib_ref] [bib_ref] The benefits of natural environments for physical activity, Shanahan [/bib_ref] , and behavioral responses [bib_ref] The role of nature-based experiences in the development and maintenance of wellness, Brymer [/bib_ref] [bib_ref] The development of a sustainable wellness service marketing strategy in Taiwan based..., Wang [/bib_ref]. Visual attention is the first response, while customers look at tourism service images (or videos, products, etc.). It reflects the positioning of wellness service images that older consumers are interested in. The second reaction is perception, which is an assessment of the subjective well-being of older consumers (e.g., the degree of well-being they perceive after participating in a yoga program). The third reaction is a behavioral response, which represents the behavioral intention of older consumers after they observe wellness service images.
## Eye tracking and visual attention
Visual attention is defined as the allocation of information processing abilities [bib_ref] Seeing the voice of the customer: Metaphor-based advertising research, Zaltman [/bib_ref] and it is used to describe an individual's selective observation activities toward images (or videos, products etc.) [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref]. Researchers can understand customers' visual behavior through analyzing their visual focus when they look at hotel advertising images using eye tracking technology. Eye tracking systems are a form of sensor technology that can be used to objectively collect data related to a customer's internal responses and that avoid the common method variance generated by collecting subjective measures through questionnaires [bib_ref] Eye tracking methodology; Theory and practice, Duchowski [/bib_ref].
Attention is a selective mechanism for determining the extent to which a customer is concerned about a specific stimulus. Attention will directly influence product selection [bib_ref] Eye tracking reveals processes that enable conjoint choices to become increasingly efficient..., Meißner [/bib_ref]. Customers do not treat all marketing images the same way; rather, they assign more visual attention to features they are interested in. Customer attention can thus be assessed by observing eye movements [bib_ref] Eye tracking reveals processes that enable conjoint choices to become increasingly efficient..., Meißner [/bib_ref]. In the marketing field, the eye tracking technology used to capture individual eye movements has been used to assess customer attention toward advertising. It has been demonstrated to be an effective tool for assessing the appeal of images [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref]. Accordingly, eye movement is thought to be an effective measure of visual attention [bib_ref] Visual attention to repeated print advertising: A test of scanpath theory, Pieters [/bib_ref] , and can also reflect the content to which viewer attention is directed [bib_ref] Space and selective attention, Rizzolatti [/bib_ref].
## The relationship between environmental clues in images and visual attention
Visual attention is a selective process that assigns limited perceptual capabilities related to specific characteristics of wellness service images, while ignoring others [bib_ref] Visual attention: The past 25 years, Carrasco [/bib_ref]. Through these processes, older consumers transfer their attention from the wellness service images as a whole to the part they are most interested in.
The results of a green exercise study showed that older consumers prefer to be close to nature rather than in indoor sports environments, because nature can satisfy their pursuit of natural experiences and convenience motivations [bib_ref] The potential of using exercise in nature as an intervention to enhance..., Calogiuri [/bib_ref]. Although indoor sports environments adopt a closed space design and provide individuals with full-length mirrors to exercise in front of them, so they can observe the physical changes resulting from physical activity, built images are less likely to attract observers' visual attention and are less able to provide psychological relaxation benefits to consumers [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref] [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref] found that natural images, as compared to built images, attract more frequent visual attention from customers. In addition, based on the attention restoration theory, as mentioned above, [bib_ref] The potential of using exercise in nature as an intervention to enhance..., Calogiuri [/bib_ref] [bib_ref] The potential of using exercise in nature as an intervention to enhance..., Calogiuri [/bib_ref] found that when individuals exercise in a natural environment, their attention will shift to the environment, rather than to their inner fatigue, leading to a decrease in perceived fatigue. In addition, consumers may have greater visual responses to pictures with natural images due to those images providing more psychological benefits [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] than pictures with built images. Therefore, compared to pictures that provide built clues, wellness service pictures that present clues related to connecting with the natural environment may be more attractive to older adults. Based on the previous discussion, the following hypothesis is proposed: Hypothesis 1 (H1). Older consumers will direct more visual attention towards images with visual clues depicting nature than to wellness service images with built clues.
## The relationship between environmental clues in images and well-being
The natural environment can be used as a wellness resource that promotes physical and psychological well-being [bib_ref] Landscape and well-being: A scoping study on the health-promoting impact of outdoor..., Abraham [/bib_ref] [bib_ref] Yoga tourism-A catalyst for transformation? Ann, Dillette [/bib_ref]. In particular, untouched surroundings are the physical environment necessary to create a transformational well-being experience [bib_ref] Conceptualizing transformative guest experience at retreat centers, Fu [/bib_ref]. When individuals are surrounded by nature, they have opportunities to reflect on the relationship between humans and the environment [bib_ref] The restorative benefits of nature: Toward an integrative framework, Kaplan [/bib_ref]. In addition, the natural environment also helps individuals recover from mental and physical trauma. According to attention restoration theory, compared to physical activity in a built environment, physical activity in a natural environment allows older consumers to experience both the beneficial effects derived from nature and from physical activity. For example, using middle-aged to elderly adults as participants, the results of Astell-Burt et al. (2013) [bib_ref] Mental health benefits of neighbourhood green space are stronger among physically active..., Astell-Burt [/bib_ref] confirmed that physically active adults living in a green environment experience a lower risk of psychological distress.
Images can convey emotional messages, in turn allowing observers to imagine what the environment provides. When individuals look at a tourism image, they can imagine themselves in the servicescape depicted in the image and can imagine experiences in that environment [bib_ref] Beyond image: Imagined experiences of a destination, Cherifi [/bib_ref]. When potential consumers see beautiful pictures of natural environments associated with a tourist hotel, their fascination for nature is aroused, and therefore positive emotions toward the hotel are increased [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. Previous research has reported that compared with wellness service images that provide clues related to an indoor sports environment, pictures with clues about nature provide individuals with more effects related to reducing fatigue and increasing relaxation and stress relief [bib_ref] Landscape and well-being: A scoping study on the health-promoting impact of outdoor..., Abraham [/bib_ref]. In addition, [bib_ref] The restorative potential of shopping malls, Rosenbaum [/bib_ref] [bib_ref] The restorative potential of shopping malls, Rosenbaum [/bib_ref] confirmed that shopping centers can promote personal and social well-being by providing shoppers with a restorative servicescape. Their research not only found that shopping centers that incorporate green elements into the retail area promote wellness, but also found that shoppers who sense recovery quality in such environments develop good attitudes and exhibit positive behavior toward shopping centers. Accordingly, older consumers who observe wellness service images with clues suggesting a natural environment will tend to have a higher sense of well-being, where these images can evoke positive emotions and even enhance expectations of an improved sense of mental and physical well-being when practicing yoga in natural spaces. Based on the previous discussion, the following hypothesis is proposed.
Hypothesis 2 (H2). Compared to observing wellness service images with built visual clues, older consumers will perceive a higher sense of well-being from wellness service images with natural visual clues.
## The relationship between environmental images and willingness to pay
Evaluations of marketing effectiveness using wellness service images are usually accompanied by the assessment of customer willingness to participate in wellness services and pay fees. A consumer's willingness to pay for a service is defined as the maximum price a consumer is willing to pay for a given service [bib_ref] Definition, measurement and determinants of the consumer's willingness to pay: A critical..., Gall-Ely [/bib_ref] [bib_ref] Measuring consumers' willingness to pay at the point of purchase, Wertenbroch [/bib_ref]. Thus, consumers may have a higher willingness to pay for wellness services that they prefer. Customers are willing to pay a relatively high price to purchase high-level service quality that maximizes consumption value [bib_ref] Willing to pay more, eager to pay less: The role of customer..., Wieseke [/bib_ref]. When older adults recognize that wellness service images with nature clues (e.g., learning yoga activities in the natural environment) can satisfy their pursuit of wellness, their willingness to pay higher fees will increase.
The attention restoration theory argues that exercise in the natural environment not only reduces physical and mental fatigue in older adults [bib_ref] The role of nature-based experiences in the development and maintenance of wellness, Brymer [/bib_ref] , but also enhances the effectiveness of physical activity [bib_ref] The benefits of natural environments for physical activity, Shanahan [/bib_ref] [bib_ref] Is physical activity in natural environments better for mental health than physical..., Mitchell [/bib_ref]. Wellness service images that provide nature clues make it possible for consumers to recognize that a tourist hotel can provide opportunities for them to interact with nature and simultaneously engage in physical activity. A high-level restorative experience can lead consumers to spend more time and money to enjoy a memorable consumer experience [bib_ref] A dose of nature and shopping: The restorative potential of biophilic lifestyle..., Rosenbaum [/bib_ref]. For example, Rosenbaum, Otalora, and Ramírez (2016) [bib_ref] The restorative potential of shopping malls, Rosenbaum [/bib_ref] found that consumers' planned shopping expenditures increased after a highly restorative experience. Thus, consumers are willing to pay more for shopping in a highly restorative retail service environment rather than in a less restorative environment [bib_ref] A dose of nature and shopping: The restorative potential of biophilic lifestyle..., Rosenbaum [/bib_ref]. Therefore, wellness service images with clues suggesting an experience in nature are expected to have a higher impact on older adults' willingness to pay more than those with indoor sports clues. Based on the previous discussion, the following hypothesis is proposed.
## Hypothesis 3 (h3).
Compared with observing wellness service images with built clues, older consumers observing images with nature clues will be willing pay more for services.
# Methods
## Participants
Older adults (age 65 and older) were the source of the study sample. The researchers contacted participants in the database of the Taichung Senior Citizens Association in Taiwan through flyers or personal visits, and invited them to participate in the experiment.
The criteria for selecting participants were as follows: (1) Individuals were over the age of 50, (2) the participants had normal vision in both eyes (more than 0.8 after correction) and had no major eye diseases (e.g., macular lesions), and (3) individuals could not physically move were excluded. This decision was made so as to ensure that the participants would not be discouraged from practicing yoga owing to physical limitations.
To effectively control the experiment, 100 candidates were contacted and agreed to participate in this study. However, 15 participants with binocular vision not reaching 0.8 or more and major eye and monocular data of 0 were excluded. A total of 85 valid participants were enrolled in this study, with an effective sampling rate of 85%. The Mini-Mental State Examination (MMSE) [bib_ref] Mini-mental state": A practical method for grading the cognitive state of patients..., Folstein [/bib_ref] is the well-established measure tool for cognition. An MMSE score above 26 was used to classify adults as not being cognitively impaired [bib_ref] Mini-mental state": A practical method for grading the cognitive state of patients..., Folstein [/bib_ref]. The average age of the participants was 66.54 years old, and the average MMSE score was 27.80. Of all participants, 42 were males and 43 were females;
A G*Power analysis was performed to determine whether the sample size was sufficient to verify the research hypothesis [bib_ref] Power 3: A flexible statistical power analysis program for the social, behavioral,..., Faul [/bib_ref]. The results of the G*Power analysis showed that the study had an 80% power to detect small effects (d = 0.27) at the 5% level of visual attention significance. The G*Power analysis results thus indicated that the sample size of this study was sufficient to test the hypotheses.
## Selection of stimuli
To select the images and minimize the confounding factors between them, the following processes were used in the present study [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. First, the pictures used as the stimuli were designed with the same yoga posture, as yoga is a low-intensity exercise mode and is suitable for promoting the wellness of older individuals and preventing diseases [bib_ref] Exercise interventions on health-related quality of life for people with cancer during..., Mishra [/bib_ref]. Second, the backgrounds of the pictures were designed with both nature clues and built clues, with the nature clues referring to the use of natural environments (e.g., grasslands, oceans, mountains, and other natural landscapes) and the built clues referring to indoorexercise-space materials (e.g., floors, carpets, mirrors, and cement walls). Third, three experts in the physical activity field were invited to remove photos with low brightness and insufficient clarity, or those that were considered unsuitable for older adults, with 36 pictures being rejected. Each picture had a single coach performing a yoga posture that can be performed by any participant in the sample. At this step, photos with specific customer characteristics were also rejected. Finally, based on the consensus of the experts, 16 pictures were selected, with eight pictures having nature clues and eight having built clues. Samples of the experimental pictures are shown in [fig_ref] Figure 1: Two samples of the experimental pictures [/fig_ref].
## Selection of stimuli
To select the images and minimize the confounding factors between them, the following processes were used in the present study [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. First, the pictures used as the stimuli were designed with the same yoga posture, as yoga is a low-intensity exercise mode and is suitable for promoting the wellness of older individuals and preventing diseases [bib_ref] Exercise interventions on health-related quality of life for people with cancer during..., Mishra [/bib_ref]. Second, the backgrounds of the pictures were designed with both nature clues and built clues, with the nature clues referring to the use of natural environments (e.g., grasslands, oceans, mountains, and other natural landscapes) and the built clues referring to indoorexercise-space materials (e.g., floors, carpets, mirrors, and cement walls). Third, three experts in the physical activity field were invited to remove photos with low brightness and insufficient clarity, or those that were considered unsuitable for older adults, with 36 pictures being rejected. Each picture had a single coach performing a yoga posture that can be performed by any participant in the sample. At this step, photos with specific customer characteristics were also rejected. Finally, based on the consensus of the experts, 16 pictures were selected, with eight pictures having nature clues and eight having built clues. Samples of the experimental pictures are shown in [fig_ref] Figure 1: Two samples of the experimental pictures [/fig_ref].
## Wellness service pictures with built clues
Wellness service pictures with nature clues [fig_ref] Figure 1: Two samples of the experimental pictures [/fig_ref]. Two samples of the experimental pictures (portraits adopted from https://www.freepik.com/free-photo/health-beautiful-female-body-peace_1057237.htm#page=8&query=gym%20background&position=8, accessed date 1 December 2021).
## Experimental procedure
The participants were asked to pretend that they were watching online marketing advertisements provided by hotels in the form of wellness service pictures, after which their vision, attitude, and willingness to pay reactions after watching these advertisements were explored. The participants were told that they had sufficient budget to purchase exercise courses. This was intended to eliminate budgetary constraints related to purchasing exercise services. The price of each exercise plan was not included in the advertisement, as this could have interfered with the service selection process.
Before the experiment, participants were asked to state their health status and to confirm that they had no mental illness or cognitive impairments, as assessed by the MMSE (i.e., scores > 24), and had normal or corrected vision greater than 0.8 [bib_ref] Differences in vision performance in different scenarios and implications for design, Goodman-Deane [/bib_ref]. All participants signed an informed consent form approved by the Human Research Ethics Committee of the National Cheng Kung University in Taiwan. The participants were then randomly assigned to either the natural environment group (n = 43) or the built environment group (n = 42). Each picture among the eight pictures was randomly selected for each group and was exhibited for 12 s to participants in an acoustically shielded room with dimmed lights (Wang et al., 2108a; Wang and Sparks, 2016). After each picture was displayed for 12 s, the next picture was displayed.
## Experimental procedure
The participants were asked to pretend that they were watching online marketing advertisements provided by hotels in the form of wellness service pictures, after which their vision, attitude, and willingness to pay reactions after watching these advertisements were explored. The participants were told that they had sufficient budget to purchase exercise courses. This was intended to eliminate budgetary constraints related to purchasing exercise services. The price of each exercise plan was not included in the advertisement, as this could have interfered with the service selection process.
Before the experiment, participants were asked to state their health status and to confirm that they had no mental illness or cognitive impairments, as assessed by the MMSE (i.e., scores > 24), and had normal or corrected vision greater than 0.8 [bib_ref] Differences in vision performance in different scenarios and implications for design, Goodman-Deane [/bib_ref]. All participants signed an informed consent form approved by the Human Research Ethics Committee of the National Cheng Kung University in Taiwan. The participants were then randomly assigned to either the natural environment group (n = 43) or the built environment group (n = 42). Each picture among the eight pictures was randomly selected for each group and was exhibited for 12 s to participants in an acoustically shielded room with dimmed lights [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref]. After each picture was displayed for 12 s, the next picture was displayed.
An eye tracker was used to record the eye movement trajectory of the participants while observing the experiment pictures. Before the eye tracker began recording eye movement data, a nine-point calibration procedure was performed. After collecting the eye movement data, the participants were asked to complete questionnaires providing information about their sense of perceived well-being and their willingness to pay to participate in the yoga program after observing the picture. The participants' gender, age, and travel frequency from the previous year were also collected. The entire experiment took approximately 1 h.
## Instruments
An eye tracker (sampling rate 60 Hz) (Tobii X2-30, Danderyd, Sweden) placed in front of a 24-inch widescreen TFT monitor (1920 × 1080 pixels) was used to present the stimuli and to capture the eye movement information from the participants. Each participant was 60 cm away from the screen monitor.
## Measurement
Visual Attention. Eye tracking technology visualizes visual attention in the form of a gaze plot, allowing researchers to explore visual attention. The key indicators used to assess individual visual attention were the fixation count and the fixation time [bib_ref] Visual attention to repeated print advertising: A test of scanpath theory, Pieters [/bib_ref] [bib_ref] Understanding Consumers' Inferences from Price and Nonprice Information in the Online Lodging..., Noone [/bib_ref]. The fixation count is the number of times the individual interacts with the stimuli, where a greater count of fixation indicates that the participant finds the information to be more attractive [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. The fixation time indicates the processing time for the individual to watch the stimuli, where a longer fixation time indicates that the individual spent more time examining the information or the relationships between the internal and external representations [bib_ref] Shining in the center: Central gaze cascade effect on product choice, Atalay [/bib_ref]. Therefore, images with a longer fixation time were observed, indicating that the participants' visual attention was attracted more during that time [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. Individual visual attention typically lasts from 200 ms to 500 ms. As neuroscience visuo-cognitive research usually uses a threshold of 200 ms [bib_ref] Defining the temporal threshold for ocular fixation in free-viewing visuocognitive tasks, Manor [/bib_ref] [bib_ref] Visual encoding and fixation target selection in free viewing: Presaccadic brain potentials, Nikolaev [/bib_ref] , values below 200 ms were removed [bib_ref] An Eye-Tracking Study of Tourism Photo Stimuli Image Characteristics and Ethnicity, Wang [/bib_ref].
Perceived well-being. The scale developed by Tseng and Shen (2014) [bib_ref] Meditation tourism: Exploring the meditation flow experience and well-being, Tseng [/bib_ref] was used to measure perceived well-being, and seven items were used. The participants were asked to express their perspectives after observing the wellness service images, including what degree of physical wellness and mental wellness they would feel if they participated in such a wellness service. These measures consisted of items with response options ranging from 1 (strongly disagree) to 7 (strongly agree).
Willingness to pay. The psychological response of willingness to pay was measured using one item. The researcher asked the participants to respond to the following statement: To assist a hotel marketing manager of a tourist hotel in the pricing of wellness services, please state the maximum price you would be willing to pay to participate in the wellness services provided by the tourist hotel in the marketing image. The participants provided the maximum amount they were willing to pay in the form of an open-ended response [bib_ref] Can, cup, or bottle? The influence of beverage vessel on taste and..., Lefebvre [/bib_ref].
# Statistical analysis
The data were analyzed using a t-test and descriptive statistics with SPSS version 21.0. The descriptive statistics were presented as means and percentages for willingness to pay in different categories. The t-test for the dependent variables was used to examine the differences in the visual attention, perceived well-being, and willingness to pay. [fig_ref] Table 1: Effects of image characteristics on older consumers' visual attention [/fig_ref] , the average time spent by the participants observing wellness service images with nature clues was higher than that spent observing images with built clues (t = 2.10, p = 0.037). Therefore, the yoga pictures with natural characteristics attracted more visual attention from the participants. Thus, the results supported Hypothesis 1.
# Results
## As indicated in
There were no significant differences found in the observations of pictures with natural and built clues in terms of perceived well-being (See [fig_ref] Table 1: Effects of image characteristics on older consumers' visual attention [/fig_ref]. Therefore, Hypothesis 2 was not supported.
As shown in [fig_ref] Table 2: Effects of image characteristics on willingness to pay in older consumers [/fig_ref] , the natural and built characteristics of wellness service images had different effects on the psychological response of willingness to pay in participants. Compared with yoga images with built characteristics, yoga pictures with natural characteristics induced more potential to evoke willingness to pay (t = 2.05, p = 0.023). Thus, this result supported Hypothesis 3. Furthermore, as indicated in [fig_ref] Table 3: Comparison of the amount older customers were willing to pay [/fig_ref] , in the nature picture group, the price range that participants were willing to pay ranged from US $10.00 to US $30.00, with the average amount being US $17.10. In terms of the percentage, participants who were willing to pay less than US $10.00 accounted for 47% of all participants, those between US $11.00 and US $20.00 accounted for 18%, and those between US $21.00 and US $30.00 accounted for 35%. In the built picture group, the average price the participants were willing to pay was US $14.80. Participants who were willing to pay less than US $10.00 accounted for 49% of all participants, those between US $11.00 and US $20.00 accounted for 29%, and those between US $21.00 and US $30.00 accounted for 22%. The t-test results showed that participants who examined the natural images had a higher level of willingness to pay than those who watched the built pictures (nature vs. built: US $17.10 vs. US $14.80, t = 2.53, p = 0.0023; see [fig_ref] Table 3: Comparison of the amount older customers were willing to pay [/fig_ref].
# Discussion
Images comprise an important medium for transmitting marketing messages and are also an important mode of evaluation for individuals prior to making accommodation decisions. A strategy using natural images in wellness services seems to be a feasible way to attract the visual attention of older consumers and to facilitate their purchase intentions. By using the power of market-oriented economies, service providers can create new business and green service models, integrating natural images and service differentiation strategies into their sports firm's overall marketing programs. The four major findings of the present study are as follows.
First, using the eye-tracking technology, it was possible to accurately capture the older consumers' visual preferences for wellness service pictures and to understand their implicit physical activity decision-making process. It was found that older customers showed a longer fixation time and larger fixation frequencies when observing wellness service pictures with nature clues as compared to those with built clues. Previous research has also reported similar results when a similar group observed wellness service pictures with nature clues as compared to indoor built clues [bib_ref] The development of a sustainable wellness service marketing strategy in Taiwan based..., Wang [/bib_ref]. Indeed, when older consumers look at wellness service pictures for a limited period of time, they selectively prefer those with nature clues [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref]. It is likely that wellness activities performed in the natural environment will strengthen the attention restoration effect and facilitate a higher restoration quality, in turn causing consumers to feel more relaxed [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] Psychological and physiological responses in patients with generalized anxiety disorder: The use..., Wang [/bib_ref]. This concurs with Li et al.'s (2016) study exploring the visually attractive characteristics of tourism and leisure sports marketing images. They found that images with nature clues captured visual attention more easily. Likewise, [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] used an eye-tracking analysis and found that marketing images with natural landscapes and artistic components yielded better advertisement effectiveness than those with built environments and artistic components in customers at an average age of approximately 55 years. Considered jointly, the previous and present results suggest that natural landscapes seem to induce more visual attraction in this group.
Second, the results of the present study indicated that observing wellness service pictures with nature clues compared with built clues did not lead to higher perceptions of well-being. This result was somewhat inconsistent with the findings of Mitchell (2013) [bib_ref] Is physical activity in natural environments better for mental health than physical..., Mitchell [/bib_ref] , who found that individuals engaging in physical activity in nature relative to other environments exhibited better mental health. [bib_ref] Psychological and physiological responses in patients with generalized anxiety disorder: The use..., Wang [/bib_ref] [bib_ref] Psychological and physiological responses in patients with generalized anxiety disorder: The use..., Wang [/bib_ref] also found that individuals exercising in an environment with natural landscapes exhibited higher levels of stressrelief, restorative quality, and personal satisfaction compared to those exercising in an environment with a virtual abstract painting. Accordingly, considering these contradictory results, we are cautious in suggesting that perceived well-being is not truly elicited through observation of pictures with nature clues. Previous research confirmed that the restorative qualities obtained from visiting nature will enhance well-being through psychological and physical benefits [bib_ref] A different way to stay in touch with 'urban nature': The perceived..., Carrus [/bib_ref]. However, observing pictures does not directly induce well-being [bib_ref] Humans and nature: How knowing and experiencing nature affect well-being, Russell [/bib_ref]. Without actually visiting these natural settings, it is difficult to perceive the benefits (psychological or physical benefits) of interacting with the natural environment [bib_ref] Humans and nature: How knowing and experiencing nature affect well-being, Russell [/bib_ref]. Furthermore, the participants who observe the pictures may only have perceived lower restorative qualities because they have not actually visited these natural environments, or the selected pictures did not provide higher perceptual restorative qualities.
Third, to the author's knowledge, this is the first study to investigate older consumers' willingness to pay after observing wellness service pictures. It was found in the current study that participants in the nature group were willing to pay an average of US $17.10 to enjoy the yoga activity, which is higher than what the participants in the built group were willing to pay. However, it is worth noting that the nature group showed two extremes (i.e., less than US $10.00 and between US $21.00 and $30.00) in terms of willingness to pay, suggesting that there are two distinct market segments when hotels provide a wellness service in a natural environment. Individuals with different characteristics will interpret and place different values on the same thing [bib_ref] Understanding Consumers' Inferences from Price and Nonprice Information in the Online Lodging..., Noone [/bib_ref]. Although this conjecture is somewhat speculative, it provides a basis for future research.
The findings show that wellness service pictures with nature clues not only elicited higher levels of visual attention in the older consumers under consideration, but also led to willingness to pay. The empirical results suggest that visual clues in hotel marketing pictures can influence customers' visual behavior and assessments of willingness to pay. In particular, images of natural servicescapes may attract more visual attention than those of built servicescapes, and natural servicescapes may also signal higher restorative quality to older potential customers. This finding was consistent with the arguments of attention restoration theory, suggesting that a natural environment can reduce mental fatigue in older consumers and provide incentives for physical activity. Therefore, if older consumers believe that they can learn yoga in an outdoor space with green trees, plants, and grass, this may induce motivation toward willingness to pay. As natural environments provide restorative resources, relaxation, leisure, social, and health benefits to individuals, they may be willing to pay more for experiences in such environments [bib_ref] Urban Green Space: Creating a Triple Win for Environmental Sustainability, Health, and..., Kruize [/bib_ref]. This study expands the concepts of attention restoration theory to the willingness to pay research field, reflecting that customer's positive responses to psychological/affective restoration may interact with purchase intention. Therefore, if hotels can satisfy customers' wellness needs or provide stress-relieving activities, they will potentially obtain a competitive advantage [bib_ref] Restorative quality in tourist hotel marketing pictures: Natural and built characteristics, Wang [/bib_ref] [bib_ref] Essential customer service factors and the segmentation of older visitors within wellness..., Chen [/bib_ref].
## Research limitations and future research suggestions
In this study, eye-motion performance, perceived well-being, and willingness to pay were used to investigate the psychological responses of older consumers to wellness service (e.g., yoga) images. Whether they would actually participant in such a physical activity was not investigated. As a result, we could not clearly confirm the actual contribution of the wellness service pictures in term of whether they actually caused the older consumers to engage in yoga during their hotel stay. It is suggested that future studies may further explore the characteristics of images that enable older consumers to feel a sense of wellbeing and how these images influence subsequent physical activity behavior and outcomes. In addition, using a longitudinal study to explore the potential relationships between different types of perceived well-being obtained via observing wellness service images (e.g., yoga) with nature clues and via practicing the similar physical activity in a hotel's outdoor natural environment is warranted.
# Conclusions
Tourists might choose a hotel based on their perception of its ability to release stress and promote wellness. The present findings show that wellness service strategies incorporating natural environments appear to make consumers more willing to use the service and willing to pay higher prices to enjoy it. Thus, hotels wanting to increase their profit can provide activities in the natural environment (e.g., allowing customers to participate in a yoga activity via interaction with the outdoor natural environment) to provide psychological and mental health benefits to their older guests.
## Institutional review board statement:
This study was carried out in accordance with the recommendations of the National Cheng Kung University's Ethics Committee, and written informed consent was obtained from all participants, in accordance with the Declaration of Helsinki. The study was approved by the National Cheng Kung University's Ethics Committee (approval no. NCKU HREC-E-107-083-2).
[fig] Figure 1: Two samples of the experimental pictures (portraits adopted from https://www.freepik.com/fr ee-photo/health-beautiful-female-body-peace_1057237.htm#page=8&query=gym%20background&po sition=8, accessed date 1 December 2021). [/fig]
[fig] Author: Contributions: Conceptualization, T.-C.W. and T.-W.T.; formal analysis, T.-W.T. and C.-L.T.; investigation, T.-C.W.; methodology, T.-W.T.; resources, C.-L.T.; software, C.-L.T.; writing-original draft, T.-C.W. and T.-W.T.; writing-review and editing, C.-L.T. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research was supported by the Ministry of Science and Technology, Taiwan. [/fig]
[table] Table 1: Effects of image characteristics on older consumers' visual attention. [/table]
[table] Table 2: Effects of image characteristics on willingness to pay in older consumers. [/table]
[table] Table 3: Comparison of the amount older customers were willing to pay. [/table]
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Vitamin C intake potentially lowers total cholesterol to improve endothelial function in diabetic patients at increased risk of cardiovascular disease: A systematic review of randomized controlled trials
Background: Vitamin C is one of the most consumed dietary compounds and contains abundant antioxidant properties that could be essential in improving metabolic function. Thus, the current systematic review analyzed evidence on the beneficial effects of vitamin C intake on cardiovascular disease (CVD)related outcomes in patients with diabetes or metabolic syndrome.Methods: To identify relevant randomized control trials (RCTs), a systematic search was run using prominent search engines like PubMed and Google Scholar, from beginning up to March 2022. The modified Black and Downs checklist was used to assess the quality of evidence.Results: Findings summarized in the current review favor the beneficial effects of vitamin C intake on improving basic metabolic parameters and lowering total cholesterol levels to reduce CVD-risk in subjects with type 2 diabetes or related metabolic diseases. Moreover, vitamin C intake could also reduce the predominant markers of inflammation and oxidative stress like C-reactive protein, interleukin-6, and malondialdehyde. Importantly, these positive outcomes were consistent with improved endothelial function or Frontiers in Nutrition 01 frontiersin.org Dludla et al. 10.3389/fnut.2022.1011002increased blood flow in these subjects. Predominantly effective doses were 1,000 mg/daily for 4 weeks up to 12 months. The included RCTs presented with the high quality of evidence.Conclusion: Clinical evidence on the beneficial effects of vitamin C intake or its impact on improving prominent markers of inflammation and oxidative stress in patients with diabetes is still limited. Thus, more RCTs are required to solidify these findings, which is essential to better manage diabetic patients at increased risk of developing CVD.
Vitamin C intake potentially lowers total cholesterol to improve endothelial function in diabetic patients at increased risk of cardiovascular disease: A systematic review of randomized controlled trials Introduction Diabetes mellitus remains one of the leading causes of deaths worldwide. Although currently used antidiabetic therapies such as metformin and insulin can manage diabetesassociated complications [bib_ref] The cardioprotective effects of metformin, El Messaoudi [/bib_ref] , their long-term therapeutic effects could be limited due to the rapid rise of diabetesrelated deaths (4). Certainly, most diabetic patients (mainly due to pathological consequences of hyperglycemia) are known to be at increased risk for cardiovascular disease (CVD)related deaths [bib_ref] Prevalence of cardiovascular disease in type 2 diabetes: a systematic literature review..., Einarson [/bib_ref]. Thus, in addition to understanding the precise pathological mechanisms implicated in diabetes-induced myocardial injury, there has been a growing need to discover novel pharmacological compounds, with strong cardioprotective properties to prolong the lives of diabetic patients.
There has been a general interest in pharmacological compounds with strong antioxidant and anti-inflammatory properties for their protective effects against diabetes-associated cardiovascular complications [bib_ref] Hyperglycemia-induced oxidative stress and heart disease-cardioprotective effects of rooibos flavonoids and phenylpyruvic..., Dludla [/bib_ref] [bib_ref] Therapeutic targeting of oxidative stress with coenzyme Q10 counteracts exaggerated diabetic cardiomyopathy..., Blasio [/bib_ref] [bib_ref] Anti-inflammatory phytochemicals for the treatment of diabetes and its complications: lessons learned..., Kong [/bib_ref]. This is important since impaired glucose intolerance in a diabetic state has been associated with aggravated pro-inflammatory response and oxidative stress-induced vascular damage [bib_ref] Oxidative stress and myocardial injury in the diabetic heart, Ansley [/bib_ref] [bib_ref] Diabetic cardiomyopathy, causes and effects, Boudina [/bib_ref]. Consistently, due to their perceived ameliorative effects against inflammation and oxidative stress, there has been a great necessity to examine the protective effects of dietary compounds against diabetesassociated complications [bib_ref] Dietary berries, insulin resistance and type 2 diabetes: an overview of human..., Calvano [/bib_ref] [bib_ref] Dietary and nutraceutical approach to type 2 diabetes, Derosa [/bib_ref] [bib_ref] Effect of induced hypoglycemia on inflammation and oxidative stress in type 2..., Kahal [/bib_ref].
There is currently a considerable interest in understanding the therapeutic role of herbs and supplements against Abbreviations: CVD, cardiovascular disease; CRP, C reactive protein; DBP, diastolic blood pressure; FPG, fasting plasma glucose; IL, interleukin; GRADE, Grading of Recommendations Assessment Development and Evaluation; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MDA, malondialdehyde; PRISMA, Preferred Reporting Items for Systematic reviews and Meta-Analysis; RCT, randomized controlled trials; SBP, systolic blood pressure; SMD, standard mean difference; T2D, type 2 diabetes. diabetes and CVD-related complications . This includes uncovering the therapeutic effects of vitamins, which are considered vital micronutrient that an organism requires for an adequate metabolic function. Indeed, various dietary compounds such as vitamin C are increasingly consumed for their envisaged benefits against metabolic complications [bib_ref] Beneficial effects of citrus flavonoids on cardiovascular and metabolic health, Mahmoud [/bib_ref] [bib_ref] Effects of vitamin C supplementation on glycaemic control: a systematic review and..., Ashor [/bib_ref]. Vitamin C is considered an essential nutrient that functions as a vital antioxidant in protecting against oxidative stress and tissue damage. Besides its availability as a dietary supplement, vitamin C can also be found in food sources including citrus fruits and vegetables. The beneficial effects of vitamin C are associated with their capacity to attenuate oxidative stress and inflammation [bib_ref] Beneficial effects of citrus flavonoids on cardiovascular and metabolic health, Mahmoud [/bib_ref]. A previously published meta-analysis showed that vitamin C intake could improve glycemic control or blood pressure in adult participants [bib_ref] Effects of vitamin C supplementation on glycaemic control: a systematic review and..., Ashor [/bib_ref]. More evidence is required to understand the health benefits of this dietary antioxidant, and to potentially curb the rising toll of CVD-related deaths in patients with diabetes or metabolic diseases. This is in support of recent reviews highlighting the gap in clinical evidence informing on the favorable outcomes of vitamin C in individuals at increased CVD-risk [bib_ref] Vitamin C and cardiovascular disease: an update, Morelli [/bib_ref] [bib_ref] Limited evidence for a beneficial effect of vitamin C supplementation on biomarkers..., Ashor [/bib_ref] [bib_ref] The effect of vitamin C supplementation on lipid profile of type 2..., Zhu [/bib_ref]. Importantly, although other reviews have reported on the potential therapeutic effects of vitamin C on controlling basic metabolic function and CVD-related outcomes (26, 30, 31), very limited information exists on the implications or link with biomarkers of inflammation and oxidative stress such as C-reactive protein (CRP) and interleukin 6 (IL-6), and malondialdehyde (MDA) levels.
# Methodology
Supplementary file 1 contains the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guidelines that were followed to prepare the manuscript. The current study does not have an approved protocol; Hyperglycemia, as a major characteristic feature of diabetes mellitus, is known be responsible for prompting metabolic abnormalities that make an individual susceptible to increased cardiovascular disease (CVD)-risk. For example, abnormal levels of serum lipids (dyslipidemia) that may eventually cause atherosclerosis through endothelial dysfunction are all implicated in the development of CVDs. Similarly, preclinical studies have implicated pathological mechanisms such as abnormal autophagy and inflammation to be involved in this process. however, the International prospective register of systematic reviews (PROSPERO) was cautiously surveyed to avoid duplicating systematic reviews or meta-analysis investigating a similar topic.
## Approach for searching randomized controlled trials
Briefly, a systematic search was run using prominent search engines like PubMed and Google Scholar, from beginning up to March 2022. This was done by two independent reviewers. To optimally cover relevant literature, a rather broad primary search strategy was applied where we explored all randomized controlled trials (RCTs) reporting on vitamin C intake in patients with diabetes or metabolic syndrome. Thereafter, the especial focus was placed on RCTs assessing the effect of vitamin C intake on outcomes related with CVD in diabetic individuals, and this was done in comparison to the placebo or comparative control. Medical Subject-Heading (MeSH) and text words "vitamin C, " "diabetes, " "metabolic syndrome, " "cardiovascular disease, " and their matching synonyms were used.
## Study selection
The study encompassed RCTs reporting on the therapeutic effects of vitamin C on outcomes related with CVD in adults (>18 years) with diabetes or metabolic syndrome. Notably, encompassed RCTs were those that assessed the use of vitamin C as an intervention, comprising the comparison group on placebo, reporting on any quantifiable outcome of CVD in patients with diabetes or metabolic syndrome. RCTs reporting on the use of vitamin C in conjunction with other therapies were excluded. Also, RCTs not describing the clear CVD-outcome or covered incomplete information were excluded. Relevant items, including participants, interventions, comparisons, and outcomes (PICO), are described below:
- Participants:
Adult patients with diabetes and at increased risk of developing CVD.
- Interventions:
Treatment intervention involved vitamin C intake in patients with diabetes or metabolic syndrome. Patients receiving placebo were used a comparative control.
- Outcomes:
The primary outcomes for this systematic review included basic metabolic profiles such as blood glucose levels, and CVD-risk measurements like lipid profiles, endothelial function, and blood pressure. Whereas the secondary outcome were biomarkers of inflammation and oxidative stress.
## Data extraction and assessment of quality
Briefly, qualifying articles were carefully selected by at least two independent investigators. The main outcome of the study was to assess the effects of vitamin C intake on outcomes related with CVD in diabetes or condition of metabolic syndrome. It remained imperative to also assess correlation between duration of intervention and improvements in CVD-related outcomes in the study population. For accurate reporting, relevant data items extracted from each RCT included the name and year of publication, the country where the study was performed, sample and gender dissemination, as well as the dose and duration of intervention. The risk of bias was independently assessed by at least two investigators using of the adapted Downs and Black checklist, which is appropriate for both randomized and non-randomized studies (32, 33).
## Overview of study findings study selection
A total of 183 RCTs were inspected for eligibility, however, only 21 studies were selected, as shown in. All encompassed studies were RCTs on the effects of vitamin C intake on outcomes related with CVD in patients with diabetes or metabolic syndrome. Disqualified studies were on the combination use of vitamin C with other pharmacological compounds, or for not having a well-defined control group. Other exclusions were related for not having a clear study design and not reporting on the effect of vitamin C on the predefined study population.
## Study characteristics
All contained RCTs were available from peer-reviewed journals, as shown in [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref]. Besides Australia (n = 3), Iran (n = 2), New Zealand (n = 1), Palestine (n = 1), most studies were from the United States (n = 4), United Kingdom (n = 5), and Europe (n = 5). Overall, the total of participants was 7,688, with an average age of 60 years, with at least 50% of them registered as males [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref]. Furthermore, approximately 90% of RCTs evaluated type 2 diabetes (T2D), while the rest were focused on patients with type 1 diabetes and the metabolic syndrome [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref]. In terms of dose selections, consistency was observed where most studies used vitamin C at 1,000 mg, taken once a day, or twice daily at doses of 500 mg [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref]. The treatment duration was consistent at 4-6 weeks [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref] , while other RCTs did evaluate short term effects of vitamin C intake in terms of hours a few days [bib_ref] Protective effects of vitamin C on endothelium damage and platelet activation during..., Morel [/bib_ref] , whereas limited studies tested the long-term effects at 4 months (37-39), 1 year (40), 4 years (41), and 9.2 years (42).
## Risk of bias assessment
Briefly, the modified Back and Downs checklist with 26 questions and four domains, which are relevant for analyzing the quality of encompassed studies [bib_ref] The feasibility of creating a checklist for the assessment of the methodological..., Downs [/bib_ref]. Out of the 21 included studies six were excellent [bib_ref] Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in..., Mason [/bib_ref] [bib_ref] Effect of ascorbic acid on microcirculation in patients with Type II diabetes:..., Lu [/bib_ref] [bib_ref] Effect of vitamin C on inflammation and metabolic markers in hypertensive and/or..., Ellulu [/bib_ref] [bib_ref] Highdose oral vitamin C partially replenishes vitamin C levels in patients with..., Chen [/bib_ref] , 12 were scored as good [bib_ref] Metabolic benefits deriving from chronic vitamin C supplementation in aged non-insulin dependent..., Paolisso [/bib_ref] [bib_ref] Administration of ascorbic acid and an aldose reductase inhibitor (tolrestat) in diabetes:..., Mcauliffe [/bib_ref] [bib_ref] Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated..., Anderson [/bib_ref] [bib_ref] Effect of vitamin C on blood glucose, serum lipids & serum insulin..., Afkhami-Ardekani [/bib_ref] [bib_ref] Unchanged renal haemodynamics following high dose ascorbic acid administration in normoalbuminuric IDDM..., Gutierrez [/bib_ref] [bib_ref] Ascorbic acid reduces blood pressure and arterial stiffness in type 2 diabetes, Mullan [/bib_ref] , and three were fair [bib_ref] Protective effects of vitamin C on endothelium damage and platelet activation during..., Morel [/bib_ref] [bib_ref] Vitamin C intake and cardiovascular disease risk factors in persons with non-insulindependent..., Mayer-Davis [/bib_ref]. Supplementary file 2 depicts that encompassed studies presented with low reporting bias with a median score of 8 out of a probable score of 10 (overall agreement 85.67%, kappa = 0.71), good external legitimacy with median score of 2 out of 3 (overall agreement 39.13%, kappa = 0.13), excellent internal validity with median score 7 out of 7 (overall agreement 65.22%, kappa = 0.34) and low risk assortment bias with median of 5 out of possible 6 (overall agreement 56.51%, kappa = 0.19). Therefore, interpretation of the findings can be trusted and applied outside the selected study population.
Evidence on the impact of vitamin C intake of metabolic and cardiovascular disease-related outcomes
The overall included studies reported on vitamin C intake and its diverse effects on basic metabolic parameters such as glycated hemoglobin (HbA1c), fasting plasma glucose and insulin levels, as well as CVD related outcomes including lipid profiles and blood pressure [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref].
In relation to basic metabolic parameters, evidence available from as early as 1995 indicated that intake of vitamin C, at 500 mg twice daily for 4 months, could improve whole body glucose disposal and non-oxidative glucose metabolism in individuals with T2D (37). Interestingly, this was consistent with enhanced plasma vitamin C levels, a decline in plasma lowdensity lipoprotein (LDL)-cholesterol, as well as reduced free radicals and insulin levels. McAuliffe [bib_ref] Administration of ascorbic acid and an aldose reductase inhibitor (tolrestat) in diabetes:..., Mcauliffe [/bib_ref] , confirmed some of these findings showing that vitamin C intake at a similar dose A summary of flow diagram presenting study selection.
(500 mg) twice daily for 12 months could increase its plasma levels while resulting in reduced albumin excretion rate in diabetic patients. As new information became available, Mullan et al. [bib_ref] Ascorbic acid reduces blood pressure and arterial stiffness in type 2 diabetes, Mullan [/bib_ref] demonstrated that vitamin C intake at 500 mg daily for 4 weeks reduced brachial systolic and diastolic blood pressure concomitant to improving arterial stiffness in patients with T2D. Whereas Morel et al. [bib_ref] Protective effects of vitamin C on endothelium damage and platelet activation during..., Morel [/bib_ref] showed that vitamin C intake at 1,000 mg daily for 5 days reduced platelet-derived microparticles in diabetic patients with myocardial infarction.
Other beneficial effects linked with vitamin C intake in patients with diabetes or metabolic syndrome extended to improving blood flow or lowering blood pressure (35, 38, 39, 52); attenuating oxidative stress and endothelial dysfunction (36, [bib_ref] Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in..., Mason [/bib_ref] [bib_ref] Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated..., Anderson [/bib_ref]. This was consistent with an effective control of fasting plasma glucose (FPG), triglycerides, LDL, HbA1c and serum insulin levels [bib_ref] Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in..., Mason [/bib_ref] [bib_ref] Effect of vitamin C on blood glucose, serum lipids & serum insulin..., Afkhami-Ardekani [/bib_ref] ; while also reducing the proinflammatory markers such as CRP and IL-6 (44). These positive effects with vitamin C intake were predominantly observed with the doses of 1,000 mg/daily, and intervention period of 4 weeks up to 12 months in patients with T2D. Alternatively, in patients with T1D, vitamin C intake could enhance its plasma levels and these effects were linked with reduced oxidative stress response (36, 40). However, very few studies have investigated the therapeutic effects of vitamin C in patients with T1D.
Opposing the advantages observed with vitamin C intake in controlling metabolic disease associated complications in diabetic patients, other studies did not report any positive effects with regular intake of this antioxidant. For example, Klein et al.
(50) showed that vitamin C at 500 mg twice a day for 4 weeks could not normalize renal hemodynamics in normoalbuminuric in normotensive diabetic patients. Mayer-Davis et al. [bib_ref] Vitamin C intake and cardiovascular disease risk factors in persons with non-insulindependent..., Mayer-Davis [/bib_ref] revealed that vitamin C intake at 275 or 133 mg daily for 4 years did not impact systolic or diastolic blood pressure nor with HDL or LDL cholesterol, or triglycerides. Similarly, Upritchard et al.and showed that vitamin C intake between the doses of 500 and 1,500 mg daily for 3-4 weeks did not significantly improve endothelial function or affect FPG, LDL oxidation, or C-reactive protein levels in patients with T2D. Consistently, other studies showed that regular intake of vitamin C at 800-1,000 mg for 2-4 weeks did not significantly affect inflammatory cytokines, oxidized LDL, FPG or insulin levels, or forearm blood flow in patients with T2D [bib_ref] Effect of ascorbic acid on microcirculation in patients with Type II diabetes:..., Lu [/bib_ref] [bib_ref] Highdose oral vitamin C partially replenishes vitamin C levels in patients with..., Chen [/bib_ref] [bib_ref] Unchanged renal haemodynamics following high dose ascorbic acid administration in normoalbuminuric IDDM..., Gutierrez [/bib_ref]. Notably, except for an RCT by Mayer-Davis et al. [bib_ref] Vitamin C intake and cardiovascular disease risk factors in persons with non-insulindependent..., Mayer-Davis [/bib_ref] which used approximately 520 participants, most of the studies that did not observe any significant results used a very low sample number, indicating this could have affected the power of the results. However, this is only a hypothesis, well-designed RCTs with adequate sample number are still required to give a better picture on the impact of vitamin C on outcomes related with CVD in patients with diabetes or metabolic syndrome.
# Discussion
## A gap in available clinical evidence
The rapid prevalence of diabetes (55), coupled with the lack of effective therapies for its management (56), has propelled research into establishing alternative approaches to prevent this calamity. Dietary supplements have become an attractive target to investigate for their health benefits, especially due to their envisaged safety profile and potential bioactive properties [bib_ref] Dietary and nutraceutical approach to type 2 diabetes, Derosa [/bib_ref] [bib_ref] Use of dietary supplements in the european prospective investigation into cancer and..., Skeie [/bib_ref]. Vitamin C is considered an essential nutrient, also forming part of the World's Health Organizations List of Essential Medicine, with abundant antioxidant effects (59, 60). Although many factors such as absorption and bioavailability profiles can influence its physiologic concentrations (61), it has long estimated that adequate intake of vitamin C of either 200 mg/day from five servings of fruits and vegetables or 100 mg/day is necessary to prevent its deficiency with a margin of safety (62). Consistently, epidemiological data support the observed improvements in CVD-related outcomes with high consumption of fruits and vegetables rich in antioxidants like vitamin C and E [bib_ref] Diet, exercise and the metabolic syndrome, Pitsavos [/bib_ref] [bib_ref] Cardiovascular health benefits of specific vegetable types: a narrative review, Riccardi [/bib_ref] [bib_ref] Fruit and vegetable intake and the risk of cardiovascular disease, total cancer..., Aune [/bib_ref]. Alternatively, while experimental data suggest vitamin C and E can protect against oxidative stress-induced cellular damage by scavenging of reactive oxygen species or by neutralizing lipid hydroperoxyl radicals [bib_ref] Criteria and recommendations for vitamin C intake, Traber [/bib_ref] , convincing evidence on the effect of multiple dietary supplements on metabolic and cardiovascular health is scarce (67). In fact, although quantitative analysis supports the positive effects of vitamin C intake on improving blood glucose control or blood pressure in patients with T2D [bib_ref] Effects of vitamin C supplementation on glycaemic control: a systematic review and..., Ashor [/bib_ref] , such evidence has not been linked with CVD-related outcomes in conditions of metabolic syndrome.
## Summary of results supporting the beneficial effects of vitamin c
The current systematic review involved 21 RCTs, with 7,688 participants, evaluating the impact of vitamin C intake on basic metabolic parameters such as HbA1c, FPG, and insulin levels, as well as CVD-related outcomes including lipid profiles and blood pressure [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref]. In fact, overwhelming studies supported the beneficial effects of vitamin C intake on improving metabolic function and reducing cholesterol levels in patients with diabetes or metabolic syndrome. In addition to reducing the pro-inflammatory markers such as CRP and IL-6 (44), vitamin C intake at an average dose of 1,000 mg daily between 3 weeks up to a period of 12 months showed consistent results lowering FPG, triglycerides, LDL, HbA1c, and serum insulin levels . Interestingly, these results were supported by significantly increased plasma levels of this antioxidant after its intake in these patients [fig_ref] TABLE 1: An overview of studies reporting on the impact of vitamin C intake... [/fig_ref]. Notably, the reduction in total cholesterol was associated with attenuation of oxidative stress and amelioration of endothelial dysfunction in these patients [bib_ref] Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in..., Mason [/bib_ref] [bib_ref] Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated..., Anderson [/bib_ref]. Further suggesting that vitamin C may exerts its therapeutic effects by terminating oxidation of lipid products that are linked with exacerbation of inflammation and endothelial dysfunction, as demonstrated elsewhere (61, 68-70). This result could also translate to improve blood flow and reduced blood pressure, as demonstrated in some RCTs (35, 38, 39, 52). However, clinical evidence on the beneficial effects of vitamin C intake or its impact on improving prominent markers of inflammation and oxidative stress in patients with diabetes is still limited. Thus, more RCTs are required to solidify these findings, which is essential to better manage diabetic patients at increased risk of developing CVD.
## Summary of results not supporting the beneficial effects of vitamin c
Other studies included within the current systematic review reported that vitamin C intake did not influence markers of metabolic function or CVD. Notably, some studies showed that intake with vitamin C at an average dose of 1,000 mg daily for 2-4 weeks does not significantly affect blood pressure nor with HDL and LDL cholesterol, as well as triglycerides [bib_ref] Vitamin C intake and cardiovascular disease risk factors in persons with non-insulindependent..., Mayer-Davis [/bib_ref]. Even when taken at a lower dose of 275 or 133 mg daily for a prolonged period of 4 years [bib_ref] Vitamin C intake and cardiovascular disease risk factors in persons with non-insulindependent..., Mayer-Davis [/bib_ref] , it was apparent this antioxidant does not affect systolic or diastolic blood pressure nor with HDL or LDL cholesterol, or triglycerides. Others showed that vitamin C intake at 800-1,000 mg for 2-4 weeks also does not impact inflammatory cytokines, oxidized LDL, FPG or insulin levels, or forearm blood flow in patients with T2D [bib_ref] Effect of ascorbic acid on microcirculation in patients with Type II diabetes:..., Lu [/bib_ref] [bib_ref] Highdose oral vitamin C partially replenishes vitamin C levels in patients with..., Chen [/bib_ref] [bib_ref] Unchanged renal haemodynamics following high dose ascorbic acid administration in normoalbuminuric IDDM..., Gutierrez [/bib_ref]. These results are like others showing failure of vitamin C intake to improve metabolic disease-related complications or Potential health benefits of vitamin C intake on cardiovascular disease-related outcomes in type 2 diabetic patients. Briefly, besides improving basic metabolic parameters such as impaired glucose/insulin levels, vitamin C intake ca also lower total cholesterol concentrations to reduce blood pressure and positively affect blood circulation. HbA1c, glycated hemoglobin; LDL, low-density lipoprotein; TG, triglycerides; GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; ROS, reactive oxygen species.
Frontiers in Nutrition 09 frontiersin.org to protect against CVDs in various clinical settings [bib_ref] Vitamins E and C in the prevention of cardiovascular disease in men:..., Lee [/bib_ref]. However, this evidence is still limited, additional studies are required to confirm any of these findings. Preclinical studies can serve as the reference model to further explore other potential benefits of vitamin C. This is essential since various preclinical models have demonstrated that this antioxidant can ameliorate the pathological consequences of inflammation and oxidative stress to alleviate diabetes-associated complications [bib_ref] Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a..., Wold [/bib_ref] [bib_ref] Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes, Jiang [/bib_ref] [bib_ref] Identification of key genes and pathways in type 2 diabetes mellitus and..., Chen [/bib_ref] [bib_ref] Distribution of spleen connective tissue fibers in diabetic and vitamin C treated..., Özerkan [/bib_ref].
## Conclusion and future perspective
Summarized evidence showed that vitamin C intake could potentially improve basic metabolic profile while markedly reducing the levels of total cholesterol in patients with T2D and the metabolic syndrome. This was consistent with improved endothelial function, which is consistent to previous reports (35, 38, 39, 52). The fact that most antioxidant therapies have been dismissal in diabetes in clinical trials [bib_ref] Oxidative stress and the use of antioxidants in diabetes: linking basic science..., Johansen [/bib_ref] , highlights the need for additional RCTs to confirm these effects, especially for patients with T1D. Indeed, by searching the PubMed engine, very few RCTs could be accessed/identified reporting on the effects of vitamin C intake in patients with diabetes (78). Thus, additional clinical trials should consider the use of larger sample sizes and lengthier intake periods that are driven to stratify effects on the basis of baseline glycemic control necessary to validate favorable outcomes of vitamin C intake. Future considerations should include making use of a qualitative approach "meta-analysis" to strengthen the presented evidence. Which is one of the limitations of the current review, since most included RCTs were too diverse and presented very substantial heterogeneity and this could have affected the interpretation of results.
# Data availability statement
The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.
[fig] Funding: This work was supported in part by baseline funding from the Biomedical Research and Innovation Platform of the South African Medical Research Council (SAMRC) and the National Research Foundation (Grant numbers: 141929 and 117829). [/fig]
[table] TABLE 1: An overview of studies reporting on the impact of vitamin C intake on cardiovascular disease (CVD)-related outcomes. [/table]
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Reviewing the relationship between machines and radiology: the application of artificial intelligence
Background: The scope and productivity of artificial intelligence applications in health science and medicine, particularly in medical imaging, are rapidly progressing, with relatively recent developments in big data and deep learning and increasingly powerful computer algorithms. Accordingly, there are a number of opportunities and challenges for the radiological community. Purpose: To provide review on the challenges and barriers experienced in diagnostic radiology on the basis of the key clinical applications of machine learning techniques. Material and Methods: Studies published in 2010-2019 were selected that report on the efficacy of machine learning models. A single contingency table was selected for each study to report the highest accuracy of radiology professionals and machine learning algorithms, and a meta-analysis of studies was conducted based on contingency tables. Results: The specificity for all the deep learning models ranged from 39% to 100%, whereas sensitivity ranged from 85% to 100%. The pooled sensitivity and specificity were 89% and 85% for the deep learning algorithms for detecting abnormalities compared to 75% and 91% for radiology experts, respectively. The pooled specificity and sensitivity for comparison between radiology professionals and deep learning algorithms were 91% and 81% for deep learning models and 85% and 73% for radiology professionals (p < 0.000), respectively. The pooled sensitivity detection was 82% for health-care professionals and 83% for deep learning algorithms (p < 0.005). Conclusion: Radiomic information extracted through machine learning programs form images that may not be discernible through visual examination, thus may improve the prognostic and diagnostic value of data sets.
# Introduction
Medical imaging is one of the first branches of health science to utilize machine learning and artificial intelligence (AI) to assist human medical practice.Machine learning allows computers to learn in an analogous way to humans, extracting patterns or classes based on input experience or a data set. This parallelism in learning is becoming increasingly close with the continuous innovations in data science and the progress of machine learning and AI.With the advancement in medical imaging technology and the incorporation of large data sets, machines can extract features that are arguably beyond the reach of human perception and cognition. In recent years, a number of machine learning algorithms have been used in content-based image retrieval systems for improving efficiency and accuracy.Several computational principles can be used to categorize machine learning algorithms, such as unsupervised learning, supervised learning, and semisupervised learning.In supervised learning, a system is provided with input and output features, and the emphasis is on understanding how these are mapped to each other. In unsupervised learning, inferences are drawn from data sets comprising input data without any labeled data. Cluster analysis is the most common unsupervised learning method, which is used for identifying trends or groups in data through exploratory data analysis.It functions by grouping sets of unlabeled data into clusters of similar features without differentiating between dependent and non-dependent variables. Semi-supervised learning is a compromise between supervised and unsupervised learning techniques, utilizing some labeled data to leverage the analysis of unlabeled data. Speech analysis is the most common application of semi-supervised learning models.Presently, a new era of AI in radiology is emerging with, focus on analyzing images which has been showing promising results for some time. Indeed, expectations the application of AI to radiological images have increased significantly. This suggests a need to review the existing literature on the application of machine learning and AI to radiological modalities, so that their potential effect can be understood. The purpose of this study is to provide a review on the challenges and barriers experienced in diagnostic radiology on the basis of the key clinical applications of machine learning techniques. The following hypothesis will be examined in the study:
H 1 ¼ The radiomic information extracted through machine learning programs form images that improves the prognostic and diagnostic value of data sets.
# Methodology
## Design and eligibility
This review has carried out a search for studies that explore the challenges and barriers in diagnostic radiology through the context of machine learning techniques. Only studies published in 2010-2019 and in English were included. The setting was hospital-based or clinical-based, and concerned reporting the effectiveness of machine learning models or AI algorithms on the ability to detect and interpret radiological findings. Narrative reviews, letters, preprints, and scientific reports were also included in the review. Interventions and findings related to home-based settings were excluded, as were studies on non-human or animal samples or duplicate data were excluded. The review assumes that expert opinion or consensus opinion and standard-of-care diagnoses are accurate.
## Sources and search strategy
## Patients and intervention
Studies that include patients diagnosed with any type of disease detected using machine learning algorithms were selected. Prospective assessments were undertaken for identifying the effect of these algorithms upon diagnostic yield and also on therapeutic yield. This review explores the implementation of machine learning algorithms and its "downstream effects" on the clinical pathway. The Consolidated Standards of Reporting Trials and Standard Protocol Items were reviewed for prospective trials.
## Data management
A manual search of citations, related articles, and bibliographies of included studies was undertaken to identify any further relevant articles that might have been missed during the automated search process. The analysis for studies providing contingency tables for both machine learning algorithm performance and
## Risk of bias
The recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement were followed throughout. Methods of analysis and inclusion criteria were specified in advance. The research question was formulated based on previously published recommendations for systematic reviews of prediction models, the CHecklist for critical Appraisal and data extraction for systematic Reviews of prediction Modeling Studies.
## Data synthesis
A single contingency table was selected for each study to report the highest accuracy of radiology professionals and machine learning algorithms. Binary diagnostic accuracy data were extracted. Contingency tables of true-negative, false-negative, true-positive, and falsepositive were used for calculating sensitivity and specificity.
## Secondary outcomes
A meta-analysis of studies was based on contingency tables to estimate the accuracy of machine learning algorithms. This review has assumed contingency tables to be independent from each other, whether a study provides various contingency tables for the same or different algorithms. A unified hierarchical model was used for the meta-analysis of diagnostic accuracy studies and the plotted summary receiver operating characteristic (ROC) curves.
# Results
The search identified 10,758 records, out of which 5035 were screened. The study found and evaluated 102 full text articles for eligibility; 23 studies 9-32 were included in this systematic review. Sixteen studies collected retrospective data and seven studies collected data prospectively. A pre-specified sample size calculation was not reported in any of the studies. The condition that health-care professionals were provided with additional clinical information alongside the image was examined in four studies. An algorithm-plus-clinician condition was undertaken with diagnostic performance in three studies. One study depended on single expert consensus, five studies used histopathology for confirmation, four studies used clinical follow-up, five studies used various models of expert consensus, two studies used clinical trial data; one study used surgical confirmation, and two studies used clinical care notes or labels.
The present review has also pooled performances of radiology professionals and deep learning models obtained from matched internally validated samples as an exploratory analysis. In addition, a single contingency table was selected to report each study with the highest accuracy.
The specificity for all the deep learning models ranged from 39% to 100%, whereas sensitivity ranged from 85% to 100%. The pooled sensitivity was 89% for the deep learning algorithms for detecting abnormalities compared to 75% for radiology experts, when averaging across studies using hierarchical summary ROC curves. Similarly, the pooled specificity was 85% for health-care professionals and 91% for deep learning algorithms (p ¼ 0.000). A comparison between radiology professionals and deep learning algorithms was made in 12 studies. Of these 12 studies, the pooled specificity for detection of abnormalities was 91% for deep learning models and 85% for radiology professionals (p < 0.000). The pooled sensitivity was 73% for radiology professionals and 81% for deep learning algorithms. The pooled sensitivity detection was 82% for health-care professionals and 83% for deep learning algorithms (p < 0.005).
# Discussion
A number of machine learning algorithms, in particular, deep neural networks, have been implemented in content-based image retrieval systems for improving query efficiency and accuracy. The processing of radiology text reports is another application of machine learning in radiology. Large text databases comprise the accumulated reports of daily radiology practice.Modern information processing technologies are used to exploit these radiology report databases to enhance retrieval, report search, and assist radiologists in making accurate diagnoses. Natural language understanding and natural language processing offer a more effective approach for managing and retrieving appropriate information hidden in the radiology reports.They can extract meaningful information as well as manage large-scale data in a more efficient way that is not possible for human readers.The importance of machine learning has grown in recent years owing to these attributes, being above all a practical way to carry out a text analysis of radiology report databases.
The advantage of rapid technological change in radiology, and the rapidly evolving field of radiomics, is similar to other fields that have benefited from transitioning to digital systems, while issues continue to present themselves, such as those surrounding the perception that machines and computers take jobs away from humans, often considered to be a cultural barrier in the implementation of AI in radiology.It has been predicted that much of the work of anatomic pathologists and radiologists will be possible through machine learning in the future, and thus, human occupations will become threatened. In addition, it is likely that machine learning techniques will become even more sophisticated in the next 5-10 years, which may threaten radiology as a thriving human discipline.Medical images are highly heterogeneous at both a population and an individual level, and so to train AI systems for a given application is a complex task if the number of available labeled images is restricted.In this regard, there may often be a risk of over-fitting the data, and there will be a loss of generalizability. Therefore, the practice of radiology may beneficially integrate AI methods, rather than replace radiologists, and improve the efficiency of digital imaging methods.A robust source of ground truth for each detection is needed to validate with AI programs trained on proven or known cases, whether the learning is supervised or unsupervised.Patient outcomes, gold standard testing results, and imaging methods can provide the source of the ground truth, but this should be comprehensively elucidated for each AI program that is established and used clinically.Fast computing systems are as yet not generally available in medical institutions for supplying results in a clinically relevant time frame for urgent or emergency diagnoses. However, this may not be a practical concern owing to the easy access to "cloud" computing solutions and the rapid development of graphic processing units at lower costs.The endeavor for generalizability of results, beyond the patient population in which the research was performed, is a broad challenge in clinical research. Data mining can be a complicated and costly process, despite the potential of big data to show the efficiency of technology with respect to patient outcomes in health care.The lack of structured reporting can be a strong hindrance behind this technology. Therefore, approved nomenclature and standards could be created by the radiology industry that provides definition and structure, to locate the same types of data across reports, irrespective of the format that each facility utilizes. Information generated on images will not be dictated quantifiably but will be directed from the image viewing solutions in prospective events.The improvement in accuracy and efficiency of machine learning emerges when location, anatomical findings, and measurements are developed as a result of the radiological viewing workflow.
The automatic generalizability of health-care knowledge from training data to future test data is one of the most significant contributions of machine learning. For instance, a computer can explain and make decisions on masses or microcalcifications of the human breast in a mammography Computer-Aided Detection (CAD) system.In this respect, then, the knowledge of radiologists of mammography diagnosis may be said to be transferred to the computer. For data, the input should be original, the associated problems should be anatomical structure of the patient and previous knowledge of the object of interest, and the objective should be segmentation of an object of interest in the image for medical image segmentation.The extraction of useful features and their identification, and designing an adequate objective function, is the second step in machine learning. Various problems can be addressed toward the task of fitting the data to the anatomical structures of the target. Training the algorithm and finding the best parameters for the graph cut model are the last step in the process. An improved scanning capability is produced through the trained machine learning segmentation algorithm for deep learning in radiology.Probabilistic models solve segmentation and image content analysis in radiological applications.Various processes are included therein, such as integration or marginalization of a complete probability model, independent and dependent variable identification, and probability density function, for making sure distributions meet the target variable or the objective function.Previous studies have addressed the segmentation problem of brain magnetic resonance images using a hidden Markov random field model 11 which is a stochastic process generated by Markov chain. Similarly, a hidden Markov random field model was used to capture the association between unidentified cluster labels and observations under spatial barriers.
Diagnostic imaging is one of the first medical disciplines that has optimally applied machine learning algorithms toward the automation of health care, while other medical fields have marked potential in this regard, particularly but not exclusively, cardiology, dermatology, gastroenterology, and pathology.Machine learning approaches may also further personalize health care to include a wider spectrum of data, such as genetic, laboratory, imaging, clinical, and laboratory information.
Linear regression (prediction of the dependent variable of the output by fitting a linear function to correlate the input/output pairs, which have a continuous range of values),logistic regression (where the prediction is carried out on dependent categorical variables),artificial neural networks (nonlinear connection of the input to the output, emulating the biological neurons found in the brain),and decision trees (in which the entry "nodes" are labeled with features that are arranged to form multiple element "classes," where a "leaf" of a decision "tree" can reach a finite discrete target in each pathway) are the algorithms used for supervised learning.AI algorithms have facilitated significant progress in image-recognition tasks, specifically through the use of deep learning approaches. These methods vary, from convolutional neural networks to variational auto-encoders, and have significant application in medical imaging analysis. In radiology practice, medical images are traditionally evaluated visually by trained physicians, for detection, characterization, and monitoring of disease. However, with the application of AI in radiology modalities, identification of images can carried out with more accuracy.
One of the major challenges of AI radiology is the lack of trust by the radiologists, when it is regarding answers related to analysis of medical images. The reason being, many radiologists perceive it as a "black box" due to doubts regarding the unclear process which gives a conclusive answer. scientific research and test running of the software in the hospital can help strengthen the radiologist's trust in AI. According to an example proposed by one study,similar cases from training databases could be depicted for rendering more information about data and providing relevant insights to the physicians. For AI radiology to survive, it is important to have the trust of its users, i.e., radiologists. Also radiologists can play a vital role in identifying targeted clinical cases for which these AI integrated tools can be implemented to test their effectiveness and sensitivity in clinical practice.They can also play a crucial role of preserving their expertise and keeping check on the drawbacks of over-reliance on technology.In the future, imaging data may be associated more readily with non-imaging data, such as those of electronic medical records or other large data sets. Indeed, when applied to electronic medical record data, deep learning can assist in extracting patient presentations that may link to clinical predictions and improve clinical decision support systems. Machine learning may thus play more of significant role in the prediction of treatment response and prognosis. Initial phases toward this type of work have already begun. For example, machine learning can accurately estimate brain tumor response to various therapies. Also, machine learning can be used in the prediction of longevity of patients by detecting characteristics representative of overall individual health.
This study has several limitations. First, it might have language bias, as only the studies that were published in English were included. Another limitation was that the studies varied in their methodology; therefore, meta-analysis was not done.
In conclusion, it is necessary for medical technologies to improve value with respect to the delivery of radiology services and medical care, for reduced time on tasks for radiologists, increased diagnostic certainty, mitigated costs of care with effective findings for patients, and faster availability of findings. Significant time and experience are required to establish whether these advantages have been met in the implemented technology and to understand comparative benefits, as with any new technological innovation. If machine learning and AI programs can be developed that are tolerant of various data acquisition protocols and work in various patient populations, they will have achieved the required outcomes. Nevertheless, success will need comprehensive understanding of the conditions under which a particular program is appropriate. Yet, the ultimate role of machine learning methods in radiology is still unclear, as is the influence these will eventually have on radiologists. What is apparent, however, is that machine learning and AI offer a powerful set of tools to analyze image data that have considerable potency. The elevated interest in AI in radiomics and radiology in recent years suggests it may have a primary role in the near future.
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Can PPAR γ Keep Cadmium in Check?
[bib_ref] Cigarette smoke exposure and alveolar macrophages: Mechanisms for lung disease, Lugg [/bib_ref] [bib_ref] Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response..., Yao [/bib_ref] [bib_ref] Cadmium and cadmium/zinc ratios and tobacco-related morbidities, Richter [/bib_ref] [bib_ref] Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response..., Yao [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response..., Yao [/bib_ref] [bib_ref] Cigarette smoke exposure and alveolar macrophages: Mechanisms for lung disease, Lugg [/bib_ref] [bib_ref] The adverse impact of cadmium on immune function and lung host defense, Knoell [/bib_ref] [bib_ref] Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response..., Yao [/bib_ref] [bib_ref] Macrophage polarization and its role in the pathogenesis of acute lung injury/acute..., Chen [/bib_ref] [bib_ref] Toxic elements in tobacco and in cigarette smoke: Inflammation and sensitization, Pappas [/bib_ref] [bib_ref] Toxic elements in tobacco and in cigarette smoke: Inflammation and sensitization, Pappas [/bib_ref] [bib_ref] Toxic elements in tobacco and in cigarette smoke: Inflammation and sensitization, Pappas [/bib_ref] [bib_ref] Peroxisome proliferator-activated receptor gamma coactivator-1α/HSF1 axis effectively alleviates lipopolysaccharide-induced acute lung injury..., Dang [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Macrophage polarization and its role in the pathogenesis of acute lung injury/acute..., Chen [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response..., Yao [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Low-dose cadmium potentiates lung inflammatory response to 2009 pandemic H1N1 influenza virus..., Chandler [/bib_ref] [bib_ref] Low-dose cadmium potentiates lung inflammatory response to 2009 pandemic H1N1 influenza virus..., Chandler [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] The adverse impact of cadmium on immune function and lung host defense, Knoell [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] The adverse impact of cadmium on immune function and lung host defense, Knoell [/bib_ref] [bib_ref] Cigarette smoke exposure and alveolar macrophages: Mechanisms for lung disease, Lugg [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] The adverse impact of cadmium on immune function and lung host defense, Knoell [/bib_ref] [bib_ref] From definition to molecular targets and therapy of lung diseases, Carvalho [/bib_ref] [bib_ref] From definition to molecular targets and therapy of lung diseases, Carvalho [/bib_ref] [bib_ref] From definition to molecular targets and therapy of lung diseases, Carvalho [/bib_ref] [bib_ref] From definition to molecular targets and therapy of lung diseases, Carvalho [/bib_ref] [bib_ref] Phosphorylation of PPARγ at Ser84 promotes glycolysis and cell proliferation in hepatocellular..., Shu [/bib_ref] [bib_ref] Phosphorylation of PPARγ at Ser84 promotes glycolysis and cell proliferation in hepatocellular..., Shu [/bib_ref] [bib_ref] Peroxisome proliferator-activated receptor gamma coactivator-1α/HSF1 axis effectively alleviates lipopolysaccharide-induced acute lung injury..., Dang [/bib_ref] [bib_ref] Maresin1 promotes M2 macrophage polarization through peroxisome proliferator-activated receptor-γ activation to expedite..., Qiao [/bib_ref] [bib_ref] Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages, Larsen-Casey [/bib_ref] [bib_ref] A new insight into the treatment of diabetes by means of pan..., Chandra [/bib_ref]
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Funding: N. Kolliputi Laboratory was funded by the Joy McCann Culverhouse endowment to the Division of Allergy and Immunology. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig]
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The effect of Liquid ice after high-intensity exercise on muscle function compared to Block ice
a b s t r a c tCryotherapy is used to recover muscle damage after exercise and to treat acute sports injuries. Liquid ice (LI) can keep cold for a long time, and is assumed more effective than block ice (BI). From this, the aim of this study was to investigate the effects of LI on the change of passive stiffness (PS) as muscle function and to validate the effectiveness of LI compared to BI. We performed the experiment as part of a case series of verification of the effects of cryotherapy. 22 healthy men (target area: right leg) were randomized to two groups: LI group and BI group. PS was measured three times during experiment protocol, pre: before exercise; post; after treating each cryotherapy after exercise; 48h: 48 hours after pre. Statistical analysis compared the PS, the amount of change in PS, and the rate of change in PS between the two groups. The rate of change between pre and 48h in LI was significantly lower compared to that in BI (p ¼ 0.03). There was no significant difference regarding other results between groups. It revealed that the difference of effect between LI and BI for PS of muscles after high-intensity exercises. These results could be helpful for the choice of intervention for reducing muscle stiffness after exercise and at sports field.
# Introduction
High-intensity exercises, especially those with eccentric contractions cause muscle damage 1e3 which leads to injuries, such as decreased muscle strength and decline in range of motion (ROM). Since these muscle injuries affect sports performance, it is important to repair muscle damage after exercise. [bib_ref] Time-course of changes in oxidative stress and antioxidant status responses following a..., Fatouros [/bib_ref] The main functions of skeletal muscles are to generate force and power, maintain posture, and produce movement. [bib_ref] Skeletal muscle: a brief review of structure and function, Frontera [/bib_ref] Among them, muscle flexibility is important for movements such as walking and sports performance. [bib_ref] Flexibility and its effects on sports injury and performance, Gleim [/bib_ref] ROM is commonly used as an objective indicator of muscle flexibility as a part of muscle function. [bib_ref] Flexibility and its effects on sports injury and performance, Gleim [/bib_ref] Passive stiffness (PS) can also be used to objectively and quantitatively assess muscle flexibility. [bib_ref] Elastography study of hamstring behaviors during passive stretching, Sant [/bib_ref] Several cryotherapy methods have been used to repair muscle damage 8e10 and treat acute sports injuries. [bib_ref] The use of ice in the treatment of acute soft-tissue injury: a..., Bleakley [/bib_ref] [bib_ref] Clinical applications OF cryotherapy among sports physical therapists, Hawkins [/bib_ref] The popular method of cryotherapy involves an ice bag. As it is easy and multipurpose, it is commonly used in the outside, such as in sports. However, maintaining the temperature of ice bag is difficult as it depends on prevailing weather and temperature condition. Liquid ice (LI), a new cryotherapy material, is gel ice made from saline water [fig_ref] Figure 1: Liquid ice can be seen on the left side and block ice... [/fig_ref]. Cryotherapy using LI may be more effective than using block ice (BI) as LI can be kept cold for a longer time and has higher contact with body surfaces. In addition to cryotherapy using BI, there have been previous studies on cryotherapy using materials such as BI and cold water or examining multiple outcomes for a single intervention, however, no study has been conducted on the effects of different types of ice using the same material.
This study aimed to investigate the effects of LI on changes in muscle flexibility and to validate its effectiveness. [bib_ref] Acute effects of different stretching durations on passive torque, mobility, and isometric..., Matsuo [/bib_ref] We focused on PS and used it as an index of flexibility as it can objectively and quantitatively assess muscle flexibility. We hypothesized that cryotherapy using LI would have a lower PS compared to that of BI after high-intensity exercise. Moreover, we performed a case series to verify of the effects of cryotherapy.
# Methods
## Participants
The participants of this study were 22 healthy men, and their right legs were used. Some participants had prior experience with cryotherapy. We confirmed that the participants did not undergo cryotherapy during the study period. They had not been diagnosed with muscle strain in their right hamstrings in the past six months and did not have diseases which prohibited cryotherapy such as Raynaud's disease. The participants were explained the experimental procedures and risks before they provided informed consent to participate in the study. The study was approved by the ethics committee and complied with the principles of the Helsinki Declaration.
## Procedures
## Experimental protocol
The subjects were randomized into two groups, depending on the intervention method, using the permuted block method: LI group (n ¼ 11) and BI group (n ¼ 11). The study was conducted for two days to measure the PS three times [fig_ref] Figure 2: Experimental protocol and measurement time points [/fig_ref]. On the first day, we measured the PS of each participant (pre). Then, each participant exercised, received cryotherapy by LI or BI for 20 minutes, 21 rested for 30 minutes, and PS was measured again (post). The PS was measured for the third time after 48 hours from the two initial tests (48h). [bib_ref] The effects of multiple daily applications of ice to the hamstrings on..., Oakley [/bib_ref] We focus on the changes in muscle flexibility under general body temperature. Therefore, we chose these three time points.
## Exercise protocol
The participants performed efferent exercises using a dynamometer (Biodex System 4.0, Biodex Medical System, Inc. Shirley, New York, USA). They were positioned on a chair, and the angle of the right knee was set from 90 to 40 . [bib_ref] The effects of multiple daily applications of ice to the hamstrings on..., Oakley [/bib_ref] They performed eight sets of 10 right maximum knee efferent flexion and the speed of knee flexion was 60 /s. [bib_ref] Interferential therapy effect on mechanical pain threshold and isometric torque after delayed..., Rocha [/bib_ref] The first set was submaximal in order to familiarize them with the machine. The rest time was 60 seconds.
## Cryotherapy intervention
The ice bags contained approximately 300 g of LI (Nippon Electric Heat Co.) or BI. After exercise, participants were provided with a cold application for 20 minutes 21 with either LI or BI and placed at the center of the right posterior thigh. The time period of the cold application has been described in a previous study. [bib_ref] The effects of multiple daily applications of ice to the hamstrings on..., Oakley [/bib_ref]
## Measurements
The PS of all participants was measured as muscle function 7 and it was assessed using the same dynamometer. The subjects lay on a bed, and their waist was held in position using belts. The right leg was tightly secured to the dynamometer leg plate, and the starting position of the right knee and hip joints was at 90 flexion. For passive testing, the subject's right knee joint was extended at a speed of 5 /s. [bib_ref] Effects of stretching velocity on passive resistance developed by the knee musculo-articular..., Nordez [/bib_ref] Participants were instructed to push a stop button just before they felt pain in their hamstrings. When the button was pushed, the passive knee flexion torque (Nm) and knee extension angle ( ) were measured. The PS was calculated as the slope of the passive knee flexion torque-angle curve. In the curve, the values of knee flexion torque were obtained at two points: the maximum knee extension angle and its half angle. Based on this, the value obtained by dividing the torque change in the torque angle curve by the angle change during that period was calculated as passive stiffness (Nm/deg). [bib_ref] Acute effects of different stretching durations on passive torque, mobility, and isometric..., Matsuo [/bib_ref] [bib_ref] Influences of tendon stiffness, joint stiffness, and electromyographic activity on jump performances..., Kubo [/bib_ref] Along with the values at each time point (pre, post, and 48h), the amount and the rate of change between pre and post, pre and 48h, as well as post and 48h were calculated. We measured the PS three times at each point and used the average values of the second and third measurements.
## Statistical analyses
The LI and BI groups were compared in terms of PS using the amount and the rate of PS. The Shapiro-Wilk test was used to determine whether the data were normally distributed. Unpaired ttests were used for the normal distribution. Mann-Whitney U tests were used for the non-normal distribution. The statistical significance level was less than 5%. The data was analyzed with JMP Pro 14 statistical software package (SAS Institute Inc., Cary, NC, USA).
# Results
The participants' data regarding age, height and weight are shown in [fig_ref] Table 1: Characteristics of 22 participants [/fig_ref] , and they revealed no significant differences (mean ± SD: age, 23.4 ± 1.0 years; height, 173.2 ± 5.9 cm; weight, 75.3 ± 1.1 kg). The rate of pre and 48h LI was significantly lower than that of BI (p ¼ 0.030), and other results did not differ significantly between LI and BI [fig_ref] Table 2: The value of passive stiffness [/fig_ref].
# Discussion
This study focused on the effectiveness of LI compared to BI for the muscle function. The results showed that cryotherapy using LI tended to reduce muscle stiffness 48 hours after high-intensity exercises; it was more effective than that the BI.
Muscle damage caused by high-intensity exercises causes inflammation and pain, leading to increased muscle stiffness. Damaged muscle during and immediately after exercise becomes stiff over time with further exercise. [bib_ref] The impact of acute and chronic resistance exercise on muscle stiffness: a..., Dankel [/bib_ref] [bib_ref] The effect of strength training on vastus lateralis' stiffness: an ultrasound quasi-static..., Santos [/bib_ref] Early and rapid cryotherapy after exercises is helpful in controlling acute inflammation; as a result, the inflamed area and deterioration of muscle stiffness are suppressed. [bib_ref] The use of ice in the treatment of acute soft-tissue injury: a..., Bleakley [/bib_ref] [bib_ref] The cold truth: the role of cryotherapy in the treatment of injury..., Kwiecien [/bib_ref] Cryotherapy tends to slow the oxygen demand of mitochondria, thereby decreasing the metabolic demand and inflammation of the muscle. [bib_ref] Blood flow after exercise-induced muscle damage, Selkow [/bib_ref] Therefore, it is useful for controlling aggravating secondary hypoxia and stiffening muscles. Furthermore, a study on cryotherapy reported that decreasing the tissue temperature by 10 e15 maximizes its effect. [bib_ref] The use of ice in the treatment of acute soft-tissue injury: a..., Bleakley [/bib_ref] It is speculation that it can effectively decrease the temperature of muscle surfaces and muscles and help muscles recover faster compared to BI via the wide range of contact and coolness due to the gel constitution of the LI and a temperature lower than that of BI. [bib_ref] Comparisons of cubed ice, crushed ice, and wetted ice on intramuscular and..., Dykstra [/bib_ref] The current study showed that LI tended to reduce muscle stiffness 48 hours after high-intensity exercises more quickly than that the BI. This suggests that acute cooling controls the further occurrence of muscle damage, and, as a result, repairs the damage faster, compared to BI, by decreasing metabolic and metabolic demands and controlling stiffening muscles.
The rate of change between pre and 48h was considered as an index of whether the muscle returned to the pre state. LI showed a significantly lower value for this rate of change. In other words, LI contributes to faster muscle recovery than BI. There was no significant difference in the rate of change between pre and post, and post and 48h. This means that there is no difference in the immediate and long-term effects between LI and BI. Some reports have shown that muscle damage is repaired 48h after exercise and cryotherapy. [bib_ref] Recovery from exercise-induced muscle damage: cold-water immersion versus whole-body cryotherapy, Abaïdia [/bib_ref] [bib_ref] The effects of multiple daily applications of ice to the hamstrings on..., Oakley [/bib_ref] However, there was no change in muscle function due to differences in the ice itself. These results show that there is a difference in the recovery speed of the muscles but no difference in the immediate or long-term effects. This revealed a difference in the effect of LI and BI on the PS of muscles after high-intensity exercises. These results could be helpful for reducing muscle stiffness in sportsperson.
This study had several limitations. First, we did not set a nontreatment group; therefore, we cannot ascertain whether cryotherapy using ice bags is better than no treatment. Second, the relationship between muscle stiffness and symptoms is unclear. Third, it is necessary to consider the reliability of PS before starting our study. However, since PS measurement was used in the previous research, [bib_ref] Determinants of musculoskeletal flexibility: viscoelastic properties, crosssectional area, EMG and stretch tolerance, Magnusson [/bib_ref] it was used in our research without validation. Fourth, we cannot refer to the physiological and performance aspects of the intervention, since we did not measure the differences in muscle temperature, blood indicators or exercise performance indicators by differences in LI and BI. Future studies should verify the physiological effects of LI and the effects of cryotherapy as a case series.
# Conclusion
The present study showed that cryotherapy using LI after highintensity exercise has a positive effect on muscle flexibility and can be useful in sports.
# Funding/support statement
No financial or material support of any kind was received for the work described in this article.
## Declaration of competing interest
The authors have no conflicts of interest relevant to this article.
[fig] Figure 1: Liquid ice can be seen on the left side and block ice on the right. [/fig]
[fig] Figure 2: Experimental protocol and measurement time points. [/fig]
[table] Table 1: Characteristics of 22 participants. Each data showed mean ± SD. [/table]
[table] Table 2: The value of passive stiffness (PS), the amount of change of PS, and the rate of change of PS. The amount of change of PS (Nm/deg) Each data showed mean ± SD. The results of t-test or Mann-Whitney U tests between LI group and BI group. *p < 0.05. N. Matsumura, S. Nagashima, K. Negoro et al. Journal of Exercise Science & Fitness 20 (2022) 23e26 [/table]
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An informatics research platform to make public gene expression time-course datasets reusable for more scientific discoveries
# Introduction
Over the past few decades, substantial funding and resources have been invested to generate biomedical datasets at many levels, ranging from nucleic acid and gene level to population level, in order to understand, treat and prevent various diseases, and protect public health. Based on data sharing policies of National Institute of Health (NIH) and other government agencies, many of aforementioned datasets are required to be shared with the general research communities. Consequently, vast amounts of biomedical data are being accumulated in databases and data repositories. However, use or reuse of these existing datasets for research by third parties is still not common as expected.
Gene expression data from various diseases under different experimental conditions are mostly deposited in the NIH/NCBI-sponsored Gene Expression Omnibus (GEO) data repository [bib_ref] Ncbi geo: archive for functional genomics data sets-update, Barrett [/bib_ref]. Like many of the biomedical databases, GEO was originally created as a data repository to comply with the data sharing policies. Often, these data sharing platforms are designed and organized for easy and convenient data submission by experimentalists, but not friendly for data retrieval and analysis. Further, it is not easy to identify the particular datasets to address a particular biological question for a specific disease from GEO, since the experimental design and study description are documented in an unstructured free text. Hence, it is necessary to use text mining and natural language processing (NLP) technologies to restructure the existing repository so that datasets can be findable, accessible and reusable.
This article describes a web-based platform that addresses the difficulties in finding, accessing, reusing biomedical datasets, specifically from GEO, as well as the difficulties in finding and forming collaborations. The novel platform, named as Gene Expression Time-Course Research (GETc) platform (http://genestudy.org/), is built on top of an analytical method based on the ordinary differential equation (ODE) model for analyzing time-course gene expression data. GETc offers the following services and functions:
- Hosts time-course gene expression datasets from GEO annotated with disease and cell types. The rest of the article is organized as follows: Section 2 discusses the background of the analytic pipeline and recommendation systems. Section 3.1 presents datasets used for developing the GETc platform. Section 3.2 describes the methodology used for analytic pipeline, recommendation systems and platform implementation. Section 4 describes and discusses the results. Finally, conclusions are presented in Section 5.
# Background
In this section, we present the three main parts of our work, (i) repositories developed for archiving datasets in the biomedical domains and their metadata, (ii) an analytic pipeline developed for analyzing gene data and (iii) dataset, literature and collaborator recommendation systems.
## Dataset repositories
It is a growing trend among the researchers to make their data publicly available for reproducibility and data reusability. Many repositories and knowledge bases have been established for different types of data in many domains. GEO(www.ncbi.nlm.nih.gov/geo/), UKBioBank(ww w.ukbiobank.ac.uk/), ImmPort(www.immport.org/home) and TCGA(portal.gdc.cancer.gov) are a few examples of repositories in the biomedical domain. DATA.GOV archives the U.S. Government's open data from agriculture, climate, education, etc. for research use. However, users from the biomedical community have to visit and search each repository separately to find data for their research, which can be time-consuming and hectic.
DataMed(datamed.org) started an initiative to solve the above issue for the biomedical community by combining biomedical repositories and enhancing the query searching using advanced NLP techniques [bib_ref] Datamed-an open source discovery index for finding biomedical datasets, Chen [/bib_ref]. DataMed indexes and searches diverse categories of biomedical datasets (3). DataCite is another data discovery index, which includes 16 187 835 works from many different domains (4). However, these repositories do not provide either insight of data or help to find collaborators, which are still challenging tasks to accomplish.
## Analytic pipelines for gene expression data
The study of gene regulation related to different biological functions is critical to understand the underlying mechanism of each function, such as cell growth, division, development and response to environmental stimulus. In addition, gene regulatory networks (GRN) have been shown useful for investigating the interaction among genes involved in a biological process, or genes responsive to an external stimulus. There are many computational approaches in the literature for inferring GRNs from gene expression data; for example, information theory models (5-7), Boolean networks [bib_ref] Inferring qualitative relations in genetic networks and metabolic pathways, Akutsu [/bib_ref] [bib_ref] Probabilistic boolean networks: a rule-based uncertainty model for gene regulatory networks, Shmulevich [/bib_ref] [bib_ref] Boolean network models of cellular regulation: prospects and limitations, Bornholdt [/bib_ref] and Bayesian networks [bib_ref] Using Bayesian networks to analyze expression data, Friedman [/bib_ref] [bib_ref] Inferring gene networks from time series microarray data using dynamic Bayesian networks, Kim [/bib_ref] [bib_ref] A new dynamic Bayesian network (dbn) approach for identifying gene regulatory networks..., Zou [/bib_ref] [bib_ref] A primer on learning in Bayesian networks for computational biology, Needham [/bib_ref]. However, these approaches are either not efficient in describing dynamic regulations between genes or restricted to small-scale networks. Meanwhile, responses to environmental stimulus, such as immune response to viral infection or response to aberrant activation of a particular pathway, are dynamic processes and require deliberate analysis of time-course gene expression data, which in turn is an ultra-high dimensional problem and needs the use of advanced statistical and computational approaches developed. Therefore, we implement an alternative comprehensive approach that exploits ODE models and gene regulatory network analysis developed in [bib_ref] High-dimensional odes coupled with mixed-effects modeling techniques for dynamic gene regulatory network..., Lu [/bib_ref] [bib_ref] Modeling genome-wide dynamic regulatory network in mouse lungs with influenza infection using..., Wu [/bib_ref] [bib_ref] Dynamic transcriptional signatures and network responses for clinical symptoms in influenzainfected human..., Linel [/bib_ref]. This model takes into account the dynamic and temporal behavior of genes, and learns the dynamic relation between genes, in the form of stimulator or inhibitor of each other. Genes (or probes) with significant expression level changes over time are identified as dynamic response genes. Then the top 3000 dynamic response genes are clustered into groups according to their expression pattern over time. Finally, a regulatory network is established by the ODE model.
## Recommendation systems
A recommendation system is an enabling mechanism to overcome information overload. Literature in this area can be broadly grouped as content-based or collaborative filtering based recommendation systems. Next, we discuss literature related to developed recommendation systems.
## Dataset recommendation
There are many dataset repositories in the biomedical domain and many datasets are added to each repository on a daily basis. For example, 34 datasets were added to GEO repository daily in 2019. Hence researchers are likely to be overwhelmed with the data available and they have to visit each repository for searching a dataset. The platforms like DataMed solved this problem and researchers only had to visit DataMed for searching the datasets. However, DataMed has not been updated recently. Again, the intent of search is always difficult to identify [bib_ref] Determining the user intent of web search engine queries, Jansen [/bib_ref]. A dataset recommendation system based on researcher's profile may be helpful for information filtering. There were a few experiments performed on data linking [bib_ref] Predicting and recommending relevant datasets in complex environments, Srivastava [/bib_ref] where similar datasets were clustered together using different semantic features. Most of these works were on linking the datasets with similar datasets rather than a dataset recommendation.
## Literature recommendation
The usefulness of the literature recommendation can be stated by the acceptance of Google Scholar, Semantic Scholar, PubMed, etc. The CiteSeer project [bib_ref] Research-paper recommender systems: a literature survey, Beel [/bib_ref] was the first of its kind to start research paper recommendation. Later, many scientific article recommendation systems were developed. Science Concierge is a content-based article recommendation system using distributional semantics (LSA) and the relevance feedback (Rocchio algorithm). It recommends articles for any number of input articles based on the 2015 Society of Neuroscience Conference articles [bib_ref] Science concierge: A fast content-based recommendation system for scientific publications, Achakulvisut [/bib_ref]. (27) proposed a citation-based collaborative filtering recommendation system for research articles using Jaccard similarity. Similar article recommendation systems have been developed using TF-IDF (28), topic modeling (29) and citation or author network analysis. TF-IDF was the most frequently applied weighting scheme for recommendation tasks [bib_ref] Research-paper recommender systems: a literature survey, Beel [/bib_ref].
SciMiner is a web-based platform for identifying gene names in text based on user input and provides literature from MEDLINE for the corresponding gene [bib_ref] Sciminer: web-based literature mining tool for target identification and functional enrichment analysis, Hur [/bib_ref]. A content-based PubMed article recommendation system, PURE, was developed using Expectation Minimization [bib_ref] Pure: a Pubmed article recommendation system based on content-based filtering, Yoneya [/bib_ref] and it recommends articles to users based on their preferred articles. (33) developed a probabilistic topic-based model for content similarity called 'pmra' on the publications from MEDLINE and this has been used as a related article search function in PubMed. Most of the proposed literature recommendation systems use embedding methods to convert text into vectors and calculate the similarity between articles.
Once a researcher finds a dataset suitable for his/her study, he/she may need literature available related to the dataset. A literature recommendation system for datasets may be a helpful tool for this scenario where researchers can get literature from PubMed for each dataset.
## Collaborator recommendation
Academic collaborator recommendation has long been regarded as a useful application in the academic environment, which aims to find potential collaborators for a given researcher by exploiting big academic data. In the past few years, several works on collaborator recommendation have been proposed [bib_ref] Collabseer: a search engine for collaboration discovery, Chen [/bib_ref] [bib_ref] Cross-domain collaboration recommendation, Tang [/bib_ref] [bib_ref] Context-aware academic collaborator recommendation, Liu [/bib_ref].
Mainly, co-author network information has been incorporated to enhance the collaboration recommendation [bib_ref] Collabseer: a search engine for collaboration discovery, Chen [/bib_ref] [bib_ref] Context-aware academic collaborator recommendation, Liu [/bib_ref] [bib_ref] Acrec: a co-authorship based random walk model for academic collaboration recommendation, Li [/bib_ref]. [bib_ref] Acrec: a co-authorship based random walk model for academic collaboration recommendation, Li [/bib_ref] proposed a random walk restart model on co-author order, latest collaboration time point and collaboration times. (37) developed a collaborator recommendation system using collaborative entity embedding developed using the topic words collected from the publications of researchers. The cross-domain collaborator recommender is another important aspect of this recommendation and (36) proposed a cross-domain collaborator recommendation using the co-author matching, topic matching and cross-domain topic learning. [bib_ref] Collabseer: a search engine for collaboration discovery, Chen [/bib_ref] proposed CollabSeer based on the co-author network and lexical similarity. However, it is difficult for new researchers or students to get recommendation using the co-author network or lexical similarity as they do not have papers. [bib_ref] Collaborator recommendation for isolated researchers, Huynh [/bib_ref] proposed a collaborator recommender for new researchers or students using input keywords, organizational relationship, ratings and activity level of the collaborators.
When a researcher finds suitable data for his/her study, the researcher may look for collaborators to work with on that dataset. In this scenario, a collaborator recommendation system for each dataset may be helpful.
# Materials and methods
Data GEO Metadata collection GEO is one of the most popular public repositories for functional genomics data. As of 18 December 2019, there were 122 222 series of datasets available in GEO. Metadata of GEO datasets such as title, summary, date of publication and name of authors was collected from the GEO using a web crawler. The PMIDs of the articles associated with each dataset were also collected. Many datasets did not have associated articles.
Time-course dataset: This study was conducted for the time-course datasets from GEO, however, the time-course datasets were not identified explicitly in the GEO websites. The time-course datasets can be identified manually by reading the dataset descriptions or scanning the associated data with it which is a time-consuming and tedious task. A keyword-based NLP method was applied for identifying time-course datasets. We implemented a regular expression-based approach to extract the time point information from the GEO metadata. For example, some phrases like '12 time points', '7 developmental stages; harvest at 10 hrs, 12 hrs', etc. were used to extract the time point information. The regular expression-based system was able to identify 167 datasets out of 200 random datasets with an accuracy of 83.5%. Further, a total of 555 datasets were filtered manually from 862 datasets identified by the above system for processing. More details on identifying time-course datasets can be found in [bib_ref] Geometadb: powerful alternative search engine for the gene expression omnibus, Zhu [/bib_ref]. Once the datasets are identified, the GSE number were fed to the pipeline (Section 3.2.1) and it automatically retrieved the data and metadata information corresponding to GSE numbers. In addition to the time points, diseases, organisms or/and cell types were identified from the title and summary of the datasets. MetaMap [bib_ref] Metamap lite: an evaluation of a new Java implementation of metamap, Demner-Fushman [/bib_ref] applied to the metadata, and the Human Disease Ontology (DOID) terms were detected from the annotated text for each dataset [bib_ref] Restructured GEO: restructuring Gene Expression Omnibus metadata for genome dynamics analysis, Chen [/bib_ref]. Further, datasets can be filtered using both the cell type and diseases.
## Medline articles
For developing dataset recommender, we collected the researcher's publications from PubMed. MEDLINE articles were collected for developing literature and collaborator recommenders. MEDLINE articles were collected from PubMed which comprises more than 29 million biomedical and life science research articles. These articles consist of information such as title, abstract, authors, affiliations, Medical Subject Headings (MeSH) terms and publisher name.
However, the articles collected from PubMed contain a variety of topics related to biomedicine and life sciences which may not be suitable for building a recommendation system for datasets in GEO. Further, the articles before 1998 were removed as the research on micro-array data started during that year [bib_ref] A content-based literature recommendation system for datasets to improve data reusability-a case..., Patra [/bib_ref]. The datasets that are related to gene expressions and articles collected from PubMed contain a variety of topics. Thus, a MeSH term-based filtering method was implemented to remove unrelated articles from the whole MEDLINE articles. The details of the filtering method can be found in [bib_ref] A content-based literature recommendation system for datasets to improve data reusability-a case..., Patra [/bib_ref]. A total of 770 537 articles were utilized for developing literature and collaborator recommendations.
# Methods
## Analytic pipeline for time-course gene expression data
We integrated the series of statistical and modeling methods for the time-course gene expression data into an analytic pipelinewhich includes eight steps as mentioned in [fig_ref] Figure 1: Time-course gene expression analytic pipeline [/fig_ref].
The final analysis results of the pipeline can be reported as the initial bioinformatics findings for narrowing down the analysis and framing scientific questions, toward new collaborative publications. We could apply the pipeline to each of the time-course gene expression datasets under one experimental or biological condition. Furthermore, simple comparison functions between two or more datasets across experimental conditions and/or from different studies are currently under development for the pipeline. We published the source code of the analytic pipeline, so others can use the pipeline and expand its functionalities.(github.com/j142857z/Pipeline (Original code)),(github.com/AutumnTail/Pipeline (Updated code)).
## Recommendation systems data recommendation:
Data recommendation is an essential part of the GETc platform. The dataset recommendation function recommends datasets to researchers based on their publications. The datasets used for this recommendation system contain data not only from GEO but also from other sources such as TCGA, ArrayExpress, SRA and Clinical Trails. We used only textual information of datasets (title and summary) and publications (title and abstract).
A researcher may have multiple research interests. To identify the research interests, we implemented a nonparametric clustering algorithm named Dirichlet process mixture model (DPMM). More details on DPMM and its parameter tuning for obtaining better number of clusters can be found in. Each researcher had to provide name and curriculum vitae (CV)/list of publications to get dataset recommendation. Researcher's names were searched in PubMed to get publications (title, abstract, year of publication). This search may result publications from other researchers with the same name which was solved by searching the title of the publication from PubMed in the CV/list of publications provided by the researcher. Finally, publications of the authors were clustered using DPMM to obtain the research topics. For each topic, datasets can be recommended by calculating cosine similarity of research field/cluster vector and dataset vectors. The detailed methodology and evaluation can be found in our previous publication on dataset recommendation.
Literature Recommendation: The literature recommendation system recommends literature for datasets. The most similar literature for a dataset can be determined simply by comparing the cosine similarity of the dataset vector and paper vectors. For developing the literature recommendation system in GETc, we used BM25 as it resulted in better precision at 10 compared to other embedding methods such as TF-IDF, word2vec and doc2vec [bib_ref] A content-based literature recommendation system for datasets to improve data reusability-a case..., Patra [/bib_ref]. Finally, we used the title based weighted re-ranking and text normalization methods to improve the retrieved results. The detailed methods, experiments and results can be found in our previous publication [bib_ref] A content-based literature recommendation system for datasets to improve data reusability-a case..., Patra [/bib_ref].
Collaborator Recommendation: For each dataset, the recommendation system suggests some collaborators based on the recommended literature. We can say that the authors of the top similar literature for a dataset can be suitable collaborators to work with on that dataset. The authors of the similar articles may have experience working on the dataset and already published articles using it. Further, the collaborators may be recommended for each dataset by ranking the unique authors of the retrieved similar articles. For a dataset (d), the score for each unique author of similar articles can be calculated using Equation (1).
[formula] AuthorScore i = n ∑ j=0 SimScore j * weight (1) weight = 0 if A i / ∈ P j 1 [/formula]
## If a i is the first or last author in p j
## If a i is not first or last author in p j
where AuthorScore i is the score for ith author calculated over all the retrieved similar articles (P = P 0 , P 1 , ...Pn) for d. n is the number of total retrieved article for d. SimScore j is the similarity score of d and jth article (P j ). Higher weights were provided to the first and last authors of each similar article whereas less weights were provided to all other authors. Finally, the authors with the highest scores were recommended as the collaborators for d.
The top 1000 recommended publications from the above literature recommender for a single dataset were used for identifying collaborators for that dataset. Furthermore, authors' affiliations provided in papers were parsed using the affiliation_parser(github.com/titipata/ affiliation_parser) package and the distance between the recommended collaborators' and the user's current location was calculated using geopy(geopy.readthedocs.io) package to show a distance-based relevance of user and collaborators.
## Getc platform
In this work, we developed an interactive web-based platform, called GETc, to facilitate collaboration and sharing of the analytic results of our pipeline on time-course gene expression data from GEO to the general research community. We have identified 555 time-course gene expression datasets with more than 7 time points from GEO. We applied our analytic pipeline on 37 of those datasets (results in Section 4). The output of the analytic pipeline for each dataset is folder of files containing intermediate and final analytic results, tables, graphics/plots and documents. The output also includes an automatically generated analysis report for each dataset.
Platform users could interactively search, browse and identify particular datasets and corresponding results of interest. They can visualize and review the analysis results including figures and tables, which can be easily downloaded via the platform web-based user-interface. For the unprocessed time-course gene expression datasets included in the platform, users can request to execute the pipeline. The platform also provides its users with recommendations by employing the recommendation systems described in Section 3.2.2. It recommends literature for timecourse gene expression datasets, potential collaborators for extracting scientific insights from the analytic results. It also recommends datasets to researchers. [fig_ref] Figure 2: High-level architecture of the GETc platform [/fig_ref] shows the overview of GETc platform. GETc platform executes the tasks mentioned inside the green box.
Users of the platform can search for a time-course dataset using keywords and phrases and see the literature available, significant gene lists, gene clusters and prospective collaborators for that dataset. A screenshot of search and view dataset functionalities is shown in [fig_ref] Figure 3: Search and view datasets in GETc research platform [/fig_ref]. The dataset can be searched if any of the searched keywords matched with the dataset id, title, abstract or platform organism. The datasets retrieved can be filtered using disease or cell type provided on the left side tree view or right side pie charts. The disease types are extracted from human disease ontology [bib_ref] Geometadb: powerful alternative search engine for the gene expression omnibus, Zhu [/bib_ref].
# Results and discussion
The results of the analytic pipeline which we applied on 37 time-course gene expression cancer datasets from GEO are presented in [fig_ref] Table A1: Results statistics from the cancer datasets [/fig_ref] line, the organism, vitro or ex vitro or in vitro or in vivo and species (human or mouse/rats species). MCF10A, MCF7, HeLa and other widely used cell lines are tested in these datasets. These cells lines are originated from various types of cancers such as breast cancer, cervical cancer and leukemia. Also, treatments in these datasets target several essential cancer pathways, such as NFkB, EGFR and hedgehog. These classifications will help researchers perform meta-analyses to identify common/key genes and GRN in a certain type of cancer.
Evaluating recommendation systems are challenging because no benchmark nor prior true annotation exists for either dataset recommendation or dataset-driven literature recommendation. For that reason, we performed a manual evaluation by asking expert human judges to rate the recommendation of systems using one to three 'stars' scale based on the relevance (1: not relevant, 2: partially relevant, 3: most relevant).
We evaluated the recommendation systems using strict and partial precision at 10 (P@10). Strict considers only 3-star, while partial considers both 2-and 3-star results. The developed dataset recommendation system was evaluated with five judges who have worked on the datasets before. The system obtained P@10 (strict) and P@10 (partial) of 0.61 and 0.78, respectively. For the literature recommendation, we considered 36 datasets for evaluation and the human judges have already worked on these datasets earlier. The proposed system obtained 0.80 and 0.87 of P@10 (strict) and P@10 (partial), respectively.
No gold standard dataset for evaluating collaborator recommendation is available to date. Similar to literature recommendation, evaluating our collaborator recommendation system was a challenging task, as it requires time to work with collaborators and only then they can provide feedback for system's output. We are currently working with additional multiple collaborators to evaluate the output of the system and generate feedback that we can use to assess the system's quality in the future.
A screenshot of literature (top right corner) and collaborator (bottom right corner) recommendations for dataset GSE14 103 is provided in [fig_ref] Figure 4: A screenshot of recommended literature and collaborators for GSE14103 [/fig_ref]. For a selected dataset on the platform UI, the literature recommendation system will generate a list of related papers recommended for users. The recommended list of collaborators can be sorted by name or distance. We have a plan to implement a search function which will allow users to search for collaborators based on the preferred city.
We believe the functions of GETc are very useful for researchers from the biomedical genomics community to present and communicate large numbers of analysis results. In addition to datasets from GEO, we are currently expanding the platform with new time-course datasets from other repositories such as TCGA, SRA and ImmPort. We applied the ODEs in the process of constructing the high-dimensional gene regularity network where having at least 8-time points was essential for the identifiability of the corresponding model. Thus, only datasets with more than or equal to 8-time points can be processed with our pipeline.
# Conclusion
In this work, we developed a novel research platform called GETc for sharing data and analytic results of timecourse gene expression datasets from GEO to improve the dataset reusability. It is built on top of an analytical method based on the ODE model for analyzing time-course gene expression data. GETc platform provides means to efficiently search and retrieve data, results, and facilitate collaboration through recommendation of related literature and potential collaborators corresponding to datasets. This platform also hosts a dataset recommendation system which will help researchers in biomedical domain to search datasets based on their publications. This will hopefully lead to better data reuse experience. We believe that the proposed novel idea and computational platform could also be applied to other types of data from different databases or data repositories.
[fig] •: User-friendly navigation and searching functions. Hosts analysis results of the time-course gene expression datasets produced by the ODE analytic pipeline. Recommends relevant datasets for users based on their research interests. Recommends relevant papers and collaborators for each dataset hosted in the platform. [/fig]
[fig] Figure 1: Time-course gene expression analytic pipeline. [/fig]
[fig] Figure 2: High-level architecture of the GETc platform. [/fig]
[fig] Figure 3: Search and view datasets in GETc research platform. [/fig]
[fig] Figure 4: A screenshot of recommended literature and collaborators for GSE14103. [/fig]
[table] Table A1: Results statistics from the cancer datasets (ORG: Organism, in vitro: ivr, ex vitro: evv, in vivo: ivv, Species: SP, Homo sapiens: HS, Rattus norvegicus: RN, Mus Musculus: MM) Jurkar or Primary T cells ivr [/table]
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Development and validation of children's mind wandering scales
# Introduction
Mind wandering emphasizes a state of consciousness that arises spontaneously in the waking state, when the content of an individual's consciousness is not determined by subjective will but is occupied by endogenous mental representations; this state can occur either in goal-directed tasks or in resting states [bib_ref] The restless mind, Smallwood [/bib_ref]. Adult studies have found that the frequency of mind wandering in daily life is as high as 46.9%, and at least 30% of the sample answered that mind wandering occurs in almost all activities [bib_ref] A wandering mind is an unhappy mind, Killingsworth [/bib_ref]. Furthermore, the frequency of mind wandering in children has also been found to reach 20 to 33% [bib_ref] Mind wandering in children: examining task-unrelated thoughts in computerized tasks and a..., Keulers [/bib_ref]. Mind wandering as a trait has significant externalities in individual differences [bib_ref] The restless mind, Smallwood [/bib_ref]. For instance, individuals with a low frequency of mind wandering show that they are focused and have good control over their thinking activities, while individuals with a high frequency show that they are often distracted during the task and cannot maintain coherent thinking, thus affecting the performance of the task. In addition, the association between the frequency of mind wandering and individuals with cognitive or emotional disorders is also receiving increasing attention. Thus, it is of great theoretical and practical importance to study mind wandering from the perspective of individual difference [bib_ref] Young and restless: validation of the Mind-Wandering Questionnaire (MWQ) reveals disruptive impact..., Mrazek [/bib_ref].
Mind wandering has been extensively studied in adults [bib_ref] Young and restless, old and focused: age-differences in mindwandering frequency and phenomenology, Moran [/bib_ref] [bib_ref] Fixation, flexibility, and creativity: The dynamics of mind wandering, Smith [/bib_ref] , but relatively limited research has been conducted in children [bib_ref] Sit still and pay attention: using the wii balance-board to detect lapses..., Cherry [/bib_ref] [bib_ref] Do children and adolescents have a future-oriented bias? a developmental study of..., Mccormack [/bib_ref] [bib_ref] Children with positive attitudes towards mind-wandering provide invalid subjective reports of mind-wandering..., Ye [/bib_ref]. In fact, mind wandering has been shown to be reliably measured in children [bib_ref] Sit still and pay attention: using the wii balance-board to detect lapses..., Cherry [/bib_ref] , and the study of its functions will help to understand the role it plays in children's cognitive and social development. In the present paper, we aim to investigate the characteristics of children's mind wandering by developing psychometric scales (Study 1a and Study 1b) and applying experience sampling method in the laboratory tasks (Study 2). In the section that follow, we review recent advances on theoretical basis and related scales of mind wandering.
Current Concerns Theory tells that once a goal is established, it becomes a current concern event, and the individual's cognitive system maintains a high level of accessibility to environmental cues that make it easy to accomplish the goal, and facilitates behavior by enhancing the accessibility of goal-related stimuli. This accessibility will continue until the goal is achieved or abandoned. Assuming that an individual's goals have a hierarchical structure, mind wandering may arise spontaneously because alternative implicit goals are automatically activated, only that the individual fails to realize it at the time. 1 thus argue that mind wandering can be incorporated into the executive control model of attention, treating the occurrence of mind wandering as goal-driven processing that simply shifts executive control from the task at hand to the processing of internal goals, and arguing that this process is cognitively resourceintensive, which further leads to the Perceptual Decoupling Hypothesis [bib_ref] Metaawareness, perceptual decoupling and the wandering mind, Schooler [/bib_ref] [bib_ref] Distinguishing how from why the mind wanders: a processoccurrence framework for self-generated..., Smallwood [/bib_ref]. Several studies of event-related potentials (ERP) provide support for this hypothesis. Some studies found that participants' P300 amplitude during self-reported mind wandering was reduced relative to the events in the task [bib_ref] Detecting and quantifying mind wandering during simulated driving, Baldwin [/bib_ref] [bib_ref] Absorbed in thought: the effect of mind wandering on the processing of..., Barron [/bib_ref] [bib_ref] Going AWOL in the brain: mind wandering reduces cortical analysis of external..., Smallwood [/bib_ref]. Because the P300 can be treated as an index of executive resources, the decreased P300 amplitude during mind wandering indicates that executive resources have been withdrawn, at least partly, from the primary task and are presumably directed toward task-unrelated thoughts [bib_ref] Why the global availability of mind wandering necessitates resource competition: reply to..., Smallwood [/bib_ref].
However, executive failure hypothesis argues that mind wandering arises as a result of executive control failure and that this process is not cognitively resource-intensive [bib_ref] Conducting the train of thought: working memory capacity, goal neglect, and mind..., Mcvay [/bib_ref] [bib_ref] Why does working memory capacity predict variation in reading comprehension? On the..., Mcvay [/bib_ref] [bib_ref] Does mind wandering reflect executive function or executive failure?, Mcvay [/bib_ref]. First, it is the default state to constantly assess the gap between the ideal state and the present state and thus to continuously generate spontaneous thoughts beyond the level of awareness. Second, mind wandering arises during the task when controlled processing is not sufficient to handle the interference generated by spontaneous thought. Studies have found that the default mode network (DMN) and executive control network (ECN) are associated with mind wandering in adults. For instance, neuroimaging studies have shown that the DMN and ECN are activated during mind wandering, and the neural activations in both networks are strongest when subjects lack meta-awareness (25-27). Similarly, evidences suggest that mind wandering in children is related to specific executive functions like inhibition and set shifting/switching (3 which could be due to dysregulation of specific brain regions, like DMN. Recent studies on healthy individuals have shown how dysregulation of action control and inhibition capacity impairs task performance that involves inhibition of actions, indicating that children may have difficulty in switching off the DMN during some tedious tasks which require focused attention and inhibit task-unrelated thoughts [bib_ref] The influence of vicarious fear-learning in "infecting" reactive action inhibition, Battaglia [/bib_ref] [bib_ref] Drifting from slow to "D'oh!": working memory capacity and mind wandering predict..., Battaglia [/bib_ref].
As can be seen from the above theories, researchers have accepted that mind wandering was determined by a combination of automatically generated thoughts in response to environmental or mental cues and the ability of the executive control system to deal with disturbing thoughts [bib_ref] Does mind wandering reflect executive function or executive failure?, Mcvay [/bib_ref] , but most of the existing scales have been designed based on one perspective only, either focusing on the individual's ability to generate spontaneous thoughts or on the individual's ability to execute control during the task, thus lacking comprehensiveness. Study1a aims to explore the dimension of the generation of children's mind wandering, and we hypothesize that both the spontaneity of individual thinking and the executive control processes are involved from the perspective of scale development.
Based on the opposing hypotheses that executive control inhibits the occurrence of mind wandering and executive control supports the continuity of mind wandering, several studies have shown that individuals with high working memory capacity (WMC) report less mind wandering in the sustained attention response task (SART) [bib_ref] Conducting the train of thought: working memory capacity, goal neglect, and mind..., Mcvay [/bib_ref] , reading task [bib_ref] Why does working memory capacity predict variation in reading comprehension? On the..., Mcvay [/bib_ref] [bib_ref] Mind wandering and reading comprehension: examining the roles of working memory capacity,..., Unsworth [/bib_ref] , and memory span task (32-34) relative to individuals with low WMC, and that individuals with high WMC also report less mind wandering when they are more focused on the task using experience sampling in everyday life [bib_ref] Myin-Germeys I. For whom the mind wanders, and when: an experience-sampling study..., Kane [/bib_ref]. In contrast, some studies have shown that individuals with high WMC report more mind wandering than those with low WMC in low cognitive demand visual search tasks and respiratory perception tasks (36), and similar findings have been found in choice reaction time tasks [bib_ref] Back to the future: autobiographical planning and the functionality of mind-wandering, Baird [/bib_ref] , where individuals with high WMC report more mind wandering about future directions. More interestingly, in these studies, when the nature of the task was divided in a different way, WMC did not predict the frequency of mind wandering, e.g., in the above-mentioned experience sampling method [bib_ref] Myin-Germeys I. For whom the mind wanders, and when: an experience-sampling study..., Kane [/bib_ref] , there was no association between WMC and mind wandering frequency when the task was divided in terms of whether it was challenging or the level of effort exerted. In addition, no association was found between WMC and mind wandering frequency in high cognitive demand visual search task (36). Indeed, researchers have suggested that individuals' executive control not only inhibits the generation of mind wanderings, but also flexibly adjusts the frequency of mind wanderings according to the demands of the task load [bib_ref] Controlling the stream of thought: working memory capacity predicts adjustment of mind-wandering..., Rummel [/bib_ref]. Thus, Study 1b aims to explore the dimension of the generation of children's mind wandering from the context of cognitive demand.
Researchers have largely acknowledged that differences in the content and frequency of mind wandering often reflect interindividual differences in trait level, yet there still lacks validated scale to measure the frequency of mind wandering in children. Through extensive reading of relevant literature, we found that the existing measurement tools have the following drawbacks.
First, some studies have used concepts such as daydreaming and task-unrelated thinking, which are close to mind wandering, as alternatives to study the characteristics of mind wandering in terms of form, content, and frequency. Such studies often change the connotation and extension of the concept of mind wandering, which not only lacks face validity but also makes the survey less relevant [e.g., Imaginal Processes Inventory Questionnaire,].
Second, attentional failure and endogenous spontaneity as the two main features of mind wandering are often taken in specific research contexts. Some scales focus only on the interference of mind wandering with specific tasks, emphasizing the characteristics of mind wandering that are not controlled by the subjective will of individuals. These instruments include Attention Related Cognitive Errors scale [bib_ref] Absent-mindedness: lapses of conscious awareness and everyday cognitive failures, Cheyne [/bib_ref] , Cognitive Failure Questionnaire [bib_ref] Everyday attention lapses and memory failures: the affective consequences of mindlessness, Broadbent [/bib_ref] and Memory Failure Scale (42), which are often used to indirectly account for the frequency of mind wandering. This type of scale focuses on the consequences of attention lapses and correlates well with behavioral task performance, and is often used as a tool to illustrate the undesirable consequences of mind wandering. However, not all mind wandering is unhelpful, and not all lapses are necessarily due to mind wandering, and researchers has also found positive implications for the existence of mind wandering [bib_ref] The mind that wanders: challenges and potential benefits of mind wandering in..., Mooneyham [/bib_ref]. The other part of the scale focuses on the content of spontaneously generated mental activities, such as resting state thought activities. These instruments include Automatic Thought Questionnaire [bib_ref] Cognitive self-statements in depression: Development of an automatic thoughts questionnaire, Hollon [/bib_ref] and Resting State Questionnaire [bib_ref] The resting state questionnaire: An introspective questionnaire for evaluation of inner experience..., Delamillieure [/bib_ref]. On the one hand, these scales were developed based on the content of mind activity, not just the frequency of occurrence. On the other hand, these scales focused on the spontaneity of mind activity, ignoring the important role played by attentional control.
Last but not the least, some researchers have used Mindful Attention Awareness Scale (MAAS), developed by Brown and Ryan (47), to indirectly explain the individual differences on mind wandering, based on the logic that mindfulness and mind wandering are opposing concepts. Although the close conceptual relationship between mindfulness and wind wandering, the strength of their association has been surprisingly low [bib_ref] Relationships between mind-wandering and attentional control abilities in young adults and adolescents, Belardi [/bib_ref]. In fact, mindfulness is considered to be a general personality trait, but mind wandering is a much more transient and fluctuating phenomenon during an ongoing task. Thus, the MAAS can be used as a reference to study an individual's level of mind wandering, but is not a substitute.
To sum up, there is no scale developed for children on the frequency of mind wandering. However, elementary school is a critical period for good behavior and a foundation for future learning development, so it is necessary to accurately screen children with excessive mind wandering and lack of mind wandering. For children with excessive mind wandering, early intervention would improve academic performance and quality of life; for children with a lack of mind wandering, a package of counseling exercises would help improve individual attention, and social and emotional functioning.
## Study a development of the frequency of children's mind wandering scale (cmws-f) item generation
We first invited 22 elementary school students for interviews, which included conceptual understanding of mind wandering and frequency of mind wandering. After that, a total of 21 relevant items were generated by combining previous relevant scales, expert opinions, and interview contents.
In order to determine the validity of the generated items, two pilot tests were conducted. The total sample included 220 children, with 94 children in the first polit test (36 boys and 58 girls) and 126 in the second test (59 boys and 67 girls). All participants are elementary school students from grades 3 to 6 in Zhejiang Province, China. The first pilot analysis revealed that there were problems such as inappropriate reverse questions, and low item-total correlations for some items. After the modification, we decided not to apply the reverse questions Frontiers in Public Health frontiersin.org . /fpubh. . and replaced the abstract concepts with life-like terms. To explore the validity of the changes, the revised scale was tested for the second time. The item analysis was good and resulted in a preliminary scale. Finally, a total of 22 items were identified (including one lie detection item, e.g., please check option 4 on this question), and all response options were on a 5-point Likert response scale (1 = strongly disagree, 2 = disagree, 3 = unsure, 4 = agree, and 5 = strongly agree).
## Exploratory factor analysis (efa) participants
The sample consisted of 332 children, of which 40 children were excluded due to randomly check and failed the lie detection item. The remaining 292 participants' data were analyzed (151 boys and 141 girls, M = 10.70 years old, SD = 1.30, 24.3% of third graders, 28.1% of fourth graders, 22.6% of fifth graders, 25.0% of sixth graders).
# Results
The structure of CMWS-F was analyzed using principal component analysis (PCA) and Promax rotation method, and the specific statistical processing was implemented with SPSS 10.0. We first conducted item analysis to assess the item appropriateness within the scale. Inappropriate items were eliminated based on the critical ratio (CR ≥ 3), the item-total correlation (r ≥ 0.3). One item was removed first as it did not meet above criteria. One main step before conducting EFA was evaluating the data appropriateness for Factor analysis using Kaiser Meyer-Olkin (KMO) and the Bartlett test of sphericity. The KMO value was 0.926, indicating that the sample size was adequate for factor analysis. The Bartlett test was significant (p < 0.001), supporting the argument that the data were appropriate for conducting EFA. Following the EFA, eight items were removed from the scale as their factor loadings fell below 0.45. The scree plot and eigenvalues < 1.0 were evaluated to determine the number of factors in this scale. In the end, two factors were extracted and the two factors accounted for 56.32% of the total variance. The two factors respectively accounted for 42.42 and 13.91% of the variance. Factor loadings ranged from 0.59 to 0.84 (see details in .
Factor 1 consisted of six items in which children frequently "absent-minded" during the process that required them to continuously devote attentional resources on the task at hand. We named this factor "attentional failure." In contrast, Factor 2 consisted of 6 items that include involuntary thoughts unrelated to the current activity, such as reflections on past events, plans for the future, or even "whimsical thoughts, " which we named "spontaneous thinking." The correlation coefficient between the two factors was 0.442, indicating the homogeneity and relative independence of the content measured by each dimension.
# Results
Considering that mind wandering might differ in frequency for boys and girls, we conducted a series of independent-sample t-tests and found that there were no gender differences for each dimension in our scales (p > 0.05, .
We then used the confirmatory factor analysis (CFA) and test-retest reliability to evaluate the scale. CFA is applied as a way to test the construct's dimensionality and to confirm the factor structure that emerged in the EFA. CFA was conducted using AMOS 4.0, and maximum likelihood estimation (MLE) was used to examine the fit of the model to the data. Evaluation of different indices has been suggested to check the fit of the model. The result revealed that the model provided an acceptable fit to the data, and the factorial structure of children's mind wandering frequency was confirmed (χ²/df = 3.14, GFI = 0.922, CFI = 0.919, TLI = 0.899, RMSEA = 0.079). All factor loadings were significant at p < 0.05.
## Test-retest reliability
Test-retest reliability reflects the stability and consistency of the scale across time. To test the degree of consistency of the scale, we assessed test-retest reliability, and the interval between the retests was 2 weeks. A total of 99 participants completed the scale twice (41 boys and 58 girls, M = 10.54 years old, SD = 1.08, 25.3% of third graders, 37.4% of fourth graders, 37.4% of fifth graders). The correlation analysis showed that the correlation coefficient of the attentional failure dimension was 0.76 (p < 0.01), the correlation coefficient of the spontaneous thinking dimension was 0.74 (p < 0.01), and the correlation coefficient of the total score was 0.80 (p < 0.01). The results showed that this scale had high test-retest reliability over time.
# Discussion
In this study, the construct validity of the scale was verified in detail in two aspects: overall model fit, and intrinsic structure of the model, and it can be concluded that the two-factor structure of the Frequency of Children's Mind Wandering Scale was supported by the data, and the test-retest reliability of the scale is robust. Early scholars believed that mind wandering arises simply as a result of failures of attentional control, and thus individual differences in attentional control could predict the frequency of mind wandering. According to this conjecture, adolescents have significantly weaker attentional control than adults, and thus adolescents should experience more mind wandering than adults. Young adolescents, however, did not report more mind wandering than adults (49). Study 1a suggests that mind wandering is the result of both attentional failure and spontaneous thinking, reflecting the individual's executive control and spontaneity of thought, respectively, which we should consider when predicting the frequency of mind wandering. It has been found that the generation of mind wandering is complex in different contexts [bib_ref] For whom the mind wanders, and when, varies across laboratory and daily-life..., Kane [/bib_ref] , and the weighting of attentional failure and spontaneous thinking is constantly changing. For this reason, we designed Study 1b to develop a contextual scale for the occurrence of children's mind wandering and to explore how the frequency of children's mind wandering changes in different contexts.
[formula] . /fpubh. . [/formula]
. /fpubh. .
## Study b development of the context of children's mind wandering scale (cmws-c)
The development of the Context of Children's Mind Wandering Scale followed the same four steps of interview and pilot test, exploratory factor analysis, confirmatory factor analysis, and test-retest analysis, each of which was strictly implemented in accordance with psychometric requirements.
## Item generation
The whole process of items generation was similar to the development of the CMWS-F. The total sample included 218 children, with a total of 92 children in the first pilot test and 126 in the second test. Through two rounds of test, a total of 22 items (including one lie detection item) were formed for the initial scale.
# Results
The structure of the CMWS-C was analyzed using PCA and Varimax rotation method, and the specific statistical processing was implemented using SPSS 10.0. Without the lie detection item, we conducted EFA on 21 items. The KMO value was 0.870, indicating that the sample size was adequate for factor analysis. The Bartlett test was significant (p < 0.001), supporting the argument that the data were appropriate for conducting EFA. Following the EFA, nine items were removed from the scale as their factor loadings fell below 0.45. Based on the results and scree plot, two factors were extracted. The results showed that the two factors accounted for 51.57% of the total variance. The two factors respectively accounted for 27.98 and 23.59% of the variance. Factor loadings ranged from 0.59 to 0.79 (see details in .
Factor 1 consisted of 6 items across which children's mind wandering invaded in contexts such as reading and taking classes, and thus we named this factor "high-demand." In contrast, Factor 2 consisted of 6 items across which children's mind wandering happened in contexts such as riding in the car and going for a walk, and this is usually a context where children are prone to mind wandering, and we named this factor "low-demand."
# Results
The result revealed that the model provided an acceptable fit to the data, and the factorial structure of CMWS-C was confirmed (χ²/df = 2.428, GFI = 0.940, CFI = 0.929, TLI = 0.911, RMSEA = 0.064).
## Test-retest reliability
A total of 100 participants completed the scale twice (42 boys and 58 girls, M = 10.52 years old, SD = 1.09, 26.0% of third graders, 37.0% of fourth graders, 37.0% of fifth graders), and the test-retest analysis showed that the correlation coefficient of the dimension of high-demand context was 0.61 (p < 0.01); the correlation coefficient of the dimension of low-demand context was 0.62 (p < 0.01); and the correlation coefficient of the total score was 0.64 (p < 0.01). The results showed that this scale had a relatively high test-retest reliability over time. In addition, a matched samples t-test showed that children reported significantly more mind wandering in low-demand contexts (M = 3.40, SD = 0.84) than in high contexts (M = 2.43, SD = 0.85), t = 18.68, p < 0.001.
## Relationship between the cmws-f and the cmws-c
After collapsing the above data, a total of 343 children completed both CMWS-F and CMWS-C (21.3% of third graders, 25.4% of fourth graders, 35.3% of fifth graders, 18.1% of sixth graders). To further examine the effects of attentional failure and spontaneous thinking on the frequency of mind wandering in different contexts, the factors of CMWS-C were used as dependent variables and the factors of CMWS-F as predictor variables in regression analysis. As seen in , both attentional failure (β = 0.194, p < 0.01) and spontaneous thinking (β = 0.514, p < 0.001) were valid predictors in the low-demand context, and spontaneous thinking had a greater weight in the regression model. In contrast, only the factor of Frontiers in Public Health frontiersin.org . /fpubh. .
## Table factor loadings of the cmws-c.
## Items factor loadings
Factor 1(high-demand) Factor 2(low-demand) [bib_ref] The restless mind, Smallwood [/bib_ref] attentional failure (β = 0.782, p < 0.001) was a valid predictor in the high-demand context.
# Discussion
Study 1b shows that the occurrence of children's mind wandering can be broadly classified into two types of contexts: low-demand contexts, which are commonly understood to be prone contexts in which individuals often let go of conscious control and allow their thoughts to drift; and high-demand contexts, in which individuals are occasionally distracted by inner thoughts. The competing relationship between mind wandering and task load has been found in adult research, i.e., mind wandering occurs less when the current task places a higher demand on an individual's cognitive resource, and conversely, mind wandering occurs more frequently. Study 1b illustrated by means of a scale that children did report significantly more frequent mind wandering in low-demand contexts than in high-demand contexts.
In combination with Study 1a and Study 1b, we investigated the psychometric properties of the CMWS-F and CMWS-C. However, it is the first time to develop a scale for children's mind wandering, and it is difficult to find an authoritative method as a Study probe-caught mind wandering in the experimental tasks Participants A power analysis was performed on children's data from a similar protocol to estimate the appropriate sample size [bib_ref] Children with positive attitudes towards mind-wandering provide invalid subjective reports of mind-wandering..., Ye [/bib_ref]. With an α = 0.05 and power = 80%, we required at least 82 participants to obtain a medium-sized effect (two-tailed, r = 0.3) for the validity of the scales in each laboratory task using the experience sampling method. Additional children were recruited in case of potential withdrawal from our tasks (e.g., inability to perform the computerized task). Finally, the total sample consisted of 365 children (all participants were from grades 4-6, 95 children in Breathing task, 105 children in Sustained Attention Response Task, 94 children in Vigilance task and 71 children in one-back task).
# Materials and procedure
In this study, we selected four typical tasks with "thought probe" inserted to study mind wandering.
In the Breathing task, participants needed to wear headphones and sit in front of a computer screen, close their eyes and pay attention to the movement of breath in and out. In addition, a "ding" sound would come from the headphones from time to time, and children needed to open their eyes to answer the questions that appeared on the screen. The question was as follows: what were you thinking before the sound appeared? The answer options were to think about something related to counting breaths (please press "F") and to think about something unrelated to counting breaths (please press "J"). Following the judgment, they were prompted to close their eyes and count breaths again. During the task, the program presented 8 times of thought probes, and the time interval between two question pages was 25, 35, 45, and 55s. A 40s buffer was set at the beginning of the program, and the entire program lasted approximately 6 min.
In the Vigilance task, there was only one stimulus "+" presented for 0.5s, and the inter-stimuli interval was 1.5, 2.5, 3.5, or 4.5s. Participants were instructed to press a button whenever they saw the "+." This task lasted 12 min and collected 30 times of thought probes on mind wandering.
In the Sustained Attention Response Task (SART), the stimuli were divided into two categories: white numbers 1-9 (non-targets) and red crosses (targets). Each stimulus presented for 0.5s and the inter-stimulus interval was 2s. Participants were asked to press the space bar to the numbers as fast and accurately as possible and to withhold the response when the red cross was presented. The ratio of the appearance of non-targets to targets was 8:1 (non-targets = 240, targets = 30), and the program would randomly probe 30 times on mind wandering. The entire program was approximately 11 min.
In the one-back task, participants were asked to wear headphones and sit in front of a computer screen. Two stimuli, black numbers 1-9 (non-target) and green question mark (target), were presented at random order, with 2s for nontarget and 3s for target on screen, followed by 1s inter-stimulus interval. Participants were asked to determine the parity of the previous number when the green question mark appeared, pressing "F" for odd numbers and "J" for even numbers. Nontarget to target ratio was approximately 7:1 (non-targets = 190, targets = 24). Similarly, the program would randomly appear 6 times on mind wandering, with a "ding" in the headset, asking the participants about their state of consciousness at that time. The entire program was approximately 11 min.
It is important to note that we used thought-sampling method to obtain participants' current state of consciousness. To help participants familiar with the procedures, we presented examples of different options to ensure all of them understood the question before the formal tests. At the end, the participants were asked to complete the CMWS-F and the CMWS-C.
# Results
We first calculated the accuracy of one-back task, and the children showed reasonable task performance (M one−back = 0.80, SD one−back = 0.22). We then evaluated how this task performance was affected by probe-caught mind wandering across subjects, and results showed a significant negative correlation between the task accuracy and the frequency of probe-caught mind wandering (calculated as the proportion of task-unrelated thoughts in probes, M = 0.37, SD = 0.30; r one−back = −0.277, p < 0.05), indicating that the participants who reported more mind wandering were associated with poor performance during a high-demand task. In addition to the breathing task (no response required), we also calculated the average coefficient of variability (CV = SD/mean) for the reaction time in our SART and Vigilance tasks (M SART = 0.34, SD SART = 0.22, M Vigilance = 0.38, SD Vigilance = 0.22), which is consistent with our previous children's study (13). Taken together, these results suggested that children are able to complete these computerized tasks.
Next, we considered the correspondence between the statelevel (probe-caught) and the trait-level mind wandering by .
/fpubh. .
## Table correlations between the frequencies of probe-caught mind wandering in tasks and the scores of the cmws-f/cmws-c in study (descriptive statistics included).
Task Type (probe-caught mind wandering) TUT probability (M ± SD)
## Cmws-f (m ± sd) cmws-c (m ± sd)
Attentional failure
## Spontaneous thinking
High-demand Low-demand calculating the correlation between the frequencies of mind wandering in laboratory tasks and the scores on CMWS-F/CMWS-C. As expected, the results showed moderate positive correlations between the frequencies of probe-caught mind wandering in the three tasks and the scores in the scales, especially in the dimension of attentional failure of CMWS-F, with correlation coefficients ranging from 0.284 to 0.316 .
# Discussion
Early scholars often wonder when children are able to correctly understand and report conscious state. Studies of young children aged 4 to 13 years suggested that the uncontrollability of comprehension awareness did not mature until 8 or 9 years of age or even later (51-53). Here, we show that children are able to correctly report their mind wandering, at least, at the age of 8 years old. Mind wandering has been shown in numerous studies in adults to be associated with sustained attention capacity [bib_ref] Construction, integration, and mind wandering in reading, Dixon [/bib_ref] [bib_ref] Thinking one thing, saying another: the behavioral correlates of mind-wandering while reading..., Feng [/bib_ref] [bib_ref] Catching the mind in flight: using behavioral indices to detect mindless reading..., Franklin [/bib_ref] , working memory [bib_ref] Why does working memory capacity predict variation in reading comprehension? On the..., Mcvay [/bib_ref] , and intelligence tests [bib_ref] Unsworth N. Working memory capacity and sustained attention: a cognitive-energetic perspective, Mrazek [/bib_ref]. Usually when individuals report more mind wandering during the task, the worse the individual performs on the task, and vice versa. Mrazek and colleagues found that the higher the frequency of mind wandering during adolescent reading, the worse the reading comprehension scores, and concluded that students aged 11-13 years already have the ability to correctly report their state of consciousness [bib_ref] Young and restless: validation of the Mind-Wandering Questionnaire (MWQ) reveals disruptive impact..., Mrazek [/bib_ref]. In all three different task conditions, we derived an agreement between mind wandering frequencies reported in the task and the mind wandering frequencies measured by the scale, confirming the criterion validity of the CMWS-F/CMWS-C.
Study 1a showed that the production of mind wandering was influenced by both attentional failure and spontaneous thinking, while study 1b showed that the contexts in which mind wandering occurred could be broadly classified into two categories: low-demand and high-demand, and the weights of the effects of attentional failure and spontaneous thinking on the frequency of mind wandering changed continuously in different contexts, constituting a dynamic and complex relational model. This would explain why the use of working memory capacity to predict mind wandering frequency under different experimental task conditions has been controversial [bib_ref] Distinguishing how from why the mind wanders: a processoccurrence framework for self-generated..., Smallwood [/bib_ref].
# General discussion
This is the first study to develop the CMWS-F and CMWS-C scales to assess the frequency of mind wandering for children. Following the steps of standard scale development (item generation and selection, EFA, CFA and test-retest analysis), we showed scales with good reliability and validity. Study 2 further provided evidence for the criterion validity of the CMWS-F and CMWS-C scales within four laboratory tasks, which showed moderate consistency between the frequencies of probe-caught mind wandering in the tasks and the scores in the scales.
Attentional failure and spontaneous thinking are two main factors that caused mind wandering in the CMWS-F. Attentional failure occurs when an individual's attention shifts from the current task to something unrelated, which arises as a result of executive control failure [bib_ref] Conducting the train of thought: working memory capacity, goal neglect, and mind..., Mcvay [/bib_ref] [bib_ref] Why does working memory capacity predict variation in reading comprehension? On the..., Mcvay [/bib_ref] [bib_ref] Does mind wandering reflect executive function or executive failure?, Mcvay [/bib_ref]. Executive control is closely related to the main characteristic of mind wandering: disengagement of the external task/environment (58). Children's capacity for attentional control is relatively immature throughout childhood and adolescence (59, 60), so their inhibitory control capacity is so weak that they are not able to focus on the current task long time. Therefore, it's not hard to explain why inhibitory control capacity is a significant predictor of children's mind wandering frequency (3).
The second factor, spontaneous thinking, means that individual's mentation is occupied by implicit goals, such as memories from the past and plans for the future [also called unintentional mind wandering, [bib_ref] Marcusson-Clavertz D. Psychometric properties of the spontaneous and deliberate mind wandering scales, Seli [/bib_ref] ]. On the one hand, [bib_ref] Children with positive attitudes towards mind-wandering provide invalid subjective reports of mind-wandering..., Ye [/bib_ref] found that there is a significant forward-looking bias of mind wandering among children. All of these suggest that only by correctly understanding the causes and the characteristics of children's mind wandering, can we give full play to the positive role of mind wandering and reasonably explore how to restrain children with high-frequency mind wandering and improve children with low-frequency mind wandering.
The CMWS-C showed that the contexts mind wandering often occurs in children can be divided into two categories: low-demand context and high-demand context. For children, the low-demand context is the leisure context like playing games and having a walk; the high-demand context is the task context like doing homework and reading books. Mind wandering can be beneficial or detrimental depending on the flexibility of cognitive resources in the specific context [bib_ref] The science of mind wandering: empirically navigating the stream of consciousness, Smallwood [/bib_ref]. In easy tasks that only demand low cognitive resources, individuals can tolerate longer delays in waiting for rewards because of mind wandering (69). However, in high cognitively demanding tasks, studies have shown that mind wandering could be detrimental for individual's performance [bib_ref] Detecting and quantifying mind wandering during simulated driving, Baldwin [/bib_ref] [bib_ref] Mind-wandering, cognition, and performance: a theory-driven meta-analysis of attention regulation, Randall [/bib_ref] [bib_ref] Driving with the wandering mind: the effect that mindwandering has on driving..., Unsworth [/bib_ref]. This reveals us to the children's mind wandering treatment should also be flexible, in low-demand context, children can be relatively unfettered when mind wandering occurs, while in high-demand context, children must be required to pay attention to the current task.
In study 2, we also found some evidence for CMWS-F and CMWS-C in experimental tasks. There was a significant correlation between the score of attentional failure dimension in CMWS-F and three tasks (p < 0.01), but no significant correlation on the spontaneous thinking. As expected, attentional failure and spontaneous thinking occur in different contexts. Because the participants were asked to complete the laboratory tasks during the experiment, they were actually in high-demand context. Just like our definitions of the two dimensions of CMWS-F, attentional failure occurs more during the task, while spontaneous thinking occurs more when the individual is free or in low-demand context. This can also explain why the scale score is significantly or nearly significantly correlated with the three tasks under the high-demand dimension in CMWS-C (p = 0.07).
As mentioned above, mind wandering has both benefits and costs [bib_ref] The mind that wanders: challenges and potential benefits of mind wandering in..., Mooneyham [/bib_ref] , so it should be viewed dialectically in the field of education. Under some conditions, mind wandering will promote students' creativity [bib_ref] Inspired by distraction: mind wandering facilitates creative incubation, Baird [/bib_ref]. But we have to admit that in many educational contexts, mind wandering could lead some negative influence. The higher the mind wandering frequency, the worse the test scores were [bib_ref] Cognitive processes in children's reading and attention: the role of working memory,..., Risko [/bib_ref]. Besides, if students fail to pay attention to the classroom or what they are learning, this may impede their chances of acquiring important knowledge or skills. The frequency of mind wandering mediates the relation between children's ratings of topic interest and learning scores [bib_ref] Sit still and pay attention: using the wii balance-board to detect lapses..., Cherry [/bib_ref] , so improving children's interest in what they have learned is a good choice to counteract the downsides of mind wandering.
Previous studies focused on the influence of divided attention on children from the cognitive process perspective (75), ignoring the spontaneous characteristics of consciousness.
Here we showed that spontaneous thinking and attentional failure are the main causes of mind wandering, and discussed the influence of context on children's mind wandering, which will be enlightening for future studies on children's learning difficulties like ADHD (76, 77). Furthermore, meta-awareness hypothesis showed that individuals who are more aware of their current mental activity could have a lower frequency of mind wandering [bib_ref] Young and restless: validation of the Mind-Wandering Questionnaire (MWQ) reveals disruptive impact..., Mrazek [/bib_ref]. Considering the uncontrollability of children's mental states, one direction for future study is to enhance children's metacognition to indirectly prevent excessive mind wandering. Similarly, mindfulness training, including practices that enhance awareness of thoughts, may modulate the occurrence of mind wandering, supported by bottom-up and top-down neural mechanisms (78).
Finally, it is not certain whether the structure of the CMWS-F/CMWS-C are different in varied cultures. Therefore, more cross-culture studies are needed to explore children's mind wandering. In conclusion, we developed and validated the CMWS-F/CMWS-C scales to explore the underlying causes and contexts of mind wandering in children. The results showed that children's mind wandering was mainly caused by attentional failure and spontaneous thinking, and the contexts could be divided into high-demand context and low-demand context. We have to admit that although our study provided a convenient measurement tool for future studies of mind wandering in children, more research is needed to complement and enrich CMWS-F/CMWS-C.
# Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
# Ethics statement
The studies involving human participants were reviewed and approved by the Ethics Committee of Zhejiang Normal University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
# Author contributions
XS and QY designed the research. ZC and QY collected the data and performed the statistical analysis. YH and QY wrote the first draft of the manuscript. All authors contributed to the research and approved the final version of the manuscript.
# Funding
[fig] .: When I'm doing my homework, I would often be miles away Before sleeping at night (when you're lying in the bed ready to sleep), things come to my mind When I finish the textbook that the teacher asks us to finish before the specified time, I can't help but think about something else0464 0599 CMWS-C, Frequency of Children's Mind Wandering Scale The bold values indicate the strongest loading for each item [/fig]
[fig] *p < 0: 05, **p < 0.01. The data in the parenttheses represented the correlation coefficient between the scores on CMWS-F/CMWS-C and probe-caght mind wandering in tasks. CMWS-F, Frequency of Children's Mind Wandering Scale; CMWS-C, Context of Children's Mind Wandering Scale. The probability of task-unrelated thought (TUT) was calculated as the proportion of task-unrelated thoughts in probes. [/fig]
[table] TABLE Factor: loadings of the CMWS-F. 3. I would try very hard to listen to the lecture or concentrate on something, but often I would still be thinking about something unrelated to the class. [/table]
[table] TABLE Gender comparisons: for CMWS-F/CMWS-C scores in study . [/table]
[table] TABLE Multiple regression: analysis of the CMWS-F on CMWS-C. [/table]
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A Case of Contact Allergic Dermatitis to Topical Minoxidil
# Introduction
Female pattern hair loss (FPHL) or androgenetic alopecia is the most common cause of hair loss in women and one of the most frequent chronic conditions encountered by dermatologists worldwide [bib_ref] Androgenetic alopecia: an evidence-based treatment update, Varothai [/bib_ref]. FPHL is nonscarring alopecia characterized by hair follicle miniaturization, a progressive transformation of pigmented terminal hair into nonpigmented villous hair [bib_ref] Female pattern hair loss: current treatment concepts, Dinh [/bib_ref] [bib_ref] Hormonal therapy in female pattern hair loss, Brough [/bib_ref] [bib_ref] Female pattern alopecia: current perspectives, Levy [/bib_ref]. The cause for miniaturization remains uncertain but is postulated to be a combination of genetic predisposition, androgen effect, and other not well-understood factors [bib_ref] Hormonal therapy in female pattern hair loss, Brough [/bib_ref] [bib_ref] Female pattern alopecia: current perspectives, Levy [/bib_ref]. Minoxidil was first introduced in the 1970s as an oral hypertensive medication and a potent vasodilator [bib_ref] Minoxidil: a review of its pharmacological properties and therapeutic use, Campese [/bib_ref]. Coincidental findings of hypertrichosis in patients on minoxidil led to the development of a topical minoxidil formulation, which has been approved by the Food and Drug Administration (FDA) for the treatment of FPHL in 1992. It is available over the counter with a good safety profile. The most common side effect of topical minoxidil is irritant contact dermatitis with the typical symptoms of itching and scaling [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref]. Most commonly, these symptoms are a result of an allergic reaction to propylene glycol, or less commonly, to minoxidil itself [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref]. We present a case of a 27-year-old woman who developed allergic contact dermatitis to minoxidil 5% foam. In addition to our case, a few other instances of allergic reactions to minoxidil itself have been reported in the literature [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref] [bib_ref] Dooms-Goossens A: Allergic contact dermatitis to minoxidil, Degreef [/bib_ref] [bib_ref] Contact dermatitis caused by topical minoxidil: case reports and review of the..., Tosti [/bib_ref].
## Case presentation
This is a case of a 27-year-old woman, not known to have any medical illnesses, who presented to our dermatology clinic with a painful swelling over the face for two days. Upon further inquiry, she used topical minoxidil 5% foam for FPHL applied once daily for four days prior to her presentation. After applying the foam, she reported itchiness over the scalp, and therefore, she stopped using it. Regardless, the itchiness continued, and she started to develop gradual swelling over the face. She presented to the emergency department one day prior to her appointment in the dermatology clinic and was given intramuscular diphenhydramine 50mg and oral prednisone 30mg. The patient had previously been prescribed minoxidil 5% spray for FPHL, and she reported itchiness over the scalp. She was then advised to substitute it with minoxidil 5% foam (propylene glycol free) after which, she presented with her current complaint. A patch test was done with a minoxidil solution containing 5% minoxidil in alcohol, and the results were interpreted by an immunologist, which showed positive sensitization [fig_ref] FIGURE 1: Positive patch test to minoxidil 5% [/fig_ref]. A patch test to alcohol and propylene glycol showed no reaction. Consequently, she was advised to avoid topical minoxidil solution or foam as a treatment for her FPHL.
# Discussion
FPHL is a nonscarring type of alopecia in which there is progressive hair thinning at the scalp vertex with a spared frontal hairline. This occurs secondary to changes in the hair cycle with simultaneous shortening and lengthening of the anagen and telogen phases, respectively. This results in hair follicle miniaturization, a progressive transformation of pigmented terminal hair into nonpigmented villous hair [bib_ref] Female pattern hair loss: current treatment concepts, Dinh [/bib_ref] [bib_ref] Hormonal therapy in female pattern hair loss, Brough [/bib_ref] [bib_ref] Female pattern alopecia: current perspectives, Levy [/bib_ref] [bib_ref] Follicular miniaturization in female pattern hair loss: clinicopathological correlations, Messenger [/bib_ref] [bib_ref] Hair loss in women: medical and cosmetic approaches to increase scalp hair..., Sinclair [/bib_ref]. Oral minoxidil is a piperidino-pyrimidine derivative, a vasodilator that is used for the treatment of hypertension. When applied topically, it has shown a direct stimulatory effect on dermal papillae and follicular hair matrix cells that is effective in arresting the process of miniaturization and producing some degree of regrowth [bib_ref] Improvement in androgenetic alopecia (stage V) using topical minoxidil in a retinoid..., Walsh [/bib_ref]. Topical minoxidil solution is currently available over the counter and has a considerably good safety profile [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref]. The most common side effects of topical minoxidil include exacerbation of seborrheic dermatitis, irritant contact dermatitis, or allergic contact dermatitis [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref]. A patch test can be provided to patients with allergic contact dermatitis to determine the causative allergen [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref]. The first case of allergic contact dermatitis to 1% minoxidil solution was reported by Weiss et al. in 1984 in a patient treated for alopecia areata [bib_ref] Alopecia areata treated with topical minoxidil, Weiss [/bib_ref]. Since then, other cases of contact dermatitis as an adverse event to topical minoxidil have been described in the literature. These instances have been commonly reported to be due to minoxidil solution vehicles such as propylene glycol and less frequently to minoxidil itself, like in our case [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref] [bib_ref] Dooms-Goossens A: Allergic contact dermatitis to minoxidil, Degreef [/bib_ref] [bib_ref] Contact dermatitis caused by topical minoxidil: case reports and review of the..., Tosti [/bib_ref]. Rarely, oral minoxidil has been associated with rashes, bullous eruptions, and Stevens-Johnson syndrome [bib_ref] Fatal toxic epidermal necrolysis associated with minoxidil, Karaoui [/bib_ref]. If patients are found to be allergic to propylene glycol, compounded formulations with alternative solvents can be used. However, patients who are found to be allergic to minoxidil itself are not candidates for using minoxidil to treat their alopecia [bib_ref] Allergic contact dermatitis to topical minoxidil solution: etiology and treatment, Friedman [/bib_ref]. Oral minoxidil has a vasodilatory effect, which may lead to angioedema as a dose-dependent side effect [bib_ref] Vasodilatory edema: a common side effect of antihypertensive therapy, Messerli [/bib_ref]. Frontal edema following application of topical minoxidil has been reported; however, it was applied following hundreds of mesotherapy injections, which most likely increased the absorption of topical minoxidil [bib_ref] Frontal edema due to topical application of %5 minoxidil solution following mesotherapy..., Güngör [/bib_ref]. In our case, the patient was previously sensitized to topical minoxidil. Two days after the application of topical minoxidil foam, she developed gradual facial edema, which mimicked angioedema; allergic contact dermatitis was proven by patch testing. An instance of the socalled pseudoangioedema, An allergic contact dermatitis mimicking angioedema, was also reported in the literature [bib_ref] How not to be misled by disorders mimicking angioedema: a review of..., Andersen [/bib_ref].
# Conclusions
Allergic contact dermatitis to minoxidil itself is increasingly reported in the literature. Therefore, patch testing should be considered if the patient reports itchiness or erythema following the application of topical minoxidil. Patients who experience an allergic reaction to topical minoxidil are commonly advised to switch to minoxidil foam as it is propylene glycol free. However, patients who are found to be allergic to minoxidil itself are not candidates for using topical minoxidil to treat their alopecia. Other treatments can be used offlabel, such as 5α-reductase inhibitors, androgen receptor antagonists, prostaglandin analogs and antagonists, laser therapy, and platelet-rich plasma injections.
[fig] FIGURE 1: Positive patch test to minoxidil 5%. [/fig]
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Identification of minor effect QTLs for plant architecture related traits using super high density genotyping and large recombinant inbred population in maize (Zea mays)
Background: Plant Architecture Related Traits (PATs) are of great importance for maize breeding, and mainly controlled by minor effect quantitative trait loci (QTLs). However, cloning or even fine-mapping of minor effect QTLs is very difficult in maize. Theoretically, large population and high density genetic map can be helpful for increasing QTL mapping resolution and accuracy, but such a possibility have not been actually tested. Results: Here, we employed a genotyping-by-sequencing (GBS) strategy to construct a linkage map with 16,769 marker bins for 1021 recombinant inbred lines (RILs). Accurately mapping of well studied genes P1, pl1 and r1 underlying silk color demonstrated the map quality. After QTL analysis, a total of 51 loci were mapped for six PATs. Although all of them belong to minor effect alleles, the lengths of the QTL intervals, with a minimum and median of 1.03 and 3.40 Mb respectively, were remarkably reduced as compared with previous reports using smaller size of population or small number of markers. Several genes with known function in maize were shown to be overlapping with or close neighboring to these QTL peaks, including na1, td1, d3 for plant height, ra1 for tassel branch number, and zfl2 for tassel length. To further confirm our mapping results, a plant height QTL, qPH1a, was verified by an introgression lines (ILs). Conclusions: We demonstrated a method for high resolution mapping of minor effect QTLs in maize, and the resulted comprehensive QTLs for PATs are valuable for maize molecular breeding in the future.
# Background
Plant architecture related traits (PATs) are of great importance for maize, as they are crucial indicators to the growth and development of the plant and finally influence the yield [bib_ref] Leaf orientation and yield of maize, Pepper [/bib_ref] [bib_ref] Post-green revolution trends in yield potential of temperate maize in the north-Central..., Duvick [/bib_ref]. Studies in past decades indicated that PATs are mainly affected by minor effect quantitative traits loci (QTLs), especially as reflected in the Nested Association Mapping (NAM) population [bib_ref] Distinct genetic architectures for male and female inflorescence traits of maize, Brown [/bib_ref] [bib_ref] The genetic architecture of maize height, Peiffer [/bib_ref] [bib_ref] Genome-wide association study of leaf architecture in the maize nested association mapping..., Tian [/bib_ref]. Fine mapping coupled with further precise cloning of these QTLs would be of great value for maize breeding. Up to now, there were only a few plant architecture related QTLs had been cloned in maize, such as tb1 [bib_ref] A distant upstream enhancer at the maize domestication gene tb1 has pleiotropic..., Clark [/bib_ref] , qPH3.1 [bib_ref] ZmGA3ox2, a candidate gene for a major QTL, qPH3.1, for plant height..., Teng [/bib_ref] , and qph1 [bib_ref] A rare SNP mutation in Brachytic2 moderately reduces plant height and increases..., Xing [/bib_ref]. However, they all are major effect QTL, with phenotype contribution larger than 10%. For minor effect QTL, the example of its cloning, even fine-mapping work has been rarely studied.
Previous QTL studies of agronomic traits in crops were mainly based on relatively small populations and a few hundred or less molecular markers. QTL intervals of these studies typically ranged from 2 to 50 cM [bib_ref] QTL mapping in testcrosses of European flint lines of maize: I. Comparison..., Lübberstedt [/bib_ref] [bib_ref] Quantitative trait locus (QTL) mapping using different testers and independent population samples..., Melchinger [/bib_ref] [bib_ref] Quantitative trait loci analysis of phenotypic traits and principal components of maize..., Upadyayula [/bib_ref] [bib_ref] Direct mapping of density response in a population of B73 x Mo17..., Gonzalo [/bib_ref] [bib_ref] Genetic analysis of agronomic traits associated with plant architecture by QTL mapping..., Zheng [/bib_ref] [bib_ref] Genetic dissection of plant height by molecular markers using a population of..., Tang [/bib_ref] [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref] [bib_ref] Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 2...., Sibov [/bib_ref] [bib_ref] Influence of dent corn genetic backgrounds on QTL detection for plant-height traits..., Wei [/bib_ref] , which was often hard to match physical map and also too large to locate candidate genes or causal polymorphisms. Small populations are easily leading to sampling bias (such as unintended assessment involved in individuals with extreme phenotype values), thus affecting the estimation of QTL effect and the accuracy of QTL location [bib_ref] Effect of population size on the estimation of QTL: a test using..., Vales [/bib_ref] [bib_ref] Quantitative trait locus mapping based on resampling in a vast maize testcross..., Schön [/bib_ref] , and unfavorable linkage event in small populations would even result in misleading inferences [bib_ref] Lessons from Dwarf8 on the strengths and weaknesses of structured association mapping, Larsson [/bib_ref]. Besides, small populations always have the drawback of limited recombination thus limit the mapping resolution [bib_ref] Quantitative trait locus mapping based on resampling in a vast maize testcross..., Schön [/bib_ref].
Marker density is another key consideration influencing the precision for recombinational breakpoints identification, and would also reduce the mapping resolution. Recent progress in high-throughput sequencing technologies has markedly enhanced and accelerated genotyping. The genotyping-by-sequencing (GBS) strategy, named "bin mapping", has been successfully applied in rice [bib_ref] High-throughput genotyping by whole-genome resequencing, Huang [/bib_ref] , Arabidopsis [bib_ref] Visualizing the genetic landscape of Arabidopsis seed performance, Joosen [/bib_ref] , sorghum [bib_ref] Identification of QTLs for eight agronomically important traits using an ultra-high-density map..., Zou [/bib_ref] , soybean [bib_ref] Pinpointing genes underlying the quantitative trait loci for root-knot nematode resistance in..., Xu [/bib_ref] , and maize [bib_ref] An ultra-high density bin-map for rapid QTL mapping for tassel and ear..., Chen [/bib_ref] [bib_ref] Genetic dissection of maize seedling root system architecture traits using an ultra-high..., Song [/bib_ref] , and re-mapped several genes or QTLs with known functions. However, almost all of these genes or QTLs verified were major effect ones. Minor effect QTLs were less investigated. And most of these studies were based on relatively small populations.
Several QTL mapping studies have been performed using large populations and increased marker density in targeted regions in plant. ZmCCT, an important photoperiod response gene in maize, was cloned by regional genotyping of key recombinants in 866 maize-teosinte BC 2 S 3 lines and analysis of other diverse maize populations [bib_ref] ZmCCT and the genetic basis of day-length adaptation underlying the postdomestication spread..., Hung [/bib_ref]. Two large RIL populations (1285 and 921 lines respectively) were used to localize an important QTL contributing to maize Sugarcane Mosaic Virus (SCMV) resistance, the key recombinants detected by Polymerase Chain Reaction (PCR) based markers could give a result of QTL interval small enough to identify candidate gene [bib_ref] Combined linkage and association mapping reveals candidates for Scmv1, a major locus..., Tao [/bib_ref]. Recent bin mapping was conducted with deeply sequenced 132 RILs in rice, PCR genotyping of QTL intervals of an expanded population size (1709 lines) enabled the fine mapping of QTLs related to flowering time down to candidates including previously cloned gene [bib_ref] Dissecting yield-associated loci in super hybrid rice by resequencing recombinant inbred lines..., Gao [/bib_ref]. These studies implied that combining large population with high density markers can be effective for QTL fine mapping. However, all these studies were performed for targeting local regions on major effect QTLs. Up to now, except the simulation, QTL studies conducted by actual data of large population (> 1000) and high density markers at the same time have not been reported.
In this work,we constructed a RIL population derived from Zhengdan958 (the most widely planted hybrid in China) of 1147 lines, generated a high density marker set using GBS strategy, constructed a bin map, and phenotyped the population in multiple environments. Fifty-one plant architecture related loci with small effect were mapped to relatively small confidence intervals, which led to a list of reasonable candidate genes. One of these QTLs was further verified with introgression lines. Thus we demonstrated a method for high resolution mapping of minor effect QTLs in maize, and the resulted comprehensive QTLs for PATs are valuable for maize molecular breeding in the future.
# Methods
## Plant materials and phenotypic evaluations
The RILs population used here includes 1147 lines, which was derived by crossing of Zheng58 and Chang7-2 (parents of the most widely planted maize hybrid Zheng-dan958 in China recently [bib_ref] Genome-wide patterns of genetic variation among elite maize inbred lines, Lai [/bib_ref] , Zheng58 is a semi-Dent germplasm, which belongs to improved Reid heterotic group. While Chang7-2 is semi-Flint germplasm, which belongs to a Chinese native heterotic group, Tangsipingtou group), followed by single-seed descend process. F 7 plants and the parental lines were phenotyped at three locations in China, Sanyuan (40°05′ N, 116°12′ E, 2010) and Shangzhuang (40°08′ N, 116°11′ E, 2011) in Beijing city, and Xinxiang (35°20′ N, 113°43′ E, 2012) in Henan Province. In Sanyuan, two replications and the randomized complete block design were applied, while for the remaining two environments, an augmented incomplete block design [bib_ref] Genome-wide association study of leaf architecture in the maize nested association mapping..., Tian [/bib_ref] with single replication was performed. Trials were performed with one row plots: row length of 3-m; row spacing of 0.5-m; and 13 plants per plot (80,000 plants per hectare) for all three locations. Traits of silk color (SC), ear height (EH), plant height (PH), tassel branch number (TBN), tassel length (TL), and upper leaf number (ULN), were measured on 10 (Sanyuan) or 5 (Shangzhuang and Xinxiang) plants per plot. PH and EH were collected at all three locations, while TL and TBN were only collected at Sanyuan and Xinxiang. SC and ULN were collected at Xinxiang only. SC was classified into five levels according to the intensity of red pigmentation. EH and PH were investigated as the distance from the node of the uppermost ear and the top of the main tassel spike to the soil surface, respectively. Relative ear height (EH/PH) was calculated as the ratio of EH to PH. TL was recorded as the length of the first branching node to the top of the main tassel spike. TBN was recorded as the number of primary tassel branches, and ULN was recorded as leaf numbers above the upper ear. Best Linear Unbiased Prediction (BLUP) was calculated for each phenotype by the lme4 package [bib_ref] Fitting linear mixed-effects models using lme4, Bates [/bib_ref] in R software across different environments, and used for subsequent analysis. The heritability estimates h 2 were calculated by R software as reported formula [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref].
An ILs population constructed by Chang7-2 as donor parent, Zheng58 as recurrent parent was used for verifying the reliability of qPH1a. In summer of 2011, 256 of BC 3 F 2 or BC 4 F 1 generation of ILs were planted in Shangzhuang in Beijing. Nineteen ILs (BC 4 F 1 ) with remarkably higher PH were picked out and planted in Shangzhuang in summer of 2012. After checked by four Indel markers (Indel_1-87, Indel_1-89, Indel_1-91, Indel_1-97, Additional file 1: that covering qPH1a interval, two of these ILs (BC 5 F 1 ), 9_5 and 10_1, that harboring the qPH1a allele descend from Chang7-2 were found and crossed with Zheng58. In summer of 2013, the progenies (BC 6 F 1 ) of 9_5 and 10_1 were phenotyped and genotyped, and 3 recombinants (BC 6 F 1 ), G36-3, G40-7 and G41-8, were found and crossed with Zheng58 again. In the summer of 2014, progenies (BC 7 F 1 ) of these 3 recombinants were planted in Shangzhuang, followed by measuring PH and genotyping by 4 Indel markers. A method named "recombinant-derived progeny testing strategy" [bib_ref] A sequential quantitative trait locus finemapping strategy using recombinant-derived progeny, Yang [/bib_ref] was invoked for inferring the location of qPH1a.
## Dna isolation, sequencing and snp identification
Shoot tissues from five seedlings per genotype were bulked for total genomic DNA isolation and extraction using the CTAB protocol [bib_ref] Rapid isolation of high molecular weight plant DNA, Murray [/bib_ref].
Zheng58 and Chang7-2 were deeply sequenced using Illumina HiSeq2000 for 28.41 (accession number, NCBI: SRX120186) and 24.95 (accession number, NCBI: SRX120903) fold coverage of the maize genome respectively [bib_ref] Genome-wide genetic changes during modern breeding of maize, Jiao [/bib_ref]. DNA of 1130 RILs was processed according to the previously published GBS method [bib_ref] An ultra-high density bin-map for rapid QTL mapping for tassel and ear..., Chen [/bib_ref] [bib_ref] A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species, Elshire [/bib_ref] , in which ApeKI was used for genomic DNA digestion followed by ligation of the barcode adapter with variable length (see Additional file 1: [fig_ref] Table 1: Summary of the bin map [/fig_ref] and a common adapter. Sets of 240 samples were pooled together and sequenced by Illumina HiSeq2000 platform to generate 100-nt paired-end reads.
SNP identification was conducted using a strict pipeline as reported [bib_ref] Genetic dissection of maize seedling root system architecture traits using an ultra-high..., Song [/bib_ref] , where BWA (version 0.5.9) [bib_ref] Fast and accurate short read alignment with burrowswheeler transform, Li [/bib_ref] were used for read alignment and GATK (version 1.0.4418) were invoked for SNP identification. For the RILs, raw reads were mapped to the position of SNPs retained from the two parents only, which reduced workload of the processing.
To ensure the map quality, only RILs with sequencing depth greater than 0.02× and genotyped SNPs more than 8000 were retained, which excluded 18 RILs from the dataset.
## Bin map construction and genetic properties analysis
The reported sliding window method [bib_ref] High-throughput genotyping by whole-genome resequencing, Huang [/bib_ref] with minor modifications was adopted to conduct genotype calling for each RIL. (1) In each 15-SNPs window, the RIL genotype was defined by the rate of alleles from Zheng58 and Chang7-2: the window was called as homozygous if > 11/15 of the sites in the window were alleles from either of the parents or heterozygous otherwise. Based on the sliding window method, recombination breakpoints are observed as a region of several heterozygous, but do not tend to exceed more than six continuous windows.Therefore, we set heterozygous regions spanning less than seven uninterrupted windows as breakpoints and divided them into two by the midpoint. Then adjacent windows with the same genotype were merged together as a block. (2) Blocks with sequenced SNPs number less than 5 or physical length less than 300 kb were set to missing to avoid false double crossover. (3) Adjacent windows and successive small blocks with frequently transient genotypes were merged into a larger heterozygous block, and heterozygous block with SNP number less than 15 or physical length less than 1 Mb were set as missing to avoid false estimation.
The RIL population was further filtered by excluding the lines with greater than 15% residual heterozygous genotypes or with more than 360 breakpoints (91 RILs), to avoid material confounding, leaving a set of 1021 lines.-Blocks without any breakpoints across the remaining population were grouped into bins, and bins with a physical length less than 5 kb were merged together. The allele frequency (Zheng58, Chang7-2 and heterozygous) of each bin was recorded to investigate instances of distorted segregation. Bins with a distorted segregation ratio larger than 2/1 or a heterozygous ratio larger than 15% were subsequently discarded (about 6% of total bins) for further improvement of the linkage map.QTL mapping software for RIL populations does not generally accept heterozygous loci, so bins with heterozygous genotype were regarded as missing followed by the "argmax" imputation method in R/qtl. Pair-wise recombinational fractions for all bins were calculated by the R/ qtl function est.rf. The R/qtl function est.map ("kosambi" method) was manipulated for linkage map calculation. A genome wide recombination ratio analysis was calculated by (linkage distance)/(physical distance) in 1 Mb windows, and the filtered gene set gene density (B73 maize reference genome V2) and SNP density were recorded at the same time.
# Qtl analysis
The QTL study of the Zhengdan958 RILs was conducted using the R/qtl package as reported [bib_ref] Genetic dissection of maize seedling root system architecture traits using an ultra-high..., Song [/bib_ref]. Briefly, composite interval mapping (CIM) method was used for QTL identification, 1000 permutations test (p < 0.05) were used for defining QTL logarithm (base 10) of odds scores (LOD) threshold, 1.5 LOD-drop method was applied for defining QTL confidence interval, and linear QTL model was used for calculating QTL additive effect and phenotypic variation explained.
# Results
## Sequencing and snp detection
Through deep sequencing of the two parents for above 24× coverage and identifying SNPs by a strict pipeline, 2,231,331 high-quality SNPs were detected between Zheng58 and Chang7-2. Approximately 67% of the SNPs were located in intergenic regions, 8% in exons, 12% in introns, and 14% within 2 kb upstream or downstream of genes (see Additional file 1: .
1130 of the 1147 RILs were sequenced as described above, yielding about 1.8 billion 100-nt reads and an average of 0.08 × sequencing depth for each line (see Additional file 1: . SNP calling generated 655,507 raw SNPs, with an average of 49,640 SNPs for each RIL and a mean of density of 1 SNP per 50 kb. While genotyping the RILs, we also genotyped the two parents using the GBS method for~0.09× to estimate the error rate associated with RILs SNP detection. The error rates of genotyping Zheng58 and Chang7-2 were found to be 0.6% and 0.9%, respectively (see Additional file 1: [fig_ref] Table 2: Comparison of detected QTLs between this study and previous studies [/fig_ref]. We examined these errors in 1 Mb windows and did not find obvious enrichment in any local regions, suggesting they can be easily excluded by the sliding window approach (see Additional file 1: [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref].
## Bin map construction
The modified sliding window method [bib_ref] High-throughput genotyping by whole-genome resequencing, Huang [/bib_ref] was applied to construct the bin map for 1021 of the 1130 sequenced RILs, and 16,769 recombinant bins were identified across the genome, which presumably captures most of recombination events that took place in the process of population construction . The physical size of these bins range from 5 to 5.354 kb, with a mean of 126 kb [fig_ref] Table 1: Summary of the bin map [/fig_ref]. 17 of 31 bins with size of more than 2 Mb were generated around 20 Mb regions flanking centromeres, where were always lacking of recombination. R/qtl [bib_ref] R/QTL: QTL mapping in experimental crosses, Broman [/bib_ref] was adopted for linkage distance calculation, resulted in a genetic map of 2508.11 cM in total. The recombination rate was 1.227 cM/Mb, which was obviously lower than 6.95 cM/Mb of the IBM Syn10 DH population, which experienced ten generations of random mating [bib_ref] An ultra-high-density map as a community resource for discerning the genetic basis..., Liu [/bib_ref].The largest interval between adjacent bins was 6.006 cM, while the average was 0.152 cM, revealing the high resolution of the bin map. Pair-wise recombinational fractions among bins were computed by est.rf in R/qtl and no obvious problems displayed (see Additional file 1: .
The 1021 RILs were produced from 6 generations of self pollination. Overall, theses RILs captured~50,040 crossover events, resulting in an average of 4.9 per chromosome [fig_ref] Table 1: Summary of the bin map [/fig_ref] , ranging from 3.5 for chromosome 10 to 7.9 for chromosome 1. We investigated the recombination ratio (expressed as the average linkage interval size across 1 Mb increments of the genome), the regional frequency of SNPs across the genome (SNP density), and the density of genes based on the filtered gene set (B73 reference genome V2) across the genome (gene density). The recombination ratio across the genome varied tremendously, ranging from 0 to 13.1 cM/ Mb, with a mean of 1.227 cM/Mb, and was significantly correlated with gene density (r = 0.678, p < 2.20 E-16), but not overall SNP density (r = − 0.027, p = 0.248, , Additional file 1: . The recombination ratio and gene density were reduced in regions surrounding centromeres and enriched near both telomeres on each chromosome , consisting with previous reports. Further analysis of the relationship between recombination and sub-regional SNP density, found that recombination ratio was positively correlated with SNP density in genic regions (exon, intron, 2 k-upstream and 2 k-downstream, r = 0.653, 0.503, 0.520, 0.415 respectively), but negatively correlated with intergenic regions (r = − 0.331, Additional file 1: . 149 regions with a 3 fold higher recombination ratio than the genomic average were identified and had an obviously elevated average gene density (37.7 for high recombination region comparing to 19.0 for genomic average) and genic region SNP density (see Additional file 2: . Consisting with previous reports that recombination events were preferentially occurring in gene regions or small multi-genic intervals [bib_ref] Genetic recombination in plants, Schnable [/bib_ref] [bib_ref] Meiotic recombination hotspots in plants, Mezard [/bib_ref] [bib_ref] Recent advances in plant recombination, Li [/bib_ref] [bib_ref] The relationship between genetic and physical distances in the cloned a1-sh2 interval..., Civardi [/bib_ref] [bib_ref] Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters, Choi [/bib_ref]. Segregation distortion was observed in seven chromosomes (Chromosome 1, 2, 5, 6, 8, 9, 10, p < 0.01, Chisquare test, after Bonferroni-correction), including 1710 bins (10.2%), with total length of 155.8 Mb which covered 7.6% of the genome (see Additional file 1: . Two of these regions contained gamete fertility related genes: ms32 (male sterility32) [bib_ref] Regulation of cell divisions and differentiation by MALE STERILITY32 is required for..., Moon [/bib_ref] on chromosome 2 and rf2 (restorer of fertility2)on chromosome 9 , suggesting a possibility that fertility related locus causing the distorted segregation in the RILs.
The bin map quality and the QTL mapping pipeline were firstly tested using the silk color phenotype. The phenotypes were scored into 5 classes according to the intensity of red pigmentation (see Additional file 1: . Totally, 5 QTLs were detected, located in chromosome 1, 4, 6, 9, 10 respectively. The most significant QTL (LOD = 13.76, was located in the interval of 46.10-50.05 Mb on chromosome 1. The bin with the second highest LOD score was just overlapping with P1 (pericarp color1) gene, which is known to contribute to the phlobaphene pigmentation of maize floral organs [bib_ref] The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by..., Grotewold [/bib_ref] [bib_ref] Isolation and molecular analysis of the maize P locus, Lechelt [/bib_ref]. The QTL on chromosome 6 was defined in the interval of 108.071-110.534 Mb with the peak just overlapping with pl1 gene, which was contributed to purple pigment in maize plant [bib_ref] Pl-bol3, a complex allele of the anthocyanin regulatory pl1 locus that arose..., Pilu [/bib_ref] [bib_ref] Allelic interactions heritably alter the activity of a metastable maize pl allele, Hollick [/bib_ref]. The chromosome 10 located QTL was in the interval of 137.325-139.207 Mb, with a bin with the second highest LOD score just encompassing r1 gene, which was known contributed to silk color variation in maize [bib_ref] An ultra-high density bin-map for rapid QTL mapping for tassel and ear..., Chen [/bib_ref] [bib_ref] Genome-wide genetic changes during modern breeding of maize, Jiao [/bib_ref]. The other two QTLs may harbor genes with unknown function responsible for silk color controlling in maize. Mapping of the well studied P1, pl1 and r1 to relative small confidence intervals (3.950 Mb for P1, 2.463 Mb for pl1 and 1.882 Mb for r1) demonstrated our map quality.
## Qtl detection for six plant architecture related traits
The parents and RILs were phenotyped for traits of PH, EH, EH/PH, TBN, TL, and ULN. Zheng58 and Chang7-2 had large differences for all of these phenotypes, except for ULN. Transgressive segregation was observed for six PATs in the RILs population (see Additional file 1: . Traits correlation coefficients were calculated (see Additional file 1: [fig_ref] Table S5: Pearson correlation coefficients among different traits in two environments [/fig_ref]. As expected, PH and EH (r = 0.755), along with EH and EH/PH (r = 0.667) were strongly correlated, while a modest correlation was observed between PH and TL (r = 0.420), and ULN and EH/PH (r = − 0.360). The heritability estimates of PH, EH, EH/PH, TBN, and TL were high with all being greater than 0.7 (see Additional file 1: .
Totally, 51 plant architecture related QTLs scattering across 10 maize chromosomes were detected above the threshold (p < 0.05, using 1000 times permutations), including 8 for PH, 9 for EH, 8 for EH/PH, 14 for TL, 6 for TBN, and 6 for ULN [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref] , Additional file 1: . The genotype profile. a The graphic genotype of 1021 RILs. Red, Zheng58 genotype; Blue, Chang7-2 genotype.Heterozygouse genotypes were set as missing data and imputed by "argmax" method in R/qtl package. b Recombination ratio (outer, pink), gene density (middle, blue) and SNPs density (inner, green) distribution across the ten chromosomes. c, d The distribution of segregation distortions in chromosome 2 and 9. Segregation distortion was test by Chi-test and -log10 (P Chi-test ) were plotted against its physical position. Candidate genes ms32, rf2 were pointed out. Threshold of no distortion (p < 0.01, after Bonferroni-correction) were showed as red dashed lines All of these QTLs were minor effect QTL, with explained phenotypic variation varied from 0.05% to 9.15%, a median of 2.01%. Seven chromosome regions, ranging in size from 2.12 to 9.53 Mb, contained QTLs (17/51 QTLs) for more than one traits, they were 86. [bib_ref] Genetic dissection of maize seedling root system architecture traits using an ultra-high..., Song [/bib_ref] Benefited from large sample size and saturated bin markers, most of the detected QTLs were defined into relatively small confidence intervals. Among the 51 QTLs, 32 were defined in intervals less than 5 Mb and 8 were located in less than 2 Mb, with a minimum of 1.03 Mb and a median interval size of 3.40 Mb for all QTLs (see Additional file 1: . Twenty-two of our QTLs overlapped with those identified in previously studies [fig_ref] Table 2: Comparison of detected QTLs between this study and previous studies [/fig_ref] , but all with intervals narrower in our study, which ranged from 1.04 to 9.72 Mb compared with other reports of intervals of 3 to 133 Mb [bib_ref] Quantitative trait loci analysis of phenotypic traits and principal components of maize..., Upadyayula [/bib_ref] [bib_ref] Direct mapping of density response in a population of B73 x Mo17..., Gonzalo [/bib_ref] [bib_ref] Genetic analysis of agronomic traits associated with plant architecture by QTL mapping..., Zheng [/bib_ref] [bib_ref] Genetic dissection of plant height by molecular markers using a population of..., Tang [/bib_ref] [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref] [bib_ref] Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 2...., Sibov [/bib_ref] [bib_ref] Influence of dent corn genetic backgrounds on QTL detection for plant-height traits..., Wei [/bib_ref] [bib_ref] Linkage mapping of domestication loci in a large maize teosinte backcross resource, Briggs [/bib_ref] [bib_ref] Genetic dissection of maize phenology using an intraspecific introgression library, Salvi [/bib_ref] [bib_ref] Dissection of the genetic architecture underlying the plant density response by mapping..., Ku [/bib_ref] [bib_ref] QTL mapping for maize resistance and yield under infestation with Sesamia nonagrioides, Samayoa [/bib_ref] [bib_ref] Genetic mapping and QTL analysis for yield and agronomic traits with an..., Park [/bib_ref] [bib_ref] Cytonuclear epistatic quantitative trait locus mapping for plant height and ear height..., Tang [/bib_ref] [bib_ref] Quantitative trait locus analysis for ear height in maize based on a..., Li [/bib_ref] [bib_ref] Quantitative trait loci for yield and morphological traits in maize under drought..., Nikolic [/bib_ref] [bib_ref] Mapping QTL for grain yield and plant traits in a tropical maize..., Lima [/bib_ref] [bib_ref] Stability of QTL across environments and QTL-by-environment interactions for plant and ear..., Zhang [/bib_ref] [bib_ref] QTL mapping of five agronomic traits in maize, Tang [/bib_ref]. Six of them were overlapped with results from the large maize-teosinte backcross population, which had 1749 progenies but limited markers [bib_ref] Linkage mapping of domestication loci in a large maize teosinte backcross resource, Briggs [/bib_ref] , with intervals ranging from 2.59 to 6.63 Mb in our RILs compared with corresponding intervals of 8-94 Mb. Four regions with closely linked QTLs for the same trait were determined (qULN3a and qULN3b, qEP3a and qEP3b, qTL1c and qTL1d, qTL8a, qTL8b and qTL8c), as adjacent two peaks were less than 25 Mb apart [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref] , Additional file 1: , but could be clearly distinguished due to the superior resolution of our panel.
# Candidate genes analysis
With the aid of high resolution bin map and large RIL population, many QTLs were located to relatively small Five silk color QTLs and three putative candidate genes. The silk color QTLs located on chromosomes 1, 4, 6, 9 and 10 were plotted. The positions of candidate genes P1, pl1 and r1 were indicated by vertical dashed lines genomic intervals that included genes with known function [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref]. For TBN, the QTL on chromosome 7 (qTBN7, LOD = 4.93, explained 7.78% phenotype variance) was located in an interval encompassing ra1 (ramosa1) [bib_ref] Architecture of floral branch systems in maize and related grasses, Vollbrecht [/bib_ref] , an important gene known for maize floral organ branching. For PH and EH/PH, the QTLs (qPH3a and qEP3a, LOD = 6.74 and 5.14, 2.20% and 5.03% variance explained for PH and EH/PH respectively) in a~3 Mb region of chromosome 3 includes na1 (nana plant1) [bib_ref] Brassinosteroid control of sex determination in maize, Hartwig [/bib_ref] , an important gene in the brassinosteroid biosynthetic pathway that influences lower internodes length and affects maize height. The PH QTL on chromosome 5 (qPH5, LOD = 7.56, 3.05% variance explained) co-localized with td1, which was involved in CLAVATA signaling pathway and harboring known impact on plant height variation in maize [bib_ref] Thick tassel dwarf1 encodes a putative maize ortholog of the Arabidopsis CLAVATA1..., Bommert [/bib_ref]. The EH/PH QTL on chromosome 9 (qEP9a, LOD = 4.72, 1.72% variance expected) encompassing d3, a gene controls cytochrome P450-mediated biosynthesis of gibberellins that involved in maize dwarfing phenotype [bib_ref] Transposon-tagging of the dwarf3 gene, which controls a cytochrome P450-mediated step early..., Winkler [/bib_ref]. A TL QTL peak on chromosome 2 (qTL2a, LOD = 4.83, 1.99% variance expected) just overlapped with zfl2, which controls inflorescence development and architecture in maize [bib_ref] Pleiotropic effects of the duplicate maize FLORICAULA/LEAFY genes zfl1 and zfl2 on..., Bomblies [/bib_ref]. An EH/PH QTL (qEP1, LOD = 11.80, 3.92% variance expected) in a 3.9 Mb region of chromosome 1 contains br1 (brachytic1, http://www.maizegdb.org) [bib_ref] Quantitative trait loci for plant height in four maize populations and their..., Beavis [/bib_ref] , which has been reported to be associated with dwarfing as well. Another EH/PH QTL (qEP2, LOD = 3.88, 0.95% variance expected) in chromosome 2 co-localized with d5 locus (dwarf plant5, http://www.maizegdb.org) [bib_ref] Genetic control of plant morphogenesis, Khavkin [/bib_ref] , another important maize dwarf mutant.
In addition to genes that had been mapped or cloned in maize, several candidate genes inferred from homologues were also identified in our QTL intervals [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref]. The largest ULN QTL, located on a tip of chromosome 2 harbored zap1, shown to be allelic to ZmMADS3, a transcription factor that influences maize internode number [bib_ref] The maize MADS box gene ZmMADS3 affects node number and spikelet development..., Heuer [/bib_ref]. The EH and EH/PH co-localized QTL on chromosome 3, encompasses myb151 which is homologous to Arabidopsis AtMYB87, the latter could suppress longitudinal elongation of multiple organs [bib_ref] Chimeric repressor analysis identifies MYB87 as a possible regulator of morphogenesis via..., Fujiwara [/bib_ref] that may contribute to plant dwarfing. The TBN QTL interval on chromosome 10 includes a gene, sbp21, homologous to Arabidopsis AtSPL10, which controls shoot-associated lateral organs development [bib_ref] Arabidopsis SBP-box genes SPL10, SPL11 and SPL2 control morphological change in association..., Shikata [/bib_ref] , may contribute to maize tassel branching. The QTL for PH that localized on chromosome 6, harbors a gene encoding a gibberellin 2-oxidase, which had been reported to participate in the pathway of gibberellin metabolism and may contribute to maize dwarfing [bib_ref] Green revolution: a mutant gibberellin-synthesis gene in rice, Sasaki [/bib_ref]. Since the ultimate goal of QTL study is to probe causal genes or polymorphisms, the ability to map loci with a narrow confidence interval containing candidates indicated the strong power of the approach used in this study.
## Verification of qph1a by introgression lines
A population of ILs constructed by Chang7-2 as donor parent, Zheng58 as recurrent parent was used for verifying the reliability of qPH1a. In the summer of 2012, two ILs, named 9_5 and 10_1 (BC 5 F 1 ), with heterozygous genotypes in the qPH1a region were chosen and backcrossed with Zheng58. In the summer of 2013, 132 and 113 progenies (BC 6 F 1 generation ILs) of 9_5 and 10_1, were planted respectively, followed by measuring plant height and genotyping by 4 Indel markers (see Additional file 1: that covering qPH1a interval. The progenies of these two ILs were segregated into two types of genotypes, heterozygous and homozygous Zheng58. The mean plant height of these two types of progenies (with genotype of homozygous Zheng58 or heterozygous) were significantly different (t-test, p-value = 0.005 and 0.007, , indicating that qPH1a was reliably located around its mapped interval on the bin map , Chr1: 90. Mb). In the summer of 2014, three recombinants (BC 6 F 1 ), named G36-3, G40-7 and G41-8, in the progenies of 9_5 and 10_1 were picked out and tested by the same method. G36-3 harbored heterozygous genotype upstream of Indel_91 and homozygous genotype downstream in the qPH1a region, while G40-7 and G41-8 just the opposite . Analysis of progenies derive from G36-3 revealed that mean plant height of these two types of progenies corresponding to two segregated genotypes that downstream of Indel_91 (91.18 Mb) was significantly different (t-test, p-value = 0.006). Meanwhile, plant height of two types of progenies corresponding to two segregated genotypes upstream of Indel_91 was non-different (t-test, p-value = 0.535 and 0.494 respectively) as revealed by progenies of G40-7 and G41-8. For QTLs would only segregate in the heterozygous regions, thus qPH1a was further confirmed to be located downstream of 91.18 Mb, given its further interval of 91.18-95. [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref] Mb on chromosome 1.
# Discussion
Accurately mapping of minor effect QTLs by combining large population and super-high density bin map Large amount of resources had been used to study agronomic QTLs in staple crops, however, relatively few underlying genes and regulatory elements have been identified. Previous reports revealed that enlarging population size could effectively enhance the efficiency of QTL mapping, and reduce the false-positive rate caused by small populations [bib_ref] Effect of population size on the estimation of QTL: a test using..., Vales [/bib_ref] [bib_ref] Quantitative trait locus mapping based on resampling in a vast maize testcross..., Schön [/bib_ref]. Besides, the utility of high-density markers was another efficient way to improve the QTL mapping resolution [bib_ref] High-throughput genotyping by whole-genome resequencing, Huang [/bib_ref]. However, for precisely location of minor effect QTLs, large population or high density marker alone is not enough [bib_ref] Linkage mapping of domestication loci in a large maize teosinte backcross resource, Briggs [/bib_ref] [bib_ref] Genetic dissection of maize seedling root system architecture traits using an ultra-high..., Song [/bib_ref]. Here, combining a panel of > 1000 RILs and GBS-based high density markers resulted in a bin map with an average physical / linkage interval of 126 kb / 0.152 cM [fig_ref] Table 1: Summary of the bin map [/fig_ref]. This resolution is greatly improved than previously linkage maps constructed with traditional molecular markers in maize which ranged from 7 to 24 cM for the average marker intervals [bib_ref] QTL mapping in testcrosses of European flint lines of maize: I. Comparison..., Lübberstedt [/bib_ref] [bib_ref] Quantitative trait locus (QTL) mapping using different testers and independent population samples..., Melchinger [/bib_ref] [bib_ref] Quantitative trait loci analysis of phenotypic traits and principal components of maize..., Upadyayula [/bib_ref] [bib_ref] Direct mapping of density response in a population of B73 x Mo17..., Gonzalo [/bib_ref] [bib_ref] Genetic analysis of agronomic traits associated with plant architecture by QTL mapping..., Zheng [/bib_ref] [bib_ref] Genetic dissection of plant height by molecular markers using a population of..., Tang [/bib_ref] [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref] [bib_ref] Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 2...., Sibov [/bib_ref] [bib_ref] Influence of dent corn genetic backgrounds on QTL detection for plant-height traits..., Wei [/bib_ref]. Among the QTLs detected, 63% (32 of 51) were defined in intervals less than 5 Mb, 8 were even located in less than 2 Mb, with a median interval size of 3.40 Mb. These intervals identified were relatively small for minor effect QTLs [bib_ref] Quantitative trait loci analysis of phenotypic traits and principal components of maize..., Upadyayula [/bib_ref] [bib_ref] Direct mapping of density response in a population of B73 x Mo17..., Gonzalo [/bib_ref] [bib_ref] Genetic analysis of agronomic traits associated with plant architecture by QTL mapping..., Zheng [/bib_ref] [bib_ref] Genetic dissection of plant height by molecular markers using a population of..., Tang [/bib_ref] [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref] [bib_ref] Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 2...., Sibov [/bib_ref] [bib_ref] Influence of dent corn genetic backgrounds on QTL detection for plant-height traits..., Wei [/bib_ref] [bib_ref] Linkage mapping of domestication loci in a large maize teosinte backcross resource, Briggs [/bib_ref] [bib_ref] Genetic dissection of maize phenology using an intraspecific introgression library, Salvi [/bib_ref] [bib_ref] Dissection of the genetic architecture underlying the plant density response by mapping..., Ku [/bib_ref] [bib_ref] QTL mapping for maize resistance and yield under infestation with Sesamia nonagrioides, Samayoa [/bib_ref] [bib_ref] Genetic mapping and QTL analysis for yield and agronomic traits with an..., Park [/bib_ref] [bib_ref] Cytonuclear epistatic quantitative trait locus mapping for plant height and ear height..., Tang [/bib_ref] [bib_ref] Quantitative trait locus analysis for ear height in maize based on a..., Li [/bib_ref] [bib_ref] Quantitative trait loci for yield and morphological traits in maize under drought..., Nikolic [/bib_ref] [bib_ref] Mapping QTL for grain yield and plant traits in a tropical maize..., Lima [/bib_ref] [bib_ref] Stability of QTL across environments and QTL-by-environment interactions for plant and ear..., Zhang [/bib_ref] [bib_ref] QTL mapping of five agronomic traits in maize, Tang [/bib_ref]. Comparing with overlapped QTLs obtained in other studies using small population sizes or sparse marker coverage found that, QTL intervals detected here were narrowed by 1.1 to 45.7 fold [fig_ref] Table 2: Comparison of detected QTLs between this study and previous studies [/fig_ref] [bib_ref] Quantitative trait loci analysis of phenotypic traits and principal components of maize..., Upadyayula [/bib_ref] [bib_ref] Direct mapping of density response in a population of B73 x Mo17..., Gonzalo [/bib_ref] [bib_ref] Genetic analysis of agronomic traits associated with plant architecture by QTL mapping..., Zheng [/bib_ref] [bib_ref] Genetic dissection of plant height by molecular markers using a population of..., Tang [/bib_ref] [bib_ref] Genetic and QTL analysis of maize tassel and ear inflorescence architecture, Upadyayula [/bib_ref] [bib_ref] Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 2...., Sibov [/bib_ref] [bib_ref] Influence of dent corn genetic backgrounds on QTL detection for plant-height traits..., Wei [/bib_ref] [bib_ref] Linkage mapping of domestication loci in a large maize teosinte backcross resource, Briggs [/bib_ref] [bib_ref] Genetic dissection of maize phenology using an intraspecific introgression library, Salvi [/bib_ref] [bib_ref] Dissection of the genetic architecture underlying the plant density response by mapping..., Ku [/bib_ref] [bib_ref] QTL mapping for maize resistance and yield under infestation with Sesamia nonagrioides, Samayoa [/bib_ref] [bib_ref] Genetic mapping and QTL analysis for yield and agronomic traits with an..., Park [/bib_ref] [bib_ref] Cytonuclear epistatic quantitative trait locus mapping for plant height and ear height..., Tang [/bib_ref] [bib_ref] Quantitative trait locus analysis for ear height in maize based on a..., Li [/bib_ref] [bib_ref] Quantitative trait loci for yield and morphological traits in maize under drought..., Nikolic [/bib_ref] [bib_ref] Mapping QTL for grain yield and plant traits in a tropical maize..., Lima [/bib_ref] [bib_ref] Stability of QTL across environments and QTL-by-environment interactions for plant and ear..., Zhang [/bib_ref] [bib_ref] QTL mapping of five agronomic traits in maize, Tang [/bib_ref].
Closely linked loci in coupling phase or repulsion phase can cause misevaluation or misdetection of QTLs, which is a major challenge in quantitative trait studies. In our study, there were 4 regions containing closely linked QTLs for the same trait (as adjacent two peaks were less than 25 Mb apart) [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref] , Additional file 1: , but could be clearly distinguished between them due to the high marker density and large population. On chromosome 3, two closely linked QTLs for ULN with different effects (qULN3a and qULN3b) were identified in the region where only a single QTL were detected by Salvi et al. [bib_ref] Genetic dissection of maize phenology using an intraspecific introgression library, Salvi [/bib_ref] [fig_ref] Table 2: Comparison of detected QTLs between this study and previous studies [/fig_ref]. These results clearly demonstrate the power of combining large population and high density markers.
Besides the increased resolution, several genes with known function were located in overlapping or close neighboring QTL peaks. For silk color, the QTL peak in chromosome 6 was just overlapping with pl1, and QTLs in chromosome 1 and 10 was mapped to P1 and r1 that both overlapping with bins with the second highest LOD scores. For agronomic traits, which were more complex than silk color, our data also has outstanding powers, as suggested by the QTL peak of qTL2a overlapped with zfl2, peak of qEP3a and qEP9a located just 23.9 and 53.6 kb away from na1 and d3 respectively. This indicated that even minor effect loci for a quantitative trait could be fine mapped. Verification of plant height QTL, qPH1a. Two BC 5 F 1 ILs and three BC 6 F 1 recombinants were genotyped by four Indels markers: Yellow, heterozygous; Green, Zheng58. The heterozygous segment of each ILs would segregate into two types of genotypes in its Zheng58-crossed progenies: homozygous Zheng58 and heterozygous. A simple t-test was performed to test PH difference between these genotypes. If significant difference (p < 0.05) was observed then qPH1a should be localized in the heterozygous region, else in the homozygous region. Finally, qPH1a was defined downstream of 91.18 Mb in chromosome 1. a Physical position for Indels. Rec. number of recombinants found in these progenies
In summary, we have demonstrated that high resolution QTL mapping can be achieved using a large RIL population together with super high density markers. However, it is worth to note that there are still some limitations if only one bi-parental population is used. For example, crop genomes like maize are often high diverse, such that two parental lines can only capture small part of overall genome diversity. For any given one bi-parental population, many QTLs will likely be missed due to lack of polymorphism between the two parental lines. Therefore, for any specific traits, it is very important to consider mapping QTLs with population of multiple parental lines.
## Minor effect qtls are important for modern maize breeding
The breeding of maize is the process of pyramiding favorable alleles. It's known that important genes or major QTLs for agronomic trait, such as tb1 [bib_ref] The evolution of apical dominance in maize, Doebley [/bib_ref] , tga1 [bib_ref] The origin of the naked grains of maize, Wang [/bib_ref] , were selected and even potentially fixed in the process of maize domestication or early breeding. Thus the subsequent commercial breeding may rely more on minor effect QTLs. The minor effect QTLs mapped for flowering time [bib_ref] The genetic architecture of maize flowering time, Buckler [/bib_ref] , leaf architecture [bib_ref] Genome-wide association study of leaf architecture in the maize nested association mapping..., Tian [/bib_ref] , inflorescence architecture [bib_ref] Distinct genetic architectures for male and female inflorescence traits of maize, Brown [/bib_ref] and height related traits [bib_ref] The genetic architecture of maize height, Peiffer [/bib_ref] in NAM population are good examples. In this study, by combining large population and high-density bin map, we mapped a list of QTLs for PATs, with all of them having minor effects. These QTLs were segregating between two parental lines of the widely adopted Chinese maize hybrid, suggesting that parental lines of modern commercial hybrid can often differ in minor effect QTLs with majority of major effect QTLs fixed.
Meanwhile, our results indicated that several genes for plant architecture are likely candidate genes, such as an1, td1, d3, and ra1. The mutation of these genes would cause extreme change in corresponding phenotypes, but were defined as minor effect QTLs in our results. It was reported that mild alleles with mutations in gene body (such as qph1 [bib_ref] A rare SNP mutation in Brachytic2 moderately reduces plant height and increases..., Xing [/bib_ref] or regulatory region (such as OsLG1 [bib_ref] OsLG1 regulates a closed panicle trait in domesticated rice, Ishii [/bib_ref] of important genes in maize and rice could cause QTL-like minor effect for the same phenotype and could be practically used in breeding. The minor effect QTLs identified in this study probably act in the same way and could be crucially useful for breeding, even though their exactly causal variations are still unknown.
Additionally, Zhengdan958 is the most widely used maize hybrid in China. Their two parental lines, Zheng58 and Chang7-2, are the top breeding materials in our current breeding system. Zheng58, an improved Reid germplasm, is derived from 5003, which was derived from an elite hybrid from the US and have being the most important dwarf germplasm in China. Chang7-2 is a classic native inbred from which more than 200 lines were derived and many of these lines were utilized as parental lines of popular hybrid varieties used in China [bib_ref] The maize inbred line Chang7-2, Cui [/bib_ref]. We believe that the QTLs identified from these two top elite lines, which were derived from the US and native Chinese germplasm respectively, would be of great value for maize breeding in China and even other regions of the world in the future.
# Conclusions
In this study, by combining large population and highdensity bin map, we mapped a list of minor effect QTLs for PATs in maize. The interval sizes of these QTLs were greatly reduced as compared with previous studies using smaller size of population or smaller number of markers. A number of well studied genes with similar function were located in these QTL intervals, and a plant height QTL were further confirmed by an introgression lines. These QTLs represent a set of reliable resource for maize molecular breeding in the future, and the method used here demonstrated the power of combining large population with high-density markers for minor effect QTL mapping.
## Additional files
Additional file 1: . Distribution of SNPs polymorphic between Zheng58 and Chang7-2 in different genomic regions. . Sequencing depth profile of Zhengdan958 RILs. [fig_ref] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits [/fig_ref]. Distribution of false GBS SNPs in 1 Mb windows by parent. . Pair-wise recombinational fractions (upper left) and LOD scores (lower right) of the bins. . The distribution of segregation distortions across ten chromosomes.Segregation distortions were tested by Chi-test, and -log 10 (P Chi-test ) were plotted against their physical positions. Threshold of no distortion (p < 0.01, after Bonferronicorrection) were showed as red dashed lines. . The 5 classes of silk color. . Phenotypic distributions of PH, EH, EH/PH, TNB, TL and ULN. [fig_ref] Table 1: Summary of the bin map [/fig_ref]. The 240 barcodes used in GBS. [fig_ref] Table 2: Comparison of detected QTLs between this study and previous studies [/fig_ref]. GBS error rate for parental lines. . Pair-wise Pearson's correlation coefficients (lower left) and p-values (upper right) among recombination, gene density, and SNP density in 1 Mb intervals by genomic region. . Regions with 3 fold higher recombinant ratio than genome average. D_Downstream, D_Exon, D_Intron, D_Upstream, D_Intergenic: SNP density in 2 k-downstream, exon, intron, 2 k-upstream of genic regions and SNP density in intergenic regions.
## Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Authors' contributions BW, ZL, WS, and JL conceived and designed the experiments. WS, ZC, BW and ZL constructed the RILs population. BW, ZL, JG, WL, JC (Jing Chen), CG, YZ and XZ performed DNA isolation and phenotype data collecting. HL and JC (Jian Chen) performed sequence library construction, BW and XD analyzed the data. BW, AH and JL wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate Not applicable
## Consent for publication not applicable
## Competing interests
The authors declare that they have no competing interests.
[fig] Figure 3: Fifty-one minor effect QTLs for 6 plant architecture traits. The ten chromosomes were displayed as grey bars according to the physical map, unit: Mb. Segments next to the chromosomes with different colors represented different QTLs for traits listed in the legend; Length of segments represented physical intervals of corresponding QTLs. Candidate genes were pointed out by short red dashed lines; red characters: reported mutants; blue characters: candidate genes inferred from homologues; black characters: genes with known function [/fig]
[table] Table 1: Summary of the bin map [/table]
[table] Table 2: Comparison of detected QTLs between this study and previous studies [/table]
[table] Table S5: Pearson correlation coefficients among different traits in two environments.Lower left, the correlation coefficients; Upper right, p-values of correlation test. Table S6. Heritability of different traits. Table S7. Summary of mapped QTLs. Table S8. Markers used for verifying qPH1a. (PDF 934 kb) [/table]
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COVID-19 length of hospital stay: a systematic review and data synthesis
Background: The COVID-19 pandemic has placed an unprecedented strain on health systems, with rapidly increasing demand for healthcare in hospitals and intensive care units (ICUs) worldwide. As the pandemic escalates, determining the resulting needs for healthcare resources (beds, staff, equipment) has become a key priority for many countries. Projecting future demand requires estimates of how long patients with COVID-19 need different levels of hospital care. Methods: We performed a systematic review of early evidence on length of stay (LoS) of patients with COVID-19 in hospital and in ICU. We subsequently developed a method to generate LoS distributions which combines summary statistics reported in multiple studies, accounting for differences in sample sizes. Applying this approach, we provide distributions for total hospital and ICU LoS from studies in China and elsewhere, for use by the community. Results: We identified 52 studies, the majority from China (46/52). Median hospital LoS ranged from 4 to 53 days within China, and 4 to 21 days outside of China, across 45 studies. ICU LoS was reported by eight studies-four each within and outside China-with median values ranging from 6 to 12 and 4 to 19 days, respectively. Our summary distributions have a median hospital LoS of 14 (IQR 10-19) days for China, compared with 5 (IQR 3-9) days outside of China. For ICU, the summary distributions are more similar (median (IQR) of 8 (5-13) days for China and 7 (4-11) days outside of China). There was a visible difference by discharge status, with patients who were discharged alive having longer LoS than those who died during their admission, but no trend associated with study date. Conclusion: Patients with COVID-19 in China appeared to remain in hospital for longer than elsewhere. This may be explained by differences in criteria for admission and discharge between countries, and different timing within the pandemic. In the absence of local data, the combined summary LoS distributions provided here can be used to model bed demands for contingency planning and then updated, with the novel method presented here, as more studies with aggregated statistics emerge outside China.
# Background
As of April 28, 2020, there have been over 3 million confirmed cases of COVID-19 and more than 200,000 deaths across 185 countries and territories. Health systems are challenged by the influx of patients as SARS-CoV-2, the pathogen causing COVID-19, has spread throughout the world since its emergence in late December 2019 [bib_ref] Critical care crisis and some recommendations during the COVID-19 epidemic in China, Xie [/bib_ref] [bib_ref] Intensive care during the coronavirus epidemic, Qiu [/bib_ref] [bib_ref] COVID-19 and Italy: what next?, Remuzzi [/bib_ref] [bib_ref] On the front lines of coronavirus: the Italian response to covid-19, Paterlini [/bib_ref] [bib_ref] The resilience of the Spanish health system against the COVID-19 pandemic, Legido-Quigley [/bib_ref]. The risks of healthcare services being overwhelmed were most dramatically illustrated in Italy, where a rapid increase of COVID-19 cases needing hospitalisation pushed a well-equipped health system of 3.2 hospital beds per 1000 people to breaking point [bib_ref] Facing Covid-19 in Italy?ethics, logistics, and therapeutics on the epidemic's front line, Rosenbaum [/bib_ref]. This raises serious concerns over the potential impact on more resource-constrained health systems in low-and middleincome countries (LMICs) as epidemics begin to expand across Africa and South America.
Understanding and predicting hospital bed demand (as well as associated staff or equipment requirements) provide crucial evidence for decision-making and contingency planning [bib_ref] Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis, Rodriguez-Morales [/bib_ref]. Predicting demand for hospital services requires an estimate of the number of patients requiring hospitalisation and an estimate of how long each person will require hospital care. It is possible to model the rate of hospitalisation in many settings based on estimated epidemic curves. However, estimating length of stay (LoS) in hospitals requires observation of individual patient pathways.
COVID-19 presents at varying levels of severity. Hospital care can vary from general ward-based care to high dependency units with oxygen support to intensive care where patients may be intubated for mechanical ventilation [bib_ref] Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis, Rodriguez-Morales [/bib_ref] [bib_ref] Review of the clinical characteristics of coronavirus disease 2019 (COVID-19), Jiang [/bib_ref]. The LoS is likely to depend on the level of care required, as well as the geographic setting due to varying COVID-19 care guidelines. For example, some hospitals in China were initially used as isolation settings [bib_ref] Fangcang shelter hospitals: a novel concept for responding to public health emergencies, Chen [/bib_ref] [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref]. As knowledge of effective treatments changes, the pathways, staff, beds, and equipment required are also likely to affect the duration and level of care needed. Moreover, patient characteristics-such as age and comorbiditiesimpact disease severity [bib_ref] Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis, Rodriguez-Morales [/bib_ref] [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref] [bib_ref] Prevalence of comorbidities and its effects in coronavirus disease 2019 patients: a..., Yang [/bib_ref] and are likely to influence LoS. If differences are significant, then capacity planning may need to account for these characteristics to provide accurate predictions of the number of beds required at each level of care. Modelling studies predicting bed occupancy published so far have broadly relied on very few sources of information for LoS estimates, which were often derived from very different settings [bib_ref] Locally informed simulation to predict hospital capacity needs during the COVID-19 pandemic, Weissman [/bib_ref] [bib_ref] Projecting hospital utilization during the COVID-19 outbreaks in the United States, Moghadas [/bib_ref] [bib_ref] Projecting demand for critical care beds during COVID-19 outbreaks in Canada, Shoukat [/bib_ref]. Estimates for LoS can be obtained from a variety of studies, but are often an incidental result rather than a study's primary outcome, and typically, only summary statistics are reported. In general, LoS distributions are right-skewed due to a minority of patients with long hospital stays and are often modelled using gamma, log-normal, or Weibull distributions [bib_ref] Fitting the distributions of length of stay by parametric models, Marazzi [/bib_ref] (although log-normal is less preferred due to its heavier tails). A particular challenge is how to synthesise appropriate LoS distributions from a range of relevant sources in similar settings, capturing the variation both within and between them. Incorporating the uncertainty and stochasticity in parameters using a distribution, rather than fixed point estimates (such as the mean over all studies), allows for more realistic model predictions.
We performed a systematic review to identify the current evidence on LoS for COVID-19 patients worldwide. We also present a method for generating LoS summary distributions by combining information from different summary statistics (mean and medians) reported in multiple studies, and accounting for differences in sample sizes. This aims to include all the variation between studies, to obtain a distribution that covers all plausible LoSs. Although similar in the sense of synthesising multiple sources, this is unlike a classic meta-analysis which aims to get a more precise estimate of a quantity assumed as being a fixed point value. In doing this work, we aim to inform the efforts of modellers and policy makers to better anticipate healthcare needs during the evolving COVID-19 pandemic.
# Methods
## Search strategy
This study was conducted following the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. We searched the bibliographic databases Embase and Medline, as well as the online pre-print archive medRxiv. The latter was expected to be an important source due to the current rapid development of this field; hence, the fully published literature would capture only a small proportion of the available information. We included articles published up to 12 April 2020 that reported a LoS for COVID-19 patients admitted to hospital. To ensure all relevant papers were captured, we examined the title, abstract, and keywords of known studies reporting LoS to identify relevant search terms. Our search combined the concepts of COVID-19 (coronavirus, COVID-19, 2019-nCov, and SARS-CoV-2) with search terms related to duration of hospital stay (length of stay, admission duration, admission length, hospital*). The search terms for hospital stay length were kept broad to capture studies that report LoS as a secondary outcome. The full search terms for Embase, Medline, and medRxiv are presented in the supplementary materials. In addition to our systematic searches, we also checked situation reports from the following organisations to see if they reported LoS estimates: UK Intensive Care National Audit and Research Centre (ICNARC), International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC), World Health Organization (WHO), the US Centers for Disease Control and Prevention (CDC), and China CDC and European CDC (ECDC).
## Inclusion and exclusion criteria
Inclusion criteria are as follows:
1 Studies that reported LoS in hospital for individuals who were admitted for confirmed COVID-19, or suspected COVID-19 which was later confirmed 2 Published (either as a pre-print or publication) between 1 January 2020 and 12 April 2020
Exclusion criteria are as follows:
1 Studies were excluded if LoS was reported for individuals only admitted to hospital for a reason other than confirmed or suspected COVID-19 2 Studies where the LoS endpoint was not death or discharge or continuing stay, for example, transfer to another hospital 3 Studies which stated that hospitalisation was used as a form of isolation 4 Studies not published in English 5 Review articles
## Screening
The screening process is summarised in [fig_ref] Figure 1: PRISMA diagram showing the results of the screening process used to identify... [/fig_ref]. All titles and abstracts were screened independently by two reviewers (EMR and SRP). Subsequently, abstracts and full texts of potentially relevant papers were independently reviewed by two reviewers (EMR and YJ).
## Data extraction and analysis
The data that was extracted from each study is presented in Supplementary Table A. Data extraction was performed by EMR, YJ, and ESN and then verified by a second member of the study team. Study characteristics (such as study dates, study population, and study design) were recorded from each study, including information on the LoS sample estimate for both total hospital LoS and intensive care unit (ICU) LoS, as well as sample size, discharge status, and completeness of follow-up. If multiple LoS estimates were reported for different study populations, these were all recorded (for example, LoS reported by disease severity, comorbidities, and treatment groups). One study specified non-ICU LoS, and this was grouped with total LoS estimates; ICU LoS was reported separately. Average patient age and sex distribution (% male) were summarised across all studies by weighted mean and standard deviation (mean (SD)), according to study sample size. Where possible, LoS estimates were recorded as median and interquartile range (IQR). Otherwise, mean and SD, or in some cases, only a point estimate was provided.
An assessment of bias was considered in these studies. Reported LoS in the majority of studies was not the primary outcome; therefore, a formal quality appraisal was not considered appropriate. Instead, we considered biases which may directly affect reported LoS. Studies were assessed as to whether follow-up had been completed for all included patients, such that an outcome of discharge or death had been observed for all and none remained in hospital at the end of the study. If this was not the case, the final summary of LoS may be biassed by including only shorter stays which were resolved within the time frame. As reported above, summaries of patient characteristics and comorbidities were recorded for each study where possible; this allowed later comparison of estimates based on features of the study population and provided some additional context with which to interpret the LoS estimates. Finally, the risk of bias related to potentially overlapping populations was also considered. Studies from the same hospital over the same time period were identified, and the study with largest sample size was selected from that group. A list of these studies is presented in Supplementary table B. LoS was then summarised including only these selected studies from the overlapping groups, along with the other non-overlapping studies. This estimate was then compared to the estimates based on all studies.
## Estimating los distributions
Overall summary distributions were created for total hospitalisation LoS and for ICU LoS. Unlike a meta-analysis, the assumption here was not that each study had estimated an average LoS with some error, but that each study had captured a portion of the overall LoS distribution across a general population. Each of the studies provides us with a "sample" which we use to generate a better idea of the "true" underlying distribution. Our aim was to therefore incorporate all the variation across studies in order to obtain a distribution covering all plausible LoS values. We included studies in the estimation of these summary distributions if they reported both the sample size along with either the median and IQR or the mean and standard deviation. If no measure of variation was provided (either IQR or standard deviation), the point estimates were included in figures but excluded from these summary results.
Weibull distributions were fitted to the summary data from each study, using Nelder-Mead optimisation (implemented in the stats package in Rfor those reporting medians and IQRs. Specifically, the shape and scale parameters were varied in order to minimise the squared distance between the distribution and study quantiles. Where estimates were presented as a mean, x, and standard deviation, s, the distribution was fitted by momentmatching using the mixdist package. The same approach was also tested using gamma distributions, but Weibull was marginally preferred with respect to total squared error in the fitted quantiles. These distributions were then discretised using the distcrete package in R. A total of 100,000 samples were then drawn from each of these distributions, with weighting according to their sample size. Specifically, the study distributions were first sampled according to a multinomial distribution defined by the studies' relative sample sizes, and LoS was then sampled from each of these sampled distributions. Due to potential important differences in the characteristics of each study population, it may not be appropriate to weight entirely on sample size without considering how representative the cohort is of the general population. Therefore, as a sensitivity analysis, we performed the same analysis without weighting in order to understand how much this influences the distribution. In some cases, studies reported LoS according to some stratification and not over the whole study population. Here, we applied the same method to summarise across the strata and obtain an estimated median and IQR across the whole population, validating the approach using examples where the overall summary statistics were also provided.
All analyses were performed using R version 3.6.3 (29 February 2020).
# Results
## Study characteristics
The results of our screening process are summarised in [fig_ref] Figure 1: PRISMA diagram showing the results of the screening process used to identify... [/fig_ref]. After removing duplicates, we found a total of 650 potentially eligible studies of which 52 studies met all the inclusion criteria. These included 32 peer-reviewed articles from the academic literature, 18 pre-print articles, and 2 reports from other sourcesand. Several studies reported LoS by specific patient subgroups, according to disease severity, comorbidities (kidney injury, liver injury, hypertension, and cardiac injury), experimental treatments (heparin, lopinavir-ritonavir), and pregnancy status. A complete description of all reported LoS estimates is provided in . The key characteristics of the included studies are summarised in [fig_ref] Table 1: Summary study characteristics of included studies [/fig_ref].
The studies were carried out between 24 December 2019 and 16 April 2020. Although the cut-off was 12 April 2020 for inclusion of published and pre-print studies, the most recent version of the ICNARC reportwas used, which included patients admitted up to 16 April 2020. The majority of studies were cohort studies (46/52), with four cross-sectional studies, one case-control, and one randomised control trial (RCT). Two articles were reports from ongoing data collections (ISARIC, 8 April 2020, and ICNARC, 16 .
Studies were mostly conducted in adults with average participant age from 19 to 76 years (mean (SD) across *Median (IQR); **mean ± SD; ***range studies, weighted by sample size, 59 (9.6) years), and overall reported only a slightly higher proportion of males to females (54 (10.9) % male). Three paediatric studies included patients from newborn to 18 years, with a weighted mean (SD) of 7 (2.8) years of age [bib_ref] Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19)..., Qiu [/bib_ref] [bib_ref] A quickly, effectively screening process of novel corona virus disease 2019 (COVID-19)..., Shi [/bib_ref] [bib_ref] Clinical and CT features in pediatric patients with COVID-19 infection: different points..., Xia [/bib_ref]. For the majority of studies, LoS was a secondary or incidental outcome rather than the primary outcome. As a result, age and sex distributions were not always specific to the LoS population, and instead reported for the overall study population. Furthermore, it was not always possible to accurately interpret the sample size of the population, nor whether the LoS estimate included still-hospitalised patients. All LoS data extracted from studies are reported in . The majority of the included studies (46/52) were based in China, with a particularly high number reported from Wuhan , and many study populations were from the same outside of China: there was one study from Italy [bib_ref] Baseline characteristics and outcomes of 1591 patients infected with SARS-CoV-2 admitted to..., Grasselli [/bib_ref] , one for the whole EU region [bib_ref] First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, Spiteri [/bib_ref] , two from the USA [bib_ref] Covid-19 in critically ill patients in the Seattle region?case series, Bhatraju [/bib_ref] , one from the UK, and one study that collated LoS estimates from multiple countries excluding China (although the majority of the data are from the UK;.
Most studies (43/52) reported LoS for total hospitalisation only, with four studies reporting LoS for ICU only, and five studies reporting both. Only 15 studies reported LoS for study populations with completed follow-up (patient discharge or death), with 37 reporting estimates for populations where some patients remained in hospital or in ICU. The majority of studies only included discharged or dead patients within their LoS estimate, even if they had incomplete follow-up of the full cohort. However, for 8 studies, it was unclear whether the reported LoS included patients who were still hospitalised [bib_ref] First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, Spiteri [/bib_ref] [bib_ref] Covid-19 in critically ill patients in the Seattle region?case series, Bhatraju [/bib_ref] [bib_ref] Clinical characteristics of coronavirus disease 2019 in China, Wj [/bib_ref] [bib_ref] Patients of COVID-19 may benefit from sustained lopinavir-combined regimen and the increase..., Liu [/bib_ref] [bib_ref] Coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on..., Wang [/bib_ref].
## Total hospital length of stay
Estimates of the total hospital LoS are summarised in [fig_ref] Figure 2: Hospital length of stay, by discharge status [/fig_ref] Where provided, the overall study estimate of LoS for each discharge status is presented. For three studies, LoS was only reported within specific patient subgroups (relating to cardiac injury [bib_ref] Association of cardiac injury with mortality in hospitalized patients with COVID-19 in..., Shi [/bib_ref] , COVID-19 recovery trajectory [bib_ref] Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a..., Shi [/bib_ref] , and treatment comparison arms; therefore, in these cases, we include both estimates. The longest stays were recorded in a study of five critically ill patients [bib_ref] Treatment of 5 critically ill patients with COVID-19 with convalescent plasma, Shen [/bib_ref] , of whom only three were discharged and all more than 50 days after admission, which does not appear representative of the overall distribution (see [fig_ref] Figure 2: Hospital length of stay, by discharge status [/fig_ref] , Shen et al. . Excluding this study, the median duration of hospitalisation ranged from 5 to 29 days. There was no observed trend with respect to when the study was conducted [fig_ref] Figure 2: Hospital length of stay, by discharge status [/fig_ref].
Estimates for LoS amongst patients who died in hospital were generally shorter than those for patients who were discharged alive, with medians between 4 and 21 days compared to 4 and 53 days, respectively. This difference is apparent in [fig_ref] Figure 2: Hospital length of stay, by discharge status [/fig_ref] , where median LoS was lower for those discharged alive in 6 out of 8 studies that reported both outcomes. In studies that reported total hospital LoS by disease severity (11 studies, , there was a trend towards more severe cases having longer LoS. However, the definition of different levels of severity was inconsistent between studies so it is not possible to draw any confident conclusion.
Visual inspection of the study estimates suggested some evidence of a difference between total hospital LoS reported within and outside China, but studies outside China were too few (5/48) for a formal comparison. However, LoS reported within the ISARIC reportin particular (which includes contributed data from 25 countries, but with the majority of patients from the UK) gave a median and IQR (4 days (1-9)) substantially lower than the weighted mean from the studies from China (15.3 days).
The patient populations observed in these studies covered a wide range of ages, including three paediatric studies [bib_ref] Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19)..., Qiu [/bib_ref] [bib_ref] A quickly, effectively screening process of novel corona virus disease 2019 (COVID-19)..., Shi [/bib_ref] [bib_ref] Clinical and CT features in pediatric patients with COVID-19 infection: different points..., Xia [/bib_ref]. Amongst patients discharged alive, there appears to be little difference in average LoS between studies with the youngest and oldest patients, but the longest estimates came from studies with average age in the upper end of the range (Wang et al. [bib_ref] Coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on..., Wang [/bib_ref] and Shi et al., with average age of 68 and 69, respectively; Supplementary . The LoS estimates which included nonsurvivors tended to come from studies with older populations, as is to be expected given the well-documented, age-dependent fatality rate.
## Icu length of stay
Median stay in ICU ranged from 5 (IQR 2-9) to 19 (no IQR reported) days. There appeared to be less of a difference according to discharge status (alive or dead) than there was for total LoS . A total of 8 studies reported ICU LoS estimates, with the same number of studies reporting LoS estimates from China and outside of China, and the resulting overall estimates are very similar. There were too few studies to conduct any comparison by age or disease severity.
## Estimated distributions
Estimated summary hospital LoS distributions for studies from China and studies outside China are shown in [fig_ref] Figure 4: Combined LOS distributions [/fig_ref]. The median and IQR for total hospital was estimated to be 14 [bib_ref] Review of the clinical characteristics of coronavirus disease 2019 (COVID-19), Jiang [/bib_ref] [bib_ref] Fangcang shelter hospitals: a novel concept for responding to public health emergencies, Chen [/bib_ref] [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref] [bib_ref] Prevalence of comorbidities and its effects in coronavirus disease 2019 patients: a..., Yang [/bib_ref] [bib_ref] Locally informed simulation to predict hospital capacity needs during the COVID-19 pandemic, Weissman [/bib_ref] for China and 5 (3-9) excluding China. This was also repeated for ICU LoS, with a median and IQR 8 (5-13) for China and 7 (4-11) outside China. Comparing patient outcomes for total LoS, patients who died had a shorter LoS distribution (median, 8; IQR, 4-12) compared with patients who were discharged (median, 14; IQR, 11-17; . Studies from China have multiple estimates for the same outcome which represent multiple treatment and comorbidity subgroups, respectively. Details of these are included in [fig_ref] Table 1: Summary study characteristics of included studies [/fig_ref] Fig. 3 ICU length of stay, by discharge status. Medians (square) are presented with interquartile range (IQR). Where estimates were reported as mean and standard deviation, equivalent quantiles have been calculated assuming a Weibull distribution (triangle); if no measure of variation was reported, only the original mean is presented (circle). The grey dashed lines represent the mean value across all point estimates within that setting, weighted by sample size. Studies are ordered by the study start date which had complete follow-up with respect to total hospital LoS were compared with studies with incomplete follow-up. A slight difference was observed, with shorter median LoS observed in studies with complete followup (median, 12; IQR, 8-17) compared with incomplete follow-up (median, 14; IQR, 10-19; . This was only performed for total hospital LoS in China, since no studies from outside China reported completed LoS for ICU. In addition, LoS estimates of all studies were compared with estimates where overlapping patient populations were removed . A list of studies included in this analysis is presented in Supplementary table B. A small difference was observed, with longer LoS estimates observed in studies where overlapping populations had been removed (n = 17; median, 16; IQR, 11-22) compared with estimates across all studies (n = 19; median, 14; IQR, [bib_ref] Review of the clinical characteristics of coronavirus disease 2019 (COVID-19), Jiang [/bib_ref] [bib_ref] Fangcang shelter hospitals: a novel concept for responding to public health emergencies, Chen [/bib_ref] [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref] [bib_ref] Prevalence of comorbidities and its effects in coronavirus disease 2019 patients: a..., Yang [/bib_ref] [bib_ref] Locally informed simulation to predict hospital capacity needs during the COVID-19 pandemic, Weissman [/bib_ref]. Again, this sensitivity analysis was only performed for total hospital LoS in China, as it was the largest group.
For total hospital LoS in China, five studies were not included since they only provided point estimates for the LoS. The point estimates from four of these studies fell within the IQR of the estimated distribution; however, for [bib_ref] Treatment of 5 critically ill patients with COVID-19 with convalescent plasma, Shen [/bib_ref] , the point estimate was much longer. For total LoS outside of China, one study reported only a point estimate [bib_ref] First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, Spiteri [/bib_ref] , and this also fell outside of the estimated IQR. Sensitivity analysis showed that weighting by sample size had minimal influence on the shape of these distributions .
# Discussion
## Summary of findings
Understanding how long patients hospitalised with COVID-19 remain in hospital is critical for planning and predicting bed occupancy as well as associated staff and equipment needs. This review found that hospital LoS observations for COVID-19 patients published in the literature to date varied from less than a week to nearly 2 months. Stay in intensive care was shorter and less variable, with studies reporting medians of 1 to 3 weeks. Where LoS was reported according to discharge status, stay was found to be shorter for those who died than for those discharged alive; however, this difference was only apparent in terms of total stay and not stay in ICU (no statistical comparison was made). With respect to practical implications, knowledge of a difference between survivors and non-survivors is of less use since the outcome will not be known in advance in order to influence decision-making. To the authors' knowledge, this is the first formal review that has been conducted on hospital LoS for COVID-19.
The included studies yield some evidence of a difference between total hospital LoS observed in China and outside of China, with shorter LoS reported in the latter group vs 5 days (3-9), respectively). However, only five studies were identified which reported LoS outside of China; therefore, this comparison is somewhat inconclusive. It may be that LoS is longer in China compared with other settings due to different criteria for hospital admission and discharge. A consensus exists across guidelines, such as ensuring resolution of symptoms and evidence of two negative PCR samples at least 24 h apart before discharge; however, differences between settings may arise as a result of local capacity and strain on the health system. We attempted to capture this difference by recording time from onset of symptoms to admission; however, only one study outside of China reported this and a comparison was not possible. It is also possible that, with foresight from witnessing the Chinese epidemic, other countries set less strict criteria for discharge, in anticipation of stretched capacity. Other countries may also have used evidence from China to improve treatment methods and hence shorten LoS. However, this unfortunately appears unlikely as we did not observe a trend when looking at the reported LoS estimates over time.
In contrast, no difference was observed between settings for ICU LoS, for which there were an equal number of studies included from within and outside China. It is important to note that there might be key differences between ICUs in China compared with other countries, yet a definition for what constituted an ICU was rarely reported. Previous studies have found that ICU characteristics varied widely across geographic regions [bib_ref] The impact of hospital and ICU organizational factors on outcome in critically..., Sakr [/bib_ref]. Further understanding of characteristics of ICUs reporting LoS for patients with COVID-19 is important in providing context on the reported estimates and should be investigated in future studies.
There appeared to be little difference of LoS observed by age in our results, apart from the fact that studies which reported deaths tended to have older patient populations. However, if there is indeed a trend, we were unlikely to observe strong evidence for it amongst these studies, since the majority include a similar mix of ages, often tending towards older cohorts, and the age distribution was not always provided for the specific subgroup who had LoS recorded. Two studieswere included in the review which reported LoS by age, and they both found longer LoS associated with older age groups. In addition, two studies of LoS from the USA which were published after the search dates also reported a trend for longer LoS in older age groups [bib_ref] Incidence, clinical outcomes, and transmission dynamics of hospitalized 2019 coronavirus disease among..., Lewnard [/bib_ref] [bib_ref] Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in..., Richardson [/bib_ref]. Studies also reported LoS by disease severity; however, the definitions of disease severity were not consistent across studies, so we did not summarise by this.
# Limitations and biases
Having been the first country to observe this novel coronavirus, published data on COVID-19 patient outcomes in China is more widely available than from countries to which the epidemic spread later on. The set of studies found in this review reflects this bias towards evidence obtained from China, particularly Wuhan. The small number of studies identified from outside of China means it is difficult to interpret comparisons across settings. Several studies have been published after our search dates which provide additional LoS estimates from outside of China. A study of 5700 patients from hospitals in the New York area reported comparable estimates for total LoS (median 4.5; IQR 2.4-8.1) [bib_ref] Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in..., Richardson [/bib_ref] ; however, studies from Northern Italy [bib_ref] Characteristics and outcomes of patients hospitalized for COVID-19 and cardiac disease in..., Inciardi [/bib_ref] , Japan [bib_ref] Presepsin in risk stratification of SARS-CoV-2 patients, Zaninotto [/bib_ref] , and California and Washington [bib_ref] Incidence, clinical outcomes, and transmission dynamics of hospitalized 2019 coronavirus disease among..., Lewnard [/bib_ref] reported longer estimates of LoS. Therefore, the total LoS outside of China may in fact be longer than what we concluded. Our code is freely available on github, and additional studies may easily be added. As more studies emerge from a broader range of settings, it would be important to re-evaluate LoS estimates, as there are likely to be between-country differences that we have not captured here.
Furthermore, a number of studies include patients from the same hospital over the same period, for example, Yang et al. [bib_ref] Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in..., Yang [/bib_ref] and Wu et al.who both reported patients from Jin Yin-Tan hospital in Wuhan, and it is possible that these studies had overlapping study populations. Furthermore, Guan et al. [bib_ref] Clinical characteristics of coronavirus disease 2019 in China, Wj [/bib_ref] was a national study conducted in China and ISARICincluded 25 countries worldwide; therefore, these studies may also include patients previously described. The effect of this double-counting would be to bias the overall summary statistics towards the LoS from these settings, and potentially reduce the total variation. Although this is acknowledged as an issue, it was not considered as a basis for exclusion since any criteria for selecting one study from the overlapping group would have been arbitrary and potentially induce another source of bias in itself. Therefore, we instead chose to conduct a sensitivity analysis based on a straightforward selection criteria of largest sample size from those studies with any potential for overlap, and found little difference between the estimates of LoS whether overlapping studies were included or excluded. The overall benefit of inclusion, particularly as many of these studies reported LoS for different subgroups, was deemed to outweigh the potential bias which may arise as a result of overlapping patient populations.
In this review, we were only able to distinguish between "total hospital LoS" and "ICU LoS", with many studies only reporting a total LoS. This total LoS will include both general hospital and ICU admissions within it. There is a need for more granularity with respect to patient pathways, distinguishing between admissions to different levels of care within one hospital episode in order to better inform healthcare contingencies. Patients may, for example, be transferred to ICU on more than one occasion during their stay, which is important to factor in when ICU capacity is particularly limited.
Changes in hospital demand may have also affected our estimates. At the beginning of the outbreak and in certain settings, hospitals were being used to isolate patients who were unable to isolate effectively at home [bib_ref] Fangcang shelter hospitals: a novel concept for responding to public health emergencies, Chen [/bib_ref] [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref]. This means that LoS for patients in some of the earlier studies within this dataset could have been longer due to this logistical reason, rather than clinical need. Studies which mentioned this explicitly were excluded, yet there may still be others which were not so transparent. In addition, it is possible that, as hospitals reach the limits of their capacity, a more stringent triage policy may be implemented and the most critical patients may not be transferred to ICU. Despite this, we did not observe a trend when looking at the reported LoS estimates over time, suggesting that this is not in fact an important issue in our data.
Finally, many studies had incomplete follow-up with respect to LoS, and as a result, patients still hospitalised at the end of the study were not included in the summary statistics (right-truncation). This will bias estimates towards shorter LoS, as patients with longer LoS will not be included. A study by Lapidus et al.investigated the bias associated with estimating average ICU LoS for COVID-19 patients based on observed LoS of discharged patients before follow-up of the entire patient cohort was completed. As expected, the authors found that the average LoS estimated at 3 months of follow-up was much longer than that estimated at 1 month. This potentially affects our estimates, given that 37 (out of 52) studies had incomplete follow-up with regard to LoS, although on comparison the difference between the groups was slight, and estimates where follow-up was complete were overall shorter. Several studies included still-hospitalised patients in their LoS summary without accounting for censoring [bib_ref] First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, Spiteri [/bib_ref] [bib_ref] Covid-19 in critically ill patients in the Seattle region?case series, Bhatraju [/bib_ref] [bib_ref] Clinical characteristics of coronavirus disease 2019 in China, Wj [/bib_ref] [bib_ref] Patients of COVID-19 may benefit from sustained lopinavir-combined regimen and the increase..., Liu [/bib_ref] [bib_ref] Coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on..., Wang [/bib_ref] , which potentially alters interpretation of the values.
## Summarising length of stay
We found that LoS is often not the primary measure of interest in studies which report it; however, it is an important parameter when it comes to forecasting bed occupancy during an outbreak. By conducting this review, we have systematically gathered a range of published estimates, providing a source from which researchers and decision-makers can obtain estimates specific to their population of interest (e.g. with respect to comorbidities) and allowing comparison of LoS between several different populations and settings.
There have been numerous previous studies which have aimed to forecast the number of hospital beds required for COVID-19 patients [bib_ref] Locally informed simulation to predict hospital capacity needs during the COVID-19 pandemic, Weissman [/bib_ref] [bib_ref] Projecting hospital utilization during the COVID-19 outbreaks in the United States, Moghadas [/bib_ref] [bib_ref] Projecting demand for critical care beds during COVID-19 outbreaks in Canada, Shoukat [/bib_ref]. Many of these studies published so far have used point estimates, only originating from one study which often does not reflect the context of interest. In particular, many used estimates from Zhou et al. [bib_ref] Clinical course and risk factors for mortality of adult inpatients with COVID-19..., Zhou [/bib_ref] which reported a shorter total hospital LoS (median 11·0, IQR 7·0-14·0; [fig_ref] Figure 2: Hospital length of stay, by discharge status [/fig_ref] , and a comparable ICU LoS (median 8·0, IQR 4·0-12·0; , compared with other studies from China. However, both of these were still longer than LoS estimates reported by studies outside of China. This means that the bed-forecasting studies relying on LoS estimates from Zhou et al. may be underestimating the number of beds required. This review has highlighted several potential sources of variation in LoS and identified common issues and biases which influence each individual estimate. This gives a motivation for considering a wider range of values than can be obtained in a single study, aiming instead to capture the overall distribution of LoS across a variety of possible patient trajectories.
Our findings provide both better estimates of the LoS distribution for patients hospitalised with COVID-19 and a method for generating these important distributions going forward. Combined with predictions of disease incidence, models forecasting bed occupancy are used to plan required hospital capacity and hence are critical for outbreak preparations. The LoS estimate is a critical parameter within such a model, and as such, any predictions are sensitive to the value or distribution being assumed, with strong implications for policy and planning. In particular, the tail of the LoS distribution must not be ignored since these few patients can block beds for a long time and form a heavy burden on capacity. Our estimates, and the proposed method for distribution collation, allow for improved predictions of this aspect of burden prediction and hence could reduce uncertainty in capacity preparedness in healthcare settings going forward. This is of value to countries still experiencing growing epidemics and those turning attention to the planning for the possibility of a second wave whilst restarting non-COVID care.
It is preferable to use data from the setting for which you are trying to forecast bed occupancy (as was done by the IHME COVID-19 health service utilisation forecasting team; however, data on completed patient stays will often not be available until well after the onset of the epidemic. Furthermore, LMICs may have reduced capacity for surveillance and monitoring in order to obtain these data. In such cases, where countries are in the early stages of an outbreak, it would be better to use a conservative (i.e. broad) distribution of LoS from another setting. As the pandemic progresses and more countries observe patients completing their hospital episodes, it will be possible to add further setting-specific summaries and improve this distribution.
As far as the authors are aware, the approach demonstrated here to summarise median and IQRs across multiple studies has not been proposed before, although there are similarities with the approach taken by others in the CMMID Working Group to pool R 0 estimates. We present an intuitive method which exploits two optimisation methods to fit parametric distributions based on reported summary statistics rather than individual data, then samples across them. In this way, we capture the central tendency and overall variation between a set of quantiles from different study populations. This allows multiple sources of evidence to be consolidated into a single distribution which can be used in bed forecasting going forward. By providing both the code for this analysis and our summary distributions, better bed occupancy predictions can be made in the future.
# Conclusion
This review summarised the available literature to provide estimates of LoS for total hospital admission and ICU which can be applied for planning and preparedness for SARS-CoV-2. We found substantial differences between China and other settings in terms of total hospital stay, but little evidence for an impact on LoS of time of study, age, or disease severity. We present summary distributions which can be used within models making predictions about bed requirements, and suggest that this may be a more robust and realistic way to characterise LoS than relying on summary data from just one setting or hospital. The majority of the data presented in this review comes from China, and as more data become available, it will be important to update this with setting-specific LoS estimates. Understanding the duration of hospitalisation of COVID-19 patients is critical for providing insights as to when hospitals will reach capacity, as well predicting associated staff or equipment requirements.
## Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.1186/s12916-020-01726-3. . Database of all studies included within the review. Available as an excel spreadsheet.
## Abbreviations
LoS: Length of stay; ICU: Intensive care unit; IQR: Interquartile range; SD: standard deviation; LMIC: low-and middle-income country; PRISMA: Preferred Reporting Items for Systematic Reviews; RCT: randomised control trial.
[fig] Figure 1: PRISMA diagram showing the results of the screening process used to identify included studies (n = 52) [/fig]
[fig] Figure 2: Hospital length of stay, by discharge status. Medians (square) are presented with interquartile range (IQR). Where estimates were reported as mean and standard deviation, equivalent quantiles have been calculated assuming a Weibull distribution (triangle); if no measure of variation was reported, only the original mean is presented (circle). The grey dashed lines represent the mean value across all point estimates within that setting, weighted by sample size. The studies are ordered by the study start date, with most recent at the top. Two studies (Shi et al.(2020-02-02) and Shi et al. [/fig]
[fig] Figure 4: Combined LOS distributions. Samples from the LoS distributions, split by location (China or rest of world) and type (ICU vs total LoS). For each subset, 100,000 draws were taken. The x-axis was cut at days = 60 [/fig]
[fig] Additional file 1: List of search terms. Supplementary Table A. Description of information extracted from included studies. Fig SA: LoS by disease severity. Fig SB: LoS by study median age. Fig SC: LoS by outcome status. Fig SD: Summary distributions for China/other and total/ICU. Fig SE: Sensitivity of length of stay (LoS) with and without overlapping populations removed. Fig SF: Sensitivity analysis of summary distributions to weighting. Additional file 2: Supplementary [/fig]
[table] Table 1: Summary study characteristics of included studies (n = 52). A total of 46 studies were identified from China, and 6 studies identified outside of China [/table]
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COVID-19 and Health Care Leaders: How Could Emotional Intelligence Be a Helpful Resource During a Pandemic?
# Background
The Coronavirus Disease 2019 (COVID-19) pandemic begun in Wuhan in December 2019 and spread rapidly across the globe, causing millions of deaths.Frontline health care workers (HCWs), including doctors, nurses, and physical therapists, have been called to face an unprecedented emergency for which society and health care systems were largely unprepared.Despite a vaccination campaign started early all around the world, hospitals remain under severe stress. Staff members have been asked to work overtime and postpone vacations to face the serious workforce shortage.Health care leaders could learn a good lesson from 2020 events: there is a need for strategies to protect HCWs' health while planning and scheduling their work in the future.
During the COVID-19 pandemic, HCWs are dealing with difficult and demanding situations (eg, the risk of infection, isolation from their families, and the higher number of daily deaths). Consequently, HCWs reported increased psychological distress (eg, anxiety, depression, insomnia, and emotional vulnerability) as described by systematic reviews with metaanalysis.The World Health Organization provided indications to protect the HCWs' psychosocial well-being,while health care leaders have been stimulated to nurture a culture of emotional intelligence (EI) towards their teams.EI is "the ability to monitor one's own and others' feelings and emotions, to discriminate among them and to use this information to guide one's thinking and actions." 10 EI is useful in helping health care teams coping with stressful situationsgiven its capacity to promote motivation, empathy, cooperation, and good communication.During a pandemic, EI should represent a resource for health care leaders to deal with the emergency: it acts as a "stress buffer" helping to recover faster from stressful situations.The aims of this Point of View article are to (1) define the EI skills-set for health care leaders, (2) describe strategies of EI implementation for health care leaders, and (3) suggest an EI training for health care leaders during COVID-19.
## Ei skills-set for health care leaders during covid-19
During the COVID-19 pandemic, health care organizations have increased their complexity.Health care leaders need to develop and apply EI skills in order to cope with higher and sustained pressures. The emotionally intelligent health care leaders should present 14 :
(1) Self-awareness (perception of emotions): having a deep understanding of personal emotions (eg, strengths, weaknesses, needs, culture, biases, and values) is useful to know how they effectively support the HCWs and influence the team performance; (2) Self-management (use of emotions): being in control of personal disruptive emotions and impulses to avoid dysfunctional behaviors (eg, aggressive or hostile outbursts) and make constructive decisions; (3) Social awareness (understanding emotions): understanding empathetically HCWs' emotions and considering their perspectives as important to communicate effectively and create a fair and trustworthy environment;
## Example of strategies to apply
Sense of unpreparedness and inability to manage a new situation, lack of time to plan but only to act (eg, "as soldiers in battle") Be aware of personal status - Find daily time to monitor and ponder on personal physical/emotional state to improve self-awareness, motivation, and resilience;
- daily practice of relaxation techniques (eg, breathing exercises, progressive muscle relaxation, autogenic training, imagery) to improve recovery and protect from stressful situations;
- daily practice of mindfulness exercises (eg, body scan, seated meditation) to reduce anxiety and allow for greater self-insight: meditation on to the present moment with a non-judgmental awareness would help to understand and regulate emotions, thoughts, and consequently behaviors Emotional distress ranging from a direct manifestation (eg, anger, loneliness, or fear) to a denial of feelings (eg, avoidance, control, or minimization)
Pay attention to team's emotions - Careful observation to HCWs' behaviors (eg, sudden manifestations of irritability, sadness, or isolation) to identify dysfunctional strategies and cope with emotional overload;
- actively listen to HCWs' words (eg, excessive use of negative words such as "fear," "anxiety," or "anger") to recognize early signs of distress and exhaustion;
- consider HCWs' cognitive appraisals of the pandemic and related emotional responses, try to put self in their shoes (eg, "if I were in their position how would I feel?", "What would I want to hear?");
Experiences of unclear communication (eg, contradictory information) or negative emotional contagious (eg, emphasized description of the pandemic, adopting pessimistic words as "trauma," "war movies," or "tragedy")
Consider personal style of interaction - Reflect on the content and style of your communication (eg, "Is your verbal/non-verbal language consistent?", "What message do you convey through your body language?")
- Provide clear and efficient routine communication (eg, daily/weekly) by sharing updated information on the pandemic and new evidence on best practice for COVID-19;
- Prefer realistic but positive words for description of the pandemic (eg, "an opportunity to learn," "an experience of change," "a possibility of growing") to stem negative emotional contagion with other HCWs
Lack of support and perception of distance between HCWs and leaders in working environments (eg, requests or suggestions are frequently ignored) Support the team - Provide empathy, kindness, and compassion by supporting HCWs in their moments of difficulty (eg, giving attention to needs and concerns especially, but not only, when declared);
- Create a positive, safe, and non-judgmental work environment where HCWs may express their point of view without fear of repercussions (eg, during weekly meetings or daily shifts); - Motivate the HCWs by making everyone feel a key player for the whole team (eg, praising them for resilience, dedication, and their commitment to fulfill the work);
Need to working as one team (eg, experiencing the mutual support and cooperation)
Promote teamwork and collaboration - Nurture a culture of unity, cohesion, humanity and respect among the HCWs based on mutual help and shared values (eg, "We work as a whole team rather than as individuals") as experienced during COVID-19 pandemic;
- Create opportunities with HCWs to discuss work issues, share concerns, and reflect on possible solutions (eg, organize regular short briefings and/or debriefings);
- Emphasize among HCWs the value of peer interaction and collaboration to improve psychological resilience (eg, give and receive)
Experiences of professional loss (eg, compromised standards of care, uncertainty) and missed reference points (eg, the need to understand the pandemic as an opportunity to learn new things)
Guide towards a new vision - Ask positive questions about the pandemic (eg, "What can we acquire from this situation?") to help HCWs reappraise negative emotions and thoughts by moving from a pessimistic to a proactive mindset;
- Develop an organizational culture that considers changes as natural elements in the transition to the future rather than obstacles (eg, events change inexorably, it is our duty to see the opportunity for improvement);
- Underline the opportunity to exit our "comfort zones" by learning and modifying tasks and activities; for instance, new professional opportunities based on new technologies (eg, tele-rehabilitation) to be adopted as an integration to the clinical routine a COVID-19 = coronavirus disease 2019; EI = emotional intelligence; HCWs = frontline health care workers.
Rossettini et al 3
## Structure of each session
Each session includes a theoretical introduction to the topic (first part) followed by a group discussion and activities (second part) to stimulate interaction between participants and favor integration between theoretical and practical skills.
## Content of sessions 1
- Introduction of the program and setting ground rules;
- Self-presentation of each participant;
- Presentation of behavioral EI models underlying the importance of EI to health care leaders at both the individual and social levels;
- Group discussion about emotional and motivational issues (personal and with teams) experienced during the COVID-19 pandemic and strategies used to address them by participants;
- Distribution of a handout on EI basics to facilitate a complete understanding of the subject 2,3,4,5 - Deeper explanation of personal and social competences required by health care leaders with particular reference to the period of the COVID-19 pandemic:
- (Session 2) topic: emotion recognition, comprehension, and acceptance; - (Session 3) topic: emotional self-control and adaptability;
- (Session 4) topic: support and effective communication;
- (Session 5) topic: teamwork - Activities to improve understanding and retention of information;
- Practice of learned skills;
- Group discussion to share experiences and foster integration of the different perspectives emerged;
## 6
- Individual activity to reinforce contents and strategies learned and experienced during program;
- Group discussion and identification of a list of behavioral guidance for health care leaders.
a COVID-19 = coronavirus disease 2019; EI = emotional intelligence.
## 4
Emotional Intelligence During COVID-19 Pandemic (4) Relationship management (emotional management): nurturing healthy relationships with others is fundamental to deal with challenges and to guide teamwork toward the desired goals with a positive attitude.
From this perspective, health care leaders should firstly develop the EI skills themselves to disseminate and implement these soft-skills also among their HCWs.
## How health care leaders might implement ei during covid-19
During the COVID-19 pandemic, time and resources are scarce, and implementing new protocols and guidelines appears unfeasible.However, even in this exceptional circumstance, using EI skills may require just a few minutes and could make a difference. Thus, health care leaders are encouraged to be emotionally intelligent toward their teams and to use their personal and social competences to foster:
(1) Self-awareness of their own status: health care leaders should listen to their emotions to acknowledge difficult days and cope adaptively and efficiently. An accurate selfassessment of emotional and physical state could help to guide wiser decisions; (2) Attention to team emotions: health care leaders should talk with their colleagues and listen and observe their verbal and non-verbal language. The HCWs' words and behaviors could provide important cues about their thoughts, emotions, and concerns, suggesting their level of emotional overload and distress; (3) Self-perception of their own style of interaction: health care leaders should pay attention to both the content of the messages delivered and the styles of communication used (eg, words and behavior) when interacting with HCWs. The quality of leaders' interaction may influence the emotions and the psychological conditions of HCWs, with potential repercussions on the organizational climate; (4) Support and understanding of the team: health care leaders should spend time with their teams to understand HCWs' needs and the appropriateness of workloads. Being sensitive and paying attention to HCWs' views and feelings is particularly crucial, especially in a context of crisis management; (5) Teamwork and collaboration: health care leaders should emphasize the role of the team and plan time for briefing and/or debriefing with their HCWs with the aim of sharing work issues and solutions. The contribution of each HCW is essential to develop a team performance and spirit based on shared values and common goals; (6) Guidance towards a new vision: health care leaders should act as a guide, knowing the feelings, strengths, and weaknesses of their HCWs, choosing a common goal to pursue and helping them to consider the pandemic also as an opportunity to learn and grow.
Based on the principles highlighted above, health care leaders should rethink their practice trying to integrate EI in their workflow. We provide a practical guide to help implement EI in a health care environment, paying particular attention to the experiences that emerged during the COVID-19 pandemic (Tab. 1).
## How to train ei for health care leaders during covid-19
Considering the challenging situations that HCWs are facing worldwide, an implementation of EI is needed and could be achieved only by providing tailored training to health care leaders. 14 EI skills can be learned and developed through programs that consider both theoretical and practical knowledge; participants should actively practice these skills in a nonjudgmental learning environment. 14 Such training programs should aim to:
(1) inform and develop EI by improving individual emotional clarity, self-awareness, and the ability to monitor one's status; (2) teach, implement, and encourage new strategies to understand and deal with colleagues' emotions; and (3) support and enable health care leaders to gain a broader perspective on EI through a guided peer-to-peer comparison.
In, we propose an example for EI training to be performed during the COVID-19 pandemic or other crisis. A psychologist facilitator would lead a 12-hour training program (6 online sessions with up to 15 participants). The virtual sessions should be held on a weekly basis and be focused on the personal and social competencies required for leaders, with particular reference to the crisis period.
During a critical period, working in groups could be an opportunity for the health care leaders to share personal experiences, discuss solutions, and feel the support of peers. To facilitate implementation of the acquired skills, participants should be encouraged to use a self-awareness worksheet/diary to record experiences during the working context. These resources would help trainees to contextualize their learning and could be analyzed during training sessions, becoming important learning opportunities for the whole group.
At the end of the programs, participants should be asked to identify a list of behavioral changes that could be applied during the COVID-19 pandemic. This could be an effective method to improve leaders' sense of responsibility, to help leaders monitor their behavior over time, and to foster a culture of EI in the workplace.
# Conclusion
The COVID-19 pandemic is not over, and the long-term psychological and physical consequences for HCWs are unknown. Health care organizations and leaders should act immediately to promote EI to protect their team in the current pandemic and prepare HCWs for future emergencies.
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Hypertonicity: Pathophysiologic Concept and Experimental Studies
# Introduction and background
Maintenance of constant volume is of great importance for proper function and survival of body cells. The regulation of cell volume under normal conditions is the consequence of two properties of cells, their ability to extrude solute into the extracellular compartment (EC), and the high permeability of the cellular membranes to water. The main mechanisms of change in cell volume are disturbances in body fluid tonicity (effective osmolality). Tonicity is the property of a solution to make cells suspended in it swell by gaining water (hypotonicity) or shrink by losing water (hypertonicity) through osmotic pressure differences between the intracellular compartment (IC) and the solution tested. Solutions are isotonic when the volume of cells suspended in them does not change by osmotic fluid transfers. Disturbances in tonicity have clinical consequences. The brain is the site of the principal clinical manifestations of both hypotonicity and hypertonicity. The neurological manifestations of either hypotonicity or hypertonicity may lead to irreparable deficits or death. Consequently, proper management of disturbances in tonicity is critical and requires an understanding of the qualitative and quantitative aspects of their pathophysiologic mechanisms.
The purpose of this review is to explore the evolution of the quantitative and qualitative aspects of the concept of hypertonicity and to summarize the experimental work, which has led to the current understanding of this topic.
## Review physicochemistry
## Definitions of osmosis, osmotic pressure, osmolality, osmolarity and tonicity
Water transfers from or into the cells and, therefore, changes in cell volume are secondary to osmotic pressure differences between the IC and the EC. Osmosis is the net movement of water across a selectively permeable (semipermeable) membrane caused by water concentration difference. Cell membranes are highly permeable to water, less permeable to electrolytes, and essentially impermeable to large organic polyanions. In a system of two compartments separated by a semipermeable membrane, osmotic pressure is measured by the hydrostatic pressure that is needed on the side of the higher solute concentration to stop net osmotic water movement across the membrane. Osmotic pressure ( ) is mathematically expressed by van't Hoff's law as follows:
## {1}
where C is the concentration of solutes in Osm/L, R is the ideal gas constant which is equal to 62.36 L×tor/(mol×K), and T is the absolute temperature in degrees Kelvin (K = 273 + actual temperature in centigrade degrees). If body temperature is considered constant at 37 o centigrade, then both T (310 o Kelvin) and R (and consequently, the product R × T) are constant and osmotic pressure is a linear function of the osmolar concentration of body fluids. In clinical practice, osmolar concentration is expressed as osmolality (mOsm/kg water in the solution) or osmolarity (mOsm/L of the solution). Osmolality provides an accurate measurement of solute concentration in biological fluids. Osmolarity is less accurate than osmolality because it is affected by the water content of the solution (the fraction of the volume of the solution that is water). Differences between osmolality and osmolarity are encountered in serum measurements. Osmotically active solutes are found only in the water phase of the serum. The average water content of the serum is 0.93 under normal conditions of hydration and serum solids. Therefore, serum osmolality exceeds serum osmolarity by approximately 7% under normal conditions. Serum osmolarity underestimates the true solute concentration in water in clinical states causing an elevation of serum solid content (e.g., hyperproteinemia, hyperlipidemia). In this case, the difference between serum osmolality and osmolarity exceeds 7%. This discrepancy is the cause of diagnostic difficulties.
The relationship between osmotic pressure, expressed in mm Hg, and osmolality, expressed in mOsm/kg, for a solution with an osmolar concentration of 1 Osm/kg can be calculated as follows: = 62.36 × 310 = 19,332 mm Hg, or (at first approximation) = 19.3 mm Hg for each mOsm/kg. A dilute solution of a completely dissociated monovalent salt, such as sodium chloride, will provide two dissolved particles per molecule. Because of interionic attraction forces, ions do not behave as entirely independent particles in biological fluids. Correction for this apparent deviation from van't Hoff's law is achieved by using an empirical factor, the osmotic coefficient, which is considered to be equal to 0.93 for monovalent sodium salts in solutions with sodium concentration equal or close to the normal serum sodium concentration. For example, isotonic saline (0.9% or 154 mmol/L sodium chloride in water) with a water content of almost 100% would have a theoretical osmolality of 308 (154 sodium + 154 chloride) mOsm/kg. Its actual osmolality is 0.93 × 308 = 286 mOsm/kg. Serum osmolarity is not measured. It is calculated as the sum of the osmotic concentrations of sodium salts, urea, and glucose in serum volume. The osmotic coefficient of sodium salts is usually taken to be equal to 2.0 in these calculations. The error in the estimate of osmolarity from this calculation is considered negligible. The total concentrations of osmotic substances in plasma, interstitial fluid, and intracellular fluid are equal in the steady state.
Osmolality and tonicity of a solution, although related, are not the same. The total concentration of dissolved solutes in a solution (the osmolality of this solution) determines its colligative properties (the degree of lowering of the freezing point, elevation of the boiling point, or depression of the vapor pressure from the corresponding value of pure water). Osmolality is measured by instruments calibrated to express changes in a colligative property (usually depression of the freezing point) in units of osmolality (mOsm/kg). The osmolality of a solution represents the sum of the osmotic concentrations (the concentrations times the corresponding osmotic coefficients) of all dissolved solutes. Tonicity of a solution is the part of its total osmolality that can cause steady state osmotic water transfers between IC and EC. The tonicity of a solution is evaluated by the photographic recording of rapid changes in the volume of cells, usually red cells, suspended in this solution. This technology has been applied only in research studies. Clinically, tonicity is estimated as the part of osmolarity attributed to solutes with extracellular distribution (see below).
## Gibbs-donnan equilibrium
The IC contains large molecular weight polyanions (e.g., proteins), to which the cell membranes are, by and large, impermeable. Although its composition varies between tissues and by distance from cell membranes, the fluid in the interstitial compartment, which is the part of EC that is in direct contact with the cells, is considered to have negligible concentrations of polyanions in general. Intracellular polyanions electrostatically attract cations and repel anions across the cell membrane. The Gibbs-Donnan relationships express the equilibrium status of electrolytes in a system of two compartments separated by a semipermeable membrane when one of the compartments contains polyanions. Understanding this equilibrium is required for analyzing water exchanges between the two compartments. The Gibbs-Donnan equilibrium predicts that the sum of anions, plus cations, which is by far the major constituent of osmolality of body fluids under normal conditions, is higher in the IC than in the EC. This is a consequence of a geometric rule relevant to the Gibbs-Donnan equilibrium. The rule states that the square has the smallest perimeter of all the rectangles that have the same surface area. For example, a square with a 6 m side and a rectangle with sides of 4 and 9 m have the same surface area (6 × 6 = 4 × 9 m 2 ). This relationship would fulfill the requirements of equation 4. However, the perimeter of the square (4 × 6 = 24 m) is less than that of the rectangle (2 × 4 + 2 × 9 = 26 m), and consequently, half of the perimeter of the square (2 x 6 = 12 m), which is an expression of equation 1, is less than that of the rectangle (4 + 9 = 13 m), which is an expression of equation 3. As a consequence of this geometric rule, the sum of the anions and cations (the osmolality) would be higher in the IC compartment than in the interstitial compartment; consequently, an osmotic pressure difference favoring translocation of interstitial fluid into the IC and cell swelling exists, creating the need for constant removal of solute from the IC in order to maintain a constant cell volume. Maintenance of a constant cell volume requires consumption of energy. Continuous sodium transfer outside the IC under the influence of Na + -K + ATPase is the main energy-consuming physiologic mechanism protecting the volume of the cells. Cells swell under anoxic conditions or under the influence of drugs inhibiting Na + -K + ATPase.
## Response of body cells to hypertonicity
## Principles governing the distribution of water between the major body compartments
The fundamental cell properties that are pertinent to the maintenance of cell volume in the normal state and responsible for clinical abnormalities in cell volume during disturbances in tonicity are the high permeability of cell membranes to water, which is universal with the exception of the membranes of the cells in the cornea and, in the absence of vasopressin, of the apical membranes of the principal cells in the renal medullary collecting ducts, and the machinery to continuously expel cations from the cells (the Na + -K + ATPase). Cell volume abnormalities develop during disturbances of body water and solute balance and can be quantitated by applying two principles that emanate from the high permeability of cell membranes to water, the osmotic principle and the principle of body water distribution.
The osmotic principle states that in the steady state osmolality is the same in the IC and EC [bib_ref] Water exchange, Peters [/bib_ref]. Experimental testing of the osmotic principle by measuring osmolality in serum and several body tissues proved to be technically difficult and proof for this principle became available approximately 15 years after Peters stated the principle [bib_ref] The freezing point depression of mammalian tissues after sudden heating in boiling..., Appelboom [/bib_ref] [bib_ref] The potential of water in mammalian tissues, Maffly [/bib_ref].
The principle of body water distribution was formulated first [bib_ref] The change in distribution of body water accompanying increase and decrease in..., Darrow [/bib_ref] , although it is a direct consequence of the osmotic principle. This principle states that, in the steady state, body water is distributed between the EC and the IC in proportion to the amount of solute in each compartment [bib_ref] The change in distribution of body water accompanying increase and decrease in..., Darrow [/bib_ref]. Quantitative analyses of abnormalities in tonicity are routinely based on the principle of body water distribution [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref] [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref] [bib_ref] A working model of the perfect osmometer hypothesis in anuria, Tzamaloukas [/bib_ref] [bib_ref] Aiding fluid prescription for the dysnatremias, Adrogué [/bib_ref] [bib_ref] Principles of management of severe hyponatremia, Tzamaloukas [/bib_ref] [bib_ref] Principles of Quantitative Fluid and Cation Replacement in Extreme Hyperglycemia, Tzamaloukas [/bib_ref] [bib_ref] Body sodium, potassium and water in peritoneal dialysis-associated hyponatremia, Sun [/bib_ref]. The formulas derived from these quantitative analyses guide quantitative treatment prescriptions for clinical states of dystonicity. The derivation, experimental testing, and limitations of these formulas should, therefore, be understood.
## General mechanisms of hypertonicity
Rises in tonicity result from changes in body water, body solute, or both water and solute. Combined gains or losses in water and solute generated through interactions between the EC and the external environment are the more common sources of hypertonic abnormalities. The loss in body water alone is seen in some clinical states (for example, in diabetes insipidus). Hypertonicity caused by net solute gain without a change in body water is uncommon. An example of this last hypertonic condition is hyperglycemia resulting from excessive hepatic glucose production during insulin deficiency states occurring in anuric subjects.
In terms of their ability to cause disturbances in tonicity, solutes are classified into two categories, those that do and those that do not cause steady state changes in cell volume. Increases in the body content of endogenous (e.g., urea) or exogenous (e.g., alcohols) solutes distributed in total body water cause rises in body fluid osmolality but do not cause changes in cell volume in the steady state. The clinical manifestations and treatment of excesses in the body content of solutes with body water distribution are different from those of hypertonicity [bib_ref] Hyperosmolar hyperglycemic nonketotic diabetic coma, Mccurdy [/bib_ref] [bib_ref] Understanding serum electrolytes. How to avoid making mistakes, Kapsner [/bib_ref] [bib_ref] Estimates of interdialytic sodium and water intake based on the balance principle:..., Ramdeen [/bib_ref] [bib_ref] Interdialytic weight gain: Role of salt and water intake, Tzamaloukas [/bib_ref]. Increases in the body content of solutes with EC distribution, whether endogenous (glucose) or exogenous (salt, mannitol), cause hypertonicity [bib_ref] The hypertonic state, Feig [/bib_ref] [bib_ref] Abnormalities in cell volume regulation and their functional consequences, Pollock [/bib_ref]. If the solute causing hypertonicity is not a sodium salt, the exit of water from the IC in hypertonic states results in dilution of the EC sodium salts, which in the normal state are the main EC solutes [bib_ref] The hypertonic state, Feig [/bib_ref]. Tonicity is estimated as the sum of serum sodium concentration times its osmotic coefficient, plus the osmotic concentration of glucose and/or any other solute with extracellular distribution.
Most solute abnormalities leading to tonicity disturbances are accompanied by changes in body water. Not taking into account the change in body water, regardless of how small it is, in the calculations of fluid shifts between the IC and EC and of osmolality changes in disturbances of tonicity can be the source of disproportionately large errors [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref].
## Mathematical models of hypertonicity
Traditionally, models of hypertonicity predicted the degree of increase in tonicity, gain in EC volume, and loss in IC volume after a hypertonic disturbance of known volume and tonicity by assuming that cells behave as perfect osmometers. Other gains or losses of water and solute, principally through the urine, routinely interfere with the magnitude of the disturbances in tonicity in clinical practice. These other gains or losses were not accounted for in the predictive models [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref] [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref] [bib_ref] A working model of the perfect osmometer hypothesis in anuria, Tzamaloukas [/bib_ref] [bib_ref] Aiding fluid prescription for the dysnatremias, Adrogué [/bib_ref] [bib_ref] Principles of management of severe hyponatremia, Tzamaloukas [/bib_ref] [bib_ref] Principles of Quantitative Fluid and Cation Replacement in Extreme Hyperglycemia, Tzamaloukas [/bib_ref] [bib_ref] Body sodium, potassium and water in peritoneal dialysis-associated hyponatremia, Sun [/bib_ref]. Therefore, the existing mathematical models of hypertonicity are strictly applicable only to anuric states. [fig_ref] explains the symbols for osmolalities and volumes in, Figure 1: Ordinates [/fig_ref] shows the determinants of the changes in tonicity, body water, and body water distribution in hypertonic states secondary to extracellular gains of hypertonic solutions in anuria. The necessary and sufficient conditions for cells to be considered as perfect osmometers are that total EC solute remains constant between stages II and III, total IC solute remains constant between stages I, II, and III in [fig_ref] explains the symbols for osmolalities and volumes in, Figure 1: Ordinates [/fig_ref] , and that equilibration of osmolalities between the IC and EC is achieved exclusively by water transfer from the IC into the EC. Under these conditions, the volumes V i1 (baseline IC volume), V e1 (baseline EC volume), V 1 (baseline body water or V i1 + V e1 ), and V 3 (volume of the infusate) and the osmolalities Os 1 (baseline osmolality) and Os 3 (osmolality of the infusate) are the independent variables, while the volume V (the difference between the final and baseline EC volumes) and the final osmolality (Os 2 )
are important dependent variables that can be calculated from the independent variables by the following formulas.
Total EC volume expansion (\(\Delta\V) = osmotic shift of water from the IC into the EC, plus volume infused):
## {5}
For expansion with water (Os 3 = 0), equation distributed, by and large, in the EC, the changes in EC sodium concentration during disturbances in tonicity are defined by total body water. This is a consequence of the osmotic principle). Whether the large amount of sodium that is bound to polyanionic proteoglycans primarily in cartilage, bone, and skin is available for rapid participation in tonicity disturbances is not clear [bib_ref] A different view on sodium balance, Titze [/bib_ref].
Formula 9 uses the same mathematical determinants of the change in serum sodium concentration as the Adrogue-Madias formula, which is currently used to calculate the increase in serum sodium concentration ([Na] 2 -[Na] 1 ) after infusion of one liter of a saline solution with a higher sodium concentration than the serum sodium concentration and is expressed by the following relationship [bib_ref] Aiding fluid prescription for the dysnatremias, Adrogué [/bib_ref] : [bib_ref] Aiding fluid prescription for the dysnatremias, Adrogué [/bib_ref] Formulas 9 and 10 provide essentially the same results [bib_ref] Principles of management of severe hyponatremia, Tzamaloukas [/bib_ref].
If final osmolality (Os 2 ) is known, the initial fluid volume that determines the final osmolality (V Osm ) after infusion of an amount of solute equal to Os 3 ×V 3 is calculated by a modification of the Fick equation that takes into account the volume infused as follows:
{11} Formula 11 calculates the so-called "osmotic volume of distribution", which, under perfect osmometer conditions, should be equal to the baseline body water (V 1 ) [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref] [bib_ref] A working model of the perfect osmometer hypothesis in anuria, Tzamaloukas [/bib_ref]. If the EC expansion is done with hypertonic saline, equation 10 can be modified to calculate the sodium volume of distribution, as follows:
## {12}
Under conditions of a perfect osmometer, V Na , V Osm , and V 1 should be equal [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref]. Thus, equations 11 and 12 allow the experimental estimation of body water. Comparison of body water estimates obtained from equation 11 and simultaneous body water measurements obtained by standard methods, such as isotopic water dilution, allowed testing of the hypothesis that cells behave as perfect osmometers [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref] [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref] [bib_ref] A working model of the perfect osmometer hypothesis in anuria, Tzamaloukas [/bib_ref].
## Experimental testing of the perfect osmometer hypothesis
Because the mathematical models of the perfect osmometer hypothesis ignored the renal contribution to body fluid balance, experimental testing of its models and of metabolic abnormalities that result from hypertonicity, such as acid-base disturbances and hyperkalemia, has usually been carried out by infusion of hypertonic solutions into anephric or anuric animals [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref] [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref] [bib_ref] The mechanism of acidosis produced by hypertonic infusions, Winters [/bib_ref] [bib_ref] Hypertonic expansion: Acidbase and electrolyte changes, Makoff [/bib_ref] [bib_ref] On the mechanism of hyperkalaemia due to hyperosmotic expansion with saline or..., Makoff [/bib_ref] [bib_ref] Hypertonic extracellular expansion in anuria, Tzamaloukas [/bib_ref]. In addition to hypertonicity, these experiments resulted in EC expansion by the volume infused and the volume osmotically transferred from the IC. The results of these early experiments are summarized below:
(a) The volume of the infused solution (V 3 ) has substantial effects on the results and must be accounted for, even when it is small as it is in hypertonic infusions [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref]. Equations 5-12 represent mathematically rigorous expressions of the perfect osmometer hypothesis because they include all the determinants of changes in osmolality and body water distribution, including the volume V 3 [bib_ref] A working model of the perfect osmometer hypothesis in anuria, Tzamaloukas [/bib_ref] [bib_ref] Principles of management of severe hyponatremia, Tzamaloukas [/bib_ref].
(b) In hypertonic saline expansion, the final serum sodium concentration is predicted accurately by equation 9 and V Na estimated from equation 12 is equal to total body water determined by reference methods. However, final osmolality is higher than the value predicted by equation 8 and, consequently, V Osm is less than either body water or V Na [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref] [bib_ref] Hypertonic extracellular expansion in anuria, Tzamaloukas [/bib_ref]. This finding led to the conclusion that hypertonic expansion produces new solute, the so-called "idiogenic osmoles" [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref]. Hypotonic expansion experiments with water confirmed the finding that V Na is equal to body water [bib_ref] The mechanism of the osmotic adjustment of body cells as determined in..., Leaf [/bib_ref] [bib_ref] The syndrome of inappropriate antidiuretic hormone secretion (SIADH): pathophysiologic mechanisms in solute..., Cooke [/bib_ref].
(c) Experimental testing of the perfect osmometer hypothesis was traditionally done by infusing hypertonic solutions into acutely anuric or anephric experimental animals [bib_ref] Apparent and osmotic volumes of distribution of sodium, chloride, sulfate and urea, Wolf [/bib_ref] [bib_ref] Osmotic volumes of distribution. Idiogenic changes in osmotic pressure associated with administration..., Mcdowell [/bib_ref] [bib_ref] Hypertonic extracellular expansion in anuria, Tzamaloukas [/bib_ref]. In addition to hypertonicity, these experiments caused substantial extracellular expansion and progressive azotemia, conditions leading to the generation of new solutes, not as a direct consequence of hypertonicity. Extracellular expansion dilutes certain solutes, for example, glucose and calcium, the extracellular concentration of which is tightly regulated by hormones. Rapid responses of the regulatory hormones cause the release of these solutes in the extracellular compartment from stores that were not osmotically active previously [bib_ref] Hypertonic extracellular expansion in anuria, Tzamaloukas [/bib_ref]. Similarly, progressive azotemia indicates the progressive generation of new solute, primarily urea. Correction for the new solute generated by extracellular expansion and azotemia accounted for only part of the "idiogenic osmoles" [bib_ref] Aiding fluid prescription for the dysnatremias, Adrogué [/bib_ref]. [fig_ref] FIGURE 2: Estimates of body water and the osmotic and sodium volume of distribution... [/fig_ref] shows the estimates of body water, V Na , and V Osm calculated after infusion of hypertonic saline in one experimental study [bib_ref] Hypertonic extracellular expansion in anuria, Tzamaloukas [/bib_ref]. Although estimates of V Na and body water are remarkably similar under a variety of experimental conditions, the discordance between V Osm and body water mandated the study of "idiogenic osmoles", which have, after their identification, been renamed "organic osmolytes".
The Nature of "Idiogenic Osmoles"
The brain was identified early as an important site of deviation from perfect osmometric behavior in acute experimental hypertonicity. Hypertonic states reduce brain volume. However, even in acute hypertonic states, the reduction in brain volume is substantially less than the reduction predicted from calculations based on perfect osmometric behavior [bib_ref] Factors that limit brain volume changes in response to acute and sustained..., Holliday [/bib_ref].
Astrocytes play a major role in the defense of brain volume in acute states of dystonicity [bib_ref] Astrocytes are an early target in osmotic demyelination syndrome, Gankam Kengne [/bib_ref]. Immediate adaptation to brain shrinking includes movement of fluid from the cerebrospinal fluid into the astrocytes. This is a limited adaptive mechanism. The main adaptation occurs by a process called regulatory volume increase (RV), which consists of intracellular solute gain.
The early component of RV in hypertonic states is accomplished within 30-120 minutes of the hypertonic stimulus by the rapid influx of ions (sodium, chloride, and potassium) from the interstitial compartment (which shrinks) into the brain IC [bib_ref] Volume regulatory influx of electrolytes from plasma to brain during acute hyperosmolality, Cserr [/bib_ref] [bib_ref] Extracellular volume decreases while cell volume is maintained by ion uptake in..., Cserr [/bib_ref]. In chronic hypertonicity, which develops in days or weeks rather than minutes or hours, brain volume is restored by the accumulation of organic compounds, the so-called "brain osmolytes" [bib_ref] Control of brain volume during hyperosmolar and hypoosmolar conditions, Cullans [/bib_ref]. shows the sequence of gains in brain osmolytes in acute and chronic hypertonicity [bib_ref] Effects of hypernatremia on organic brain osmoles, Lien [/bib_ref] [bib_ref] Study of brain electrolytes and organic osmolytes during correction of chronic hyponatremia...., Lien [/bib_ref].
## Figure 3: time course of brain organic osmolytes in hypertonicity.
From experimental studies in rats subjected to acute and chronic hypernatremia [bib_ref] Effects of hypernatremia on organic brain osmoles, Lien [/bib_ref] and from the stage of correction of experimental chronic hyponatremia [bib_ref] Study of brain electrolytes and organic osmolytes during correction of chronic hyponatremia...., Lien [/bib_ref].
A number of major and minor organic osmolytes has been identified in the brain [bib_ref] Control of brain volume during hyperosmolar and hypoosmolar conditions, Cullans [/bib_ref]. Glutamate, glutamine, taurine, and myoinositol are considered as major osmolytes while aspartate, alanine, glycine, choline, lysine, serine, threonine, glycerophosphorylcholine (GPC), betaine, gamma-aminobutyric acid (GABA), and phosphocreatine are classified as minor osmolytes [bib_ref] Control of brain volume during hyperosmolar and hypoosmolar conditions, Cullans [/bib_ref]. Some of these osmolytes are amino acids acquired from the circulation while others are polyols (myoinositol) or methylamines (betaine, GPC) formed locally. At least one osmolyte (GABA) is a neurotransmitter. Organic osmolyte acquisition accounts for 30-50% of the solute accumulation in the brain IC in chronic hypertonicity [bib_ref] Control of brain volume during hyperosmolar and hypoosmolar conditions, Cullans [/bib_ref] [bib_ref] Effects of hypernatremia on organic brain osmoles, Lien [/bib_ref] [bib_ref] Metabolism of glucose, amino acids, and some related metabolites in the brain..., Shank [/bib_ref] [bib_ref] Elevation of rat brain amino acids, ammonia and idiogenic osmoles induced by..., Chan [/bib_ref] [bib_ref] Taurine: a role in osmotic regulation of mammalian brain and possible clinical..., Thurston [/bib_ref] [bib_ref] Effect of acute and chronic hypernatremia on myoinositol and sorbitol concentration in..., Lohr [/bib_ref] [bib_ref] Taurine and osmoregulation: Taurine is a cerebral osmoprotective molecule in chronic hypernatremic..., Trachtman [/bib_ref] [bib_ref] Characterization of the major brain osmolytes that accumulate in salt-loaded rats, Heilig [/bib_ref] [bib_ref] The role of polyols in cerebral cell volume regulation in hypernatremic and..., Trachtman [/bib_ref] and is slow, requiring two days or more to reach a steady state. Renal medulla is physiologically subjected to rapid variations in interstitial tonicity and is endowed with a rapid mechanism of modifying its intracellular osmolyte content. The main osmolytes in the renal medulla are sorbitol, myoinositol, GPC, and betaine [bib_ref] Renal medullary organic osmolytes, Garcia-Perez [/bib_ref].
Water administration in a state of acute hypertonicity leads to rapid loss of the excess electrolytes and rapid restoration of the brain cell volume to normal. After correction of chronic hypertonic states by water administration, organic osmolyte brain content declines over two days [bib_ref] Effects of hypernatremia on organic brain osmoles, Lien [/bib_ref].
## Mechanisms of intracellular solute gain in hypertonicity
Hypertonicity causes activation of several ion transporters in cell membranes. Activation of the Na + , K + , 2Clcotransporter leads to the intracellular transfer of sodium chloride and potassium chloride. Tandem activation of Na + /H + exchange and Cl -/HCO 3 exchange leads also to sodium chloride entry into the cells. Sodium ion entering the cells is extruded through Na + /K + ATPase in exchange for potassium. Potassium chloride is the final salt gained intracellularly in hypertonicity [bib_ref] Cell volume regulatory mechanisms. An introduction . Mechanisms and Significance of Cell..., Friedrich [/bib_ref].
The mechanisms responsible for the intracellular accumulation of organic osmolytes in hypertonic states include intracellular formation and transport. Examples of solutes acquired by intracellular formation include sorbitol formed from glucose under the catalytic action of activated aldose reductase, GPC formed from phosphatidylcholine under the influence of phospholipase A 2 , and amino acids derived from autophagic proteolysis. Examples of organic osmolytes acquired by intracellular transport include myoinositol, betaine, and taurine, which are acquired through the action of Na + coupled transporters [bib_ref] Cell volume regulatory mechanisms. An introduction . Mechanisms and Significance of Cell..., Friedrich [/bib_ref]. Hypertonicity activates a variety of genes regulating metabolic functions and transporters responsible for the intracellular accumulation of organic osmolytes [bib_ref] Tonicity-dependent regulation of osmoprotective genes in mammalian cells. Mechanisms and Significance of..., Ferraris [/bib_ref]. Tonicity enhancer binding protein (TonEBP) is a transcription factor found in abundance in the brain, renal medulla, heart, liver, and activated T cells that plays a major role in hypertonicity and immune phenomena. This factor is activated by hypertonicity and regulates the expression of several osmolyte transporters [bib_ref] Large discrepancies in cellular distribution of the tonicity-induced expression of osmoprotective genes..., Maallem [/bib_ref].
The alterations of metabolism induced by hypertonicity have dire consequences. Severe hypertonicity leads to cell death by apoptosis. Apoptotic pathways activated by hypertonicity include activation of caspase-8 and release of cytochrome 3 with activation of caspase-3 and caspase-9 [bib_ref] Multiple cell death pathways are independently activated by lethal hypertonicity in renal..., Choi [/bib_ref]. Elevated levels of the tumor suppressor p53, which initiates apoptotic pathways in hypertonicity, is an important apoptotic mechanism activated by hypertonicity but apparently not the only one [bib_ref] Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress, Lambert [/bib_ref].
# Conclusions
Formulas used to calculate the volume of hypotonic solutions needed to correct the hypertonic states do not take into account ongoing fluid losses during treatment, which should be replaced. The same formulas also fail to account for the intracellular acquisition of new solute created by the hypertonicity. This new solute is the source of serum osmolality values substantially higher than those predicted by the formulas and may cause severe complications during correction of the hypertonic states. When the hypertonic states are secondary to conditions of known magnitude causing hypernatremia, the degree of observed elevation in the serum sodium concentration is equal to the degree predicted by the formulas. This last observation justifies the application of predictive formulas in the treatment of hypernatremia, provided ongoing fluid losses are also addressed.
## Additional information
[fig] The: Gibbs-Donnan equilibrium states that if there are impermeable anions in one of two compartments separated by a semipermeable membrane: (a) the concentration of electrolyte cations is higher and the concentration of electrolyte anions is lower in the compartment with the impermeable anions; (b) the concentration of anions and cations is equal in the compartment that does not contain impermeable anions; and (c) the products of the molar concentrations (anions times cations) are equal in the two compartments. mathematical expression of the Gibbs-Donnan equilibrium between the EC and the interstitial compartment is as followsinterstitial cation and anion electrolyte concentrations, respectively. [/fig]
[fig] FIGURE 1: Stages of hypertonicity caused by extracellular gain of a hypertonic solution.Stage I: Addition of a hypertonic solution to the EC prior to any mixing between the EC and the hypertonic solution. Stage II: Hypothetical stage of complete mixing of EC fluid and infused hypertonic solution prior to any interaction with the IC. Stage IIII: Final steady state after osmotic transfer of water from the IC into the EC that led to equal osmolalities in the two compartments. Ordinates: volumes. Abscissae: osmolalities. The figure was constructed assuming that V i1 = 2xV e1 , V 3 = 0.5xV e1 , and Os 3 = 2xOs 1 . The numbers within the boxes show osmolalities. [/fig]
[fig] FIGURE 2: Estimates of body water and the osmotic and sodium volume of distribution in acute hypernatremia.Shown here are the average estimates of body water (VH 2 O) measured by tritiated water dilution, the osmotic volume of distribution (VOsm) calculated by formula 11, and sodium volume of distribution (VNa) calculated by formula 12 in a study of anuric dogs infused with hypertonic saline[26]. The figure makes two points: (a) Even in acute hypertonicity, VOsm is substantially lower than VH 2 O and (b) VNa is equal to VH 2 O. The equality of VNa and VH 2 O provides the basis for applying equations 9 or 10 in the management of dysnatremias. [/fig]
[table] 2016: Argyropoulos et al. Cureus 8(5): e596. DOI 10.7759/cureus.596 [/table]
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Acute bilateral myopia induced by Triplixam: a case report
Background: Side effects of the systemic drugs used to treat eyes are not common. Triplixam is used to treat systemic hypertension and contains amlodipine, indapamide and perindopril arginine as active ingredients which might have induced the sudden myopia. The transient myopia with objective findings disappeared after the discontinuation of the drug. Case presentation: A 33-year-old male presented to the emergency department with a history of blurred vision in both eyes. Development of myopia, lens thickening, choroidal effusion and retinal striae at the macula with the increase in macular thickness was observed in both eyes. These symptoms cleared completely after the drug was discontinued. Myopisation could have been caused by lens thickening and changing its refractive index as a result of allergic or idiosyncratic reaction of the ciliary body. Retinal striae may be caused by the volume effect of the choroidal effusion. Conclusion: Our report describes the adverse effect of Triplixam, probably resulting from its ingredient indapamide. Although indapamide is a common drug used in the treatment of systemic hypertension, it is important for cardiologists, general practitioners and other physicians to be aware of the possible adverse effect of Triplixam.
# Background
Ocular side-effects induced by drugs are rare. Transient myopia induced by drugs has been reported to be caused by the ingestion of sulphonamide-derived drugs, such as methazolamide, sulfasalazine, indapamide, acetazolamide, hydrochlorothiazide, ethoxzolamide, psychotropic drugs, etc.. In this paper a case of sudden vision blurring in a young male who used Triplixam is presented. Triplixam is a drug used to treat systemic hypertension that combines three active ingredients: amlodipine, indapamide and perindopril arginine. Indapamide is a sulphonamide-derived diuretic which might have induced the myopia. The transient myopia disappeared after the discontinuation of the drug.
## Case presentation
A 33-year-old male presented to the emergency department with a history of blurred vision in both eyes. The patient started experiencing blurred vision in the morning after using a computer during the night before. The patient did not feel any pain or other symptoms and the patient had never worn glasses. The medical history of the patient revealed uncontrolled hypertension that had lasted for several years for which he had started taking medications recently. He was taking Triplixam (amlodipine 5 mg, indapamide 2.5 mg, perindopril arginine 5 mg) (Les Laboratoires Servier, France) once per day, prescribed by his physician for his systemic hypertension. He started this medication 4 days prior to the appearance of vision blurring. The visual acuity at presentation was 20/40 in the right eye and 20/100 in the left eye. The intraocular pressure was 14.6 mmHg in both eyes (determined by using a Schiøtz indentation tonometer).
The results of slit lamp biomicroscopy showed that the cornea, lens, and vitreous were normal, however, a retinal striae radiated from the fovea in the both eyes. Fundus photography was performed to document the retinal striae. After performing a refraction test, the visual acuity of the patient improved to 20/20 in both eyes with a spherical correction of − 1.75 dioptre sphere (DS) in the right eye and − 2.00 DS in the left eye. Objective refraction was − 1.75 DS/− 0.50 dioptre cylinder (DC) × 21°in the right eye and − 2.00 DS/− 0.25 DC × 165°in the left eye. A cycloplegic refraction revealed a value of − 1.50 DS/− 0.25 DC × 36°in the right eye and − 2.00 DS/− 0.25 DC × 136°in the left eye. The central corneal thickness and corneal endothelial morphology were evaluated using non-contact specular microscopy and there were no pathological changes observed. The initial B-scan ultrasonography showed diffuse choroidal thickening. Optical biometry was performed to measure the axial length which was 23.70 mm and 23.76 mm in the right and the left eye, respectively. The lens thickness was 4.19 mm and 4.18 mm in the right and the left eye, respectively. The anterior chamber depth was similar in both eyes at 3.60 mm. An optical coherence tomography (OCT) showed a mild thickening of the macula at 240 and 243 μm in the right and the left eye, respectively.
The patient was advised to stop taking a triple drug combination, Triplixam, and was referred to a cardiologist for a further investigation. The patient reported that blurred vision reversed 4 days after discontinuing the medication. After 16 days, the patient was examined once again as he was travelling abroad due to a planned vacation. His visual acuity had improved to 20/20 without any refractive error. Intraocular pressure was 15.9 mmHg in both eyes (determined by using a Schiøtz indentation tonometer). Refraction results revealed a value of 0.00 DS/− 0.25 DC × 15°in the right eye and 0.00 DS/ − 0.25 DC × 165°in the left eye. Repeated measurements of endothelial morphology and corneal thickness showed no significant differences. Optical biometry revealed no difference in axial length. The lens thickness decreased to 3.97 mm and 3.96 mm in the right and the left eye, respectively. The anterior chamber depth increased to 3.68 mm and 3.69 mm in the right and the left eye, respectively. Fundus examination revealed the disappearance of retinal striae at the macula and peripheral choroidal effusion had also resolved in B-scan.. The OCT was repeated which showed a reduction in the thickening of the macula, 230 μm in both eyes. A presumptive diagnosis of drug-induced acute myopia due to an ingredient of Triplixamdiuretic indapamidewas made. Electrocardiography, veloergometry and echocardiography tests were performed, and no pathology was revealed. Patient was prescribed by a cardiologist to take
# Discussion and conclusions
We report a case of acute myopia caused by oral consumption of Triplixam which is a combination of three active ingredients: amlodipine, indapamide and perindopril arginine. Triplixam is used to treat systemic hypertension. There are reports that diuretics can be the cause of acute myopia; the active ingredient indapamide was the most likely cause of acute myopisation.
Other authors have reported a case of an acute myopia when patient was treated for arterial hypertension with a combination of indapamide and amlodipine and all symptoms reversed when only amlodipine was left to control hypertension. These findings exclude the possible myopic effect of amlodipine. Also, there is no evidence that perindopril arginine could cause acute myopia. Indapamide is a sulphonamide diuretic and is generally used to treat hypertension. The mechanism of myopisation is not completely understood. Literature describes paradoxical cases of idiopathic oedema caused by diuretic therapy. Myopisation might have been caused by a lens thickening and changing its refractive index as a result of an allergic or idiosyncratic reaction of the ciliary body. Previously mentioned authors revealed that a more precise ultrasound biomicroscopy and mode B ultrasonography disclosed bilateral ciliochoroidal effusion with anterior rotation of the ciliary body and iridocorneal angle narrowing, possibly resulting in sudden myopia. The reported retinal striae may have been caused by vitreoretinal traction.
Other authors report the presence of striae due to internal limiting membrane folds caused by the volume effect of the choroidal effusion. In the analysed case, cycloplegics had no effect and the diagnosis of myopia in association with ciliary spasm was withdrawn. The patient did not report severe eye pain, his intraocular pressure was within the normal range and no other acute angle-closure glaucoma symptoms were observed. After discontinuing Triplixam, we believe that no further treatment is required. Patient was referred to a cardiologist to change the drug group in order to control arterial hypertension. Indapamide in combination with other ingredients of Triplixam was probably the sole cause of this adverse reaction. Although indapamide is a common drug used to treat systemic hypertension, it is important that cardiologists, general practitioners and other physicians are aware of possible adverse effects of Triplixam.
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Unbiased Taxonomic Annotation of Metagenomic Samples
The classification of reads from a metagenomic sample using a reference taxonomy is usually based on first mapping the reads to the reference sequences and then classifying each read at a node under the lowest common ancestor of the candidate sequences in the reference taxonomy with the least classification error. However, this taxonomic annotation can be biased by an imbalanced taxonomy and also by the presence of multiple nodes in the taxonomy with the least classification error for a given read. In this article, we show that the Rand index is a better indicator of classification error than the often used area under the receiver operating characteristic (ROC) curve and F-measure for both balanced and imbalanced reference taxonomies, and we also address the second source of bias by reducing the taxonomic annotation problem for a whole metagenomic sample to a set cover problem, for which a logarithmic approximation can be obtained in linear time and an exact solution can be obtained by integer linear programming. Experimental results with a proof-of-concept implementation of the set cover approach to taxonomic annotation in a next release of the TANGO software show that the set cover approach further reduces ambiguity in the taxonomic annotation obtained with TANGO without distorting the relative abundance profile of the metagenomic sample.
# Introduction
N ext-generation sequencing technologies have moved forward the development of metagenomics, a new field of science devoted to the study of microbial communities by the analysis of their genomic content, directly sequenced from the environment [bib_ref] A bioinformatician's guide to metagenomics. Microbiol, Kunin [/bib_ref] [bib_ref] 6, e1000667. Youden, W.J. 1950. Index for rating diagnostic tests, Wooley [/bib_ref] [bib_ref] Metagenomics: A guide from sampling to data analysis. Microb, Thomas [/bib_ref]. A sequenced metagenomic sample consists of a large number of relatively short DNA or RNA fragments, called reads, and one of the first steps in the computational analysis of a metagenomic sample is the identification of the organisms present in the sequenced environment and their relative abundance, that is, the classification of the metagenomic sample.
In this article, we focus on the taxonomic annotation problem, that is, the classification of the reads from a metagenomic sample using a reference taxonomy, for which we adapt some basic notions from statistical classification in machine learning. We abstract away from the computational problem of mapping reads to reference sequences, and assume that a set of candidate sequences in a reference taxonomy is given for each read in the metagenomic sample to be classified. These candidate sequences are usually obtained either by sequence composition methods (those reference sequences with oligonucleotide frequencies within a given distance threshold to the oligonucleotide frequencies of the read) or by sequence similarity methods (those reference sequences that the read can be aligned to within a given threshold of sequence similarity, or those reference sequences that the read can be mapped to with at most a given number of mismatches).
In a statistical binary classification problem, the confusion matrix [fig_ref] Table 1: Confusion Matrix for a Binary Classification Problem FN, false negative [/fig_ref] shows the number of correctly and incorrectly classified instances of each class. True positives (TP) are the correctly classified positive instances, true negatives (TN) are the correctly classified negative instances, false positives (FP) are the misclassified negative instances, and false negatives (FN) are the misclassified positive instances. The TP rate, sensitivity, or recall R of a classification is the ratio TPR = TP=(TP + FN) of TP to the total number of positive instances, the FP rate is the ratio FPR = FP=(FP + TN) of FP to the total number of negative instances, the TN rate or specificity is the ratio TNR = TN=(FP + TN) of TN to the total number of negative instances, and the FN rate is the ratio FNR = FN=(TP + FN) of FN to the total number of positive instances. Furthermore, the precision of a classification is the ratio P = TP=(TP + FP) of TP to the total number of positive predictions. They are usually combined into a single indicator of classification error as either the area under the receiver operating characteristic (ROC) curve AUC = (TPR -FPR + 1)=2 or the F-measure, which is the harmonic mean F = 2=(1=P + 1=R) of precision and recall [bib_ref] Evaluation: From precision, recall and F-measure to ROC, informedness, markedness and correlation, Powers [/bib_ref].
In a metagenomic classification problem, the annotation of a read as coming from a particular sequence in a reference taxonomy often involves solving the ambiguity of multiple candidate sequences, caused among other factors by reads being not long enough to ensure a unique identification of the reference sequences they come from. Reference taxonomies are rooted trees, with the leaves labeled by sequences at the taxonomic rank of species or strain, and these ambiguities are solved by annotating reads as coming from internal nodes, at higher taxonomic ranks in the reference taxonomy. When classifying a read as coming from an internal node in a reference taxonomy [fig_ref] Definition 1: Let TP, TN, FP, and FN be the number of TP, FP,... [/fig_ref] , the leaves under the internal node are TP if they are labeled by candidate sequences, otherwise they are FP, and the remaining leaves under the lowest common ancestor (LCA) of the candidate sequences are FN if they are labeled by candidate sequences; otherwise, they are TN. Annotating a read as coming from the LCA of the candidate sequences in a reference taxonomy [bib_ref] Microbial community analysis using MEGAN, Huson [/bib_ref] maximizes precision, as in that case there are no TN and no FN, but at the expense of specificity, because the number of FP in a reference taxonomy can be very large. Annotating a read as coming from an internal node with the largest F-measure value [bib_ref] Flexible taxonomic assignment of ambiguous sequencing reads, Clemente [/bib_ref] [bib_ref] Further steps in TANGO: Improved taxonomic assignment in metagenomics, Alonso [/bib_ref] [bib_ref] BioMaS: A modular pipeline for bioinformatic analysis of metagenomic amplicons, Fosso [/bib_ref] [bib_ref] MetaShot: An accurate workflow for taxon classification of hostassociated microbiome from shotgun..., Fosso [/bib_ref] minimizes the classification error as a combination of precision and sensitivity.
However, there are at least two sources of bias in the taxonomic annotation of a metagenomic sample. One the one hand, reference taxonomies are imbalanced, that is, the instances of one class significantly outnumber the instances of the other classes, and this can be observed at any taxonomic rank. For example, the NCBI Taxonomy [bib_ref] Type material in the NCBI taxonomy database, Federhen [/bib_ref] , which is the most comprehensive taxonomic reference to date, includes as of March 13, 2017, an imbalanced number of sequences for Bacteria (1,412,065), . Within the Bacteria, for example, there is also an imbalanced number of sequences for the Actinobacteria (593,837), Proteobacteria (440,315), Firmicutes (245,632), Bacteroidetes (77,866), Planctomycetes (8899), Fusobacteria (7789), and others (37,727).
In a statistical binary classification problem, imbalanced data sets result in a good coverage of the positive instances and a frequent misclassification of the negative instances, since most of the standard machine learning algorithms consider a balanced training set [bib_ref] An insight into classification with imbalanced data: Empirical results and current trends..., López [/bib_ref]. In a metagenomic classification problem, an imbalanced reference taxonomy may also yield an imbalance between the positive and negative classes, because the larger the clade of the LCA in a reference taxonomy of the candidate sequences for a read, the larger the negative class for the classification of the read. In this article, we show that this is in general not the case, and we also show that the Rand index is a better indicator of classification error than the often used area under the ROC curve and F-measure, when the reference taxonomy is imbalanced and also for balanced reference taxonomies. Another source of bias in the taxonomic annotation of a metagenomic sample lies in the existence of multiple candidate nodes in a reference taxonomy with the least classification error for a given read, one of which is usually chosen arbitrarily for the taxonomic annotation of the read [bib_ref] Flexible taxonomic assignment of ambiguous sequencing reads, Clemente [/bib_ref] [bib_ref] Further steps in TANGO: Improved taxonomic assignment in metagenomics, Alonso [/bib_ref]. Instead of breaking ties independently for each read in a metagenomic sample, we show in this article that the shift from a one-sequence-read-at-a-time view to a whole-set-of-sequence-reads view yields a better resolution of any remaining ambiguities in the taxonomic annotation of a metagenomic sample.
## Taxonomic annotation using imbalanced reference taxonomies
Recall that in a metagenomic classification problem, an imbalanced reference taxonomy yields an imbalance between the positive and negative classes. Let us define the balance ratio of a classification problem as the ratio of the size of the positive class to the size of the negative class. Recall also that the reference taxonomies used in metagenomic classification are highly imbalanced. It turns out that balanced and imbalanced reference taxonomies yield exactly the same metagenomic classification problems, as long as they have the same number of internal nodes. Some evidence supporting this observation follows.
The topology of the most possible balanced binary reference taxonomy is a complete binary tree, as every internal node (and also the root) has two descendant clades of exactly the same size. On the other hand, the topology of the least possible balanced binary reference taxonomy is a rooted caterpillar, as every internal node (and also the root) has one big descendant clade and one small (with only one node) descendant clade. Borrowing the notion of total cophenetic index for phylogenetic trees [bib_ref] A new balance index for phylogenetic trees, Mir [/bib_ref] to measure the balance of reference taxonomies, complete binary trees have indeed the minimum value while rooted caterpillars have the maximum value. Notice, however, that the total cophenetic index of the NCBI Taxonomy [bib_ref] Type material in the NCBI taxonomy database, Federhen [/bib_ref] restricted to the standard taxonomic ranks (Kingdom, Phylum, Class, Order, Family, Genus, and Species) is 206,110,330,551, which represents only 0.00032060% of the interval between the minimum value (727,931) and the maximum value for the number of taxa in the restricted NCBI Taxonomy. Now, in a metagenomic classification problem, any subset of the leaves of a reference taxonomy may be labeled by the candidate sequences for the classification of a given read. For a given subset of the leaves of a reference taxonomy, each candidate internal node (at or under the LCA of the subset of the leaves) for the taxonomic annotation of the read yields a certain number of TP, FP, TN, and FN. For example, for the reference taxonomy in [fig_ref] Definition 1: Let TP, TN, FP, and FN be the number of TP, FP,... [/fig_ref] , the subset of grayed leaves yields, for the candidate internal node j, a metagenomic classification problem with TP = 3, FP = 1, TN = 3, FN = 1, and thus, balance ratio (3 + 1)=(1 + 3) = 1. [fig_ref] Table 2: Distribution of TP, FP, TN, FN Values [/fig_ref] shows the distribution of the number of TP, FP, TN, and FN for all subsets of the leaves of a reference taxonomy and for every candidate internal node for the taxonomic annotation of a read having as candidate sequences the subset of the leaves, for both a complete binary tree and a rooted caterpillar with 8 leaves.
The resulting distribution of TP + FN values [fig_ref] Table 2: Distribution of TP, FP, TN, FN Values [/fig_ref] , right) is exactly the same in both cases, and thus, a complete binary tree and a rooted caterpillar with the same number of leaves have the same balance ratio. In fact, any two reference taxonomies for the same taxa have the same balance ratio as long as they have the same number of internal nodes, because they yield a metagenomic classification problem for any subset of the leaves and for any candidate internal node, and TP + FN equals the number of leaves in the subset.
Let us assume that the reads in a metagenomic sample to be classified come from known sequences in a reference taxonomy, as it is usually the case in the taxonomic annotation of metagenomic samples, whereas reads coming from novel sequences are annotated by using clustering methods instead. Given a read and a set of candidate sequences in a reference taxonomy, the taxonomic annotation of the read at a certain node in the clade of the LCA in the reference taxonomy of the set of candidate sequences can then be taken to be correct if, and only if, the candidate sequence that the read comes from lies in the clade of the node at which it is annotated.
Based on this observation, we have studied the performance of some of the most often used indicators of classification error: the Yule / [bib_ref] On the methods of measuring association between two attributes, Yule [/bib_ref] , also known as Matthews correlation coefficient [bib_ref] Comparison of the predicted and observed secondary structure of T4 phage lysozyme, Matthews [/bib_ref] , the area under the ROC curve, the Youden J (Youden, 1950), the F-measure [bib_ref] Evaluation: From precision, recall and F-measure to ROC, informedness, markedness and correlation, Powers [/bib_ref] , the Jaccard similarity coefficient [bib_ref] Etude comparative de la distribution florale dans une portion des Alpes et..., Jaccard [/bib_ref] , and the Rand index [bib_ref] Objective criteria for the evaluation of clustering methods, Rand [/bib_ref] , in the taxonomic annotation of metagenomic samples. The Yule / is given by
[formula] / = TP TN -FP FN ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (TP + FP)(TP + FN)(TN + FP)(TN + FN) p [/formula]
The Youden J is given by The area under the ROC curve is given by
[formula] J = TP TN -FP FN (TP + FN)(FP + TN)AUC = 1 2 TP TP + FN + TN FP + TN [/formula]
The F-measure is given by
[formula] F = 2 TP 2 TP + FP + FN [/formula]
The Jaccard similarity coefficient is given by
[formula] C = TP TP + FP + FN [/formula]
The Rand index is given by
[formula] R = TP + TN TP + FP + TN + FN [/formula]
If the denominator in any of these formulas is zero, the value of the indicator is arbitrarily set to zero.
We have computed the value of all these indicators of classification error for each possible set of candidate sequences in a reference taxonomy and for each possible candidate node for the taxonomic annotation of a read coming from each of the candidate sequences, for different taxonomic reference topologies: complete binary trees that have the largest possible balance but yield the least balanced metagenomic classification problems, and rooted caterpillars that have the smallest possible balance but yield the most balanced metagenomic classification problems. For these classification problems, we have counted the number of times the taxonomic annotation is correct, that is, the number of times the candidate sequence that the read comes from lies in the clade of the node in the reference taxonomy at which it is annotated.
The results [fig_ref] Table 3: Total Number of Correct Taxonomic Annotations Under the Yule [/fig_ref] show that the worst indicator of classification error is the Yule /, followed by AUC and the Youden J (which are equivalent, as J = 2 AUC -1), the F-measure and the Jaccard similarity coefficient C (which are also equivalent, as C = F=(2 -F)), and that the Rand index R is the best indicator of classification error for the taxonomic annotation of metagenomic samples. This can be explained by the fact that in a metagenomic classification problem, we focus on the correct classification of a correct taxonomic annotation while in a statistical classification problem in machine learning, where both positive and negative instances are taken into account, correlation measures such as the Yule / (which is equivalent to the Pearson correlation coefficient for binary classification problems) often are the best indicators of classification error. Now, the taxonomic annotation of a metagenomic sample involves obtaining the candidate nodes in a reference taxonomy with the least classification error (for a given indicator) for each of the reads in the metagenomic sample. We have proved in [bib_ref] Flexible taxonomic assignment of ambiguous sequencing reads, Clemente [/bib_ref] that, when the F-measure is taken as indicator, it suffices to consider candidate nodes that are either candidate sequences themselves, or the LCA of two or more candidate sequences in the reference taxonomy. That is, it suffices to consider as candidate nodes the LCA skeleton tree [bib_ref] New common ancestor problems in trees and directed acyclic graphs, Fischer [/bib_ref] of the set of candidate sequences for a given read.
We prove below that it also suffices to consider the LCA skeleton tree when the Yule /, the Youden J, the area under the ROC curve, the Jaccard similarity coefficient, or the Rand index is taken as indicator of classification error.
Let T be a reference taxonomy, let M i be the set of candidate sequences for the classification of read i, and let T i be the subtree of T rooted at the LCA of M i . See [fig_ref] Definition 1: Let TP, TN, FP, and FN be the number of TP, FP,... [/fig_ref] for a schematic view.
Definition 3. A node j in T i is called relevant if it is equal to a candidate sequence in M i or equal to the LCA of two or more candidate sequences in M i .
Also, for every node j in T i , let T i‚ j be the subtree of T i rooted at j, let L i be the set of all candidate sequences in T i , and let N i be the set of all candidate sequences in T i that do not belong to M i (hence,
[formula] L i = M i [ N i ). [/formula]
Similarly, let M i‚ j be the set of all candidate sequences in T i‚ j that belong to M i , let N i‚ j be the set of all candidate sequences in T i‚ j that do not belong to M i‚ j , and let L i‚ j = M i‚ j [ N i‚ j . Using this notation, for the taxonomic annotation at node j of a read i with candidate sequences M i [fig_ref] Definition 1: Let TP, TN, FP, and FN be the number of TP, FP,... [/fig_ref] , the TP are TP i‚ j = M i‚ j , the FP are FP i‚ j = N i‚ j , the TN are TN i‚ j = N i nN i‚ j , and the FN are FN i‚ j = M i nM i‚ j . Let C i‚ j be the Jaccard correlation coefficient for node j in T i , that is, C i‚ j = TP i‚ j =(TP i‚ j + FP i‚ j + FN i‚ j ). Similarly, let Y i‚ j , J i‚ j , A i‚ j , and R i‚ j be the Yule /, the Youden J, the area under the ROC curve, and the Rand index for node j in T i , respectively. We have:
Theorem 1. For each node j in T i , there exists a relevant node j 0 such that Y i‚ j 0 ! Y i‚ j , J i‚ j 0 ! J i‚ j , A i‚ j 0 ! A i‚ j ,C i‚ j 0 ! C i‚ j , and R i‚ j 0 ! R i‚ j .
Proof. Suppose that j is a node in T i that is not relevant. In particular, j is not the root of T i . Let j 0 be the LCA of the candidate sequences in M i‚ j . Clearly, j 0 is relevant and it is a strict descendant of j, and therefore, since T i‚ j 0 is a strict subtree of T i‚ j , jM i‚ j j = jM i‚ j 0 j while jN i‚ j j > jN i‚ j 0 j.
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[formula] Let TP = jM i‚ j j, FP = jN i‚ j j, FN = jM i j -jM i‚ j j, TN = jN i j -jN i‚ j j and, similarly, let TP 0 = jM i‚ j 0 j, FP 0 = jN i‚ j 0 j, FN 0 = jM i j -jM i‚ j 0 j, TN 0 = jN i j -jN i‚ j 0 j. We have that TP 0 = TP, FP 0 FP, FN 0 = FN, TN 0 ! TN, and TN 0 + FP 0 = TN + FP. [/formula]
Yule /: It has to be proved that
[formula] TP 0 TN 0 -FP 0 FN 0 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi (TP 0 + FP 0 )(TP 0 + FN 0 )(TN 0 + FP 0 )(TN 0 + FN 0 ) p ! TP TN -FP FN ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (TP + FP)(TP + FN)(TN + FP)(TN + FN) p [/formula]
Since TN 0 + FP 0 = TN + FP, TP 0 + FN 0 = TP + FN, TP 0 = TP, and FN 0 = FN, it suffices to prove that
[formula] TP TN 0 -FP 0 FN ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi (TN 0 + FN)(TP 0 + FP 0 ) p > TP TN -FP FN ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (TN + FN)(TP + FP) p(1) [/formula]
where TN 0 > TN and FP 0 < FP. We shall rewrite the numerators. It is straightforward to check that if we denote TP + FN = P 0 , FP + TN = FP 0 + TN 0 = N 0 , P 0 + N 0 = M, TP + FP = P, and TP + FP 0 = P 0 , then
[formula] TP TN 0 -FP 0 FN ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi (TN 0 + FN)(TP 0 + FP 0 ) p = M TP -P 0 P 0 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P 0 (M -P 0 ) p TP TN -FP FN ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (TN + FN)(TP + FP) p = M TP -P 0 P ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P(M -P) p [/formula]
and, therefore, Equation (1) becomes
[formula] M Á TP -P 0 P ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P(M -P) p < M Á TP -P 0 P 0 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P 0 (M -P 0 ) p(2) [/formula]
where 0 < TP < P 0 < P < M. Moreover, notice that TP < P 0 because j is not the root of T i . Consider the function
[formula] u(x) = M Á TP -P 0 x ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi x(M -x) p [/formula]
Equation (2) says that u(P) < u(P 0 ) if 0 < P 0 < P < M. So, to complete the proof of the statement, it is enough to prove that the function u(x) is decreasing on 0 < x < M. Its first derivative is
[formula] u 0 (x) = M(2TP -P 0 )x -M 2 Á TP ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi (x(M -x)) 3 p Then u 0 (x) < 05(2TP -P 0 )x -M Á TP < 0 Now, if 2TP P 0 , then u 0 (x) < 0 for every x, while if 2TP > P 0 , then u 0 (x) < 05x < M Á TP 2TP -P 0 and in this case M < M Á TP 2TP -P 0 because M < M Á TP 2TP -P 0 52M Á TP -MP 0 < M Á TP5M(TP -P 0 ) < 0 [/formula]
and the latter inequality holds because TP < P 0 . This implies that, also in this case, if x < M, then u 0 (x) < 0.
Area under the ROC curve: It has to be proved that
[formula] TP 0 (FP 0 + TN 0 ) + TN 0 (TP 0 + FN 0 ) (TP 0 + FN 0 )(FP 0 + TN 0 ) ! TP(FP + TN) + TN(TP + FN) (TP + FN)(FP + TN) [/formula]
We have that (TP 0 + FN 0 )(FP 0 + TN 0 ) = (TP + FN)(FP + TN) and TP 0 (FP 0 + TN 0 ) = TP(FP + TN). Then, it suffices to prove that TN 0 (TP 0 + FN 0 ) ! TN(TP + FN). However, TP 0 = TP, FN 0 = FN, TN 0 ! TN and thus, the inequality follows.
Rand index: It has to be proved that
[formula] TP 0 + TN 0 TP 0 + FP 0 + TN 0 + FN 0 ! TP + TN TP + FP + TN + FN [/formula]
We have that TP 0 = TP, FN 0 = FN, TN 0 ! TN, FP 0 + TN 0 = FP + TN and thus, the inequality follows.
Corollary 1. The Yule Y i‚ j , the Youden J i‚ j , the area under the ROC curve A i‚ j , the Jaccard correlation coefficient C i‚ j , and the Rand index R i‚ j only need to be computed for nodes j in T i that are relevant.
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## A set cover approach to taxonomic annotation
Let us recall fromthat an instance of the set cover problem is a collection C of subsets of a finite set X whose union is X, and a solution to the set cover problem is a smallest subset C 0 C such that every element in X belongs to at least one member of C 0 . The set cover problem is nondeterministic polynomial time complete (NP-complete), but a logarithmic approximation can be computed in linear time [bib_ref] Approximation algorithms for combinatorial problems, Johnson [/bib_ref] [bib_ref] A linear-time approximation algorithm for the weighted vertex cover problem, Bar-Yehuda [/bib_ref] and an exact solution can be obtained by integer linear programming.
Recall also that in a metagenomic classification problem, there are often multiple candidate nodes in a reference taxonomy with the least classification error for a given read. As a set cover problem, the set of elements X is the set of candidate nodes in a reference taxonomy with the least classification error for the reads in a metagenomic sample, and the collection C of subsets of X is the collection of sets of candidate nodes in the reference taxonomy with the least classification error for each read.
The following example is adapted from ; see [fig_ref] Definition 2: Let TP, TN, FP, and FN be the number of TP, FP,... [/fig_ref].
Example 1. Consider a metagenomic sample with reads x 1 ‚ . . . ‚ x 12 and candidate nodes in a reference taxonomy with the least classification error as follows: fy 1 ‚ y 3 g for x 1 , fy 1 ‚ y 4 g for x 2 , fy 1 ‚ y 5 g for x 3 , fy 1 ‚ y 3 g for x 4 , fy 1 ‚ y 2 ‚ y 4 g for x 5 , fy 1 ‚ y 2 ‚ y 5 g for x 6 , fy 3 ‚ y 4 g for x 7 , fy 2 ‚ y 4 g for x 8 , fy 2 ‚ y 5 g for x 9 , fy 3 ‚ y 6 g for x 10 , fy 4 ‚ y 6 g for x 11 , and fy 5 g for x 12 : Then, as an instance of the set cover problem, X = fx 1 ‚ . . . ‚ x 12 g and C = fy 1 . . . ‚ y 6 g, where y 1 = fx 1 ‚ x 2 ‚ x 3 ‚ x 4 ‚ x 5 ‚ x 6 g, y 2 = fx 5 ‚ x 6 ‚ x 8 ‚ x 9 g, y 3 = fx 1 ‚ x 4 ‚ x 7 ‚ x 10 g, y 4 = fx 2 ‚ x 5 ‚ x 7 ‚ x 8 ‚ x 11 g, y 5 = fx 3 ‚ x 6 ‚ x 9 ‚ x 12 g, and y 6 = fx 10 ‚ x 11 g.
In a solution C 0 to a metagenomic classification problem viewed as a set cover problem (X‚ C), each read in X is annotated to a node in C 0 C. Such a taxonomic annotation is not necessarily unique, and there may still be ambiguities in the classification of the metagenomic sample. For the problem instance from Example 1, the smallest solution is fy 3 ‚ y 4 ‚ y 5 g, which implies the taxonomic annotation of reads x 1 , x 4 , and x 10 to node y 3 , reads x 2 , x 5 , x 8 , and x 11 to node y 4 , reads x 3 , x 6 , x 9 , and x 12 to node y 5 , and read x 7 to either node y 3 or node y 4 in the reference taxonomy. The greedy algorithm of [bib_ref] Approximation algorithms for combinatorial problems, Johnson [/bib_ref] yields the approximate solutions fy 1 ‚ y 4 ‚ y 5 ‚ y 3 g and fy 1 ‚ y 4 ‚ y 5 ‚ y 6 g.
The taxonomic annotation of a metagenomic sample can thus be seen as the reduction, and ideally the removal, of ambiguity in the identification of the reads in the metagenomic sample, where a read is ambiguous if it is annotated to more than one node in a reference taxonomy. Viewing the metagenomic classification problem as a set cover problem, an element of X is ambiguous if it belongs to more than one subset of the collection C 0 C. The subsets of a set cover overlap on ambiguous elements.
Definition 4. Let X be a finite set and let C be a collection of subsets of X whose union is X. The overlap of a set cover C 0 C is the total size of the subsets minus the size of X.
## Fig. 2.
Left: A metagenomic classification problem viewed as a set cover problem. X is the set of reads from a metagenomic sample, and C is the collection of candidate nodes in the reference taxonomy with the least classification error for some reads from the metagenomic sample. Right: The smallest solution to the set cover problem instance.
Let the size of a set cover be the number of subsets of X that it contains, and let the total size of a set cover be the total size of the subsets of X that it contains. This corresponds to set cover problems I and II in [bib_ref] Approximation algorithms for combinatorial problems, Johnson [/bib_ref]. It turns out that a set cover of smallest size does not necessarily have the least overlap, while a set cover of smallest total size always has the least overlap.
Proposition 1. A set cover with the least number of subsets does not necessarily have the least overlap.
Proof. Let X = f1‚ . . . ‚ ng and assume, without loss of generality, that n = 2k for k ! 3. Let S be the following collection of subsets of X:
[formula] f1‚ 2g‚ f3‚ 4g‚ . . . ‚ fn -1‚ ng‚ f1‚ . . . ‚ n -1g‚ f2‚ . . . ‚ ng [/formula]
The set cover f1‚ . . . ‚ n -1g‚ f2‚ . . . ‚ ng has size 2, which is the smallest possible for S and X, and overlap n. The set cover f1‚ . . . ‚ n -1g‚ fn -1‚ ng also has size 2, but it has overlap 1. Same for the set cover f1‚ 2g‚ f2‚ . . . ‚ ng, and S and X have no other set cover of size 2. However, the set cover f1‚ 2g‚ f3‚ 4g‚ . . . ‚ fn -1‚ ng has size n=2 and overlap 0, which is the least possible overlap.
-The following result follows directly from Definition 4.
Corollary 2. A set cover with the least total size of subsets has the least overlap.
Based on the solution of a set cover problem with the least total size of subsets, the abundance profile of a metagenomic sample is given by the proportion of reads mapped to each node in the set cover, adjusted by a uniform distribution of any still ambiguous reads among all the nodes in the set cover that they are mapped to.
Example 2. The relative abundance profile of the solution to the set cover view of the metagenomic classification problem of Example 1 is as follows: y 3 has a relative abundance of (1 + 1 + 0:5 + 1)=12 = 29:17% y 4 has a relative abundance of (1 + 1 + 0:5 + 1 + 1)=12 = 37:50% y 5 has a relative abundance of (1 + 1 + 1 + 1)=12 = 33:33%
# Experimental results
We have implemented the set cover approach to taxonomic annotation in a next release of the TANGO software [bib_ref] Flexible taxonomic assignment of ambiguous sequencing reads, Clemente [/bib_ref] [bib_ref] Further steps in TANGO: Improved taxonomic assignment in metagenomics, Alonso [/bib_ref] , which belongs in the BioMaS [bib_ref] BioMaS: A modular pipeline for bioinformatic analysis of metagenomic amplicons, Fosso [/bib_ref] and MetaShot [bib_ref] MetaShot: An accurate workflow for taxon classification of hostassociated microbiome from shotgun..., Fosso [/bib_ref] pipelines. The new implementation of TANGO consists of the following: a first Python script for extracting the candidates matches for each read from the BLAST output, a second Python script for taxonomic annotation using the NCBI Taxonomy [bib_ref] Type material in the NCBI taxonomy database, Federhen [/bib_ref] , based on the ETE Toolkit (Huerta-Cepas et al., 2016), a third Python script for taxonomic annotation using the Greengenes taxonomy [bib_ref] An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses..., Mcdonald [/bib_ref] , fourth Python script for resolving any remaining ambiguities by finding an exact solution to a set cover problem with the least total size of subsets, based on Gurobi Optimizer (Gurobi Optimization, Inc., 2017), and a fifth Python script for obtaining the relative abundance profile of the metagenomic sample.
While the second and third scripts process the input metagenomic sample one-sequence-read-at-a-time, the fourth script processes the output of the second or third script for the whole set of reads.
When using BLAST to map the reads to target sequences in the chosen reference taxonomy, the candidate matches for a read are those with the same E-value as the top hit. Notice that TANGO can be used with any read mapping tool alternative to BLAST [see [bib_ref] A survey of sequence alignment algorithms for next-generation sequencing, Li [/bib_ref] [bib_ref] Mapping reads on a genomic sequence: An algorithmic overview and a practical..., Schbath [/bib_ref] for a survey] by adapting the first script to the output format of the particular tool. Notice also that TANGO can be used for the taxonomic annotation of both amplicon reads [bib_ref] A renaissance for the pioneering 16s rRNA gene, Tringe [/bib_ref] , with an amplicon reference taxonomy such as RDP [bib_ref] Ribosomal Database Project: Data and tools for high throughput rRNA analysis, Cole [/bib_ref] , , or SILVA [bib_ref] The SILVA ribosomal RNA gene database project: Improved data processing and web-based..., Quast [/bib_ref] , and shotgun reads [bib_ref] Sequencing technologies-the next generation, Metzker [/bib_ref] , with a whole-genome reference taxonomy such as the NCBI Reference Sequence database [bib_ref] Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional..., O'leary [/bib_ref].
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To assess the reduction in ambiguity of the set cover approach as opposed to the TANGO approach to taxonomic annotation, we have classified a representative subset of 302,581 reads from the human microbiome metagenomic data set of [bib_ref] Moving pictures of the human microbiome, Caporaso [/bib_ref] (available from ftp://ftp.microbio.me/qiime/tutorial_files/mov-ing_ pictures_tutorial-1.9.0.tgz) using the plain TANGO approach, the plain set cover approach, and the combined TANGO plus set cover approach. As illustrated in , when mapping the 302,581 reads from the human microbiome metagenomic data set to the 99,322 microbial sequences in release 13.5 of the Greengenes taxonomy clustered at 97% identity and classifying them with TANGO, there are no reads with more than three candidate annotations and, when refining the TANGO output with the set cover approach, the number of unambiguous reads raises from 300,907 to 301,101, the number of reads with two candidate annotations drops from 200 to only 9, and the number of reads with three candidate annotations drops from 3 to 0.
Furthermore, to also assess the influence of the reference taxonomy in the taxonomic annotation of the metagenomic data set, we have mapped these 302,581 reads using release 2.2.31 of BLAST [bib_ref] Basic local alignment search tool, Altschul [/bib_ref] to the microbial sequences in release 13.5 of the Greengenes taxonomy [bib_ref] An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses..., Mcdonald [/bib_ref] clustered at various identity percent values, ranging from 61% to 100% [fig_ref] Table 4: Average Ambiguity of BLAST Matches, TANGO Taxonomic Annotations, Nontaxonomic Annotations with the... [/fig_ref]. The reduction in ambiguity follows a similar pattern: at 99% identity, the number of unambiguous reads raises from 300,916 to 301,111, the number of reads with two candidate annotations drops from 193 to only 1, and the number of reads with three candidate annotations drops from 3 to 0, and, at 100% identity, the number of unambiguous reads raises from 300,941 to 301,109, the number of reads with two candidate annotations drops from 171 to only 6, and the number of reads with three candidate annotations drops again from 3 to 0.
Finally, we have computed the relative abundance profiles at the phylum rank of the BLAST matches, the TANGO taxonomic annotations, the nontaxonomic annotations with the set cover approach, the TANGO taxonomic annotations refined with the set cover approach and, for reference, the QIIME taxonomy assignment using open-reference OTU picking (Navas- [bib_ref] Advancing our understanding of the human microbiome using QIIME, Navas-Molina [/bib_ref] , for the 302,581 reads from the human microbiome metagenomic data set and the 99,322 microbial sequences of the Greengenes taxonomy clustered at 97% identity. As can be seen in [fig_ref] Table 5: Relative Abundance Profile of BLAST Matches, TANGO Taxonomic Annotations, Nontaxonomic Annotations with... [/fig_ref] , the four relative abundance profiles are consistent, with only minor differences between them and the QIIME relative abundance profile. Histogram of BLAST matches, TANGO taxonomic annotations, nontaxonomic annotations with the set cover approach, and TANGO taxonomic annotations refined with the set cover approach, for the 302,581 reads from the human microbiome metagenomic data set and the 99,322 target sequences of the Greengenes taxonomy clustered at 97% identity. The rightmost bars correspond to 16 or more candidate annotations.
# Conclusion
We have addressed two potential sources of bias in the taxonomic annotation of metagenomic samples, which is usually done by first mapping the reads to the reference sequences and then classifying each read at a node in the clade of the LCA of the candidate sequences in the reference taxonomy with the least classification error. On the one hand, we have shown that the reference taxonomy being balanced or imbalanced does not affect the balance of the metagenomic classification problem, and we also shown that the Rand index is a better indicator of classification error for metagenomic classification problems than the often used area under the ROC curve and F-measure. On the other hand, we have reduced the taxonomic annotation problem for a whole metagenomic sample to a set cover problem, for which a logarithmic approximation can be obtained in linear time and an exact solution can be obtained by integer linear programming, and we have shown that a solution to the set cover problem with the least total size of subsets minimizes the ambiguity in the taxonomic annotation of the reads in a metagenomic sample.
We have also developed a proof-of-concept implementation of the set cover approach to taxonomic annotation in a next release of the TANGO software, as a series of Python scripts. Experimental results on a human microbiome metagenomic data set using BLAST and the latest release of the Greengenes taxonomy show that the set cover approach further reduces ambiguity in the taxonomic annotation obtained with TANGO without distorting the relative abundance profile of the metagenomic sample.
Future work includes extending the computation of balance ratio and total number of correct taxonomic annotations to the NCBI Taxonomy, taking ancestry relationships among the nodes in the reference taxonomy into account in the set cover formulation of the taxonomic annotation problem and last, but not least, extending the set cover problem formulation of the taxonomic annotation problem to a nontaxonomic metagenomic classification problem, with reference sequences but without a reference taxonomy.
# Acknowledgments
[fig] Definition 1: Let TP, TN, FP, and FN be the number of TP, FP, TN, and FN in a binary classification problem. The balance ratio of the classification problem is (TP + FN)=(FP + TN). [/fig]
[fig] FIG. 1: Classifying a read using a reference taxonomy. The grayed leaves are the candidate sequences for the classification of the read, and node i is their lowest common ancestor in the reference taxonomy. The taxonomic annotation of the read at node i implies the absence of TN and FN. With a taxonomic annotation of the read at node j, however, the grayed leaves under node j are the true positives, the remaining grayed leaves are the FN, the remaining leaves under node j are the false positives, and the still remaining leaves under node i are the TN of the metagenomic classification problem. FN, false negatives; FP, false positives; TN, true negatives; TP, true positives 350 FOSSO ET AL. [/fig]
[fig] Definition 2: Let TP, TN, FP, and FN be the number of TP, FP, TN, and FN in a binary classification problem. [/fig]
[table] Table 1: Confusion Matrix for a Binary Classification Problem FN, false negative; FP, false positive; TN, true negative; TP, true positive. [/table]
[table] Table 2: Distribution of TP, FP, TN, FN Values (Left) and Distribution of TP + FN Values (Right) in Metagenomic Classification Problems for Different Taxonomic Reference Topologies: Complete Binary Tree (B) [/table]
[table] Table 3: Total Number of Correct Taxonomic Annotations Under the Yule (/), the Area Under the Receiver Operating Characteristic (ROC) Curve (A) or the Youden J, the F-Measure (F) or the Jaccard Similarity Coefficient, and the Rand Index (R) for Reads Coming from Known Sequences, for Different Taxonomic Reference Topologies (Complete Binary Tree and Rooted Caterpillar) with n Leaves [/table]
[table] Table 4: Average Ambiguity of BLAST Matches, TANGO Taxonomic Annotations, Nontaxonomic Annotations with the Set Cover Approach, and TANGO Taxonomic Annotations Refined with the Set Cover Approach, for the 302,581 Reads from the Human Microbiome Metagenomic Data Set and the Greengenes Taxonomy Clustered at Various Identity Percent ValuesThe number of target sequences and the number of BLAST matches are also shown. [/table]
[table] Table 5: Relative Abundance Profile of BLAST Matches, TANGO Taxonomic Annotations, Nontaxonomic Annotations with the Set Cover Approach, TANGO Taxonomic Annotations Refined with the Set Cover Approach, and QIIME (Open-Reference OTU Picking) for the 302,581 Reads from the Human Microbiome Metagenomic Data Set and the Greengenes Taxonomy Clustered at 97% Identity [/table]
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The Use of Both Therapeutic and Prophylactic Vaccines in the Therapy of Papillomavirus Disease
Approximately 50% of penile cancers can be attributed to HPV infections, although HPV also infects healthy subjects without progressing to neoplasia. In a British study carried out among 43 couples, 69% of the men were HPV-positive in the uro-genital tract (15). Another study in the USA showed similar high levels of 5 https
Human papillomavirus (HPV) is the most common sexually transmitted virus. The high-risk HPV types (i.e., HPV16,are considered to be the main etiological agents of genital tract cancers, such as cervical, vulvar, vaginal, penile, and anal cancers, and of a subset of head and neck cancers. Three prophylactic HPV vaccines are available that are bivalent (vs. HPV16,, tetravalent (vs. HPV6,, and non-avalent (vs. HPV6,. All of these vaccines are based on recombinant DNA technology, and they are prepared from the purified L1 protein that self-assembles to form the HPV type-specific empty shells (i.e., virus-like particles). These vaccines are highly immunogenic and induce specific antibodies. Therapeutic vaccines differ from prophylactic vaccines, as they are designed to generate cell-mediated immunity against transformed cells, rather than neutralizing antibodies. Among the HPV proteins, the E6 and E7 oncoproteins are considered almost ideal as targets for immunotherapy of cervical cancer, as they are essential for the onset and evolution of malignancy and are constitutively expressed in both premalignant and invasive lesions. Several strategies have been investigated for HPV therapeutic vaccines designed to enhance CD4 + and CD8 + T-cell responses, including genetic vaccines (i.e., DNA/ RNA/virus/ bacterial), and protein-based, peptide-based or dendritic-cell-based vaccines. However, no vaccine has yet been licensed for therapeutic use. Several studies have suggested that administration of prophylactic vaccines immediately after surgical treatment of CIN2 cervical lesions can be considered as an adjuvant to prevent reactivation or reinfection, and other studies have described the relevance of prophylactic vaccines in the management of genital warts. This review summarizes the leading features of therapeutic vaccines, which mainly target the early oncoproteins E6 and E7, and prophylactic vaccines, which are based on the L1 capsid protein. Through an analysis of the specific immunogenic properties of these two types of vaccines, we discuss why and how prophylactic vaccines can be effective in the treatment of HPV-related lesions and relapse.
Keywords: human papillomavirus, immune response, cancer, prophylactic vaccine, therapeutic vaccine INTRODUCTION Despite the introduction of prophylactic vaccines, the incidence of human papillomavirus (HPV)-related tumors remains high, particularly in developing countries of the sub-Saharan region. The current HPV prophylactic vaccines have indications for use in women up to the age of 45 years, but they are predominantly administered to adolescent of 9-15 years. As most cancers develop decades after an initial HPV infection, the impact of this vaccination program will be seen in the longterm. Therefore, setting-up a therapeutic vaccine that can provide results similar to surgical treatment or chemotherapy represents a challenge for the eradication of HPV-induced tumors. However, therapeutic vaccines are not yet available for clinical practice.
Several studies have suggested that administration of HPV prophylactic vaccines after surgical treatment of high-grade cervical intraepithelial neoplasia (CIN2-3) can be considered as an adjuvant to prevent HPV reactivation or reinfection. The relevance of prophylactic vaccines has also been demonstrated in the management of genital warts, although clinical studies have not delivered univocal results to date.
In this review, the HPV-related tumors and the life cycle of HPV are described, to better understand the characteristics of the different viral proteins that are targeted by prophylactic and therapeutic vaccines. Then, we summarize the leading features of prophylactic and therapeutic vaccines that target the L1 and E6, E7 oncoproteins, respectively. Finally, through an analysis of the specific immunogenic properties of these two types of vaccines, we discuss how prophylactic vaccines can be effective for the treatment of HPV-related lesions, and for prevention of HPV-related relapse.
## Hpv prevalence
Human papillomavirus is the main agent of sexually transmitted diseases, and it can cause cancer in different anatomical districts with different prevalences. The highest proportion of HPV attribution as responsible for a cancer is for the cervix, where >99% of specimens are HPV-positive. In 2012, HPV-invasive cervical cancers reached >500,000 cases, which resulted in ∼250,000 deaths around the world 1 . HPV-related cancers are differently distributed across genders: among women, 8.6% of cancers are linked to HPV infections, while in men, <1% of cancers are attributable to HPV 2,3 . Differences are also observed in the geographic distributions of invasive cervical cancers: more than 85% occur in developing countries, where cervical screening programs and HPV vaccination campaigns are rarely available 4. HPV infection in a cervical site is frequently asymptomatic, and >90% of these resolve within 2 years without medical intervention (5), apparently through rapid immune clearance. However, the protective power of natural anti-HPV antibodies and the duration of immunity after infection are not fully understood. Also, only 50-60% of women show detectable anti-HPV antibodies after infection.
There are 15 high-risk (HR)-HPV genotypes that can lead to cancers of the cervix, anus, penis, vagina, vulva, and oropharynx (i.e., HPV16, . The relevance of HPV to each of these individual cancers is now considered.
## Cervical cancer
Overall, 90% of cervical cancers are attributed to HR-HPV types. HPV16 and HPV18 are the most prevalent in invasive cervical cancer, where they account for 62.5 and 15.7% of cases, respectively. HPV-associated cancers include cervical squamous cell carcinoma (70%), cervical adenocarcinoma (25%), and mixed histology tumors (7) 5 . An immunocompromised status represents a risk factor for cervical dysplasia, as well as for latent reactivation of HPV at genital sites. Patients with human immunodeficiency virus (HIV) infection have a 5-fold greater risk of acquiring HPV-associated cervical cancer than those without HIV infection. Precancerous (squamous) intraepithelial lesions are categorized as low-grade (LSIL) and high-grade (HSIL) (9).
## Anal cancer
Anal intraepithelial neoplasia (AIN1-3) represents the precursor of invasive anal cancers, where 65% are cervical squamous cell carcinomas. For both sexes, 88-94% of these cancerous lesions are positive for HPV DNA, with HPV16 as the most commonly detected (∼87% of HPV-positive tumors), while only 9% of these anal cancers harbors HR-HPV18.
Annually, about 18,000 women are diagnosed with anal cancer worldwide, and this cancer is more frequent in women than in men. Furthermore, anal cancer incidence is increasing, which appears to be due to changes in sexual risk factors for HPV transmission. Persistent anal HPV infection is the major cause of anal cancer 7. Women with a history of cervical cancer and cervical intraepithelial neoplasia grade 3 (HSIL) are also at increased risk of anal cancer. Cervical HR-HPVpositivity is associated with anal HR-HPV prevalence. In a study carried out by Lin, anal HR-HPV prevalence was significantly higher in cervical HR-HPV-positive women (43%) vs. cervical HR-HPV-negative women (9%). These associations were even stronger for HPV16-positivity: in cervical HPV16-positive women, anal HPV16 prevalence was 41%, while in the HPV16negative cervical group, anal HPV16 prevalence was 2%.
In men, the risk of anal cancer development is strictly related to sexual behavior and HIV immune status.
HPV prevalence, with 65.5% of the men HPV-positive: 51.2% of them harbored at least one typed HPV, and 14.3% of them were positive for an unclassified HPV infection. HPV clearance is observed in 75% of cases within 1 year. HPV-positivity is greater in penile intraepithelial neoplasia (PIN 1,2,3), which is considered the precursor of penile cancer, and in basaloid histological neoplasia (range, 75-80%) than in invasive cervical squamous cell carcinoma (range, 30-60%). HPV16 and HPV18 are the HPV types that are most frequently associated with all types of penile cancers 2 .
## Vulvar cancer
It is estimated that 40-50% of vulvar cancers are associated with HPV. Overall, vulvar cancers represented 3% of gynecologic cancers in 2002, and 60% of were observed in developed countries. Vulvar intraepithelial neoplasia (VIN) is considered a precursor of vulvar squamous cell carcinoma, which represents >90% of vulvar cancers. The World Health Organization recognizes a three-grade system of VIN (i.e., VIN1-3), and VIN3 is considered as a precursor of invasive vulvar cancer. However, until recently, VIN2-3 had been considered as HSIL, and VIN1 (or LSIL) is no longer used, as there is misclassification of these low-grade lesions: these are often actually condilomata acuminata with transient HPV infection, or an inflammatory status of the vulva. VIN can be caused by two distinct etiological agents: HPV, which is linked to the usual form of VIN (uVIN), and differentiated VIN (dVIN) is associated with lichen sclerosus . Generally, uVIN is common among young women, while dVIN is frequently seen for post-menopausal women. In VIN3/HSIL lesions, HPV16 had been detected in >91% of cases.
## Vaginal cancer
Cancer of the vagina is rare, and it accounts for only 2% of gynecological neoplasia. Nevertheless, from 2000 to 2015 there was an increase in vaginal cancer, which corresponded to 0.4% of vulvar carcinomas in the USA 9 . Vaginal intraepithelial neoplasia (VAIN) is considered a pre-malignant lesion. Previously, a threetiered classified was used (VAIN1-3) according to epithelial involvement. In 2014, the World Health Organization revised this classification by substituting VAIN2-3 with HSIL, and VAIN1 with LSIL. About 82% of high-grade lesions (i.e., vaginal intraepithelial lesions, VAIN3, HSIL) and 91% of invasive vaginal carcinomas test positive for HPV DNA and/or HPV antibodies. HPV16 is the most prevalent HPV type in HSIL and vaginal cancers 2.
## Head and neck cancer
The most common head and neck cancers are squamous cell carcinomas (HNC) and they include neoplasia of the oral cavity, tongue, tonsils, oropharynx, hypopharynx, and larynx. The HR-HPV types most frequently detected in head and neck cancers are HPV16, followed by HPV18 2. Head and neck cancers are frequent in southern-central Asia; however, an increase in head and neck cancer incidence has been seen over recent years in developed countriesand among Caucasian men. Tonsillectomies can increase the overall survival rates of patients with diagnosis of tonsil carcinoma, but it does not influence the overall risk of oropharyngeal cancer.
A reduction in HPV-positive oropharyngeal cancer is observed in people with a specific genetic locus in the human leukocyte antigen region (HLA-DRB1 * 1301-HLA-DQA1 * 0103-HLA-DQB1 * 0603). This protective effect might involve increased immune targeting of HPV-infected cells through the major histocompatibility complex haplotype binding to HPV peptides, resulting in a strong CD4 + T-cell response.
## Respiratory papillomatosis
Different annual incidences of respiratory papillomatosis have been reported in different countries. For example, in Denmark, similar incidence has been reported in children (juvenile onset) (3.6/100,000 children) and in adults (3.9/100,000 adults), while in the USA the annual respiratory papillomatosis incidence is 3-fold higher in children than in adults (4.6 vs. 3.9/100,000 children/adults).
HPV6 and HPV11 are the main genotypes detected in respiratory papillomatosis. As spontaneous regression is rarely observed, surgical treatment is necessary to prevent progression of the lesions. Moreover, recurrence of papillomatosis is often observed, and retreatment is needed in most cases, which comes at a high economic burden. Although no structured trials have been carried out to date, HPV vaccine administration prior to the onset of sexual behavior might have a positive impact on prevention of respiratory papillomatosis in adulthood.
## Genital warts
Human papillomavirus infection can not only cause cancer, but also benign genital warts. These are very diffuse in the young and in adults, with prevalence from 4 to 11% (41-43). Treatment of genital warts includes therapies with imiquimod and podophyllotoxin, or surgical procedures, or cryotherapy and tricloroacetic acid. These medical interventions represent high costs for both private insuranceand health systems.
Condylomas were classically considered a benign lesion, with the exception of Buscke-Lowenstein tumors. This large tumor can undergo local invasion and can transform into anal cervical squamous cell carcinoma.
## Viral characteristics and immune responses life cycle of hpv
Human papillomaviruses are non-enveloped icosahedral viruses that consist of 72 capsomers and are 55 nm in diameter. The viral genome is a circular double stranded DNA of ∼8,000 bp in length.
According to the time-regulated expression of proteins during the viral cycle, three functional genome regions can be distinguished: (i) the early region that encodes the E1, E2, E4, E5, E6, E7, E8 viral proteins that have regulatory functions in infected epithelial cells; (ii) the late region that encodes the two viral capsid proteins L1 and L2; and (iii) the long control regions (also known as upstream regulatory regions) that contain cisacting regulatory sequences that are involved in the control of viral replication and post-transcriptional phases.
Different viral proteins are expressed in different layers of the epithelium. The early proteins are expressed in basal epithelial cells, while the late proteins are expressed in the granular layer that includes more differentiated cells, and from where the virus is assembled and released. E1 is an ATPase helicase that is involved with E2 in viral replication and transcription regulation. The E1 and E2 complex interacts with the long control region ori site, which is considered to be the origin of HPV DNA replication. In the initial phase of infection, the HPV DNA genome is in the episomal form. It shows low amplification activity and there are ∼100 copies/cell. E2 ensures episomal maintenance of the HPV genome through interactions with other cellular factors. For example, Bromo-domain protein 4 (BRD4) is a mitotic chromosome-associated protein that is a critical binding partner for E2 for this activity. BRD4 and E2 co-localize on condensed mitotic chromosomes, and mediate episome segregation. E2 also regulates transcription of the E6 and E7 oncoproteins, the expression of which depends on an early promoter. E1 to E4 are encoded by a spliced RNA, and along with E5, they are translated under early promoter control in undifferentiated cells, and they appear to facilitate efficient productive replication in differentiating cells.
E6 and E7 are small proteins of about 150 and 100 amino acids, respectively. The E6 oncoprotein acts through its PDZbinding motif, which promotes its interactions with PDZ domains in multidomain proteins, to alter their functionality. These PDZ domains are present in many multidomain proteins that regulate key steps in the cellular processes of apoptosis, adhesion, and polarity . E6 also impairs the activity of the p53 protein, which prevents DNA damage accumulation through induction of DNA repair, cell-cycle arrest, or apoptosis, which leads to transformation of HPV chronically infected cells.
The main targets of E7 are the pRB, p107, and p130 proteins, which are components of a complex that can repress the E2F transcription factor. When E7 interacts with pRB, p107, and p130, it induces their degradation, and so E2F is free to activate genes such as cyclins A and E, to promote transition from G1 to S-phase of the cell cycle. The productive viral cycle also includes the synthesis of the late proteins L1 and L2 in the suprabasal epithelial cell layers, and this step is characterized by a change in mRNA splicing. Icosahedral virions are composed of 360 L1 proteins that are organized in pentamers, each of which is associated with one monomer of L2. The productive life cycle is completed when the virions self-assemble, after packaging of the amplified HPV DNA genome, with the viral particles then shed from the epithelial cell layers.
## Natural immune response
The immune response has an important role in clearing most HPV infections, although sometime the virus cannot be eliminated and can persist for several years, which represents a risk factor for neoplasia development. HPV-associated neoplastic progression is linked to dysregulated expression of the early viral genes. Specifically, increased expression of the E6 and E7 proteins in the basal epithelium leads to increased cellcycle entry and loss of differentiation across the epithelium. The main cause of dysregulated HPV gene expression is integration of the viral genome into the host chromosomes. HPV DNA integrates randomly into the host DNA. During this process, the viral DNA can often be broken at any position within the E1-E2 region, with the loss of E2 function, and the consequent overexpression of E6 and E7 that promotes cellular transformation. However, a proportion of cervical cancers are associated with episomal DNA only. In such cases, the E2 open reading frame integrity is maintained, and this protein is expressed throughout the progression of the malignancy.
In natural infections, both humoral-and cell-mediated immune responses are induced. Genital infection with oncogenic HPV is common, but only a minority of infected patients develop epithelial lesions or cancer. Spontaneous clearance of an established infection is likely to be mediated by the cellular immune responses. Indeed, strong Th1 CD4 + T-cell responses that are specific for HPV16 E6, E7, and E2 have been frequently detected in peripheral blood mononuclear cells of healthy individuals. In contrast, responses against HPV16 E6, E7, and E2 have rarely been detected in patients with HPV16positive genital lesions or antigen-specific proliferative responses that show a non-inflammatory cytokine profile.
Similarly, effective HPV18-specific T-cell responses are only seen in healthy controls, and not in HPV18-positive patients. For the role of CD8 + T-cells in disease regression, a comparison of CD8 + T-cell responses to E6 and E7 using enzyme-linked immunospot assays in individuals with incident or prevalent HPV 16 or 18 infections did not show any significant difference in the frequency of positivity between these two patient groups (33 vs. 40%). At variance with this, in CIN2/3 lesions, more CD8 + T-cells were seen for the epidermis of tissues that went on to regress. Also, large numbers of intraepithelial CD8 + tumor-infiltrating lymphocytes have been associated with an absence of lymph-node metastases in patients with large early stage cervical cancer. Taken together, these findings indicate that the development of high-risk HPVpositive cervical cancer is associated with failure of HPV-specific T-cell responses.
The humoral immune response to HPV infection is mainly directed against conformational epitopes in the variable regions of the major coat protein L1. This develops slowly, and is usually weak. Indeed, seroconversion appears to occur 6-18 months after infection, and type-specific antibodies to L1 are detected in 60-70% of women who acquire HPV infection. HPV-seroprevalence is considerably lower in men than women, and it has been suggested that HPV-seropositive women might have higher antibody levels than HPV-seropositive men.
IgG and IgA are the most abundant isotypes in sera from natural infections. Other HPV antigens (e.g., E1, E2, E6, L2) do not commonly induce antibody responses in patients with acute or persistent HPV infections.
Studies that have investigated whether naturally acquired HPV antibodies can protect against subsequent HPV infections Frontiers in Immunology | www.frontiersin.org have reported mixed results. More recently a systematic review and meta-analysis that included >24,000 individuals showed that natural HPV antibodies provide protection against subsequent type-specific genital HPV infections in females. However, given that the antibody titers in natural immunity are considerably lower than those observed with vaccination, and that antibody responses are preferentially induced in women and are not induced in all infected individuals, it is likely that protection through the development of natural immunity is inferior to protection obtained from HPV vaccination.
## Hpv vaccines
## Prophylactic vaccines
Three prophylactic vaccines for prevention of HPV infection are available at present: a bivalent vaccine against HPV16 and HPV18 (Cervarix) that was approved in 2007; a tetravalent vaccine against HPV6, 11, 16, and 18 (Gardasil) that was approved in 2006; and a nonavalent vaccine against HPV6, that was approved in 2016. However, the non-avalent vaccine is the only HPV vaccine that is currently available in the USA, and it was approved for males and females from 9 to 45 years by the US Food and Drug Administration in late 2018.
Initially, the administration of the HPV vaccines was in three doses, with the more recent change to a two-dose schedule driven by evaluation of girls aged 9-13 years who had received either two or three doses. The antibody responses of the young women (aged 16-26 years) who had followed a two-dose schedule were similar to those who received all three doses. Therefore, in 2016, the Advisory Committee on Immunization Practice declared that there was only the need for two doses of vaccine for those under 15 years of age. However, for females who start the vaccination between 15 and 45 years old, a three-dose schedule is recommended (at 0, 1-2, and 6 months). Also immunocompromised patients should follow the three-dose schedule regardless of sex and age at vaccination (86).https://www.cdc.gov/hpv/dowloads/9vhpv-guidance.pdf All three of these vaccines use recombinant DNA technology and are prepared from the purified L1 protein, which self-assembles to form HPV type-specific empty shells (virus-like particles; VLPs). Only intact VLPs can generate protective antibodies, which supports the evidence that conformational epitopes of L1 are required to generate neutralizing antibodies.
The evidence that HPV VLP vaccines protect against high viral challenges through induction of neutralizing anti-L1 antibodies was obtained in preclinical studies in animals, which thus provided the strong rationale for development of VLP-based vaccines. In particular, in a canine model of experimentally induced oral papillomas, it was demonstrated that dogs vaccinated with the major capsid protein, L1, of canine oral papillomavirus developed antibodies against canine oral papillomavirus and became completely resistant to the viral challenge. Similarly, vaccination of rabbits with L1 VLPs protected them against papillomas induced by cottontail rabbit papillomavirus. In addition, in both of these animal models, passive transfer of immune serum protected the dogs and rabbits against the canine oral papillomavirus and cottontail rabbit papillomavirus challenges, respectively.
In humans, analysis of vaccine-induced antibody responses measured by several methods has demonstrated that almost 100% of vaccinated individuals generate a strong type-restricted serum antibody response to L1 VLP. These methods have included conventional enzyme-linked immunosorbent assays, competitive radioimmunoassays, competitive Luminex-based immunoassays, and pseudovirion-based neutralization assays.
Initial and follow-up studies that assessed the immunogenicity of the HPV 16/18 AS04-adjuvant vaccine in 15-to 25-year-old women showed that after vaccination, anti-HPV16 and anti-HPV18 total IgG antibodies peaked at month 7, reached a plateau between months 18 and 24, and remained constant for up to 76 months. Measurement of the neutralizing antibodies using pseudovirion-based neutralization assays confirmed high levels of functional antibodies as well. Then evaluation of long-term immunogenicity of the HPV16/18 vaccine in the serum of 15to 55-year-old females revealed that the seropositivity for anti-HPV16 remained high in all of the age groups 10 years after the first vaccination. For anti-HPV18, there were more seropositive females in the 15-to 25-year-old group (99.2%) than for the 26to 45-year-olds (93.7%) and 46-to 55-year-olds (83.8%). In these studies, the anti-HPV16 and anti-HPV18 titers remained above natural infection levels in all of the age groups, and more interestingly, they were predicted to persist for more than 30 years after vaccination.
Comparisons of the immunogenicities of the HPV16/18 and HPV6/11/16/18 vaccines in healthy women aged 18-45 years revealed that 7 months after vaccination, the serum neutralizing antibody responses elicited by the bivalent vaccine were significantly higher than those for the HPV6/11/16/18 vaccine. The differences in these responses for HPV16 and HPV18 were maintained at month 24, and also up to month 60 in women aged 18-45 years.
Antibody titers induced by vaccination are higher than those produced by natural infection. Responses to HPV vaccination is weakly influenced by gender, with higher seroconversion in males than females (99 vs. 93%), and by age, with higher antibody titers in women aged 9-15 years. The FUTURE I trial demonstrated that the efficacy of the tetravalent HPV vaccine was 100% against condyloma in HPV-naïve women, and 70% (vaginal condyloma) to 78% (vulvar condyloma) in the overall population. The efficacy of the non-avalent vaccine is comparable to that of the tetravalent vaccine against condyloma. Prophylactic HPV vaccines show excellent protection against high-grade CIN (i.e., CIN2, CIN3) and adenocarcinoma in situ for HR-HPV-naïve women. In particular, the non-avalent vaccine showed the highest efficacy for prevention of onset of CIN1 (relative risk reduction, 98.9%), CIN2 (97.1%), and CIN3 (100%) neoplasia.
Data for vaccine prevention against AIN are more limited. In the Guanacaste study, the tetravalent HPV vaccine prevented HPV16/18 infection in anal anatomic sites in 84% of women who were HPV-seronegative at baseline. Palefsky reported 77.5% prevention of AIN among HPV-naïve men aged 16-26 years who had sex with men (MSM). The tetravalent vaccine also protects heterosexual naïve men from both anogenital HPV infections and HPV lesions, with an efficacy against infections and associated lesions of >90% (98). Also a Finnish randomized trial reported significant reduction of genital HPV infections in men following HPV16/18 vaccine administration.
For oropharyngeal cancer prevention, a risk reduction of 93.3% for precursor lesions of HPV-induced oral cancer was reported for the Guanacaste study. However, further studies are needed to demonstrate the efficacy of these vaccines on oropharyngeal cancer development.
## Therapeutic vaccines
The therapeutic vaccines differ from the prophylactic vaccines as they are aimed at the generation of cell-mediated immunity, rather than neutralizing antibodies. Although prophylactic vaccines can prevent HPV infections in 100% of cases, and precancerous cervical lesions (i.e., CIN) caused by the HPV 11 http://www.rho.org/files/WHO_HPV_tech_info_nocover_2007.pdf genotypes included in the vaccine, HPV-related lesions remain a public problem worldwide for several reasons: (i) only 8% of low and middle income countries have introduced HPV vaccination programs 12 ; (ii) HPV types that are not included in vaccines might be responsible for cancers (100); (iii) the cost of requirements for a cold chain and the absence of sanitary infrastructure limits HPV vaccine deployment in developing countries; and (iv) HPV vaccines are recommended for young women (9-26 years old), and as women older than 26 years are not vaccinated, they can develop cancers. It is also estimated that the impact of HPV vaccination on cancer incidence might not be appreciated for at least 20 years from any mass vaccination. Currently, the treatment of high-grade disease (CIN2-3) includes electrosurgical excision of the transformation zone, with carbon dioxide lasers or knives used to perform conization, where the entire transformation zone is removed (101, 102). Incomplete excision, however, can occur, and HPV transformed cells can remain, which will facilitate recurrent neoplasia. Hence, there is the need for a therapeutic vaccine that can fully eliminate malignant cells.
The aim of a therapeutic vaccine against HPV is to induce in-vivo virus-specific T-cell responses against established HPV infections and lesions. For therapeutic vaccination to deliver unequivocal clinical benefits, improvements must be achieved at two levels: by maximizing the induction of T-cell responses with optimal amplitude, specificity and effector profile; and by ensuring that vaccine-induced Tcells can reach the tumor site and perform their functions without restraint.
Among the HPV proteins, the E6 and E7 oncoproteins are considered to be almost ideal targets for immunotherapy of cervical cancer, as these proteins are essential for the onset and evolution of malignancy, and are constitutively expressed in both premalignant and invasive lesions, while being absent in healthy cells. E6 and E7 have therefore been included in most therapeutic vaccines developed to date. Usually, a DNA sequence that encodes a fusion protein of E6 and E7 is inserted into a vector, and mutations are introduced into the regions that are responsible for the E6 interactions with p53, and the E7 interactions with pRB, to reduce their oncogenic power. The E1 and E2 viral proteins are also attractive candidates for therapeutic vaccines that target early viral infections, as they are highly expressed before viral genome integration.
Several strategies have been investigated for HPV therapeutic vaccines designed to enhance CD4 + and CD8 + T-cell responses, including genetic vaccines (e.g., DNA/ RNA/ virus/ bacterial), and protein-based, peptide-based or dendritic-cell-based vaccines. Among the bacterial vectors, live attenuated Listeria monocytogenes has been used to generate a promising HPV therapeutic vaccine. L. monocytogenes is considered a potent vaccine vector because it enters professional antigen-presenting cells and induces antigen-presenting cell maturation, and strong innate and adaptive immunity. In addition, L. monocytogenes grows very efficiently in vitro and lacks lipopolysaccharides, which are a major toxicity factor with Gram-negative bacteria. The safety of a recombinant live attenuated L. monocytogenes secreting E7 as a fusion protein joined to non-hemolytic listeriolysin O (Lm-LLO-E7) was demonstrated in a phase I clinical study that was conducted with 15 patients with late-stage metastatic cervical cancer. Evaluation of the efficacy of Lm-LLO-E7 (also known as ADXS11-001) in a prospective phase II clinical trial as second-line and third line for patients with recurrent metastatic cervical cancer showed that 12-month overall survival was 38%, which exceeded the historical overall survival of such patients, of 25%. A phase III clinical trial of Lm-LLO-E7 for high-grade cervical cancer is being conducted at the time of writing (see NCT02853604).
Encouraging data have also been obtained in clinical studies that have tested DNA-based vaccines. DNA vaccination consists of direct introduction into tissues of a plasmid that contains the DNA sequence that encodes the antigen(s) against which an immune response is sought. This relies on in-situ production of the antigen(s) as a result of the transfection of antigen-presenting cells and non-antigen-presenting cells, with the presentation of the expressed antigen(s) by both MHC class I and class II molecules. Furthermore, this results in activation of all three arms of the adaptive immune response (i.e., helper T cells, cytotoxic T cells, antibodies).
However, although DNA vaccines have been shown to induce balanced CD4 + and CD8 + T cells as well as humoral immune responses in small animal models, clinical data from multiple studies have demonstrated that they induce poor T-cell responses.
Many strategies to facilitate antigen processing and presentation, and also antigen delivery, have been adopted to ameliorate the immunogenicity of DNA vaccines against HPV.
A phase I study was carried out using the DNA vaccine VGX-3100 that consists of a mixture of two plasmids that encode the optimized consensus of the E6 and E7 genes of HPV genotypes 16 and 18. These were delivered via intramuscular injection, followed by electroporation, with 18 patients who had been previously treated for cervical intraepithelial neoplasia (CIN2/3). This study showed that 78% of the patients developed CD8+ T-cell responses, and 100% showed antibody positivity to at least two vaccine antigens. Notably, the peripheral blood T-cell responses elicited by VGX-3100 were an order of magnitude greater than naturally occurring responses, and a log unit greater than those previously reported for HPV therapeutic vaccines.
In 2015, the efficacy, safety, and immunogenicity of VGX-3100 was assessed in a phase II clinical trial in patients with CIN2/3. In the per-protocol analysis, 30.6% of the placebo recipients and 49.5% of the VGX-3100 recipients showed histological regression. Concomitant histopathological regression and viral clearance occurred in 14.3% of placebo recipients compared with 40.2% of vaccinated recipients. Post-hoc immunological analysis here demonstrated that VGX-3100 elicited significantly increased frequency of T-cell responses against HPV16/18 E6 and E7, and that the magnitude of the Tcell response against E6 was associated with clinical outcome. Humoral immune responses were also lower in placebo recipients than in VGX-3100 recipients, and the antibody responses against HPV16, HPV18, and E7 were significantly higher in the patients who had concomitant histopathological regression and viral clearance, compared to those who did not. A phase III clinical trial of VGX-3100 for women with CIN was initiated in 2017, and it is expected to end in 2021 (see NCT03185013).
Viral vectors including adenoviruses, adeno-associated viruses, alphaviruses, and vaccinia viruses (e.g., modified vaccinia Ankara virus; MVA) can be used to express the E2, E6, and E7 oncoproteins, and they can stimulate CD4 + and CD8 + T-cell responses. A MVA vector was used to produce the Tipapkinogen Sovacivec vaccine, which includes three exogenous genes that encode the human cytokine interleukin-2, and non-oncogenic E6 and E7. This vaccinia virus can induce interferon-α production and express HPV16 E6 and E7, which are presented by dendritic cells to activate naïve T cells in lymph nodes. At a follow-up of 2.5 years, compared to the placebo cohort at 10% viral clearance, the administration of Tipapkinogen Sovacivec vaccine provided complete resolution for 24% of patients with CIN2/3, irrespective of their HR-HPV baseline infection (i.e., HPV16, . However, despite this significantly improved HPV viral clearance with this vaccine, it has still not been licensed for clinical use because of the modest efficacy.
Finally, a vaccine designed on recombinant MVA that contained the bovine papillomavirus E2 protein (MVA E2) was used to treat HPV-induced ano-genital intraepithelial lesions. A phase III study showed that 90% of female patients had complete elimination of lesions after treatment with MVA E2, with 100% seen for men. All of these patients treated with MVA E2 developed antibodies against the MVA E2 vaccine and generated a specific cytotoxic response against the papillomatransformed cells.
Interestingly, novel vaccination strategies aimed to maximize systemic as well as genital resident memory T cell responses to treat sexually transmitted infections and human papilloma virus neoplasia are being developed. In this context several studies have investigated the effect of either the topical delivery of host and pathogen derived immunomodulatory molecules or the delivery route of immunization in the induction of cervicovaginal long lived CD8+ T cell responses.
## Hpv prophylactic vaccines used as therapeutic vaccines
The treatment of premalignant lesions (CIN2,3) by LLEP or conization sometimes fails to prevent lesion recurrence. This is often linked to incomplete excision of transformation zone consciously carried out by gynecologists. In fact, evidence shows that large excision of the cervix can compromise cervix integrity and can cause adverse neonatal outcome with preterm risk. Moreover, recurrence risk is greater in presence of HR HPV infection.
A systematic review of studies of the treatment of high-grade lesions (HSIL/ CIN2-3) reported that a median of 28% of the women remained positive for oncogenic HPV types 3 months after treatment. A decrease in this HPV persistence was seen during follow-up, as it fell to 21% after 6 months (102). Also, higher risks for the development of cervical and vaginal neoplasia have been reported for women who had previously been treated for CIN3, in comparison to the general female population, with this higher risk persisting for 20-25 years, and possibly longer. The risk of cervical cancer after treatment also increases with age. A large study with long-term follow-up for women treated for CIN3 reported standard incidence and mortality ratios (i.e., treated vs. placebo) for cervical and vaginal cancers of 10.58 and 7.60, respectively, for women aged 60-69 years, and 2.03 and 1.52, respectively, for women aged 30-39 years. Also, women who had previously reported CIN3 lesions showed greater probability of developing other HPV-related neoplasia of the genital tract (e.g., vaginal, vulvar, anal) or the oropharyngeal district.
As no vaccine has yet been licensed for therapeutic use, the prophylactic vaccines have been tested in several trials to determine their effectiveness for prevention of HPV disease recurrence or reinfection after CIN2-3 treatment. The recurrence for MSM who undergo treatment for high-grade anal intraepithelial neoplasia (HGAIN) is particularly high, as 50% show recurrence within 1 year. This makes it essential to find a treatment that can reduce the development of highgrade lesions in treated patients. In 2011, the effectiveness of the tetravalent HPV vaccine for the prevention of recurrent HGAIN was evaluated in HIV-negative, self-identified MSM with a history of biopsy-proven and treated HGAIN. In the 340.4 person-years of follow-up, 30.7% of the non-vaccinated patients developed recurrent HGAIN, compared to 13.6% of the vaccinated patients. Among these patients who were infected with HR-HPV types, the tetravalent vaccine was associated with significantly decreased risk of recurrent HGAIN at 2 years from study entry (hazard ratio, 0.47). To explain the partial effectiveness of the tetravalent vaccine in this study, it was speculated that some of these patients might have developed diseases that were related to the HPV genotypes not covered by the tetravalent vaccine or to multiple HPV infections. Further, some HGAIN might not have been identified and treated before the vaccinations, or the viral integration into the host genome had already occurred. Unfortunately, these aspects were not investigated.
In 2013, Kang et al. investigated the effectiveness of the tetravalent HPV vaccine to prevent recurrence of CIN2-3 in patients with high-grade CIN treated by the loop electrosurgical excision procedure. Recurrence was seen for 7.2% of the non-vaccinated patients and by 2.5% of the vaccinated patients. In patients infected with HPV16 and/or HPV18, 8.5% of the non-vaccinated patients and 2.5% of the vaccinated patients developed recurrent disease related to these HPV types. Although encouraging, these data indicate that the prophylactic HPV vaccine had weak activity against such HPV16/18-related high-grade lesions. Recently, a prospective clinical project, the SPERANZA study, was carried out to determine the effectiveness of the tetravalent vaccine for reduction of the risk of clinical relapse in women treated for CIN2. Overall, 344 women were included in the study, and 6.4% of the nonvaccinated women showed clinical disease recurrence, while for the vaccinated women, there was only 1.2% recurrence. Vaccination here was associated with significantly reduced risk of subsequent HPV-related high-grade CIN after cervical surgery, at 81.2%. For the non-vaccinated women, the recurrent clinical disease was attributed to HPV11,, while for the vaccinated women, the two cases of clinical disease recurrence were associated with HPV33 and HPV82. In this study, about 40% of the patients enrolled were >36 years old, although neither the age range nor the age of women with recurrent clinical disease were reported, and thus it cannot be determined if the efficacy of the tetravalent HPV vaccine was influenced by the age of the patients at the time of their vaccination. At variance with this, a study by Hildesheim et al. included 1,711 women with carcinogenic human HPV infection and 311 women who received loop electrosurgical excision for cervical precancer. Here, there was no evidence that HPV16/18 vaccination alters the fate of an HPV infection present at the time of vaccination, or the rates of cervical infections and lesions after loop electrosurgical excision. For these HPV16/18 infections, in the cohort of women with HPV infection but without precancer, the efficacy of clearance was 5.4%, with progression to CIN1 seen for 15.5%, and to CIN2, for 0.3%. Moreover, after the loop electrosurgical excision, the vaccination had no significant effects on HPV16/18 infections and/or HPV16/18-associated cytological and histological lesions.
The data obtained on the efficacy of the tetravalent HPV vaccine for the prevention of anal condylomas are, however, more encouraging. Three hundred and thirteen MSM (mean age, 42 years) were enrolled for a median of 981 days. During the follow-up, condyloma developed in 18.8% of non-vaccinated patients, and in 8.6% of vaccinated patients. Moreover, several clinical studies have demonstrated activity for HPV vaccination in the treatment of genital warts.
Altogether, these data suggest that there is possibility that prophylactic vaccines reduce the risk of HSIL recurrence in previously infected patients, although the exact protective mechanisms in infected individuals is not understood. The high risk of recurring infections is consistent with either auto-inoculation across anatomic sites or new inoculation or episodic reactivation of latent infection. As HPV vaccines prevent infection by induction of L1-specific antibodies that block viral entry, and L1 is not generally expressed during the oncogenic process, it is expected that these vaccines will be effective in the prevention of auto-inoculation or new infections.
The greater effectiveness obtained with prophylactic vaccines in the prevention and regression of genital warts might be related to the integration state of the HPV genome. In genital warts, the virus is not generally integrated into the host genome, and therefore viral particles are produced. In this case, the prophylactic vaccines that block viral entry through induction of L1-specific antibodies can prevent reinfections, which will favor the elimination of the virus. Conversely, in high-grade lesions, the virus genome is often integrated into the host genome, and infected cells do not express L1 and do not produce viral particles. Thus, as transformed cells are frequently in the basal layer of the derma, they will not be recognized by vaccine-induced antibodies, which are ineffective in the control of the disease course.
Furthermore, there are some cases in the treatment of HPVrelated cancers where the use of prophylactic vaccines might not be recommended:
(1) Anal and cervical cancers that are not attributable to the HPV types that are included in the non-avalent HPV vaccine. Several studies have demonstrated that half of the HPV infections in MSM are caused by HPV types that are not included in the non-avalent HPV vaccine . Here, over 2 years of observation, only about 30% of HIV-positive MSM had incidents of HR-HPV infections that were covered by the nonavalent vaccine. This situation can be also observed for women .
(2) HPV DNA-negative cervical tumors. Over recent decades, several studies have reported that some cervical cancers are HPV-negative. Often, HPV DNA negativity is due to the sensitivity of the methods used in the HPV DNA detection, and so samples that have tested as HPV-negative might show as HPV-positive when retested with more sensitive assays (e.g., nested PCR). The Cancer Genome and Molecular Characterization of Cervical Cancer Study used next-generation sequencing to characterize primary cervical cancers, and it established that 5% of the specimens were HPV-negative. This subset of HPV DNA-negative cancers is mainly observed among adenocarcinoma cancers, and predominantly in gastric-type adenocarcinomas. The pattern of immunostaining of gastric-type adenocarcinomas shows strong and diffuse positivity for MUC-6 and HIK1083 antibodies, which recognize epitopes of gastric pyloric glycoproteins, although they are p16 negative, which is a cell-cycle regulatory protein. Gastric-type adenocarcinomas have significantly higher rates of recurrence and mortality than HPV-positive cancers. Furthermore, progression and regression of gastric-type adenocarcinomas are independent of HPV infection, and thus HPV vaccine administration here would be inappropriate.
# Conclusions
The data reported in this review highlight the significant efforts that have been carried out to set-up therapeutic vaccines against HPV-related malignancies. Although several approaches to produce an effective vaccine have been attempted, including the use of proteins, synthetic peptides, and viral proteins expressed in different vectors, and although some of the data appear encouraging, no therapeutic vaccines have been licensed in clinical practice yet. Recently, prophylactic vaccines have been used for treatment of recurrent forms or reinfections in subjects who have previously undergone surgical resection. However, the trials here have offered conflicting results, and vaccination did not guarantee 100% effectiveness. This is probably due to a residual burden of transformed cells that can persist after the surgical treatment, and that are not targeted by the humoral L1-specific immune response induced by the prophylactic vaccines. Although it cannot be excluded that the therapeutic potential of prophylactic vaccines could be improved by using different adjuvants or route of immunization, an additional limit in using prophylactic vaccines for therapeutic purposes is seen by the evidence that the non-avalent vaccine does not include all of the HR-HPV types. As the real extent of protection given by the non-avalent vaccine against other HPV types is not known, its use in the treatment of tumors related to these other HR-HPV types is questionable. Furthermore, for endometrial adenocarcinomas, such as gastrictype adenocarcinomas, which are HPV DNA-negative, careful virological and histological diagnosis must be made before administration of HPV prophylactic vaccines to treat HPV recurrence or reinfection.
# Author contributions
AG and PD wrote the original version and edited versions. MC edited versions. DL and CS prepared figures and tables.
# Funding
This work was supported by Ricerca Corrente Funding from Italian Ministry of Health.
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Incidence and extent of TDP-43 accumulation in aging human brain
Introduction:The transactivation response element DNA-binding protein 43 kDa (TDP-43) is a major component of the ubiquitin-positive and tau-negative inclusions in frontotemporal lobar degeneration and sporadic amyotrophic lateral sclerosis (ALS). TDP-43 may accumulate in cases of Alzheimer's disease (AD), Lewy body disease (LBD), and argyrophilic grain disease (AGD). However, few studies have focused on the incidence and extent of TDP-43 deposition in aging.Results:We analyzed 286 consecutive autopsy brains neuropathologically. Of these, 136 brains with pathologically minimal senile changes were designated as control elderly brains (78.5 ± 9.7 y). For comparison, we selected 29 AD, 11 LBD, and 11 AGD patients from this series of autopsy brains. Sections of the hippocampus, amygdala, medulla oblongata, and lumbar spinal cord were immunostained with anti-phosphorylated TDP-43 antibody (PSer409/ 410). TDP-43 immunoreactive structures were classified into four types: dystrophic neurites (DNs), neuronal or glial cytoplasmic inclusions, and intranuclear inclusions. TDP-43 immunoreactive structures were observed in 55/136 control elderly (40.0 %), 21/29 AD (72.4 %), 8/11 LBD (72.7 %), and 6/11 AGD (54.5 %) brains. TDP-43 immunoreactive structures in control elderly brains were mostly DNs. These DNs were predominantly present in the uncus of the anterior hippocampus over age 65. The frequency of cases with DNs in the amygdala of control elderly brains was less than that of AD, LBD, and AGD brains. The mean age at death was significantly higher in cases with TDP-43 immunoreactive structures than cases without them.Conclusions:In conclusion, TDP-43 immunoreactive DNs may develop as a consequence of aging processes in the human brain. In particular, the uncus of the anterior hippocampus is an area highly susceptible to TDP-43 accumulation over age 65.
# Introduction
The transactivation response element DNA-binding protein 43 kDa (TDP-43) is a major component of the ubiquitin-positive and tau-negative inclusions in frontotemporal lobar degeneration (FTLD-TDP) and sporadic amyotrophic lateral sclerosis (ALS) [bib_ref] TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration..., Arai [/bib_ref] [bib_ref] Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis, Neumann [/bib_ref] [bib_ref] A harmonized classification system for FTLD-TDP pathology, Mackenzie [/bib_ref]. However, the cytopathology and distribution pattern of TDP-43 immunoreactive deposits generally differ between FTLD-TDP and ALS [bib_ref] A harmonized classification system for FTLD-TDP pathology, Mackenzie [/bib_ref] [bib_ref] Stages of pTDP-43 pathology in amyotrophic lateral sclerosis, Brettschneider [/bib_ref].
Previous studies have indicated that TDP-43 immunoreactive inclusion bodies can be detected in other neurodegenerative disorders as well, including Alzheimer's disease (AD) [bib_ref] TDP-43 immunoreactivity in hippocampal sclerosis and Alzheimer's disease, Amador-Ortiz [/bib_ref] [bib_ref] Concurrence of TDP-43, tau and alpha-synuclein pathology in brains of Alzheimer's disease..., Higashi [/bib_ref] [bib_ref] Concomitant TAR-DNA-binding protein 43 pathology is present in Alzheimer disease and corticobasal..., Uryu [/bib_ref] [bib_ref] Phosphorylated TDP-43 in Alzheimer's disease and dementia with Lewy bodies, Arai [/bib_ref] [bib_ref] Abnormal TDP-43 immunoreactivity in AD modifies clinicopathologic and radiologic phenotype, Josephs [/bib_ref] , Lewy body disease (LBD) [bib_ref] Concurrence of TDP-43, tau and alpha-synuclein pathology in brains of Alzheimer's disease..., Higashi [/bib_ref] [bib_ref] Phosphorylated TDP-43 in Alzheimer's disease and dementia with Lewy bodies, Arai [/bib_ref] [bib_ref] Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases, Nakashima-Yasuda [/bib_ref] , and argyrophilic grain disease (AGD) [bib_ref] Accumulation of phosphorylated TDP-43 in brains of patients with argyrophilic grain disease, Fujishiro [/bib_ref]. Furthermore, several studies have focused on TDP-43 deposition in the absence of neurodegenerative disorders [bib_ref] Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases, Nakashima-Yasuda [/bib_ref] [bib_ref] Pathological 43-kDa transactivation response DNA-binding protein in older adults with and without..., Geser [/bib_ref] [bib_ref] TDP-43 in aging and Alzheimer's disease -a review, Wilson [/bib_ref] [bib_ref] TDP-43 deposition in prospectively followed, cognitively normal elderly individuals: correlation with argyrophilic..., Arnold [/bib_ref]. One such study demonstrated that TDP-43 deposition increased with age and did not occur in individuals younger than 65 years [bib_ref] Pathological 43-kDa transactivation response DNA-binding protein in older adults with and without..., Geser [/bib_ref]. Another study indicated that the incidence of TDP-43 deposition in the amygdala and hippocampus of cognitively normal elderly individuals was 36 % [bib_ref] TDP-43 deposition in prospectively followed, cognitively normal elderly individuals: correlation with argyrophilic..., Arnold [/bib_ref]. These previous studies suggest that TDP-43 deposition may be associated not only with specific neurodegenerative disorders but also with aging and specific anatomical areas. To further analyze the functional role of TDP-43, it will be important to understand the anatomical distribution of TDP-43 immunoreactive deposits in a large number of elderly individuals with neuropathologically minimal changes. The aim of this study was to clarify the incidence and distribution of TDP-43 deposition in control elderly brains by immunohistochemical analysis of a series of autopsied individuals. We also analyzed TDP-43 immunoreactive deposits in individuals with AD, LBD, and AGD for comparison.
# Materials and methods
## Brain samples
Tissue samples were collected at the Tokyo Metropolitan Geriatric Hospital, which provides community-based medical service to the elderly population. From February 2008 to July 2012, we obtained the 286 consecutive autopsy brains (165 men and 121 women) used for this study. Patient ages ranged from 43 to 104 years (mean ± SD, 82.0 ± 9.6 y). Clinical information was retrospectively obtained from the medical chart. The Mini-Mental State examination (MMSE) [bib_ref] A practical method for grading the cognitive state of patients for the..., Folstein [/bib_ref] was used for evaluation of cognitive function, and the clinical dementia scale (CDR) [bib_ref] A new clinical scale for the staging of dementia, Hughes [/bib_ref] was employed for the grading of cognitive decline as previously reported [bib_ref] Staging of argyrophilic grains: an age-associated tauopathy, Saito [/bib_ref]. The brain samples used in this study were registered to the Brain Bank for Aging Research (BBAR) with the deceased's relatives' informed consent to carry out comprehensive research. The BBAR is approved by the ethics committee of the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.
## Neuropathology and immunohistochemistry
The brains and spinal cords were examined according to routine protocols used in our laboratory [bib_ref] Neuropathologic analysis of Lewy-related alpha-synucleinopathy in olfactory mucosa, Funabe [/bib_ref]. Briefly, the cerebral and cerebellar hemispheres as well as the brainstem were dissected in the midsagittal plane at the time of autopsy. In each case, part of the brain was stored at −80°C for further biochemical and molecular analyses. The brains and spinal cords were fixed in 20 % buffered formalin (WAKO, Osaka, Japan) for 7-13 days. After fixation in formalin, the cerebral hemispheres, brainstem, and cerebellum were dissected along the coronal, axial, and sagittal planes, respectively. Representative anatomical areas were embedded in paraffin, and 6-μm-thick sections were obtained to diagnose each case neuropathologically. Sections were stained with hematoxylin and eosin (H&E) and the Klüver-Barrera methods. Selected sections were further examined with modified methenamine and Gallyas-Braak silver staining for senile changes, Congo red for amyloid deposition, and Elastica Masson trichrome stain for vascular changes. Selected sections were immunostained with a Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) and an I-View Universal DAB Detection Kit (Roche, Basel, Switzerland) in accordance with the manufacturer's instructions. The antibodies employed are shown in [fig_ref] Table 1: Antibodies used for immunohistochemistry [/fig_ref]. Histologic sections were observed under a research microscope (Eclipse 90i; Nikon, Tokyo, Japan).
## Case selection
The case selection procedure in this study is shown in [fig_ref] Figure 1: Case selection procedure [/fig_ref]. First, of the 286 consecutive autopsy cases, we selected cases that met all of the following conditions and defined them as control elderly cases that have minimal senile neuropathologic changes: Braak's neurofibrillary tangle (NFT) stage II or less, senile plaque (SP) stage [bib_ref] Neuropathological stageing of Alzheimer-related changes, Braak [/bib_ref] A or less, AGD stage [bib_ref] Staging of argyrophilic grains: an age-associated tauopathy, Saito [/bib_ref] II or less, and Lewy body stage [bib_ref] Lewy body-related alpha-synucleinopathy in aging, Saito [/bib_ref] II or less. For comparison, we selected cases with AGD stage III as AGD and those with Lewy body stage above III as LBD. We also defined AD according to our brain bank definition (Braak NFT stage above IV and Braak's SP stage C) [bib_ref] Neuropathological diagnostic criteria for Alzheimer's disease, Murayama [/bib_ref].
As to the cases with comorbid pathologies and other neurological disorders, we also provided the results of TDP-43 pathology. However, they were not used for comparison with control elderly cases because they were limited in number: AD plus LBD (n = 6), AD plus AGD (n = 3), AD plus progressive supranuclear palsy (PSP) [bib_ref] Preliminary NINDS neuropathologic criteria for Steele-Richardson-Olszewski syndrome (progressive supranuclear palsy), Hauw [/bib_ref] (n = 1), AD plus hippocampal sclerosis (HS) (n = 1), LBD plus AGD (n = 1), LBD plus PSP (n = 1), AGD plus HS (n = 1), senile dementia with tangles (NFTD) (n = 6) [bib_ref] Senile dementia with tangles (tangle predominant form of senile dementia), Jellinger [/bib_ref] , Creutzfeldt-Jakob disease (CJD) (n = 4), ALS/ALS with dementia (n = 4), multiple system atrophy (MSA) (n = 3), spinocerebellar ataxia type 6 (SCA6) (n = 2), spinocerebellar ataxia type 3 (SCA3) (n = 1), PSP (n = 2), HS (n = 2), dentatorubral-pallidoluysian atrophy (DRPLA) (n = 1), corticobasal degeneration (CBD) (n = 1), FTLD-TDP (n = 1), and mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) (n = 1
## Evaluation of severity and distribution of tdp-43 immunoreactivity
The presence and severity of TDP-43 immunoreactive structures were assessed in four brain sections including the anterior hippocampus, amygdala, medulla oblongata, and lumbar spinal cord. In fact, the anterior hippocampus is key anatomical structures of FTLD-TDP. In addition, TDP-43 immunoreactive deposits have been well analyzed in AD, DLB and AGD in the limbic regions [bib_ref] TDP-43 immunoreactivity in hippocampal sclerosis and Alzheimer's disease, Amador-Ortiz [/bib_ref] [bib_ref] Concurrence of TDP-43, tau and alpha-synuclein pathology in brains of Alzheimer's disease..., Higashi [/bib_ref] [bib_ref] Phosphorylated TDP-43 in Alzheimer's disease and dementia with Lewy bodies, Arai [/bib_ref] [bib_ref] Abnormal TDP-43 immunoreactivity in AD modifies clinicopathologic and radiologic phenotype, Josephs [/bib_ref] [bib_ref] Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases, Nakashima-Yasuda [/bib_ref] [bib_ref] Accumulation of phosphorylated TDP-43 in brains of patients with argyrophilic grain disease, Fujishiro [/bib_ref]. The medulla oblongata and spinal cord are important areas in association with ALS with TDP-43 [bib_ref] Stages of pTDP-43 pathology in amyotrophic lateral sclerosis, Brettschneider [/bib_ref]. Therefore, we considered that those four areas are appropriate for the study about TDP-43 pathology in control elderly brains. We assessed TDP-43 immunoreactive structures in each anatomical region shown in [fig_ref] Table 2: Brain regions analyzed in this studyFig [/fig_ref] and under a 10× objective. Each anatomical region was identified using the neuroanatomy atlas. The uncus was identified and analyzed at the level of amygdala and anterior hippocampus. Neuronal cytoplasmic inclusions (NCIs), glial cytoplasmic inclusions (GCIs), and neuronal intranuclear inclusions (NIIs) immunoreactive for TDP-43 were quantitatively analyzed and scored as 0 to 3 depending on the total number of TDP-43 immunoreactive NCIs, GCIs, or NIIs: 0, none; 1, 1-3; 2, 4-9; and 3, ≥10. TDP-43 immunoreactive dystrophic neurites (DNs) were semi-quantitatively scored as 0 to 3: 0, absent; 1, sparse; 2, moderate; and 3, severe.
# Statistical analysis
The Mann-Whitney U test was used to compare the age at death and brain weight between TDP-43-positive and TDP-43-negative groups. Fisher's exact test was used to compare the male-to-female ratio and number of TDP-43 structures. A p value of less than 0.05 was considered statistically significant.
# Results
The demographics of the cases in this study are shown in [fig_ref] Table 3: Demographics of cases included in this study [/fig_ref]. We selected 136 cases as control elderly (mean age at death ± 1 SD, 78.5 ± 9.7 y). For comparison, we selected 29 AD, 11 LBD, and 11 AGD cases from the series of autopsied individuals. Compared with the control elderly group, the mean age at death was higher for AD, LBD, and AGD groups [fig_ref] Table 3: Demographics of cases included in this study [/fig_ref].
Distribution of TDP-43 pathology in control elderly brains TDP-43 immunoreactive structures were found in 55/136 (40.0 %) cases [fig_ref] Table 4: Presence of TDP-43 pathology [/fig_ref]. Most of these structures were DNs (53/136 cases, 39.0 %; [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref] ; they were predominantly detected at the uncus of the anterior hippocampus (29/136 cases, 21.3 %; [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref] , [fig_ref] Figure 3: TDP-43 immunoreactive structures in representative cases [/fig_ref]. In rare instances, DNs were present in other limbic areas such as the uncus of the amygdala (5/134 cases, 3.7 %; two cases of amygdala were unavailable, [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref]. In control elderly brains, TDP-43 immunoreactive GCIs and NCIs were rare. In fact, GCIs were observed only in a 94-year-old woman's entorhinal cortex and NCIs only in a 98-year-old woman's subiculum and pyramidal layer [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref].
No NIIs were present in control elderly brains. TDP-43 immunoreactive DNs were also found in the inferior olivary complex (14/136 cases, 10.3 %), anterior horn (7/136 cases, 5.1 %), and white matter of the lumbar spinal cord (11/136 cases, 8.1 %), as shown in [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref].
No NCIs or GCIs were found in the medulla oblongata or lumbar spinal cord. [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref]. There were no DNs in the spinal cord. No NCIs or GCIs were found in any case of LBD.
## Agd brains
TDP-43 immunoreactive structures were found in 6/11 (54.5 %) AGD brains and observed in the uncus and amygdaloid nuclei [fig_ref] Table 4: Presence of TDP-43 pathology [/fig_ref]. These structures were DNs (6/11 cases, 54.5 %). TDP-43 immunoreactive NCIs or GCIs were also found in the amygdaloid nuclei (3/11 cases, 27.3 %). DNs were observed in the uncus of the anterior hippocampus (5/11 cases, 45.5 %) and amygdala (3/10 cases, 33.3 %; one case was unavailable). No TDP-43-positive NIIs were found in these brains. In the medulla oblongata, we found TDP-43 immunoreactive DNs [fig_ref] Table 5: TDP-43 distribution in this study [/fig_ref]. In the lumbar spinal cord, we found TDP-43 immunoreactive DNs in the white matter in an 85-year-old male. Neither NCIs nor GCIs were found in any of the AGD brains.
## Age and frequencies of dns in four groups
The mean age at death was significantly higher in cases with TDP-43 immunoreactive structures than in those without TDP-43 immunoreactive structures in control elderly and AGD [fig_ref] Table 4: Presence of TDP-43 pathology [/fig_ref]. However, there were no significant differences in the mean age at death in AD and LBD [fig_ref] Table 4: Presence of TDP-43 pathology [/fig_ref]. Neither the brain weight nor sex ratio differed significantly among the four groups. Because DNs were the predominant form of TDP-43 immunoreactive structure and observed mostly in the uncus in control elderly, we analyzed the frequencies of DNs in the uncus of the anterior hippocampus and amygdala in comparison with those of AD, LBD, and AGD. There were no significant differences in the frequency of cases with DNs in the uncus of the anterior hippocampus between control elderly and disease controls . In contrast, the frequency of cases with DNs observed in the uncus of the amygdala was higher in AD, LBD, and AGD compared with control elderly .
In addition, we focused on the association between the percentage of cases carrying DNs in the uncus of the anterior hippocampus and aging in control elderly. DNs were observed from age 65 to 94. In these individuals, there was no statistical association between the rate of DNs and aging . However, the rate of DNs may increase beyond 90 years of age .
The relationship between TDP-43 immunoreactivity and cognitive function, cerebrovascular disease in control elderly brains
In control elderly brains, the rates of cases with cognitive impairment (CDR ≥ 0.5) among cases with and without TDP-43 immunoreactive structures were 38.3 % (18/47 cases, CDR was unavailable in 8 cases) and 34.3 % (28/70 cases, CDR was unavailable in 11 cases), respectively. In addition, we examined the influence of cerebrovascular pathology. The rates of cases with cerebrovascular pathology among cases with and without TDP-43 immunoreactive structures were 61.2 % (34/55 cases) and 67.9 % (55/81 cases), respectively. There were no significant differences between cases with and without TDP-43 immunoreacrive structures.
TDP-43 pathology in comorbid pathologies, other neurological disorders and intermediate pathologies.
TDP-43 immunoreactive structures were also found in the following cases: AD plus LBD (5/6 cases, 83.3 %); AD plus AGD (3/3 cases, 100 %); AD plus PSP (1/1 case, 100 %); AD plus HS (1/1 case, 100 %); LBD plus AGD (1/1 case, 100 %); LBD plus PSP (0/1 case, 0 %); AGD plus HS (1/1 case, 100 %); NFTD (3/6 cases, 50 %); CJD (0/4 cases, 0 %); ALS/ALS with dementia (4/4 cases, 100 %); MSA (2/3 cases, 66.7 %); SCA6 (2/2 cases, 100 %); SCA3 (0/1 case, 0 %); PSP (2/2 cases, 100 %); HS (2/2 cases, 100 %); DRPLA (0/1 case, 0 %); CBD (1/1 case, 100 %); FTLD-TDP (1/1 case, 100 %); MELAS (0/1 case, 0 %); NFTC (13/27 cases, 48.1 %); ADC (9/18 cases, 50 %); and PSC (6/12 cases, 50 %).
# Discussion
Our study is the first neuropathologic analysis that has clarified the anatomical distribution of TDP-43 immunoreactive structures in elderly brains with minimal senile neuropathologic changes in large numbers. We revealed the following findings. First, TDP-43 immunoreactive structures were present in 40 % of control elderly. Second, these structures were predominantly observed at the uncus of the anterior hippocampus as DNs over age 65. Third, the number of cases with DNs in the amygdala of control elderly was less than that of AD, LBD, or AGD. Fourth, no NCIs or GCIs were found in the medulla oblongata and lumbar spinal cord in all four groups. Finally, the mean age at death was significantly higher in cases with TDP-43 immunoreactive structures than those without such structures.
A few studies have assessed the frequency of TDP-43 immunoreactive structures in normal brain. Wilson et al. reported that 2/63 (3 %) were positive for TDP-43 in neurologically normal individuals ranging from 23 to 94 years old at death [bib_ref] TDP-43 in aging and Alzheimer's disease -a review, Wilson [/bib_ref]. Another study of 110 cognitively normal elderly brains (mean age at death 86 ± 6.0) revealed that 40 cases (36.4 %) showed TDP-43 immunoreactivity in the limbic area [bib_ref] TDP-43 deposition in prospectively followed, cognitively normal elderly individuals: correlation with argyrophilic..., Arnold [/bib_ref]. In yet another study, 12 of 60 brains (20 %) without severe mental illness (mean age; 68) were reported as TDP-43 immunopositive in the amygdala or hippocampus [bib_ref] Pathological 43-kDa transactivation response DNA-binding protein in older adults with and without..., Geser [/bib_ref]. The wide variation in the rate of TDP-43 immunoreactivity may depend on the age distribution of the sample population and the definition of a normal brain. In fact, the definition of a normal brain was based on the clinical information in previous studies. Because we defined the control elderly brain by using a neuropathologic staging system in association with neurodegenerative disorders and aging, the present study could control the effects of other protein deposits, including tau and α-synuclein, on TDP-43.
The distribution of TDP-43 immunoreactive structures in control elderly was different from those in AD. The structures in AD were widely present in the amygdala and hippocampus. Furthermore, in most cases, the TDP-43 immunoreactive structures in AD took the form of NCIs/GCIs, whereas only two cases of control elderly had GCIs or NCIs. These findings suggest that the pathological mechanism of TDP-43 accumulation in control elderly is different from that in AD. Although the amygdala was considered to be the first affected region of TDP-43 deposition in AD [bib_ref] Staging TDP-43 pathology in Alzheimer's disease, Josephs [/bib_ref] , TDP-43 immunoreactive structures in the amygdala may be non-specific. In this study, the frequency of cases with TDP-43 immunoreactive DNs observed in the uncus of the amygdala was significantly higher in AD than that in control elderly. This finding confirms that the sequential TDP-43 progression begins in the amygdala in AD. Interestingly, two control elderly cases over 90 showed NCIs or GCIs in the hippocampus and parahippocampus. Therefore, a portion of the TDP-43 immunoreactive NCIs and GCIs may be associated with aging. In addition, there is a possibility that these cases had neurodegenerative disorders at the earliest stages. Further cases need to be investigated to confirm these possibilities.
Previous studies that used a small number of cases also reported that TDP-43 immunoreactive structures were frequently observed in the amygdala in AD, LBD, and AGD [bib_ref] TDP-43 immunoreactivity in hippocampal sclerosis and Alzheimer's disease, Amador-Ortiz [/bib_ref] [bib_ref] Concurrence of TDP-43, tau and alpha-synuclein pathology in brains of Alzheimer's disease..., Higashi [/bib_ref] [bib_ref] Phosphorylated TDP-43 in Alzheimer's disease and dementia with Lewy bodies, Arai [/bib_ref] [bib_ref] Abnormal TDP-43 immunoreactivity in AD modifies clinicopathologic and radiologic phenotype, Josephs [/bib_ref] [bib_ref] Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases, Nakashima-Yasuda [/bib_ref] [bib_ref] Accumulation of phosphorylated TDP-43 in brains of patients with argyrophilic grain disease, Fujishiro [/bib_ref]. In the present study, TDP-43 immunoreactive DNs in the amygdala of control elderly brains were less numerous than in that of AD, LBD or AGD brains. Therefore, TDP-43 immunoreactive deposits in the amygdala are associated with deposits of Correlation between age and incidence of TDP-43 immunoreactive structures in the uncus of the anterior hippocampus in control elderly brains. The incidence of TDP-43 positive brains ranged between 13 % and 27 % between ages 65 and 89 years. Although there were no significant differences, the incidence of TDP-43-positive brains increased to 50 % at ages 90 to 94 years Incidence of TDP-43 immunoreactive dystrophic neurites (DNs) in the uncus of the anterior hippocampus and amygdala. a There were no significant differences in the number of cases with DNs in the uncus of the anterior hippocampus between control elderly brains and brains with Alzheimer's disease (AD), Lewy body disease (LBD), and argyrophilic grain disease (AGD). b The incidence of DNs in the amygdala was significantly higher in the AD, LBD, and AGD brains than in control elderly brains proteins such as tau and α-synuclein, which are related to other neurodegenerative disorders. In contrast, the number of cases with TDP-43 immunoreactive DNs in the uncus of the anterior hippocampus of control elderly showed no significant differences compared with AD, LBD, and AGD. Importantly, these DNs were present from age 65 to 94 years in our study. In particular, the proportion of TDP-43-positive cases was 13 % to 27 % in the age range of 65-89 years and 50 % in the age range of 90-94 years . These findings suggest that TDP-43 accumulation in the uncus of the anterior hippocampus in the form of DNs is predominantly associated with physiological aging and not with neurodegenerative disorders.
We found TDP-43 immunoreactive DNs in the medulla oblongata and the spinal cord in control elderly brains and there were no NCIs or GCIs in all groups. These structures were predominantly located in the inferior olivary nuclei and the white matter of the spinal cord. The cases of AD, LBD, and AGD brains also showed TDP-43 immunoreactive DNs in those anatomical regions. We believe that the TDP-43 immunoreactive DNs in those specific anatomical regions may be associated with human physiological aging. In fact, the distribution pattern of TDP-43 deposits in this study clearly differs from that in ALS and FTLD-TDP brains that show many NCIs [bib_ref] Stages of pTDP-43 pathology in amyotrophic lateral sclerosis, Brettschneider [/bib_ref] [bib_ref] Sequential distribution of pTDP-43 pathology in behavioral variant frontotemporal dementia (bvFTD), Brettschneider [/bib_ref].
Our study has some limitations. Because many aging brains have comorbid pathologies, the numbers of pure AD, LBD, and AGD brains in this study were relatively small. In addition, a part of TDP-43 accumulation of in AD, LBD, and AGD brains may be influenced by the aging process. In fact, for AD, LBD, and AGD, the mean age of death was significantly higher than that for control elderly.
Although TDP-43 has been associated with memory loss and medial temporal atrophy in AD, the role of TDP-43 accumulation in control elderly brains remains unresolved. The present study, we could not find the relationship between cognitive function and TDP-43 pathology in control elderly cases. Since the present study has not focused on neuropsychological examination, further studies in large numbers are needed to determine whether TDP-43 pathology might lead to subtle changes in cognitive function.
# Conclusions
In summary, our study clearly revealed that TDP-43 immunoreactive structures may develop as a consequence of aging processes in the human brain. Most of them were DNs not NCIs and GCIs, and were observed in the uncus of anterior hippocampus. Additional studies may be important to understand the role of TDP-43 accumulation in normal human aging. Besides those control elderly cases, we believe that the cases without TDP-43 immunoreactive deposits are also substantial for analysis of pathomechanism of TDP-43 accumulation.
## Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files.
[fig] Figure 1: Case selection procedure. NFT, neurofibrillary tangle; SP, senile plaque; AGD, argyrophilic grain disease; LB, Lewy body; AD, Alzheimer's disease; LBD, Lewy body disease (Parkinson's disease and dementia with Lewy bodies); PSP, progressive supranuclear palsy; HS, hippocampal sclerosis; NFTD, NFT-predominant form of senile dementia; CJD, Creutzfeldt-Jakob disease; ALS, amyotrophic lateral sclerosis; MSA, multiple system atrophy; SCA, spinocerebellar ataxia; DRPLA, dentatorubral-pallidoluysian atrophy; CBD, corticobasal degeneration; FTLD, frontotemporal lobar degeneration; MELAS, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes; NFTC, NFT-predominant change; ADC, AD-type change; PSC, plaque dominant senile change [/fig]
[fig] Figure 3: TDP-43 immunoreactive structures in representative cases. a Dystrophic neurites in the uncus of the anterior hippocampus in the control elderly brain. b Neuronal cytoplasmic inclusions (arrows) in the dentate gyrus in Alzheimer's disease (AD). c Glial cytoplasmic inclusions (arrows) in the entorhinal cortex in AD. d Neuronal intranuclear inclusion (arrow) in the dentate gyrus in AD. Scale bar = 20 μm [/fig]
[table] Table 1: Antibodies used for immunohistochemistry [/table]
[table] Table 2: Brain regions analyzed in this studyFig. 2 Evaluated anatomical regions in this study. a anterior hippocampus; b amygdala; c medulla oblongata; d lumbar spinal cord. Each anatomical region was identified using the neuroanatomy atlas[24]. The uncus was defined and identified using the human hippocampus, third edition[25] Fig. 3b, c). DNs were observed in the uncus of the anterior hippocampus (11/28 cases, 39.3 %; in one case, the anterior hippocampus was unavailable) as well as in the amygdala (14/29 cases, 48.3 %). In 7/29 cases (24.1 %), TDP-43 immunoreactive NIIs were also present in the amygdala and hippocampus(Fig. 3d). To a lesser degree, TDP-43-positive DNs were found in the medulla oblongata (5/29 cases; 17.2 %) and lumbar spinal cord (3/29 cases; 10.3 %). There were neither NCIs nor GCIs in any case of AD(Table 5). [/table]
[table] Table 3: Demographics of cases included in this study [/table]
[table] Table 4: Presence of TDP-43 pathology [/table]
[table] Table 5: TDP-43 distribution in this study [/table]
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Gluteal Subcutaneous Atrophy After Depot Steroid Injection for Allergic Rhinitis
Allergic rhinitis is a common and often distressing condition. Currently, treatment with nonsedating antihistamines, topical therapy, and immunotherapy are very effective. Despite this, intramuscular depot steroids are commonly used in clinical practice. Here, we present the case of a young woman who developed disfiguring scarring after a depot steroid injection. This case highlights the risks of this form of treatment for allergic rhinitis.
Systemic steroids have played a role in the management of allergic rhinitis in the past 5 decades. Early reports confirmed an excellent response to systemic steroids. In recent years, the use of oral steroids has not been recommended for the management of allergic rhinitis in view of the efficacy of topical therapy and concerns about adverse effects.
However, oral and intramuscular depot steroids remain popular in primary care because of their efficacy and the expense of allergen-specific immunotherapy. Here, we present the case of a patient who suffered disfiguring subcutaneous atrophy in the gluteal region from a depot triamcinolone injection given for allergic rhinitis.
## Case report
The patient is a 29-year-old immigrant. Shortly after moving to New Zealand, she suffered disabling allergic rhinitis symptoms. Her symptoms were perennial. She was clinically assessed by her family physician and was given a depot 40-mg triamcinolone injection into the gluteal region. She experienced rapid relief of her nasal obstruction and rhinorrhea. She had received prior injections of depot steroids for her rhinitis. Within a few weeks, she developed a large 5-cm scar at the injection site [fig_ref] FIGURE 2: Close-up view of the 5-cm scar after a 40-mg triamcinolone injection given... [/fig_ref].
She was reviewed in the immunology clinic at the Auckland Hospital. Skin prick testing confirmed strongly positive reactions to dust mites. She was treated with a combination of loratadine 10 mg and Budesonide 100 mg nasal spray twice daily. She declined the offer of immunotherapy on account of expense. She was reviewed by a plastics and reconstructive surgeon, but no operative intervention was offered.
# Discussion
Depot steroid injections for seasonal allergic rhinitis have been commonly used in some parts of the world, particularly Denmark. One study estimated that 0.66% of the entire Danish population received depot steroid injections each year. [bib_ref] Hay fever and a single intramuscular injection of corticosteroid: a systematic review, Stergaard [/bib_ref] The efficacy of depot steroid injections has been established in several randomized controlled trials. [bib_ref] Systemic corticosteroid treatment for seasonal allergic rhinitis: a common but poorly documented..., Mygind [/bib_ref] Depot steroids appear to be more effective than topical beclomethasone [bib_ref] Intramuscular betamethasone dipropionate vs. topical beclomethasone dipropionate and placebo in hay fever, Laursen [/bib_ref] and are as effective as 7.5 mg of oral prednisone given for 3 weeks. [bib_ref] Intramuscular betamethasone dipropionate vs. oral prednisolone in hay fever patients, Laursen [/bib_ref] Most patients experience 4 to 5 weeks of symptoms relief after a single injection.
Generally, nasal obstruction is more likely to improve than rhinorrhea or sneezing. It is of interest that most of these The author has no funding or conflicts of interest to disclose. Correspondence to: Rohan Ameratunga, MBChB, Clinical Immunologist, Auckland Hospital, Park Road, Grafton, Auckland 1023, New Zealand. Telephone: 64-9-3797-440 (ext: 6113). Fax: 64-9-307-2826. E-mail: [email protected]. Copyright Ó 2012 by World Allergy Organization randomized trials of depot steroids were undertaken in Denmark. All these randomized trials of depot steroids for allergic rhinitis were undertaken before 1988. It was around this time that effective nonsedating antihistamines became widely available.
In recent years, there has been increasing concern about the use of depot steroids in the management of allergic rhinitis. [bib_ref] Primum non nocere, Bousquet [/bib_ref] Patients with severe symptoms unresponsive to antihistamines and topical steroid therapy should be referred to an allergy specialist for consideration of immunotherapy. [bib_ref] Allergen injection immunotherapy for seasonal allergic rhinitis, Calderon [/bib_ref] The lack of allergy services in many parts of the world, including New Zealand, has hindered such referrals. [bib_ref] Lesson of the week: depot corticosteroid treatment for hay fever causing avascular..., Nasser [/bib_ref] Although relatively uncommon, adverse effects from depot steroids are a source of increasing concern. Bilateral avascular necrosis after a depot steroid injection has been reported. [bib_ref] Lesson of the week: depot corticosteroid treatment for hay fever causing avascular..., Nasser [/bib_ref] There is concern about hypothalamic pituitary adrenal axis suppression and long-lasting effects on bone mineral density. [bib_ref] Primum non nocere, Bousquet [/bib_ref] Subcutaneous atrophy of the deltoid has been described after depot steroid injections for allergic rhinitis. This has been termed pseudomorphea. [bib_ref] Pseudomorphoea': a side effect of subcutaneous corticosteroid injection, Holt [/bib_ref] One case of subcutaneous atrophy has been estimated to occur after 11,000 injections. [bib_ref] Hay fever and a single intramuscular injection of corticosteroid: a systematic review, Stergaard [/bib_ref] Although this may be a rare event, there is concern about underreporting. [bib_ref] Primum non nocere, Bousquet [/bib_ref] In some patients, the area of scarring improves slowly after several years. This case illustrates the small but significant risk of using this form of treatment for a non-life-threatening disorder. Before this case, it was previously thought that intramuscular injections into the gluteal region were associated with a low risk of subcutaneous atrophy. [bib_ref] Hay fever and a single intramuscular injection of corticosteroid: a systematic review, Stergaard [/bib_ref] It is uncertain if this patient received an intramuscular injection or whether the triamcinolone was injected into the subcutaneous tissues.
Depot steroid injections remain very poplar among primary care physicians and patients. Their low cost and effectiveness underscore this popularity. The pressure to use this form of treatment should be resisted given the small but significant risk of disfiguring adverse effects.
A short course of oral steroids could be justified for patients attending an important function such as a wedding or before surgery in unresponsive patients. Given the availability of effective therapy, depot steroid injections should be considered an obsolete form of treatment for allergic rhinitis. This from of treatment, particularly repeated doses, should no longer be accepted as the standard of care of allergic rhinitis. 10
[fig] FIGURE 2: Close-up view of the 5-cm scar after a 40-mg triamcinolone injection given for allergic rhinitis. [/fig]
[fig] FIGURE 1: Subcutaneous atrophy of the gluteal region after a 40-mg depot triamcinolone injection. [/fig]
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A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR
Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCYexpressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.
# Introduction
Allogeneic stem cell transplantation (SCT) has become the treatment of choice for patients with various hematological malignancies and some studies have also shown that metastatic renal cell carcinoma (RCC) does respond to this therapy [bib_ref] Immunotherapy for renal cell cancer, Yang [/bib_ref]. Several studies have explored allogeneic SCT after nonmyeloablative or reduced intensity conditioning (RIC) with or without donor lymphocyte infusions (DLI) as curative treatment for metastatic RCC, and objective response rates varied from 0% to 53% [bib_ref] Immunotherapy for renal cell cancer, Yang [/bib_ref] [bib_ref] Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation, Childs [/bib_ref] [bib_ref] Allogeneic blood stem cell transplantation after a reduced-intensity, preparative regimen -A pilot..., Pedrazzoli [/bib_ref] [bib_ref] Allogeneic stem-cell transplantation of renal cell cancer after nonmyeloablative chemotherapy: feasibility, engraftment,..., Rini [/bib_ref] [bib_ref] Allogeneic hematopoietic cell transplantation for metastatic renal cell carcinoma after nonmyeloablative conditioning:..., Tykodi [/bib_ref] [bib_ref] Allogeneic peripheral-blood progenitor-cell transplantation for poor-risk patients with metastatic breast cancer, Ueno [/bib_ref]. However, substantial transplantation-related mortality and toxicity due to graft-versus-host-disease (GVHD) has been observed. Therefore, further development of allogeneic SCT for solid tumors demands a more specific approach to selectively boost graft-versus-tumor (GVT) reactivity without enhancement of GVHD.
Minor histocompatibility antigens (MiHA) are the target antigens of the GVT response, and expansion of MiHA-specific cytotoxic T lymphocytes (CTL) usually precedes clinical remission of the malignancy in patients treated with DLI [bib_ref] Induction of HA-1-specific cytotoxic T-cell clones parallels the therapeutic effect of donor..., Kircher [/bib_ref] [bib_ref] Hematopoiesis-restricted minor histocompatibility antigens HA-1-or HA-2-specific T cells can induce complete remissions..., Marijt [/bib_ref] [bib_ref] Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy, Goulmy [/bib_ref]. However, alloreactive CTL responses induced upon DLI generally lack tumor specificity and are often accompanied by GVHD. Therefore, it would be highly beneficial to direct T cell immunity towards MiHA that are selectively expressed on malignant cells. Only a few tissue-restricted MiHA have been described that are aberrantly expressed on solid tumors, including HA-1, ECGF-1, BCL2A1, LRH-1 and C19orf48 [bib_ref] Treatment of post-transplanted, relapsed patients with hematological malignancies by infusion of HLA-matched,..., Fujii [/bib_ref] [bib_ref] Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human..., Hambach [/bib_ref] [bib_ref] Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors..., Overes [/bib_ref] [bib_ref] Aberrant expression of BCL2A1-restricted minor histocompatibility antigens in melanoma cells: application for..., Torikai [/bib_ref] [bib_ref] C19orf48 encodes a minor histocompatibility antigen recognized by CD8+ cytotoxic T cells..., Tykodi [/bib_ref]. In addition to a MiHA, an immunogenic antigen has been described expressed on metastatic RCC, HERV-E which is derived from a human endogenous retrovirus [bib_ref] Regression of human kidney cancer following allogeneic stem cell transplantation is associated..., Takahashi [/bib_ref]. However, further characterization of the target antigens of alloreactive T cells on RCC tumor cells is of great importance for the development of specific post-transplant immunotherapy for metastatic RCC.
Here, we characterized MiHA-specific T cell responses in two patients with metastatic RCC treated with partial T cell-depleted RIC-SCT followed by DLI. Presence of MiHA-specific CTL targeting RCC tumor cells in vitro could be demonstrated in both patients who experienced partial regression or stable disease following RIC-SCT and DLI without clinical evidence of GVHD. In one patient the T cell response was directed against the HLA-A2-restricted SCMY peptide FIDSYICQV, and in the other patient a MiHA-specific CTL clone emerged with the capability of targeting RCC cell lines. We found that this CTL recognizes an HLA-B7-restricted MiHA resulting from a polymorphism in the splice donor site within the ZNF419 gene. This novel MiHA, designated ZNF419 alternatively spliced polymorphic histocompatibility antigen in RCC (ZAPHIR), is co-expressed by RCC and transformed B cells, but is not presented by non-hematopoietic fibroblasts. Furthermore, ZAPHIR-specific CD8+ T cells were detectable in peripheral blood post-transplant using tetramer analysis. These findings exemplify that ZAPHIR represents a new target antigen for the development of adjuvant immunotherapy for RCC and hematological malignancies after allogeneic SCT.
# Results
Assessing for alloreactive MiHA-specific CD8+ CTL after transplantation of metastatic RCC patients Four patients with progressive metastatic RCC after treatment with at least one line of therapy were treated with RIC-SCT from an HLA identical sibling donor [fig_ref] Table 1: Patient characteristics, GVHD, DLI and outcome [/fig_ref]. These patients had undergone tumor nefrectomy at a median of 4.9 years (range 2.8-11.1 years) before transplantation. After RIC-SCT, all patients reached eventually complete donor chimerism in the T cell fraction. The first tumor evaluation in UPN677 at three months after RIC-SCT showed stable disease, paralleled by expansion of CD8+ T cells during tapering of cyclosporine A (CsA). One month later this patient developed encephalopathy attributed to EBVreactivation. Following DLI, a second expansion of CD8+ T cells as well as circulating EBV-specific CD8+ T cells was detected up to 11 weeks using tetramers against the GLCTLVAML epitope. At seven months after RIC-SCT a second DLI was administered and the tumor in the abdomen remained stable without occurrence of GVHD [fig_ref] Figure 1: Longitudinal follow-up of CD8+ T cells in peripheral blood from metastatic RCC... [/fig_ref]. Evaluation of disease response in UPN686 3 months after RIC-SCT showed partial regression of pulmonary metastases that coincided with expansion of CD8+ T cells during tapering of CsA and before the first DLI. This patient received DLI at 3 months after RIC-SCT inducing a second CD8+ T cell expansion and conversion to complete donor T cell chimerism [fig_ref] Figure 1: Longitudinal follow-up of CD8+ T cells in peripheral blood from metastatic RCC... [/fig_ref]. Three months later (i.e. 6 months after RIC-SCT) CT-scan evaluation showed stable disease. Unfortunately, this patient died from invasive fungal infection 9 months after RIC-SCT. Post-mortal examination showed multiple histological-confirmed fungal lesions and two pulmonary metastasis were found, but no signs of GVHD. Collectively, the chimerism kinetics and expansions of CD8+ T cells in UPN677 and UPN686 indicated T cell alloreactivity, but episodes with infections may have interfered.
To investigate whether MiHA-specific CD8+ T cells targeting RCC tumor cells could be isolated from these two patients post DLI-1, IFN-c+ CD8+ T cells (<2%) were sorted and weekly restimulated with irradiated recipient peripheral blood mononuclear cells (PBMC) obtained pre-SCT. The resulting CD8+ T cell lines, termed CTL line H (UPN677) and CTL line B (UPN686), displayed significant IFN-c production against recipient EBV-LCL, but not towards donor EBV-LCL, indicating the recognition of a disparate MiHA [fig_ref] Figure 1: Longitudinal follow-up of CD8+ T cells in peripheral blood from metastatic RCC... [/fig_ref]. These data indicate that MiHAspecific CD8+ T cells were present in vivo in RCC patients UPN677 and UPN686 following allogeneic RIC-SCT and DLI.
Emergence of SMCY-specific CTLs in a RCC patient after DLI Next, we revealed TCR-Vb1+CD8+ cells to be the dominant T cell population in CTL line H using TCR-Vb PCR and flow cytometry (data not shown). Functional analysis showed that specific IFN-c production by CTL line H was substantially inhibited by anti-HLA class I and anti-HLA-A2 antibodies, but not by antibodies against anti-HLA-B/C and anti-HLA class II [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. Furthermore, testing EBV-LCL from unrelated HLA-A2+ individuals revealed that 3 out of 9 individuals were recognized, which were all of male origin [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. These observations suggested that the dominant TCR-Vb1+CD8+ T cells in CTL line H recognize an HLA-A2-restricted HY antigen. Therefore, we stained CTL line H with PE-and APC-conjugated tetramers for HLA-A2-restricted SMCY.A2 epitope FIDSYICQV and found around 12.5% tetramer+ CD8+ T cells [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. These SMCY.A2 tetramer+ T cells confirmed to be TCR-Vb1+ by flow cytometry (data not shown). A greater than 95% pure population of SMCY.A2 tetramer+ CTLs was isolated by flow cytometry allowing further characterization of its cytotoxic potential [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. Flow cytometry-based cytotoxicity assays revealed that the TCR-Vb1+ SMCY.A2 CTL induced high levels of cytotoxicity against HLA-A2+ peptide-loaded SMCY-donor EBV-LCL as well as SMCY+ recipient EBV-LCL [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. Tetramer analysis showed that SMCY.A2-specific CD8+ T cells became detectable in vivo in the post-DLI setting, constituting 3.6%, 2.1% and 2.4% of the CD3+CD8+ T cell population collected at week +24, +28 and +31, respectively [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. Collectively, these data show that SMCY.A2 CD8+ T cells expanded in RCC patient UPN677 after DLI with the potential of targeting SMCY-expressing RCC tumor cells in the absence of GVHD.
Identification of a novel HLA-B7-restricted MiHA resulting from a polymorphism in the splice donor site of ZNF419 TCR receptor analysis using PCR and flow cytometry showed a predominant TCR-Vb4+CD8+ population in CTL line B, which were sorted and cultured resulting in a pure population (data not shown). This TCR-Vb4+CD8+ CTL, designated CTL B1, mediated specific IFN-c production against recipient EBV-LCL, but not towards donor EBV-LCL [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref]. Release of IFN-c could be completely inhibited by anti-HLA class I and anti-HLA-B/C antibodies, but not by antibodies against anti-HLA-A2 and anti-HLA class II [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref]. Testing of EBV-LCL from unrelated individuals sharing expression of HLA-B7 with the recipient, and EBV-LCL from an HLA class I-mismatched individual that were transduced with HLA-B*0702, revealed that CTL B1 recognizes an HLA-B7-restricted MiHA [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref].
Next, we investigated whether the MiHA recognized by CTL B1 was selectively expressed by hematopoietic cells. Therefore, EBV-LCL, PHA-stimulated T cells and non-hematopoietic fibroblasts from two MiHA+ HLA-B7+ healthy donors were used as target cells. CTL B1 produced IFN-c upon co-culture with EBV-LCL, whereas PHA-stimulated T cells and TNF-a/IFN-c pre-treated bone marrow-derived fibroblasts were not recognized [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref]. In contrast, all target cell types were significantly recognized by allo-HLA-B7 CTL, indicating their susceptibility to CTL-mediated recognition [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref]. To investigate whether the MiHA targeted by CTL B1 is expressed by RCC tumor cells, we performed flow cytometry-based cytotoxicity assay. We observed that CTL B1 significantly targets the HLA-B7+ RCC cell line SKRC33 and not SKRC18, showing MiHA mediated cytotoxicity [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref]. In addition to these RCC cell lines, CTL B1 also recognizes and kills other solid tumor cell lines such as the HLA-B7+ brain cancer cell line DAOY, but not pharynx cell line FaDu. Allo-HLA-B7-specific CTL efficiently lysed all solid tumor cell lines tested [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref].
To map the chromosomal region containing the gene encoding the MiHA recognized by CTL B1, we performed whole genome association scanning (WGAs). A total of 87 endogenous HLA-B7+ EBV-LCL were tested and 42 were recognized by CTL B1 [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. Comparing CTL recognition with SNP genotypes showed a strong association for a cluster of 5 SNPs on chromosome 19 [fig_ref] Figure 2: CD8+ T cells reactive with the SMCY [/fig_ref]. This genetic region included the ZNF419 gene which is located at 19q13.43. There are 7 different splice variants of the ZNF419 gene [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref] , from which several are expressed by patient UPN686 and its related donor [fig_ref] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines [/fig_ref]. To confirm that the MiHA is encoded by the ZNF419 gene, we cloned the different splice variants of the ZNF419 gene from both the donor and recipient EBV-LCL. The various constructs were transiently transfected into 293T-HLA-B*0702/ICAM/CD80 cells and tested for CTL B1 recognition. CTL B1 efficiently recognized cells transfected with a construct encoding an alternative ZNF419 isoform of the MiHA-positive recipient EBV-LCL [fig_ref] Figure 4: A SNP in the splice donor site between exon 4 and 5... [/fig_ref] , whereas none of the ZNF419 isoforms cloned from the donor EBV-LCL stimulated IFN-c production by CTL B1 (data not shown). Sequence analysis revealed that the recognized ZNF419 isoform results from the A/G SNP rs2074071 located in the splice donor site involved in the joining of exon 4 to intron 4. As a result of the G to A substitution in the splice donor site, the alternative ZNF419 transcript of the recipient contains a disparate amino acid sequence compared with that of the donor. By analyzing this disparate sequence for HLA-B7 binding peptides using the NetMHC server, we identified the strong HLA-B7 binding peptide IPRDSWWVEL that is located over the exon4-intron4 boundary [fig_ref] Figure 4: A SNP in the splice donor site between exon 4 and 5... [/fig_ref]. We synthesized this 10-mer IPRDSWWVEL peptide (encoded by rs2074071 A variant), the 9-mer IPRDSWWVE peptide and the 8mer IPRDSWWVEL peptide. We tested the HLA-B7 decamer peptide IPRGSWWVEL that would be encoded by the G genotype as well. In addition, we synthesized the HLA-B7 9-mer peptide IPRGEWHGA that is located on the exon4-exon5 boundary of the full length ZNF419 transcript. Their ability to stimulate IFN-c release by CTL B1 upon loading of synthetic peptide on donor EBV-LCL was tested and only the IPRDSWWVEL epitope was recognized [fig_ref] Figure 4: A SNP in the splice donor site between exon 4 and 5... [/fig_ref]. Flow cytometry analysis of CTL B1 showed that only the tetramer containing the 10-mer peptide was positive as opposed to tetramers containing the 8-, or 9-mer peptide [fig_ref] Figure 4: A SNP in the splice donor site between exon 4 and 5... [/fig_ref]. These data demonstrate that the 10-mer peptide IPRDSWWVEL is the naturally presented epitope that is recognized by CTL B1. Based on these findings, we designated this novel MiHA as ZAPHIR for ZNF419 alternatively spliced polymorphic histocompatibility antigen in RCC, which is the result of a polymorphism in a splice donor site.
Expression of ZNF419 in various (malignant) hematopoietic and non-hematopoietic cell samples was performed by microarray gene expression analysis. Analysis of primary hematopoietic tissues [fig_ref] Figure 5: ZNF419 microarray gene expression analysis of [/fig_ref] show that the overall expression of the gene is low, as is the case for hematopoietic malignancies [fig_ref] Figure 5: ZNF419 microarray gene expression analysis of [/fig_ref]. Although fibroblast, keratinocytes, proximal tubular epithelial cells (PTEC) and RCC show low expression levels as well, the keratinocytes show an upregulation of ZNF419 when cultured in the presence of IFN-c [fig_ref] Figure 5: ZNF419 microarray gene expression analysis of [/fig_ref].
## Detection of zaphir-specific cd8+ t cells in vivo
Next, we determined the rate of ZAPHIR disparity in HLA-B7matched SCT donor-recipient pairs using a competitive allele-specific PCR. In 98 donor-recipient pairs (including RCC patient UPN686), we identified 13 ZAPHIR-positive patients (i.e. 13.3%) that had received a transplant from a ZAPHIR-negative donor. To detect ZAPHIR-specific CD8 + T cells in these mismatched patients in vivo, we constructed an HLA-B7 tetramer using the 10-mer peptide IPRDSWWVEL. First, we analyzed PBMCs samples of RCC patient UPN686 from which the ZAPHIR-specific CTL B1 was isolated. In addition, specific detection of ZAPHIR-tetramer + CD8+ T cells was assessed after in vitro stimulation of PBMC samples with peptide-pulsed donor EBV-LCL. At fourteen weeks post-SCT (i.e. pre-DLI) ,0.01% ZAPHIR-tetramer + cells could be directly detected in the PBMC sample, but after stimulation the percentage increased to 0.26% within the total CD8+ T cell population [fig_ref] Figure 6: Detection of ZAPHIR-specific CD8+ T cells in peripheral blood of RCC patient... [/fig_ref]. Interestingly, the percentage ZAPHIR-tetramer + CD8+ T cells increased at 4 weeks after DLI to a level of 0.11%, and persisted at a low frequency (,0.01%) up to 20 weeks post-DLI. In vitro stimulation resulted in an increase of ZAPHIR-specific CD8+ T cells up to 1.37% for week 4, 0.46% for week 9, and 0.70% for week 20 post-DLI [fig_ref] Figure 6: Detection of ZAPHIR-specific CD8+ T cells in peripheral blood of RCC patient... [/fig_ref]. These data show that ZAPHIR-specific CD8+ T cells with the capability of targeting RCC metastases emerged in RCC patient UPN686 following SCT and DLI in the absence of GVHD.
Specific detection of ZAPHIR-tetramer + CD8+ T cells was also performed in the peripheral blood of 12 additional ZAPHIRmismatched patients. In 1 of these 12 patients, chronic myeloid leukemia (CML) patient UPN 539, 0.04% and 0.36% ZAPHIR-tetramer+ CD8+ T cells could be detected in the in vitro-stimulated T cell cultures obtained from PBMC samples at 7 and 40 weeks post- therapeutic DLI, while no (,0.01%) tetramer+ cells could be detected directly in the PBMC [fig_ref] Figure 6: Detection of ZAPHIR-specific CD8+ T cells in peripheral blood of RCC patient... [/fig_ref]. Furthermore, ZAPHIR-specific CTL could be cloned from PBMC collected 40 weeks post-therapeutic DLI [fig_ref] Figure 4: A SNP in the splice donor site between exon 4 and 5... [/fig_ref]. These data indicate that ZAPHIR-specific CD8+ T cell responses can also be detected in transplanted CML patients.
# Discussion
MiHA are considered to play a dominant role in mediating GVT reactivity after HLA-identical allogeneic SCT for both hematological malignancies and solid tumors. Especially MiHA with a hematopoietic-or tumor-restricted distribution are promising targets to boost GVT reactivity without enhancing GVHD. For the development of tumor-specific immunotherapy it is important to enlarge the spectrum of molecularly identified MiHA that are selectively expressed. In this study, we characterized antigens targeted by allogeneic donor T cells in metastatic RCC patients treated with partial T cell-depleted RIC-SCT followed by DLI. Using PBMCs collected from two patients, showing either stable disease or partial response, we succeeded in expanding CD8+ CTLs with the capability of targeting MiHA expressed by RCC cells. Interestingly, a TCR-Vb4+CD8+ CTL targeting a novel HLA-B7-restricted MiHA, designated ZAPHIR, was isolated from RCC patient UPN686 showing partial regression of lung metastases in the absence of GVHD. Flow cytometry-based cytotoxicity assays revealed that ZAPHIRspecific CTL B1 mediated efficient lysis of HLA-B7+ RCC cell lines. In addition, ZAPHIR-specific CTL also mediated cytotoxicity against an HLA-B7+ brain tumor cell line. Furthermore, while EBV-LCL were recognized by ZAPHIR-specific CTL, PHA-stimulated T cells and cytokine-treated bone marrow (BM)derived fibroblasts did no induce IFN-c production. These data indicate that the HLA-B7-restricted MiHA ZAPHIR is coexpressed by solid tumors and transformed B cells. Notably, emergence of ZAPHIR-specific CD8+ T cells in RCC patient UPN686 as well as CML patient UPN 539 was observed in the absence of GVHD. Microarray gene expression analysis of several (malignant) hematopoietic cells and normal tissues show that expression of ZNF419 is low. This might explain the absence of an immune response, as is the case in vivo in non-malignant tissues, and therefore no GVHD could be observed. However, as the keratinocytes cocultured with IFNc show that under inflammatory conditions ZNF419 is upregulated and could lead to an immune response. Since the tumor tissues are surrounded by an inflammatory environment, a GVT response mediated by MiHA-specific T cells could occur in vivo.
In order to identify the novel MiHA recognized by CTL B1, we used the WGAs method that was recently described by Van Bergen et al [bib_ref] High-throughput characterization of 10 new minor histocompatibility antigens by whole genome association..., Van Bergen [/bib_ref]. This method makes use of the SNP-genotyping of unrelated HLA-B7+ individuals who were divided in a MiHA+ and MiHA-group and yielded a highly specific genomic location on chromosome 19q13.43. This led to the rapid identification of ZNF419 as the gene of interest showing WGAs to be a very suitable method for MiHA identification. Further analysis revealed that the novel ZAPHIR epitope is located on the exon4-intron4 boundary which is the result of a G to A substitution in the splice donor site leading to an alternative spliced ZNF419 variant. The mechanism that MiHA immunogenicity can arise from alternative splicing due to a single intronic SNP, has been previously described for the MiHA ACC-6 [bib_ref] Alternative splicing due to an intronic SNP in HMSD generates a novel..., Kawase [/bib_ref].
In RCC patient UPN677, we demonstrated emergence of SMCY.A2-specific CD8+ T cells after DLI without the clinical manifestations of GVHD. Interestingly, Hambach et al. showed that SMCY.A2-specific CTL are capable of efficiently targeting solid tumor cells in a three-dimensional micro tumor model [bib_ref] Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human..., Hambach [/bib_ref]. Therefore, our clinical findings suggest that emergence of SMCY.A2 CTL may have played a role in stabilization of the tumor growth after allogeneic SCT and DLI in patient UPN677. Interestingly, high numbers of SMCY.A2 CD8+ T cells up to 3.6% of the total CD8+ T cell population in patient UPN677 did not induce clinical signs of GVHD to either skin, liver or gut. Earlier studies have shown that SMCY is ubiquitously expressed, and SMCY-specific CTL responses have been associated with GVHD [bib_ref] Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy, Goulmy [/bib_ref] [bib_ref] Minors come of age: Minor histocompatibility antigens and graft-versus-host disease, Chao [/bib_ref] [bib_ref] Graft-versus-leukemia reactions after bone marrow transplantation, Horowitz [/bib_ref] [bib_ref] Molecules and mechanisms of the graft-versusleukaemia effect, Bleakley [/bib_ref]. However, GVHD is a multi-factorial disease process to which many factors contribute, and we speculate that lack of other inflammatory triggers prevented the development of acute GVHD (reviewed by Ferrara et al [bib_ref] Graft-versus-host disease, Ferrara [/bib_ref].
Currently, the role of allogeneic RIC-SCT for the treatment of metastatic RCC patients is still unclear. New developments in treatment of metastatic RCC have significantly changed the treatment strategies for these patients. However, the novel drugs that are currently used do not cure patients and the incidence of complete remissions is low. Therefore, new strategies should continue to be explored and the discovery of antigenic targets of alloreactive T cells in transplanted RCC patients may allow the development of tumor-specific post-transplantation strategies such as vaccination or adoptive T cell transfer. Two earlier studies have identified target antigens at the molecular level using CD8+ CTL isolated from patients with metastatic RCC treated with allogeneic SCT. Tykodi et al. identified an HLA-A*0201-restricted MiHA, which is encoded by the C19orf48 gene located on chromosome 19q13 [bib_ref] C19orf48 encodes a minor histocompatibility antigen recognized by CD8+ cytotoxic T cells..., Tykodi [/bib_ref]. In addition, recognition of the non-polymorphic RCC over-expressed human endogenous retrovirus type E (HERV-E) antigen by alloreactive T cells was described by Takahashi et al [bib_ref] Regression of human kidney cancer following allogeneic stem cell transplantation is associated..., Takahashi [/bib_ref]. Not only tumor-specific MiHA are interesting for these purposes, but also hematopoietic-restricted MiHA with aberrant expression in solid tumor cells may be useful for this strategy. Genotype frequency analysis in HLA-B7 allo-SCT couples revealed a disparity rate of 13.3%, making the ZAPHIR MiHA therapeutically relevant as is the case for the other, commonly mismatched MiHA HA-1, LRH-1 and BCL2A1 [bib_ref] A frameshift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response..., De Rijke [/bib_ref] [bib_ref] Identification of a polymorphic gene, BCL2A1, encoding two novel hematopoietic lineage-specific minor..., Akatsuka [/bib_ref] [bib_ref] The minor histocompatibility antigen HA-1: a diallelic gene with a single amino..., Den Haan [/bib_ref]. Adoptive transfer of a single dose of HA-1 CTLs was effective in eradicating disseminated solid tumors in a mouse model [bib_ref] Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human..., Hambach [/bib_ref]. Therefore, adoptive immunotherapy with ex vivo-generated CTLs directed against MiHA expressed by RCC might be an attractive adjuvant approach as has also been studied in patients with leukemia [bib_ref] Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes..., Mutis [/bib_ref]. Alternatively, patients could be vaccinated with peptides or DCs loaded with peptides or electroporated with MiHA-encoding mRNA [bib_ref] siRNA silencing of PD-L1 and PD-L2 on dendritic cells augments expansion and..., Hobo [/bib_ref] [bib_ref] Efficient activation of LRH-1-specific CD8+ T-cell responses from transplanted leukemia patients by..., Overes [/bib_ref]. In conclusion, this study describes T cell responses after partial T cell-depleted RIC-SCT followed by DLI in patients with metastatic RCC. Our transplant procedure resulted in stable engraftment, manageable GVHD and T cell responses in two patients. SMCY-specific CD8+ T cells were identified in a patient with stable disease, and CD8+ T cells targeting the novel HLA-B7restricted MiHA ZAPHIR in the second patient with a partial response. Although the role of allogeneic RIC-SCT in RCC may be limited in the near future, this study shows that the approach of partial T cell-depleted SCT followed by DLI induces MiHAspecific T cell responses potentially targeting RCC tumor cells. The significance for cellular immunotherapy of the novel MiHA ZAPHIR is demonstrated by the emergence of ZAPHIR-specific CTL after allo-SCT, and its ex vivo cytotoxic activity against RCC and transformed B cell lines. Furthermore, identification of the antigenic targets of alloreactive T cells remains important for the further understanding of the GVT response in different malignancies and for development of strategies to selectively target tumor cells after allogeneic SCT.
# Materials and methods
## Patient eligibility
## Conditioning regimen, gvhd prophylaxis and dli
Patients received partial T cell-depleted RIC-SCT. The conditioning regimen consisted of total lymph node irradiation on each of three consecutive days followed by cyclophosphamide 50 mg/kg body weight intravenously on each of four consecutive days (total dose 200 mg/kg bodyweight). Inguinal and femoral lymph node regions were also irradiated. A total dose of 12 Gy was delivered in 2 Gy fractions, twice daily, on three consecutive days.
Donors were HLA-identical siblings. Following T cell depletion, CD3+ T cells were added back to generate a stem cell graft containing a fixed number of 0.5610 6 T cells/kg body weight of recipient. All patients received CsA 3 mg/kg/day by continuous intravenous infusion from days -1 to +14. Thereafter CsA dose was reduced to 2 mg/kg/day and continued until day 21, when CsA was tapered off for 8-10 weeks.
Patients underwent CT-scan of abdomen and chest before, and every 3 months after RIC-SCT to assess disease response. Patients without acute GVHD grade .II, without chronic GVHD and with persistent disease received DLI four weeks after discontinuation of immunosuppression. If no GVHD occurred and residual disease persisted, a second DLI was administered two months later. The first DLI dose consisted of 0.1610 8 T cells/kg bodyweight of the recipient, and the second DLI dose was 0.7610 8 T cells/kg.
## Cell isolation and culture
CD8+ CTL lines H and B were isolated from PBMC obtained one and three months after DLI-1, respectively, by weekly stimulation with PBMC obtained before SCT in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA) supplemented with 10% human serum (HS; Sanquin blood bank, Nijmegen, the Netherlands). After initial stimulation, CTL line H, CTL line B, the HLA-B7 alloreactive CTL Kor18 and the HLA-A2 alloreactive CTL 1E2 (0.5*10 6 ) were cultured in IMDM/10% HS containing irradiated (80 Gy) recipient EBV-LCL (0.5*10 6 ), irradiated (60 Gy) allogeneic PBMC (0.5*10 6 ) from two donors, 100 IU/ml IL-2 (Chiron, Emeryville, CA) and 1 mg/ml PHA-M (Boehringer, Alkmaar, the Netherlands). All cell lines and primary cells were cultured in IMDM/10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands). Fibroblasts were cultured from BM aspirates obtained from healthy stem cell donors. BM was resuspended in 20 ml IMDM/20% FCS and incubated overnight at 37uC in tissue culture flasks. The non-adherent fraction was removed and fibroblasts were further cultured in IMDM/20% FCS to passage 3 before analysis. T cell blasts were generated by stimulating PBMC in IMDM/10% HS with 20 mg/ml PHA-M for 3 days. Thereafter, PHA-activated T cells were washed and further cultured with 100 IU/ml IL-2 for 2 additional days.
## Ifn-c secretion assay
IFN-c producing CTLs were detected and isolated using the IFN-c secretion assay (Miltenyi Biotec). Briefly, 1610 6 CTLs were incubated with 1610 6 irradiated (30 Gy) recipient EBV-LCL in a total volume of 2 ml IMDM/10% HS. After 16 hours of incubation at 37uC, cells were harvested, washed with PBS/ 0.5% FCS and 5 mM EDTA, and labeled at a concentration of 10 8 cells/ml with 50 mg/ml Ab-Ab conjugates directed against CD45 and IFN-c for 10 minutes on ice. Subsequently, cells were diluted with IMDM/10% FCS at 1610 5 cells/ml and allowed to secrete IFN-c for 45 minutes at 37uC. After the cytokine-capturing period, cells were collected, resuspended at a concentration of 10 8 cells/ml in PBS/0.5% FCS/5 mM EDTA, and stained with 5 mg/ ml PE-conjugated anti-IFN-c mAb and FITC-conjugated CD8 mAb for 20 minutes at 4uC. Finally, cells were analyzed and sorted using an Epics Elite flow cytometer (Beckman Coulter).
## Flow cytometry
PE-and APC-labeled SMCY.A2 tetramers containing peptide FIDSYICQV and ZAPHIR.B7 tetramers containing peptide IPRDSWWVE were produced as described previously [bib_ref] Peptide-MHC class I tetrameric complexes display exquisite ligand specificity, Burrows [/bib_ref]. PElabeled tetramer for the HLA-A2-restricted EBV peptide GLCTLVAML were purchased from Beckman Coulter. PBMC or CTL lines were incubated with 20 mg/ml tetramer for 15 min at room temperature. After washing with PBS/0.5% bovine serum albumin (BSA; Sigma,St Louis, MO, USA), cells were labeled with
## Retroviral transduction of hla-b*0702 in cell lines and ctl stimulation assay
LZRS-HLA-B*0702-IRES-EGFP vector was used to generate a stable producer cell line. Retroviral transduction was performed using non-tissue culture-treated 35-mm 2 dishes (Becton Dickinson) coated with 10 mg/ml retronectin (Takara Biomedicals, Shiga, Japan). In brief, 10 6 target cells were resuspended in 2 ml virus supernatant and transferred to retronectin-coated dishes. After 24 h of incubation, cells were collected and incubated with fresh virus supernatant and used in CTL stimulation assays [bib_ref] A human minor histocompatibility antigen specific for B cell acute lymphoblastic leukemia, Dolstra [/bib_ref]. Release of IFNc was determined by ELISA (Pierce Endogen, Rockford, IL).
## Flow cytometry-based cytotoxicity studies
Flow cytometry-based cytotoxicity assays were performed as previously described [bib_ref] New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells..., Jedema [/bib_ref]. HLA-B7 + cell lines were labeled with 2.
## Whole genome association scanning (wgas)
A panel of 87 HLA-B*0702 expressing EBV-LCL was selected to perform WGAs. To test recognition of the EBV-LCL panel by the T cell clone, triplicate samples of 6610 4 EBV-LCL were thawed, washed and cultured for 2 days, and 5610 3 MiHA specific T cells were added to each well. After 24 hours, culture supernatant was used for IFN-c ELISA. Of each EBV-LCL, PCRfree whole genome amplification was performed and analyzed by Illumina Human1M-duo arrays containing probes for 1.1 million SNPs (Illumina, San Diego, CA) [bib_ref] High Throughput Characterization of 10 New Minor Histocompatibility Antigens by Whole Genome..., Van Bergen [/bib_ref]. EBV-LCL panel was divided into MiHA pos and MiHA neg groups using 5 times the level of IFN-c production in the absence of EBV-LCL as a threshold for recognition. WGAs was performed by combining T cell recognition with SNP-genotyping data. The level of matching between both patterns was calculated according to Fisher's exact test.
## Genotyping using kaspar system
Genotyping of the HLA-B7-matched SCT donor-recipient pairs was conducted by KASPar (KBioscience, Herts, UK), a fluores-chromosome 19q13.43. The nucleotide and deduced amino acid sequences of the alternatively and correctly spliced transcripts present in recipient and donor, respectively, are shown. Disparity between the recipient and donor ZNF419 protein sequence is due to a G to A polymorphism in de splice donor site of intron 4 leading to the ZAPHIR epitope (underlined). (C) ZAPHIR epitope reconstitution with synthetic peptides corresponding to HLA-B7 10-mer peptide IPRDSWWVEL, 9-mer peptide IPRDSWWVE, 8-mer peptide IPRDSWWV, HLA-B7 10-mer peptide IPRGSWWVEL and HLA-B7 9mer peptide IPRGEWHGA. Donor EBV-LCL was pulsed with 10 mM peptide and tested for recognition by CTL B1 in the presence of 25 U/ml IL-2. Data are displayed as mean 6 SD of triplicate wells. (D) Flow cytometry analysis of CTL B1 with tetramers containing the 8-, 9-, or 10-mer peptide. Cells were stained with PE-and ACP-conjugated tetramer, CD8 AlexaFluor 700, CD4 FITC and Sytox Blue. Cells were analyzed by flow cytometry by gating on CD8+CD4-Sytox Blue-lymphocytes. The percentage of tetramer+ cells among CD8+ T cells is depicted. doi:10.1371/journal.pone.0021699.g004 cence-based competitive allele specific PCR which utilizes nonlabeled primers. Details of the process and primer sequence of SNP rs2074071 can be obtained from KBioscience.
## Microarray gene expression analysis
Total RNA was isolated from various (malignant) hematopoietic and non-hematopoietic cell samples using small and micro scale RNAqueous isolation kits (Ambion, Inc., Austin, TX). Malignant hematopoietic cell samples included CML, AML and ALL cells isolated by flow cytometry based on expression of CD34, CD33, and CD19/CD34, respectively. Non-malignant hematopoietic cells included total PBMC, and B-cells, T-cells and monocytes isolated from total PBMC by flow cytometry based on expression of CD19, CD3 and CD14, respectively. In addition, hematopoietic stem cells were isolated from G-CSF-mobilized peripheral blood by flow cytometry based on expression of CD34. Nonhematopoietic cell samples included cultured skin-derived fibroblasts, keratinocytes and PTEC (kindly provided by Dr. C. van Kooten, Leiden, Netherlands). Finally, total RNA was isolated from EBV-B cells, PHA-T blasts and RCC cell lines. (kindly provided by Dr. E.M.E. Verdegaal, Leiden, Netherlands). RNA samples were amplified for microarray gene expression analysis using the Illumina TotalPrep RNA amplification kit (Ambion). Briefly, total RNA was treated with DNase I using the TURBO DNA-free kit (Ambion), and converted to cDNA by reverse transcription. After purification of cDNA, cRNA was synthesized by in vitro transcription. cRNA samples were prepared following the whole-genome gene expression direct hybridization assay (Illumina, Inc., San Diego, CA), and resuspended cRNA samples were dispensed onto Human HT-12 v3 Expression BeadChips (Illumina). Hybridization was performed in the Illumina hybridization oven for 17 hours at 58uC. Microarray gene expression data were analyzed using Rosetta Resolver 7.2 gene expression data analysis system.
## Construction of full-length and truncated znf419 constructs
The full-length ZNF419 transcript in pCMV6-XL was obtained by Origene, Rockville, MD (sc 316397). The different splice variants of the ZNF419 transcript containing exon 1 to 5 and the exon 4/intron 4 boundary were generated by RT-PCR of the MiHA+ recipient and MiHA-donor EBV-LCL. The following primers were used to amplify ZNF419 transcript variants: ZNF419-F 59-AAAGGTACCTCTACTCACAGGCTCCGAT-GG-39 and ZNF419-R 59-AAACCTAGGGAGGTTGAAACGC-TGGCTAAAG-39. These primers contained BamHI and KPNI digestion sites. PCR products were purified from 1.5% agarose gel using the Qiagen gel extraction kit, and cloned into vector pcDNA3.1(+) (Invitrogen) using the BamHI and KPNI digestions. Constructs were verified by sequencing, and used for transfection into 293T-HLA-B*0702/ICAM/CD80 cells. In brief, 3*10 4 293T-HLA-B*0702/ICAM/CD80 cells were plated into poly-Dlysine coated 96-wells flat bottom wells (Becton Dickinson, Franklin Lakes, NY), cultured overnight at 37uC, and then transfected with 200 ng plasmid DNA using lipofectamin reagent (Invitrogen). Transfected cells were cultured for 2 additional days and used in CTL stimulation assays.
Epitope prediction and epitope reconstitution assay HLA-B7 binding peptides were predicted using the NetMHC 3.2 server (http://www.cbs.dtu.dk/services/NetMHC/). Peptides were synthesized by Proimmune and dissolved in DMSO. Peptide stock solutions were diluted in IMDM to a concentration of 1 mM. In peptide recognition assays, target cells were preincubated with peptide for 1 hour in a volume of 1 ml prior to the addition of CTL.
## Supporting information
[fig] Figure 1: Longitudinal follow-up of CD8+ T cells in peripheral blood from metastatic RCC patients UPN677 and UPN686 in relation to clinical outcome. (A) UPN677: percentages of recipient T cells (right y axis) are compared with CD8+ T cell count 610 6 per liter peripheral blood (left y axis). Administration of SCT and DLI 1-2 are indicated by m and ., respectively. Treatment interval with CsA is shown by the dotted line. Time points of confirmed EBV infection, occurrence of EBV-specific T cells (depicted as % from total CD8+ T cell population) and death are indicated. (B) UPN686: for legend see (A). Time points of confirmed candidemia and death are indicated. doi:10.1371/journal.pone.0021699.g001 [/fig]
[fig] Figure 2: CD8+ T cells reactive with the SMCY.A2 peptide developed after DLI in RCC patient UPN677. (A) HLA-restriction was determined by the production of IFN-c released by CTL line H upon stimulation with recipient EBV-LCL in the presence of HLA blocking antibodies. Data are displayed as mean IFN-c release 6 SD of triplicate wells. (B) Production of IFN-c by CTL line H stimulated with EBV-LCL of 9 HLA-A2+ unrelated individuals, showing recognition of 3 out of 9 EBV-LCL of male origin. (C) Flow cytometry analysis of CD8+ CTL line H simultaneously stained PE-and APC-conjugated SMCY.A2 tetramer, CD8 AlexaFluor 700, CD4, CD14, CD16 and CD19 FITC and Sytox Blue. CD8+ T cells were gated on FITC-and Sytox Blue-cells. The percentage of SMCY.A2 tetramer-binding cells among viable CD8+ T cells was 12.6%. These SMCY.A2 tetramer+ CD8+ T cells were sorted and expanded resulting in a .95% pure population. (D) Survival of the recipient and donor EBV-LCL, and donor EBV-LCL pulsed with peptide FIDSYICQV was determined in a flow cytometry-based cytotoxicity assay after incubation with SMCY.A2-specific CTL from patient UPN677 (&), allo-HLA-A2 CTL (.; positive control) or medium only ( N ) at an E:T ratio of 3:1 in the presence of 25 U/ml IL-2. Data are depicted as mean 6SD of triplicate wells. (E) Detection of SMCY.A2-specific CD8+ T cells in peripheral blood of RCC patient UPN677. PBMC collected 24, 28 and 31 weeks post DLI-1 were simultaneously stained with PE-and APC-conjugated SMCY.A2 tetramer, CD8 AlexaFluor 700, CD4, CD14, CD16 and CD19 FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Cells were gated on CD8+FITC-Sytox Blue-lymphocytes, and the percentage of tetramer-binding cells among CD8+ T cells is depicted. doi:10.1371/journal.pone.0021699.g002 [/fig]
[fig] From: December 2002 till August 2003, four consecutive patients with metastatic RCC have been treated in our transplantation program. The eligibility criteria for patients included histological proven, clinically progressive metastatic RCC, failure on earlier cytokine based systemic therapy and no other curative treatment available. This study was approved by the institutional review board of RUNMC, and all patients gave written informed consent. [/fig]
[fig] Figure 3: Cytotoxicity of HLA-B7-restricted CTL B1 against RCC and hematological tumor cell lines. (A) HLA-restriction was determined by the production of IFN-c released by CTL B1 upon stimulation with recipient EBV-LCL in the presence of HLA blocking antibodies. (B) Production of IFN-c by CTL B1 stimulated with rt EBV-LCL, EBV-LCL of an unrelated individual (#2) sharing HLA-B7 with the recipient, and an EBV-LCL of an HLA class I-mismatched individual (#3) that was transduced with HLA-B*0702. (C) Production of IFN-c by CTL B1 and allo-HLA-B7 CTL stimulated with MiHA+ EBV-LCL and PHA-stimulated T cells and bone marrow-derived fibroblasts. Fibroblasts were pretreated with 10 ng/ml TNFa and 200 U/ml IFNc for 2 days before coculture with CTL B1 (E:T ratio 10:1). (D) MiHA mediated cytotoxicity on the HLA-B7+ RCC cell lines SKRC33 (rs2074071 genotype AA) and SKRC18 (rs2074071 genotype GG), the brain tumor cell line DAOY (rs2074071 genotype AG), and pharynx cell line FaDu (rs2074071 genotype GG), in flow cytometry-based cytotoxicity assays was determined after incubation with CTL B1 (&), allo-HLA-B7 CTL (.; positive control) or medium only ( N ) at an E:T ratio of 3:1 in the presence of 25 U/ml IL-2. Data are depicted as mean 6SD of triplicate wells. doi:10.1371/journal.pone.0021699.g003 [/fig]
[fig] Figure 4: A SNP in the splice donor site between exon 4 and 5 of the ZNF419 gene determines CTL B1 recognition. (A) IFN-c production by CTL B1 upon stimulation with 293T-HLA-B*0702/ICAM/CD80 cells transfected with ZNF419 full length, cloned splice variant containing the exon4/exon5 boundary and the alternative ZNF419 splice variant containing intron 4. (B) Schematic representation of the ZNF419 gene on AlexaFluor700-conjugated anti-CD8 (Invitrogen) in combination with FITC-conjugated CD4, CD14, CD16 and CD19 (Beckman Coulter) for 30 min at 4uC. Finally, cells were washed and resuspended in PBS/0.5% BSA containing 0.2 mM Sytox Blue (Invitrogen) marking dead cells and analyzed using the Cyan flow cytometer (Beckman Coulter). Tetramer staining using both APC and PE showed double positive events, allowing optimal discrimination between background staining and positive cells. All FITC-positive cells were gated into a dump channel and played no role in further analysis. [/fig]
[fig] 5: mM carboxyfluorescein diacetate succimidyl ester (CFSE; Molecular Probes Europe). Target cells (1610 4 ) were co-cultured with unlabelled effector cells (3610 4 ) at an E:T ratio of 3:1 in a total volume of 200 ml IMDM/10% FCS containing 25 U/ml IL-2 in 96-wells flat-bottom plates. After 1-3 days of co-culture, cells were harvested and 7-amino-actinomycin D (7AAD; Sigma-Aldrich, St Louis, MO) was added. Numbers of viable target cells were quantified by flow cytometry. [/fig]
[fig] Figure 6: Detection of ZAPHIR-specific CD8+ T cells in peripheral blood of RCC patient UPN686 and CML patient UPN539. (A) PBMC collected before DLI and 4, 9, and 20 weeks post DLI were stained with PE-and ACP-conjugated ZAPHIR/HLA-B7 tetramer, CD8 AlexaFluor 700, CD4, CD14, CD16 and CD19 FITC and Sytox Blue. Cells were analyzed by flow cytometry by gating on CD8+FITC-Sytox Blue-lymphocytes. The percentage of tetramer+ cells among CD8+ T cells is depicted. The remaining PBMCs were stimulated once with ZAPHIR peptide pulsed (10 mM) EBV-LCL of the donor and assayed on day 7 for ZAPHIR-tetramer+ CD8+ T cells. (B) PBMC collected pre-DLI, 7 and 40 weeks post-DLI were stained and gated as described under (A). The remaining PBMCs were stimulated once with ZAPHIR peptide-pulsed (10 mM) EBV-LCL of the donor and assayed on day 7 for ZAPHIR-tetramer+ CD8+ T cells. doi:10.1371/journal.pone.0021699.g006 [/fig]
[fig] Figure S1: Generation of MiHA-reactive CD8+ CLT lines from patients with RCC after allogeneic RIC-SCT. (A) Detection of IFN-c secreting CD8+ T cells in T cell lines generated from patient UPN677 and UPN686 after co-culture with EBV-LCL of the recipient. Stimulated T cells were stained with PE-conjugated IFN-c detection reagent and FITC-conjugated anti-CD8 mAb, and analyzed by flow cytometry. (B) Production of IFN-c by CTL lines H and B upon stimulation with recipient (Rt) EBV-LCL, donor (Do) EBV-LCL or medium. Data are displayed as mean IFN-c release 6 SD of triplicate wells. (TIF) Figure S2 WGAs using 87 HLA-B7 positive EBV-LCL revealed a cluster of 5 SNPs to associate on chromosome 19 cluster of SNP. (A) Detection of IFN-c-secretion by CTL B1, here depicted as OD450, upon stimulation with 87 HLA-B7+ EBV-LCL showed 42 EBV-LCL to be recognized. The threshold for recognition was set at 5 times the level of IFN-c production in the absence of EBV-LCL. (B) Detail of chromosome 19 showing all SNP that cluster according to Fisher's exact test. No other SNP cluster was found at any other chromosome. (TIF) Figure S3 ZNF419 splice variant. (A-B) The full length transcript of ZNF419 (NM_001098491) consists of 5 exons which are spliced into an additional 6 splice variants (NM001098492 through NM_001098496 and NM_024691). Due to SNP rs2074074 in the splice donor site an additional transcript occurs in patient UPN686 which includes exon 4 (grey) leading to a stopcodon (*). (C) The different splice variants that were expressed by patient UPN686 and its related donor were amplified as a pool, using the primerset as depicted (R) and cloned into pcDNA3.1(+). (TIF) Figure S4 Generation of ZAPHIR-reactive CD8+ CTL Lines from CML patient UPN539 40 weeks post DLI. (A) PBMC collected 40 weeks post DLI were sorted for TET+CD8+ T cells (inside square) and cloned into single cells. (B) Production of IFN-c by ZAPHIR-specific CTL clones with ZAPHIR-peptide pulsed donor EBV-LCL, donor EBV-LCL or medium. Data are displayed as mean IFN-c release 6 SD of triplicate wells. (TIF) [/fig]
[table] Table 1: Patient characteristics, GVHD, DLI and outcome.Abbreviations: IFN-a, interferon-a; IL-2, interleukin-2, G250 mAb, monoclonal antibody against G250; RT, radiotherapy; 5FU, 5-fluorouracil; GVHD, graft-versus-hostdisease; DLI, donor lymphocyte infusion; PD, progressive disease, SD stable disease; na, not applicable; ne, not evaluated; PR partial remission. doi:10.1371/journal.pone.0021699.t001 [/table]
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Anticipatory threat responses mediate the relationship between mindfulness and anxiety: A cross-sectional study
# Introduction
Anxiety is a salient feeling of worry, nervousness, or unease when facing a threatening situation. People with pathological anxiety experience hypervigilance and increased behavioral responsivity in the absence of rationally fearful stimuli [bib_ref] From normal fear to pathological anxiety, Rosen [/bib_ref]. A lifetime prevalence estimate of 28.8% places anxiety disorders as the most common class of mental disorders, causing serious damage to patients' social functioning [bib_ref] Resolving the neural circuits of anxiety, Calhoon [/bib_ref]. Furthermore, the COVID-19 pandemic has led to an 11.2% increase (95% uncertainty interval: 5.3-17.3) in cases of anxiety disorders in China in 2020, which heavily burdens both families and society.
Mindfulness-based interventions (MBIs) are a promising category of anxiety treatment. Mindfulness is the basic attentional stance underlying various Buddhist traditions, such as Theravada, Vajrayana and Mahayana (Zen), and has been called "the heart" (or "dharma" in Sansfrit) of Buddhist meditation, which was historically developed as a method to suspend personal suffering [bib_ref] Mindfulness-Based interventions in context: past, present, and future, Kabat-Zinn [/bib_ref]. Since it spread to the West, it has been increasingly applied in clinical settings. Mindfulness is commonly defined as the perception and acceptance of constantly changing experiences (6), which may include thoughts, emotions, somatic sensations, and responses to the external stimuli . Evidence of its validity in alleviating anxiety comes from studies demonstrating negative relationships between mindfulness and anxiety (9, 10), intervention research (11- [bib_ref] Randomized trial of cognitive behaviour group therapy and a mindfulness-based intervention for..., Koszycki [/bib_ref] , and meta-analysis [bib_ref] The effect of mindfulness-based therapy on anxiety and depression: a meta-analytic review, Hofmann [/bib_ref] [bib_ref] Mindfulnessbased interventions for the improvement of well-being in people with multiple sclerosis:..., Carletto [/bib_ref]. Over the past several decades, MBIs have been increasingly utilized among groups of relatively healthy individuals to promote wellbeing, as well as in a wide variety of clinical disorders, as a complement to cognitive or behavioral techniques to relieve mental distress [bib_ref] Mindfulnessbased stress reduction and health benefits, Grossman [/bib_ref] [bib_ref] Mindfulness-based intervention improves residual negative symptoms and cognitive impairment in schizophrenia: a..., Shen [/bib_ref]. The most common and well-studied MBIs are mindfulness-based cognitive therapy (MBCT) and mindfulness-based stress reduction (MBSR). MBSR is a manualized treatment program widely used to reduce psychological morbidity associated with chronic illnesses and treat emotional and behavioral disorders (6). MBCT is similar to and involves MBSR but emphasizes the ability to self-manage, control, and improve (17). Although empirical research on the effectiveness of mindfulness is increasing, the mechanisms through which mindfulness improves anxiety have rarely been investigated. One potential mechanism is an anticipatory response to uncertain threats [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref].
Immoderate reactions to uncertain stimuli have been regarded as an intolerance of uncertainty (IU) [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref]. People with high IU are inclined to consider ambiguity a threat and are prone to overestimate the likelihood of an uncertain event and the cost of responding, thus resulting in maladaptive behaviors such as vigilance (i.e., paying more early phasic and sustained attention to uncertain target cueing) (21) and avoidance, which aims to decrease uncertainty (22). Recent perspectives consider anxiety to be a future-oriented emotional state; abnormal and excessive anticipatory responses under unpredictable threats explain the unique variance in anxiety psychopathology that contributes to stress and anxious behaviors [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref] [bib_ref] Uncertainty and anticipation in anxiety: an integrated neurobiological and psychological perspective, Grupe [/bib_ref]. Consistent with this perspective, a meta-analysis showed that IU is strongly associated with a range of symptoms in disorders, such as obsessive-compulsive disorder, panic disorder, social anxiety disorder, and agoraphobia, thus validating IU's trans-diagnostic importance [bib_ref] The impact of methodological and measurement factors on transdiagnostic associations with intolerance..., Mcevoy [/bib_ref].
Unlike the excessive anticipation reaction to potential future threats experienced in a state of anxiety, mindfulness refers to the non-judgmental awareness of the present moment's real experience, which is contrary to the future-oriented thinking involved in anxiety-driven cognition and its relevant emotion regulation strategies . The anxiety-attenuating effects of MBIs have already been observed in the anticipatory phase for negative emotions, cortisol, and the autonomic nervous system [bib_ref] Effects of two mindfulness based interventions on the distinct phases of the..., Gamaiunova [/bib_ref] , especially in the parasympathetic nervous system (27). Kim's research on panic disorders claimed that there was a significant correlation between the reduction in IU and relief of panic symptoms after MBCT (12). In line with this research, another cross-sectional study indicated that the benefits of mindfulness on anxiety symptoms are mediated by self-reported IU. However, this mediating effect was not confirmed in physiological responses (i.e., the startle reflex) to uncertain threats [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref]. One possible interpretation of this finding is that the IU questionnaire measured a higher-order cognitive process response to uncertainty while the startle reflex measured a lower-order defensive response to uncertainty.
Recent electrophysiological (EEG) studies have focused on the modulation of anxiety over threat anticipation by the intensity of uncertainty and found that, compared with certain cues, uncertain aversive cues elicit significantly larger stimuluspreceding negativity (SPN), P2 (a positive posterior deflection peaking at 200-250 ms), and late positive potential (LPP) (21, 28). The LPP is a centroparietal slow wave that seems to be modulated by higher-level cognitive processing (29) and has been demonstrated to be sensitive to stimulus predictability [bib_ref] The p300 wave of the human Event-Related potential, Picton [/bib_ref]. With respect to uncertainty processing, studies have reported increased LPP amplitude for uncertain aversive cues compared with certain safe cues in threat-of-shock designs (21, 31). According to Grupe's "uncertainty and anticipation model of anxiety" (UAMA) theory [bib_ref] Uncertainty and anticipation in anxiety: an integrated neurobiological and psychological perspective, Grupe [/bib_ref] , the overestimation of threat costs and probabilities causes exaggerated emotional and behavioral reactivity in anxious individuals. Thus, we chose LPP as an indicator of sustained cognitive processing in our research, which explores the potential mediating effects of uncertain threat responses on the association between mindfulness and anxiety and further investigates whether the relationships would vary according to the degree of threat exhibited by the stimuli [bib_ref] Anomalous static and dynamic functional connectivity of amygdala subregions in individuals with..., Wang [/bib_ref].
Ongoing research has shown that the neural correlates of trait anxiety can predict pathological anxiety-driven behaviors [bib_ref] Anomalous static and dynamic functional connectivity of amygdala subregions in individuals with..., Wang [/bib_ref]. Thus, the present study focused on highly anxious individuals to explore the underlying mechanisms through which mindfulness alleviates anxiety. It was hypothesized that (1) both trait-anxious (TA) individuals and low-anxious (LA) individuals would express excessive anticipatory responses (including self-reported IU, behavioral reaction time (RT) and EEG responses) under conditions of threat uncertainty, and that the TA group's response would be more intense; (2) mindfulness would be negatively correlated with anxiety and IU; and (3) reactions to uncertainty might be the possible mechanism by which mindfulness alleviates anxiety.
## Materials and methods participants
To recruit individuals with different levels of anxiety, recruitment advertisements were posted at the Army Medical University and three affiliated hospitals. Volunteers who scanned the quick response (QR) codes on the advertisements to sign up for the study were asked to complete the screening questionnaires (n = 191). The presence of psychiatric and neurological diseases was assessed through this screening questionnaires. We applied the following inclusion criteria: (1) aged 18-45 years, (2) right-handed individuals, (3) no history of neurological or psychiatric diagnosis, and (4) normal visual acuity or corrected visual acuity. In total, 162 subjects completed all questionnaire items and met all inclusion criteria. Based on their scores on the Trait Anxiety Inventory (TAI) in the State-Trait Anxiety Inventory (33), participants who scored 44 and above were classified as TA individuals while those with a score of 34 and below were classified as LA individuals [bib_ref] The neural correlates of cognitive effort in anxiety: effects on processing efficiency, Ansari [/bib_ref]. Thus, we invited 35 TA individuals and 36 LA individuals to participate in further EEG research. The intentions and project procedure of this study were then provided to qualified participants, and their written informed consent was obtained. This study was conducted in accordance with the Declaration of Helsinki. The regional ethics committee of the Army Medical University approved this consent procedure and the study protocol (reference number: 2020-019-02). Participants were paid 50 RMB after they finished this study. shows the participants' flowchart. A total of 71 participants (TA group = 35, LA group = 36) were eligible to participate in the EEG experiments. Eight subjects were excluded because of excessive EEG artifacts. The final sample comprised 63 participants (TA group = 32, LA group = 31). G * Power 3.1.9.7. was used to determine the sample size. Based on the input parameters specifying a medium effect size of f = 0.25, α = 0.05, 1-β = 0.95, number of groups = 2, and number of measurements = 4, we obtained a total sample size of N = 36. Thus, the sample size of our study (n = 63) was sufficient for statistical analysis.
## Task design
Participants completed self-report questionnaires and EEG assessments under predictable and unpredictable conditions. The EEG recordings were conducted in a sound-attenuated room. The task was a modified version of a published threat test (35) that included four conditions: (1) predictable positive events (PP), (2) predictable negative events (PN), (3) unpredictable positive events (UP), and (4) unpredictable negative events (UN). Each condition contained 80 trials, with each trial consisting of a 300-ms fixation point presented at the center of the computer screen, followed by a 600-ms cue (i.e., "positive" or "negative"), after which a sequence of numbers would appear. Participants were told that a pleasant or aversive image (their valence was in accordance with the cues) would be shown on the screen after the sequence of numbers. During predictable conditions, the numbers would count down from any number between 10 and 6 to 1, at which point an image would appear. Under unpredictable conditions, the sequence of numbers would appear randomly, and pleasant or aversive images would be presented at any time. Pictures were presented for 1,500 ms and subjects were required to determine whether the scenes in the image occurred indoors (press "F" on the keyboard) or outdoors (press "J" on the keyboard). The image disappeared after the keystroke. After a 100-ms blank screen, feedback (correct or false) for participants' responses appeared (see . The RT of the images was collected only for the correct response and then averaged for each condition.
The task consisted of two predictable runs and two unpredictable runs with a counterbalanced sequence. Each run consisted of 40 positive and 40 negative trials, and the order of the images was randomized. At the end of each run, a mandatory 30-s rest was provided. Before the formal experiment began, eight practice trials were performed to familiarize the participants with the paradigm. To ensure that all participants received the same information, instructions on the experimental procedures were standardized and displayed on a computer screen before the practice stage. The same researcher answered all questions throughout the study process.
# Materials
A total of 320 images were selected from the native Chinese Affective Picture System (CAPS) [bib_ref] The development of netive Chinese affective picture system -a pretest in 46..., Bai [/bib_ref]. A 9-point scale ranging from 1 (negative/calm) to 9 (positive/arousal) was used to evaluate each picture's valence and arousal degree. In the present study, the mean valence and arousal ratings of the selected 160 positive images were 6.71 ± 0.37 and 5.76 ± 0.58, respectively. The selected 160 negative images had a mean valence and arousal rating of 2.84 ± 0.83 and 5.01 ± 0.55, respectively. The valence and arousal ratings of the 160 pictures following the Frontiers in Public Health frontiersin.org . /fpubh. .
## Questionnaires
Hospital anxiety and depression scale (HADS)
The HADS was constructed using the 7-item anxiety subscale and the 7-item depression subscale. A 4-point Likert .
/fpubh. . scale ranging from 0 (not at all) to 3 (almost all the time) was used to evaluate participants' emotional states in the preceding month. The total scores ranged from 0 to 21, with a critical value of 9 for each subscale [bib_ref] The effects of tolerance of uncertain on risk preferences and it's context-dependency, Zigmond [/bib_ref]. The internal consistency coefficients of the anxiety subscale and the depression subscale for our sample were 0.90 and 0.89, respectively.
## Trait anxiety inventory (tai)
The 20-item TAI was administered to measure participants' general feelings of anxiety using a 4-point Likert scale (1 = barely, 4 = almost always). The total scores range from 20 to 80, with higher scores indicating greater trait anxiety (33). The Cronbach's alpha for our sample was 0.95.
## Intolerance of uncertainty scale (ius)
The IUS includes 11 items that assess how people react to uncertain situations in their lives. Participants rated the items on a 5-point Likert scale ranging from 1= "not at all characteristic of me" to 5= "entirely characteristic of me". The total scores ranged from 11 to 55, with higher scores indicating less tolerance to uncertainty (38). The Cronbach's alpha for our sample was 0.96.
## Five facet mindfulness questionnaire (ffmq)
The 20-item FFMQ was used to measure five facets of mindfulness: observing, describing, acting with awareness, non-judging to inner experience, and non-reacting to inner experience. Participants rated the items on a 5-point Likert scale (1 = never or very rarely true, 5 = very often or always true) [bib_ref] Validation of a Chinese version of the five facet mindfulness questionnaire in..., Hou [/bib_ref]. The Cronbach's alpha for our sample was 0.84.
## Eeg recording and data reduction
Continuous EEG data were collected using a 64 channel NuAmp acquisition system (Neuroscan Inc.) according to the international 10-20 system, with a reference at Cz and the ground placed between Fz and Fpz. Horizontal and vertical EEG activity was recorded from positions next to the outer rims of each eye and from above and below the right eye, respectively. The sampling rate was 1,000 Hz, and the impedances of all electrodes were below 5kΩ.
Offline, a digital average mastoid reference, (M1+M2)/2, was performed. The raw EEG data were bandpass filtered from 0.01 Hz to 30 Hz and manually scored for muscle and eye movement artifacts. They were segmented from 100 ms before cue onset to 2,000 ms afterward, referred to as baseline −100 to 0 ms before cue onset. Six electrodes-CP3, CPz, CP4, P3, Pz, and P4-were selected for further analysis, since the LPP in existing research was detected mostly in the centro-parietal region of the scalp (21). The inspection of the EEG data revealed a late positive component (with an onset of approximately 400 ms and an offset of approximately 1,100 ms) over parietal occipital sites. The mean amplitude of the LPP was extracted for further analysis (see .
## Data analyses
Data analyses were performed using SPSS 19.0 and AMOS 20.0. The Shapiro-Wilk normality test was used to assess data or variable distribution. Age, HADS_A, TAI, and IUS did not show a normal distribution; therefore, comparisons were made by the Mann-Whitney U test. The differences in demographic variables (except Age) and FFMQ between TA group and LA group were analyzed by independent samples t-tests and chi-square tests. After splitting the data by group, a 2 (Groups: TA, LA) × 4 (Conditions: PP, UP, PN, and UN) repeated-measures analysis of variance (rmANOVA) was applied to examine the discrepancies in behavioral RT and LPP amplitude with respect to the four stimuli conditions for the TA and LA groups. Greenhouse-Geisser correction was applied to correct all ANOVA results. The Sidak correction was used to correct alpha for multiple comparisons. The Spearman rank-order correlation was used to calculate the relationships between variables. The cocor was used to conduct statistical comparisons between correlations (40). The mediation hypothesis was tested with structural equation modeling (SEM). Indices including CMIN/DF (a value between 1 and 3 indicates acceptable fit between the hypothetical model and the sample data), adjusted goodness-of-fit index (AGFI, a value >0.90 indicates acceptable model fit) [bib_ref] Cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus..., Hu [/bib_ref] , and root-meansquare error of approximation (RMSEA, a value between 0.05 and 0.08 reflects reasonable model fit) [bib_ref] Anticipation of uncertain threat modulates subsequent affective responses and covariation bias, Browne [/bib_ref] were calculated to assess the overall model fit.
# Results
## Self-report measures
According to the parametric and non-parametric test results, group differences in the demographic variables were not significant (see . shows the means and standard deviations for all self-reported variables for the TA and LA groups. According to the independent samples t-test and Mann-Whitney U test, the grouping effects for all selfreported variables were significant, with lower scores for FFMQ and subscales (except for the observing subscale), and higher scores for HADS-A, TAI, and IUS in the TA group. The results indicated that the participants in the TA group were more intolerant of uncertainty, tended to feel more stress, and had less mindfulness than emotionally healthy participants.
. /fpubh. . presents the results of the rmANOVA after splitting the RT to analyze the TA and LA groups across the four conditions. Although no significant group × condition interaction was found, there was a significant main effect of the condition. For all participants, the mean RT during the UN condition was significantly longer than those during the other three conditions, and the mean RT during PN was significantly longer than that during PP. The rmANOVA for RT also revealed a significant main effect of the group. The mean RT of TA group was significantly longer than that of the LA group, particularly with UN stimuli. Based on the results, we found that participants waited longer to press keys on uncertain and negative stimuli, especially in the TA group. presents the significant main effect of condition and group × condition interactions at the three occipital electrodes (i.e., CP3, PZ, and P4). Follow-up planned comparisons indicated that for the TA group, the amplitude of LPP during trials signaling unpredictable negative stimuli was greater than that of LPP during trials signaling certain positive stimuli (p < 0.009). Although there were upward trends from certain conditions to unpredictable conditions and from positive to negative stimuli on the amplitude of LPP for the LA group, the changes were not statistically significant. In addition, within the unpredictable negative condition, the TA group demonstrated significantly higher LPP than the LA group (p = 0.02, CP4 and 0.01, P4), whereas in the other three conditions (PP, UP, PN), the amplitude of LPP to cues did not differ significantly between groups (see , .
## Rt to the images
## Lpp
## Mediation model of anticipatory response to uncertain threats on mindfulness improving anxiety
The intercorrelations between all variables are summarized in . IUS scores, RT, and LPP amplitude to uncertain threats were all significantly related to scores on measures of anxiety symptoms and were inversely related to FFMQ scores. These results support the supposed correlations and allow for further mediation analyses. Self-reported IUS was significantly associated with RT to the images in both anxiety groups. No significant relationship between IUS scores and LPP amplitude was found.
The SEM results demonstrated that the overall model yielded a satisfactory fit, CMIN/DF = 1.68, RMSEA<0.08, and AGFI = 0.99. All specific indirect effects on anxiety via anticipatory responses to uncertain threats, including IUS scores, RT, and LPP amplitude to uncertain threats, were significant (all p < 0.05, see . There was no significant direct effect of mindfulness on anxiety. However, anticipatory responses to uncertain threats were found to mediate the association between mindfulness and anxiety. The standardized indirect effects of IU scores, RT, and LPP amplitude on uncertain threats were −0.40, −0.05, and −0.05, respectively (all p < 0.05).
## Frontiers in public health
frontiersin.org . /fpubh. .
# Discussion
Uncertainty about future threats is disruptive in anxiety. The current research aimed to explore whether an excessive threat response (both self-reported IU and behavioral and EEG responses to uncertain threats) significantly mediates the negative relationship between mindfulness and anxiety and to further investigate whether the relationship would vary according to various degrees of anxiety.
## Excessive anticipatory response to uncertain threats
Substantial research has shown that excessive reactions to uncertainty play a crucial role in pathological anxiety (21, [bib_ref] Uncertainty and anticipation in anxiety: an integrated neurobiological and psychological perspective, Grupe [/bib_ref]. According to the study results, in comparison with LA individuals, there was greater self-reported IU in TA group, which supports the previous findings of a positive relationship between IU and anxiety [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref]. Longer RT before key presses in the . /fpubh. . uncertain negative stimuli was found in both groups, suggesting that individuals might be more involved in uncertain threats. Moreover, according to the attentional control theory, increased attention consumes limited cognitive resources and less impairs the performance efficiency of concurrent task processing (44, 45). In addition, LPP was recorded while the subjects were exposed to predictable and unpredictable conditions. As hypothesized, the LPP amplitude of the TA group regarding uncertain threats was significantly higher than that of the LA group. Enhanced LPP during trials signals unpredictable threats, suggesting increased salience and sustained higherlevel cognitive processing for these cues. Previous EEG research has investigated uncertainty-related dynamics in attentional allocation and sustained stimulus elaboration in a cued-picture paradigm. The results showed larger P2 and LPP amplitudes in uncertain conditions, suggesting that the threat uncertainty context could enhance individuals' ability for early attentional capture and late top-down allocation of attention to stimuli (21).
Further, the present study contrasted participants with high and low TA to demonstrate that this trend of excessive anticipatory response to uncertain threats is more pronounced in the TA group. It seems that people with higher TA may demonstrate lower cognitive flexibility. They tend to prioritize uncertain threats and experience more difficulty adapting to new information [bib_ref] Specificity of trait anxiety in anxiety and depression: Meta-analysis of the State-Trait..., Knowles [/bib_ref] , which may predispose them to pathological anxiety-driven behaviors [bib_ref] Anomalous static and dynamic functional connectivity of amygdala subregions in individuals with..., Wang [/bib_ref]. Correlation analysis showed that self-reported IUS was significantly associated with RT to the images in both anxiety groups, which is consistent with a previous research [bib_ref] What do I do now? Intolerance of uncertainty is associated with discrete..., Morriss [/bib_ref]. However, the relationship between IUS and LPP amplitude was not statistically significant. The result was inconsistent with Nelson et al.'s research (48). In their study, participants were invited to complete a passive fear generalization paradigm, and the research found that prospective IU (IUS-P, and not inhibitory IUS) was negatively correlated with LPP amplitude in the face of uncertainty, suggesting that individuals high in IUS-P might engage in cognitive avoidance during the threat uncertainty condition. Nevertheless the present study used a different version of IUS and conducted a different task that required participants' feedback. Thus, future research should adopt an experimental paradigm that includes trials that (1) only need participants' passive observation and (2) need their active feedback to further investigate the relationship between IUS and LPP amplitude.
## Anticipatory threat responses mediating the benefits of mindfulness on anxiety symptoms
A previous cross-sectional study conducted by Kraemer et al. affirmed that self-reported IU mediates the relationship between mindfulness and health anxiety [bib_ref] Randomized controlled trial of mindfulness meditation for generalized anxiety disorder: Effects on..., Kraemer [/bib_ref]. However, research on the relationship between mindfulness, anxiety, and physiological responses (i.e., the startle reflex) in the unpredictable threat condition has shown mixed results [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref]. The authors explained that this might be due to the IU scale measuring a higher-order cognitive response to uncertainty involving cognitive processes such as attention, working memory, and metacognition (49) while the startle magnitude measures a lower-order defensive response to uncertainty [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref] , and the latter does not seem necessary for the conscious experience of any emotional cognitive state (50). Thus, in the current study, the LPP amplitude to uncertain negative stimuli was chosen as an indicator of higher-level cognitive processing (29). As we assumed, the results demonstrated significant mediating effects of excessive threat response (both self-reported IU as well as RT and LPP amplitudes to uncertain threats) on the beneficial effects of mindfulness on anxiety.
Over the past few decades, MBIs have become increasingly ideal therapeutic strategies for relieving anxiety [bib_ref] The effect of mindfulness-based therapy on anxiety and depression: a meta-analytic review, Hofmann [/bib_ref] [bib_ref] Mindfulnessbased interventions for the improvement of well-being in people with multiple sclerosis:..., Carletto [/bib_ref]. Of the mindfulness elements, the non-judging awareness of the present moment's real experience was strongly associated with anxiety symptoms . This mindful awareness of the present moment could allow anxious individuals to avoid futureoriented thinking and the overestimation of a threat's costs and related possibilities [bib_ref] Uncertainty and anticipation in anxiety: an integrated neurobiological and psychological perspective, Grupe [/bib_ref] , thereby mitigating individuals' threat anticipation of an uncertain event from excessive expectations to more reasonable expectations and eventually to non-judgmental acceptance [bib_ref] Uncertainty and anticipation in anxiety: an integrated neurobiological and psychological perspective, Grupe [/bib_ref]. Moreover, mindful acceptance might help decrease the defensive motivation elicited by uncertainty and instead strengthen one's ability to allow an experience to be as it is, thereby relieving intolerance and inflated anxiety about potential threats [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref].
Smaller amplitudes of LPP during trials signaling unpredictable negative stimuli and shorter RT before key presses in uncertain threats were observed in people with higher degrees of mindfulness. It could be speculated that people with higher mindfulness would appear less blocked by uncertain threats and that under uncertainty, their cognitive resources would be more flexibly deployed according to circumstantial demands (21). Thus, higher mindfulness would contribute to alleviating negative reactions to unpredictable stimuli, in that uncertainty would be less likely to be identified as something that is unacceptable or needs to be stopped [bib_ref] Evaluating the unique contribution of intolerance of uncertainty relative to other cognitive..., Papenfuss [/bib_ref] , ultimately relieving anxiety.
This study provides insight into mindfulness interventions for individuals with anxiety. Mindfulness practices would work well on higher-level cognitive processing by guiding anxious individuals to: (1) observe the present moment rather than worry about the future so they are less involved in the potential threat that they imagine might happen and (2) act with awareness and allow everything (including the thoughts in the mind) to just be as it is rather than trying to control them. This approach does not mean that there is no coping with mental distress, but that there is a way to respond consciously in a state of awareness without judgment [bib_ref] Mindfulness-based intervention improves residual negative symptoms and cognitive impairment in schizophrenia: a..., Shen [/bib_ref] , which could help individuals with anxiety reduce their automatic avoidance of pain since mental discomfort is often unavoidable and a failure to cope often brings more anxiety.
## Limitations and future research
First, the mediating effect of the anticipatory threat responses on the association between mindfulness and anxiety was based on a cross-sectional survey. Further intervention studies are warranted, and responders and non-responders should be compared to measure the causal nature of these relationships. Second, the findings were based on 63 individuals who were either emotionally healthy or trait anxious and did Frontiers in Public Health frontiersin.org . /fpubh. . not include anyone with clinical anxiety disorders, which may undermine the significance of several relationships between variables. For example, the correlation between IU and LPP amplitude in response to uncertain threats was only marginally significant in the present study (p = 0.056). Third, we did not inquire about previous contemplative/meditative or bodymind practices. Such practices may affect the responses of the participants, entailing a risk of reporting bias. Given the role of social desirability in self-reported measures, the study itself may also entail a risk of self-reporting bias, which may have affected the selection of TA and LA respondents, as well as the response of the participants to other measures (e.g., IUS and HADS). In addition, the sample size was small, which limits the power of the study to detect possible relationships and mediating effects. Therefore, a large-scale intervention study involving adequately-powered samples with comparable experiences of contemplative/meditative or bodymind practices and heightened symptom levels on multiple anxiety dimensions is necessary to replicate these findings in future research.
## Contribution to the field
Anxiety disorders are associated with substantial functional impairment and imposes a heavy burden on both families and society. Many studies have shown that MBIs can effectively alleviate anxiety; however, the underlying neural mechanism has not yet been elucidated. Research based on self-reported IU suggests that alleviating higher-order cognitive responses to uncertainty might mediate the effect of mindfulness on anxiety symptoms. Accordingly, the current study collected the LPP amplitudes in response to uncertain negative stimuli as the physiological indicators of higher-level cognitive processing. The results demonstrated significant mediating effects of LPP amplitude and RT on uncertain threats in the relationship between mindfulness and anxiety. The results provide further evidence that reactions to uncertain threats may play a role in the association between mindfulness and anxiety and suggest that interventions are needed to specifically target excessive anticipatory responses to uncertain threats.
# Conclusion
In summary, the current research demonstrated that unpredictable, high-threat conditions might trigger a more intense anticipatory response (including self-reported IU, behavioral RT, and LPP amplitude) in TA. It further verified that mitigating anticipatory threat responses might be the potential mechanism by which mindfulness alleviates anxiety. These findings lay important groundwork for understanding the role of strong intolerance of potential threats in the development and maintenance of anxiety and may have practical implications for informing the development and optimization of mindfulness treatments for anxiety.
# Data availability statement
Data used in the present analysis are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.
# Ethics statement
The studies involving human participants were reviewed and approved by the Ethics Committee of Army Medical University. The patients/participants provided their written informed consent to participate in this study.
# Author contributions
YX: designed and executed the study, assisted with the data analyses, gained ethical approval, and wrote the paper. WH: recruited the subjects, collaborated with the design, and writing of the study. XY: collaborated with the recruiting of the subjects and analyzed the data. FL: collaborated with material preparation and data collection. ML: collaborated in the writing and editing of the final manuscript. All author approved the final version of the manuscript for submission.
# Funding
This study was financially supported by National Natural Science Foundation of China (No. 31700958).
. /fpubh. . of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
[fig] FIGURE: Flow of participants. [/fig]
[table] TABLE Between -: group di erences regarding demographic data.Using Mann-Whitney U tests and Chi-square tests, between-group differences in the demographic variables were not statistically significant. LA, Low-anxious individuals; TA, Trait-anxious individuals. [/table]
[table] TABLE Scores on: self-report scales for the LA (n = ) and TA (n = ). LA, Low-anxious individuals; TA, Trait-anxious individuals; M, Means; SD, Standard Deviations; HADS-A, Anxiety subscale of Hospital Anxiety and Depression Scale; TAI, Trait Anxiety Inventory; FFMQ, Five Facet Mindfulness Questionnaire; IUS, Intolerance of Uncertainty Scale. [/table]
[table] TABLE Results of: the rmANOVA on two groups and four conditions for LPP. [/table]
[table] TABLE Correlations between: all variables. [/table]
|
18F-Fluciclovine (18F-FACBC) PET imaging of recurrent brain tumors
Purpose The aim of our study was to investigate the efficacy of 18 F-Fluciclovine brain PET imaging in recurrent gliomas, and to compare the utility of these images to that of contrast enhanced magnetic resonance imaging (MRI) and to [ 11 C-methyl]-L-methionine ( 11 C-Methionine) PET imaging. We also sought to gain insight into the factors affecting the uptake of 18 F-FACBC in both tumors and normal brain, and specifically to evaluate how the uptake in these tissues varied over an extended period of time post injection. Methods Twenty-seven patients with recurrent or progressive primary brain tumor (based on clinical and MRI/CT data) were studied using dynamic 18 F-Fluciclovine brain imaging for up to 4 h. Of these, 16 patients also had 11 C-Methionine brain scans. Visual findings, semi-quantitative analyses and pharmacokinetic modeling of a subset of the 18 F-Fluciclovine images was conducted. The information derived from these analyses were compared to data from 11 C-Methionine and to contrast-enhanced MRI. Results 18 F-Fluciclovine was positive for all 27 patients, whereas contrast MRI was indeterminate for three patients. Tumor 18 F-Fluciclovine SUVmax ranged from 1.5 to 10.5 (average: 4.5 ± 2.3), while 11 C-Methionine's tumor SUVmax ranged from 2.2 to 10.2 (average: 5.0 ± 2.2). Image contrast was higher with 18 F-Fluciclovine compared to 11 C-Methionine (p < 0.0001). This was due to 18 F-Fluciclovine's lower background in normal brain tissue (0.5 ± 0.2 compared to 1.3 ± 0.4 for 11 C-Methionine). 18 F-Fluciclovine uptake in both normal brain and tumors was well described by a simple one-compartment (three-parameter: V b ,k 1 ,k 2 ) model. Normal brain was found to approach transient equilibrium with a half-time that varied greatly, ranging from 1.5 to 8.3 h (mean 2.7 ± 2.3 h), and achieving a consistent final distribution volume averaging 1.4 ± 0.2 ml/cc. Tumors equilibrated more rapidly (t 1/2 ranging from 4 to 148 min, average 57 ± 51 min), with an average distribution volume of 3.2 ± 1.1 ml/cc. A qualitative comparison showed that the rate of normal brain uptake of 11 C-Methionine was much faster than that of 18 F-Fluciclovine. Conclusion Tumor uptake of 18 F-Fluciclovine correlated well with the established brain tumor imaging agent 11 C-Methionine but provided significantly higher image contrast. 18 F-Fluciclovine may be particularly useful when the contrast MRI is non-diagnostic. Based on the data gathered, we were unable to determine whether Fluciclovine uptake was due solely to recurrent tumor or if inflammation or other processes also contributed.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
# Introduction
Various amino acid PET tracers have been studied in brain tumors. They include [methyl-11 C]-L-methionine ( 11 C-Methionine), O-(2-[ 18 F]-fluoroethyl)-L -tyrosine ( 18 F-FET) and 6-[ 18 F] -fluoro-L-DOPA ( 18 F-DOPA). Amino acid accumulation in brain tumors is a function of both increased transport across tumor blood vessels and across tumor cell membranes due to overexpression of amino acid transporter systems. Studies have shown that tumor detection by amino acid PET imaging is more sensitive than 18 F-FDG PET imaging owing to the better tumor-to-normal brain image contrast seen with the amino acids. Also, withF-FDG, the appearance of tumor-associated inflammation can mimic that of tumor progression, but this is much less of a problem for amino acids. Recent studies showed that amino acid PET imaging, including 11 C-Methionine and 18 F-FET, has greater accuracy than MRI for the evaluation of post-therapeutic effectsand of tumor recurrence. The European Association of Nuclear Medicineand Neuro-oncologyrecommend using amino acid PET rather than MRI for these indications.
The diagnostic accuracies of current amino acid PET tracers ( 11 C-Methionine and 18 F-FET), however, remain suboptimal and the need for further improvement in neuro-imaging approaches persists.
Pre-clinical studies have shown thatF-Fluciclovine could be a potential PET tracer for brain tumor imaging, and the results from clinical studies evaluating 18 F-Fluciclovine PET for primary staging of gliomas are encouraging. Recently, showed in a preliminary study of six patients thatF-Fluciclovine may provide better assessment thanC-Methionine for the initial detection of glioma. Furthermore,F-Fluciclovine may be a promising tracer to detect high grade recurrent gliomas.
The objective of the present study was to investigate the efficacy of 18 F-Fluciclovine brain PET imaging in the setting of recurrent or progressive disease and to compare it to contrast-enhanced MRI imaging and toC-Methionine brain PET imaging.
# Methods
## Patients
This prospective clinical study was performed after Institutional Review Board approval (Memorial Sloan-Kettering Cancer Center IRB#03-028). Twenty-seven patients who underwent 18 F-Fluciclovine brain PET for suspicion of recurrent or progressive gliomas between August 2004 and January 2008 were recruited under two different protocols (16 in the first, 11 in the second) with different funding sources (NCI and an internal source). The inclusion criteria, however, were identical and the data acquisition procedures were very similar with the primary difference being that theC-methionine study was left off of the second protocol. All patients fasted for at least 4 h before PET imaging. Written informed consent was obtained from all patients.
## Synthesis of 18 f-fluciclovine and 11 c-methionine
Clinical-gradeF-labeled Fluciclovine and 11 C-labeled methionine were produced by the Radiochemistry and Molecular Imaging Probes Core at MSKCC.F-Fluciclovine andC-methionine PET image acquisition Following a 1-min IV infusion of 370 MBq of 18 F-Fluciclovine, dynamic images of the brain were acquired in 2D mode (i.e. with septa) for 45 min on a GE Advance PET (first 21 patients) or a GE Discovery STE PET/CT (last 6 patients) scanner. The patients were subsequently removed from the scanner but returned up to three times for 20-min static images of the brain between 90 and 240 min post injection. No adverse effects were reported following the injection of
[formula] F-Fluciclovine. [/formula]
For a subset of patients who agreed to the procedure, a heated-hand arterialization of the venous blood was performed using chemical heating pads. Starting at 30 s post initiation of the 18 F-Fluciclovine infusion,~1-mL blood samples were acquired approximately every 20 s, with the precise time (synchronized to the PET computer's clock) recorded for each sample. As time progressed, the spacing between samples was increased (at most, a total of 25 samples were acquired for each patient). The later samples were taken at the same times as the respective late static imaging sessions (one sample at or soon after each session). For each blood sample, a portion was used to obtain plasma, and aliquots of whole blood and plasma were assayed for 18 F activity concentration using a calibrated gamma counter [LKB Wallac]. This procedure in many cases failed (e.g., hand insufficiently heated) but was judged to have been successful in 8 (out of 20 attempted) patients, whose resulting blood time-activity curves achieved a peak SUV greater than 8.
For 16 of the patients, 11 C-Methionine PET was performed on the same day, 2.5 or more hours prior to the initiation of theF-Fluciclovine PET imaging. In these scans, dynamic images of the brain were acquired over 45 min, following a 1 min IV infusion of 370 MBq of [ 11 C-methyl]-L-methionine. No adverse effects were reported after the injection of 11 C-Methionine.
All PET images were reconstructed with an iterative ordered subsets expectation maximization (OSEM) algorithm available from the PET camera manufacturer. All emission scans were accompanied by a 2-to 5-min germanium-68 transmission scan (on the GE Advance) or low dose CT scan (on the GE DSTE) for attenuation correction purposes. Manufacturer-provided corrections for scatter, randoms and detector inhomogeneity were also applied.
# Image analysis
All 18 F-Fluciclovine and 11 C-Methionine visual and SUV analyses were conducted using the Hermes HybridViewer imaging software (Hermes Medical Solutions, Stockholm, Sweden) based on a summed 25-45 min PET static image set registered to a contrast-enhanced MR (or in one case CT) image acquired within 30 days of the PET image. We chose to base our analysis upon a summed 25-45 min image because the uptake was relatively stable over this time period; it was the latest time consistently acquired across patients for bothF-Fluciclovine and 11 C-Methionine and it was similar to time points used in other studies of 18 F-Fluciclovine uptake into brain tumors.
The reader, an experienced nuclear medicine physician (LM), interpreted the scans visually and classified them as positive (high or low tumor uptake) or negative (no uptake), and manually defined the borders of the tumor on each of the scans (independently and blinded to the patient clinical and imaging outcomes). Tumor SUVmax of each patient was recorded along with its ratio to the SUVmeans taken from two different normal-brain reference regions. One reference region was placed over the contralateral normal brain, and the other placed within the cerebellum. The degree of overlap between all paired modality combinations (MRI-Fluciclovine, MRI-Methionine and Fluciclovine-Methionine) were assessed using Sorensen-Dice coefficients.
For the kinetic analysis, time-activity curves describing theF-Fluciclovine uptake were derived from large regions of interest placed within the normal cerebellum and small (~1-3 cc) regions covering the most intense uniform portion of the lesion, both initially based solely on their appearance within the summed 25-45 min image. The uptake within the tumor region was also assessed visually at all other time-points to assure homogeneity within the region and throughout the entire time-course of the study. Adjustments to the volume were made if this criterion was not met. We chose not to do modeling on the contralateral normal brain out of concern that the regions would contain a mixture of white and gray matter thereby violating the homogeneous tissue assumption implicit in the use of a compartmental model.
## Kinetic analysis of dynamic 18 f-fluciclovine pet images
The static 18 F-Fluciclovine PET images were each registered to the summed 25-45 min frame of the dynamic image set using a rigid-body transform that maximized a normalized cross-correlation target function. The resultant resampled image sets were then appended to the end of the initial 45-min dynamic series resulting in a single dynamic PET image set extending up to 4-h in duration. All PET and blood activity concentration measures were converted to standard uptake values (SUVs) normalized by patient body mass.
Blood sample data was fitted to a sum of three exponentials convolved with a square pulse mimicking the 18 F-Fluciclovine infusion. The resulting sum of exponentials was then used as the input function, I, when fitting the tumor and normal cerebellum time-activity tissue curves, T, using a nonlinear search algorithm that minimized the weighted leastsquares difference between the data and the following model equation:
[formula] T ¼ V b I þ k 1 e −k 2 t *I [/formula]
where * denotes convolution, t is time, V b is the blood volume and k 1 and k 2 are the first-order rate constants describing the transport of 18 F-Fluciclovine between the plasma and the tissue.
During the fitting process the above equation was averaged over the durations of the individual PET frames from which T was determined. The weights used in the fitting were chosen based on an estimate of the total counts contributing to the tissue measurement (i.e. a function of the frame duration and the tissue activity concentration). Models involving an additional exponential were also evaluated based on a comparison of their Bayesian Information Criterion (BIC) values.
## Reference standard
To establish the ground truth, tumor pathology results were used whenever a biopsy or a surgery was performed. In patients for which no tissue samples were available, the clinical and imaging follow-up was used.
# Statistical analysis
All statistical comparisons of the 25-45 min uptake metrics, and ratios thereof, were conducted using a Spearman correlation. All tumor volume comparisons made use of a Wilcoxon paired test. Kaplan-Meier survival analysis was performed and the relation between risk of death and tumor SUVmax (Tmax), tumor SUVmax to contralateral SUVmean (Tmax/Co_mean), tumor SUVmax to cerebellar SUVmean (Tmax/Ce_mean) and tumor volume were evaluated using a Wald test in a Cox model. All analyses were performed using R version 3.5.0 (www.r-project.org). A p value of less than 0. 05 was considered statistically significant for all analyses.
# Results
## Patients
Twenty-seven 18 F-Fluciclovine PET studies were performed. Patient characteristics are summarized in. All patients had contrast-enhanced conventional imaging (26 MRI and 1 CT) within 30 days of FACBC PET (mean 9 days) to evaluate recurrent or progressive disease: based on the MRI results 22 patients were considered to have recurrence or progressive disease, two patients stable disease and three patients had equivocal results by MRI.
All patients were considered to have an active tumor based upon the clinical and/or MRI assessment at the time of 18 F-Fluciclovine imaging. Thirteen patients had a biopsy or a surgical resection after the 18 F-Fluciclovine imaging providing a proof of the recurrence. Pathology at this stage revealed: nine patients with GBM (5 initially with GBM, 2 initially with astrocytoma grade III and 2 initially with astrocytoma grade II). In addition, there were two low grade gliomas (initially oligodendroglioma grade II and astrocytoma grade II), one high grade astrocytoma (initially an astrocytoma grade III), one high grade glioma (initially an astrocytoma grade II). In 13 other patients without a second pathological diagnosis (5 patients had initial low-grade gliomas and 8 had high grade gliomas), the diagnosis of recurrent/progressive disease was based on clinical follow up. For one patient (patient 4, with an initial oligodendroglioma grade III), there was no medical record after the 18 F-Fluciclovine PET, but MRI and PET imaging suggested tumor recurrence. That patient died 9 months later, suggesting an active tumor.
Twenty-five patients are known to have died during the follow up period. For two patients (patients 24 and 25), only a single post-PET imaging follow-up note was available; both had clinical disease progression at that time . The median overall survival was 31.6 months and the 5-year overall survival rate was 33% (95% CI: 16-51%).
## F-fluciclovine and 11 c-methionine uptake: analysis of 25-45 min static images
The tumor uptake and tumor-to-normal brain ratio results are summarized in, and representative PET scans are shown for two patients. All 27 tumors were clearly delineated with 18 F-Fluciclovine PET.F-Fluciclovine tumor uptake ranged from 1.5 to 10.4 SUVmax (average ± standard deviation 4.5 ± 2.3), Co_mean ranged from 0.14 to 1.08 (0.4 ± 0.2), Ce_mean ranged from 0.2 to 1.75 (0.6 ± 0.3) and Tmax/Co_mean and Tmax/Ce_mean ratios from 3.6 to 23.8 (10.8 ± 4.5) and from 4.3 to 15.2 (7.9 ± 2.8), respectively. In three patients with equivocal MRI results (patients 5, 9 and 25),F-Fluciclovine PET confirmed tumor progression (all GBM, including two biopsy-proven after imaging). Two patients had visually low 18 F-Fluciclovine tumor uptake. One of these two patients (patient 20) had an astrocytoma grade II at time of diagnosis, and five years later, clinical suspicion of recurrence. The MRI showed recurrent disease with a small tumor volume (4.4 cm 3 ).F-Fluciclovine tumor SUVmax and tumor/brain ratio were low at 2.06 and at 3.6 and 4.3 for ratios to contralateral and cerebellum, respectively, and PET tumor volume was 2.5 cm 3. A resection of this tumor after Fluciclovine imaging showed a recurrent low-grade glioma. Eleven years after initial presentation (6 years after the 18 F-Fluciclovine PET scan), this patient progressed again. Re-resection of the recurrent tumor showed a high-grade astrocytoma. The second patient (patient 23) had an initial GBM. The MRI showed stable disease and a small tumor volume (5.7 cm 3 ).F-Fluciclovine tumor SUVmax and tumor-to-cerebellum ratio were low (2.4 and 4.5, respectively), but the ratio of tumor to contralateral normal brain was relatively high (7.1). The tumor was not re-resected, and the patient died 6 months after the PET imaging.
We did not attempt to determine the correlation between tumor uptake and tumor grade because too few patients had a biopsy or a surgical resection at the time of the 18 F-Fluciclovine PET imaging (and therefore there was no histology on which to base an assessment of grade) and because too few patients had low-grade tumors (2/13 among the patients with re-resection and 5/14 based on initial grading).
Sixteen patients had both 18 F-Fluciclovine and 11 C-Methionine PET imaging. Six of 16 patients had low tumor uptake of 11 C-Methionine, and extremely low tumor contrast. 11 C-Methionine PET tumor SUVmax ranged from 2.2 to 10.2 (average 5.0 ± 2.2), Co_mean ranged from 0.48 to 2.03 (1.3 ± 0.4), and Ce_mean ranged from 0.67 to 3.1 (1.7 ± 0.6). SUVmax values were lower with 18 F-Fluciclovine than with 11 C-Methionine (p = 0.03), but there was a very high correlation between tumor 18 F-Fluciclovine and 11 C-Methionine SUVmax values (r = 0.92). This correlation decreased somewhat when the tumor values were normalized by either cerebellar (r = 0.52) or contralateral (r = 0.76) normal brain mean values.
Visual analysis clearly showed higher tumor-tonormal brain image contrast with 18 F-Fluciclovine than with 11 C-Methionine, due to the lower background (lower normal brain uptake) in the 18 F-Fluciclovine studies. Uptake in the contralateral brain was about 30% below that of the cerebellum, for both 18 F-Fluciclovine and 11 C-Methionine, but the two regions were highly correlated across patients ( 18 F-Fluciclovine r = 0.78 and 11 C-Methionine r = 0.88). The higher contrast of the 18 F-Fluciclovine images was quantitatively confirmed when we compared the Tmax/Co_mean and Tmax/Ce_mean ratios between 18 F-Fluciclovine and Both ratio values were significantly higher with 18 F-Fluciclovine (p < 0.0001). An intra-patient comparison of 18 F-Fluciclovine and 11 C-Methionine uptake in normal brain (contralateral and cerebellar combined) showed the uptake of the two tracers to be functionally, though perhaps not completely linearly related (Supplement S-4), with 11 C-Methionine uptake over three times that of 18 F-Fluciclovine. A slight but persistent negative correlation of normal brain uptake and patient age was seen for both 18 F-Fluciclovine and 11 C-Methionine, consistent with similar findings for 11 C-Methionine seen in other studies (Supplement S-3). No consistent trends were seen between uptake and either weight or height (Supplement S-1 and S-2).
## Comparison of 18 f-fluciclovine, 11 c-methionine and mri determined tumor volumes
Tumor volumes, as determined by 18 F-Fluciclovine PET and contrast-enhanced MRI were highly correlated (r = 0.91) . Overlap in the volumes as measured by the Dice coefficient ranged from 36% to 87%, with a mean of 64 ± 13%. 11 C-Methionine volumes were somewhat less well correlated to the MRI (r = 0.82) with a tendency of the 11 C-Methionine volumes to be smaller than those based on MRI contrast for smaller lesions (<10 cc). Dice coefficients in this case varied between 7% (a lesion barely seen with 11 C-Methionine) and 81% with a mean of 50 ± 19%. The overlap between 18 F-Fluciclovine and MRI volumes was significantly higher than the overlap between 11 C-Methionine and MRI was found to have a high correlation (r = 0.87). The Dice values for the overlap between 11 C-Methionine and 18 F-Fluciclovine determined volumes ranged from 10% to 82%, with a mean of 59 ± 18% (patient's example.
## Analysis of dynamic data
Typical examples of the dynamic time-activity profiles (TACs) of 18 F-Fluciclovine in both tumor and normal cerebellum along with the modeled estimates are shown in. The uptake of 18 F-Fluciclovine into both tumor and normal brain regions was generally well described by a first-order compartmental model that included a blood space and a single reversible tissue compartment. In some instances, adding an additional exponential to the model improved the BIC value, but this generally reverted back to a single exponential if the volume of interest was adjusted so as to reduce heterogeneity within the volume. BIC values were further improved by fixing the blood volume at 7.5% for both tumors and normal brain, thereby avoiding covariance with the rate constants.
The parameter values determined by the compartmental model fits of tumor and normal cerebellar 18 F-Fluciclovine uptake for individual patients are shown in. Not surprisingly, the tumor rate constants varied from patient to patient considerably more than did the normal brain. In general, the 18 F-Fluciclovine tumor equilibration half-times were The analysis of the dynamic 11 C-Methionine data was limited because blood samples were not obtained (due to patient comfort and logistical reasons related to the short half-life of c. Shows the correlation between the tumor SUVmax and the same patient's cerebellar mean for 18 F-Fluciclovine (black circles) and 11 C-Methionine (gray circles). d. Relationship between the brain uptake forF-Fluciclovine and the same patient's brain uptake of 11 C-Methionine in the normal cerebellum (black circles) and in the normal contralateral brain (gray circles) Comparison of tumor volumes as assessed on each of three image sets. a. Correlation between volumes determined based on T1 contrast enhanced MRI and 18 F-Fluciclovine PET (black circles) and between T1c MRI and 11 C-Methionine PET (gray circles). Fitted lines are forced through the origin. b. Comparison between the two PET derived volumes ( 18 F-Fluciclovine vs. 11 C-Methionine). Again, fitted line is forced through the origin 11 C). Therefore, we did not perform any kinetic modeling and were unable to make a direct comparison of the rate constants. However, a qualitative assessment of the relative shapes of the time-activity curves can still provide some insight. The uptake of 11 C-Methionine into normal brain was extremely fast (consistent with a rapidly equilibrating blood space) but extended to levels well above that of 18 F-Fluciclovine, rapidly reaching a plateau. These observations suggest a much faster transport across the blood-brain-barrier (BBB) and equilibration with normal brain tissue of 11 C-Methionine (relative to 18 F-Fluciclovine)and Supplement S1).
Regardless of the relative tumor uptake level, 11 C-Methionine quickly (< 5 min) reached a constant degree of contrast relative to cerebellum that extended over the majority of the 45 min imaging period. The ratio-versus-time plots for 18 F-Fluciclovine, on the other hand, show a time dependence of image contrast with a large variation between patients.
# Discussion
18 F-Fluciclovine ( 18 F-FACBC) is an alicyclic, non-natural amino acid (anti-1-amino-3-[ 18 F]fluorocyclobutane-1-carboxylic acid) containing a 4-membered ring. It was originally developed as a stable leucine analogue for brain tumor imaging. The mechanism of 18 F-Fluciclovine uptake differs from that of other radiolabeled amino acids that are widely used for tumor imaging. Most amino acid transport substrates, including L-methionine, O-(2-fluoroethyl)-L-tyrosine (FET), and 3,4-dihydroxy-6-fluoro-L-phenylalanine (F-DOPA)make use of the system L transporters, LAT1 (SLC7A5) and LAT2 (SLC7A8). In contrast, the major mediator of Fluciclovine uptake is ASCT2 (SLC1A5), with LAT1 also contributing but generally to a much lesser extentthough perhaps modulated by pH. Unlike naturally occurring amino acids, Fluciclovine is not metabolized and it is not incorporated into protein (a feature which simplifies interpretation of the images and modeling of its kinetics).
Our study is one of the first evaluating 18 F-Fluciclovine imaging in recurrent or progressive gliomas. Tumor accumulation of 18 F-Fluciclovine was observed in all patients considered to have active disease (recurrent or progressive gliomas), even when MRI was not diagnostic. This suggests that 18 F-Fluciclovine PET may be able to differentiate tumor recurrence/progression from post-treatment necrosis and/or inflammation (i.e. pseudo-progression on contrast MRI. However, we could not test this hypothesis because none of the latter were confirmed by lesion pathology, since all lesions were recurrent gliomas.F-Fluciclovine images showed high contrast, confirmed by the high tumor-to-brain ratios (tumor SUVmax-to-SUVmean of a reference normal-brain region), concordant with the first clinical studies. Our results showed that the two ratios calculated with two different reference regions (either the contralateral normal brain tissue or the cerebellum) are highly correlated, as was previously described by Kondo et al..F-Fluciclovine tumor volumes were correlated with volumes based on contrast-enhanced MRI (or CT) with extensive overlap. Compared to MRI volumes, the 18 F-Fluciclovine PET volume was about equal for 1 patient, clearly larger for 13 patients (2 patients volumes <10% MRI volumes and 11 patients volumes >10% MRI volumes) and smaller in 13 patients (3 patients volumes < −10% MRI volumes and 11 patients volumes > −10% MRI volumes). This was a surprising finding. We were expecting that the 18 F-Fluciclovine PET volumes would be higher than MRI volumes because in previous imaging studies in gliomas, the PET-derived amino-acid tumor volumes tended to be larger than the region of contrast enhancement, including in one study involving 18 F-Fluciclovine. However, in our population (recurrent/progressive disease) we did not find this to be the case. We speculate that the contrast MRI sometimes over-estimated active tumor volume in our patients, because it included treatmentinduced necrosis (e.g., radionecrosis), whereas 18 F-FACBC PET imaging primarily reflected viable progressive tumor. Unfortunately, all patients did not undergo post-imaging re-resection or biopsies of their tumors to confirm this hypothesis. The extensive BBB disruption in these patients is clearly also a potential confound (given Fluciclovine's slow rate of transport across the intact BBB) tending to improve the correlation between the contrast MRI and Fluciclovine derived volumes.
It has been reported that most amino acid tracers (e.g., FET, MET, FACBC) do not accurately separate tumor grades based on histological subtypes. Nevertheless, Parent et al. recently showed that 18 F-Fluciclovine PET may discriminate highand low-grade gliomas. In our study we chose not to test the correlation between tumor grading and Fluciclovine uptake because the overwhelming majority of patients presented with high-grade glioma, as one might expect in a recurrent/ progressive disease setting. For 16 patients we were able to directly compare 18 F-Fluciclovine and 11 C-Methionine PET. The rate of 11 C-Methionine uptake into normal brain was found to be significantly higher than that of 18 F-Fluciclovine. This is consistent with 11 C-Methionine's relatively high affinity for the LAT1 transporter which is highly expressed in the endothelial cells of the brain compared to ASCT2, 18 F-Fluciclovine's preferred transporter. 11 C-Methionine uptake levels achieved in the normal brain were between 1.5 and 3-fold higher than that of 18 F-Fluciclovine. This difference in background was a major factor driving the difference in tumor contrast between 18 F-Fluciclovine and 11 C-Methionine, as reflected by their tumor-to-normal brain ratios. This difference was previously described by with initial staging of gliomas. Because of this difference in contrast,F-Fluciclovine tumor volumes were easier to delineate and this too may account for 18 F-Fluciclovine's higher correlation and overlap with conventional imaging-derived volumes, compared to that for 11 C-Methionine PET. Although 11 C-Methionine is capable of being incorporated into proteins and trapped within cells, it is also conceivable that if imaging were performed at a later time post-injection, considerable washout would have occurred from the normal brain tissues to levels below that of 18 F-Fluciclovine. Given carbon-11's 20-min half-life, however, late imaging with 11 C-Methionine is not practical.
An important advantage of 11 C-Methionine PET over contrast enhanced MRI is its ability to detect infiltrating tumor even in the absence of BBB disruption. To the extent that 18 F-Fluciclovine's transport is hampered by an intact BBB (and conversely enhanced by a disrupted BBB), this may pose significant challenges when interpreting a Fluciclovine study. However, because the vast majority of our subjects were found to have progressive disease, we were unable to evaluate whether heightened washout from benign tissue having a disrupted BBB would have mitigated this confound.
Since LAT1 and ASCT2 are frequently upregulated in primary tumorswe were not surprised to find that the tumor SUVmax values were correlated between these two amino-acid tracers. However, it was somewhat surprising to see this correlation diminish when the tumor SUV values were normalized by either the contralateral or cerebellar normal brain mean values. The average SUV of these relatively large regions of interest should be robust, low-noise measures. Therefore, using the normal brain as a normalizer should inject a minimal amount of noise and this should more than compensate for the random biases inserted by a less than perfect body habitus normalizer. One might conclude therefore, that patient body mass (the normalizer implicit to the SUV metric we used) was near perfect. However, we also found the uptake of 18 F-FACBC and 11 C-Methionine to be highly correlated in the normal brain between patients. Moreover, we found that the tumor uptake of each of these tracers was correlated with that same patient's normal brain uptake. Neither of these results is at all expected and might typically be indicative of a poor normalizer. However, if body mass were indeed a poor normalizer, one would expect there to be some residual correlation to body mass, but this was not supported by our data (Supplement S-1). Taken together, this evidence suggests that there may be some as yet unidentified parameter (e.g. endogenous amino acid blood levels) that is influencing (at least partially) the uptake of both 18 F-Fluciclovine and 11 C-Methionine.
We found that the 18 F-Fluciclovine uptake into the normal brain when measured at 25-45 min post injection varied greatly (~8-fold with~50% CoV's, see, much more than we had expected. Similarly, our kinetic analysis of 18 F-Fluciclovine uptake in these same tissues also showed equilibration half-times that varied greatly, ranging between 1.5 and 10 h with a mean of 2.7 ± 2.3 h (CoV = 86%). However, because we imaged these patients over an extended period of time (well beyond that of any previous patient study involvingF-Fluciclovine), our kinetic analysis was able to find that the normal cerebellum reached a remarkably consistent distribution volume of 1.4 ± 0.2 mL/cc (CoV = 11%). This finding is potentially explained by transport rates varying as a function Parameter values determined by the compartmental model fits of variations in endogenous amino acids that are near saturation. Thus the 25-45 min time period might be too early to make a robust measurement of 18 F-Fluciclovine uptake. Kinetic analysis of the tumors showed the tumors to have a k1:k2 ratio (3.2 ± 1.1) that was significantly higher (p = 0.001) than that of normal brain (1.4 ± 0.2). Although very variable, an overall higher rate of 18 F-Fluciclovine transport (k1) into tumors (ranging from 0.02 to 0.73 min −1 , with a mean of 0.14 ± 0.24 min −1 ) was observed, compared to that in normal brain (0.008 ± 0.003 min −1 ). This is consistent with a higher expression of the ASCT2 transporter in gliomas compared to normal brain. It has yet to be determined which, if either, of these parameters (k1 or the k1:k2 ratio) provides more useful information. It is clear, however, that both the tumor uptake and its contrast to normal brain vary considerably as a function of the time post injection, as seen in the tumor-to-cerebellum ratioversus-time plots. If it is determined that the k1:k2 ratio is a prognostic parameter, then it may be necessary to image later (perhaps 3 or 4 h post injection) before a simple SUV metric can be used as a robust correlate of progression or survival. Conversely, if k1 is the better parameter of interest, earlier imaging is more likely appropriate. Either way, the timing of the scan may explain the difficulty we found in demonstrating a correlation between 18 F-FACBC uptake and survival.
Overall, 18 F-Fluciclovine PET can detect recurrent and progressing gliomas. Compared to 11 C-Methionine,F-Fluciclovine images have better contrast, largely due to the lower uptake of 18 F-Fluciclovine in normal brain (lower background). The kinetic analysis of 18 F-Fluciclovine is consistent with a linear single-compartmental model and highlights the dependence of SUV and the contrast achieved by
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Zebrafish as an Emerging Model for Bioassay-Guided Natural Product Drug Discovery for Neurological Disorders
Most neurodegenerative diseases are currently incurable, with large social and economic impacts. Recently, there has been renewed interest in investigating natural products in the modern drug discovery paradigm as novel, bioactive small molecules. Moreover, the discovery of potential therapies for neurological disorders is challenging and involves developing optimized animal models for drug screening. In contemporary biomedicine, the growing need to develop experimental models to obtain a detailed understanding of malady conditions and to portray pioneering treatments has resulted in the application of zebrafish to close the gap between in vitro and in vivo assays. Zebrafish in pharmacogenetics and neuropharmacology are rapidly becoming a widely used organism. Brain function, dysfunction, genetic, and pharmacological modulation considerations are enhanced by both larval and adult zebrafish. Bioassay-guided identification of natural products using zebrafish presents as an attractive strategy for generating new lead compounds. Here, we see evidence that the zebrafish's central nervous system is suitable for modeling human neurological disease and we review and evaluate natural product research using zebrafish as a vertebrate model platform to systematically identify bioactive natural products. Finally, we review recently developed zebrafish models of neurological disorders that have the potential to be applied in this field of research.
# Introduction
Central nervous system (CNS) diseases and disorders, including Alzheimer's disease (AD), schizophrenia (SCZ), Huntington's disease (HD), and Parkinson's disease (PD), signify a global burden on society in terms of disability, economic loss, and human suffering. Globally, more than a million people have CNS disorders. CNS disorders are multifaceted diseases with unclear causes and often ineffective therapies, with only a few therapeutic drugs being clinically effective. Natural products (NPs) are small molecules synthesized from living organisms (plants, bacteria, and fungi) and are similar to secondary metabolites. Among all existing sources for drug discovery against single targets of new lead compounds, NPs are most promising but are underutilized. Crude extracts from NPs are a complex mixture of mostly uncharacterized compounds, some of which may have unwanted effects. Worldwide, nearly 30% of all top-selling drugs are NPs or their derivatives. NPs are an excellent source of new drug-like compounds to be discovered, and their diversity of chemicals has helped to develop drugs for a wide range of neurodegenerative disorders. Most new drugs have been authorized from either NPs themselves or NPs over the past 30 years. In complex NP extracts, the isolation and structural characterization of bioactive small molecules involves several new methodologies that need considerable time and effort. Furthermore, there are several methods involved in testing NPs in high-throughput screening (HTS). Combinatorial libraries with NP-like compounds have been recently used in HTS.
A vital component of the drug discovery program for NPs is bioassay-guided separation. In bioassay-guided separation, each chromatographic fractionation undertakes biological evaluation for further fractionation, and only biologically active fractions are selected. The crude extracts are fractionated and evaluated in bioassays. Further fractionation is repeated until the chosen activity isolates pure compounds and then characterizes them structurally. Novel pharmaceutically active NPs had been identified through screening and fractionation of crude extracts using several presently regarded in vitro assays, collectively with (i) cell fractions, (ii) entire cellular assays, or (iii) recombinant enzymes as target molecules. Notwithstanding its application for HTS identification, the biomedical relevance of the isolated active metabolites can be limited when using only enzymatic or in vitro assays. To overcome this limitation, high-resolution micro-fractionation can be coupled with high-content bioassays to further analyze the separate constituents. In contrast to cell-based reporter or enzymatic assays, high-content bioassays (e.g., phenotypic assays using some cells or organisms) allow for an impartial investigation of pharmacological activity. Many in vivo animal models offer the possibility of independent screening of biomedically relevant bioactivities. However, milligrams of compounds are required for mammalian models and are thus not ideal for in vivo platforms for micro-fractionation and rapid HPLC profiling approaches.
Moreover, many naturally derived active compounds not only play a role as drugs but also help in the development of many new model structures for synthetic molecules through combinatorial chemistry. During the last 20 years, about 50% of drugs introduced to the market have been derived indirectly or directly from small bioactive molecules. As a source of chemical diversity, unfulfilled expectations from current R&D strategies and emerging trends have led to interest in NPs. NPs have attracted considerable attention in the treatment of CNS diseases due to their neuroprotective and therapeutic effects. NPs are excellent sources of safe, precise, and effective anti-neurotherapeutic agents and thus are useful in the development of safer substitutes to pharmaceuticals. Recent literature suggests that many bioactive compounds have both neurotrophic and neuroprotective actions; therefore, for peripheral neuropathy early treatment using phytochemical approaches could be one of the important strategies in preventing many neurological disorders.
Many presently known bioactive NPs have been previously recognized for their activity-guided extract isolation through the use of in vitro assays. Biologically active NPs have been identified by physical characteristics using chromatography, mass spectrometry, and NMR spectroscopy analysis. In vivo bioassay-guided fractionation has not widely been used for the discovery of drug-like NPs, as traditional in vivo models (e.g., mice and rats) are low-throughput systems and require much larger quantities of compounds for testing in these systems.
The zebrafish (Danio rerio) provides a complementary integrative biological model for the discovery of natural drug-like products through in vivo bioassay-guided chromatographic fractions requiring only microgram quantities of individual components. Zebrafish are vertebrates, and thus are more evolutionary similar to humans compared to non-vertebrate models. Logistically speaking, zebrafish are tiny and can be kept in a small space in high numbers. Zebrafish are currently emerging as an in vivo vertebrate model system for drug discovery and functional genomics. In addition to their many pharmacological and physiological similarities with mammals, zebrafish have many added advantages including small size of embryos and larvae (0.5 to 5 mm depending on the stage of development), optical transparency, rapid ex vivo development, and high fecundity (up to hundreds of offspring per day). These characteristics makes zebrafish a standalone versatile experimental in vivo model compatible with HTS and NP discovery micro-fraction techniques. Furthermore, zebrafish embryos and larvae provide the convenience of using microtiter plates (96-well and even 384-well plates) to test the activity of micro-fraction isolated natural compounds. Depending on the performance of these isolated compounds, the need for only microgram quantities to initiate an initial biological reaction represents another advantage of using zebrafish as a model organism in comparison to other vertebrates (e.g., rodents, where the energy dose requirements are typically one thousand times higher). This latter feature is prime to NP discovery, as many high-resolution HPLC-based separation techniques, particularly micro-fractionation, bring about very limited pattern quantities that could otherwise be inadequate for in vivo activity analysis.
The neuroprotective activity of bioactive compounds from herbal drugs has been proven by using cellular or animal models. However, effective delivery of drugs to the brain remains the main task in the discovery and development of new CNS disease treatments. This review focuses on neurological disorders with an emphasis on neurodegenerative diseases, use of zebrafish for bioassay-guided isolation of neuroactive small molecule from NPs, and new methods to develop zebrafish neurodegenerative models that have the potential for expansion into NP drug discovery applications.
## Neurodegenerative diseases
Neurodegenerative diseases lead to a rapid loss of brain processes such as cognitive and/or motor neuron function, and are a major challenge facing aging populations. AD, PD, HD, and amyotrophic lateral sclerosis (ALS) are common neurodegenerative diseases. Neurodegenerative diseases share common characteristics and mechanisms despite their different clinical forms. One of these features is regional cytosolic or nuclear protein aggregation. Specific features include extra cell deposition of plaques of amyloid-beta (Aβ), intracellular accumulation of inclusions of hyperphosphorylated microtubule-binding tau in AD, intracellular storage of α-synuclein in PD, inclusion of TAR DNA-binding protein (TDP)-43 transactive response in ALS, frontotemporal dementia, and polyglutamine protein aggregates in HD and other repeat CAG-polyglutamine diseases. While for some cases genetic causes have been identified, the main driver is a complex interaction of predisposition factors in genetics and the environment. In every common neurodegenerative disease condition, there is usually a mixture of hereditary and "sporadic" forms. While the identity of many mutated genes in family forms of AD, PD, and ALS is known, the function of such genes and how their mutations induce neuronal degeneration is not fully understood. Processes that cause degeneration and the death of particular neuron types are probably the most important discovery goals in the field, shaping the disease's manifestations and defining the characteristics of all neurodegenerative diseases.
## Using the zebrafish model for neurological disorders
The zebrafish is being progressively used to model neurodegenerative diseases and neurological disorders successfully, with promises to test potential treatments for diseases and disorders. The zebrafish CNS is similarly arranged to that of other vertebrates, and is traditionally separated into the hindbrain, midbrain, forebrain, ascending and descending spinal cord, cranial nerves, motor spinal cord, and sensory nerves. Zebrafish neuroanatomy has been examined and described in detail elsewhere during development, as well as in adults. The genome of the zebrafish is widely annotated. The evolutionary lineage of zebrafish (teleost-bonyfish) separated about 450 million years ago from the human lineage (tetrapod). Zebrafish pairs can produce large number of embryos that make it possible to achieve relatively high-throughput screening drug studies and behavioral testingwith simple methods for modulating gene expression available. Many human-associated neurodegenerative disease proteins in zebrafish are homologous, highlighting potentially preserved molecular cellular functions that can be easily examined.
## Zebrafish and alzheimer's disease
The most common form of irreversible neurodegenerative disorder and dementia is Alzheimer's disease (AD). Fifty million people were estimated to live with AD in 2018, and this figure is predicted to increase to 152 million by 2050. AD's main clinical feature is progressive memory loss, motor and speech impairment, deception, depression, and aggressive behavior. There is significant neuronal damage in AD patients in numerous brain regions. This is usually caused by extracellular deposition of amyloid-beta peptide and tau protein aggregates called neurofibrillary tangles (NFTs). Several risk factors are identified or under investigation, including both genetic and environmental factors, as potential triggers of AD. AD may be classified as familial AD (FAD, at < 65 years of age) or sporadic AD (SAD, at > 65 years of age). Most of the knowledge of AD pathogenesis has been defined by studying FAD mutations. Some of the genetic targets are precursor protein amyloid-ß (APP) and presenilins (PSEN1 and PSEN2) associated with increased FAD risk. The more common form of AD occurs sporadically (representing >90% of cases). Multi-faceted pathogenesis of SAD is associated with several risk factors such as old age and the presence of the apolipoprotein (APOE) gene ε4 allele and/or the recently identified genetic risk factor sortilin-related receptor (SORL1). SORL1 is an APOE receptor with primary expression in neurons of the brain.
Zebrafish have human orthologous genes that play key roles in AD. The zebrafish genes psen1and psen2are human PSEN1 and PSEN2 orthologs, respectively, whereas the genes appa and appb are human APP co-orthologs. The zebrafish genome also contains orthologous genes for gamma-secretase's complex components, PSENEN (psenen), NCTN (ncstn), and APH1b (aph1b). In addition, β-secretase orthologs (BACE1 and BACE2) were also identified (bace1and bace2in zebrafish. The zebrafish genome contains co-orthologs of the microtubule-associated tau protein (MAPT) gene, which encodes tau protein (mapta, and maptb). The human APOE and SORL1 co-orthologs apoea and apoeb are also present in the zebrafish genomeand sorl1, respectively.
## Zebrafish and parkinson's disease
Dopaminergic neuron degeneration, as well as the presence of Lewy bodies called intracytoplasmic inclusions, are neuropathological lesions associated with Parkinson's disease (PD). Six genes associated with Parkinsonism have been identified, including Parkin, DJ-1, PINK1, α-Synuclein, UCHL-1, and LRRK2. Although predominantly a motion disorder, PD is a mixed group of neurological conditions that are not capable of producing or controlling movement and cognitive impairment. The human PARK2 ortholog in zebrafish (park2) resides on chromosome 13, and encodes a protein of 458 amino acids (465 in humans). The PINK1 zebrafish ortholog has 54% similarity, and an initial study reported a severe developmental phenotype in pink1 k/d zebrafish. The PARK7 zebrafish ortholog encodes a protein of 189 amino acids with 83% human DJ-1 identity.
## Zebrafish and huntington's and other polyglutamine diseases
Huntington's disease (HD) is a monogenic neurodegenerative disease that follows an autosomal dominant pattern of huntingtin gene mutant form (HTT) inheritance. The mutation encodes for an abnormal trinucleotide that leads to glutamine (CAG) expansion at the HTT protein amino terminal and arises in an extended polyglutamine tract of the Huntingtin protein. This causes cell death by gain of function mechanisms, like protein accumulation, mitochondrial dysfunction, and caspase activation. A decline in normal Huntingtin can also make a significant contribution to pathogenesis. To try to elucidate the loss as well as the gain of function mechanisms, zebrafish models are being used. The HD cDNA homology in zebrafish was isolated as the first step towards discovering the possible role of the HD gene in initial vertebrate development. This cDNA codes a predicted protein product of 3121 amino acids with a human HTT identity of 70%. Loss of developmental expression of 15/hd1 caused noticeable morphological abnormalities, including pericardial edema microcephalus and CNS necrosis. Zebrafish htt is also necessary for normal pharyngeal arch cartilage development.
## Zebrafish and amyotrophic lateral sclerosis (als)
Amyotrophic lateral sclerosis (ALS) is characterized by protein inclusions present in the affected neurons. These protein inclusions are linked to spinal cord motor neuron loss and downward motor tracts in the brain and spinal cord. Familial ALS is fairly rare, but a gene-encoding mutation of superoxide dismutase (SOD1) inherited in an autosomal dominant motif causes 20% of such cases. The mutations are usually prescribed by gain of function mechanisms. Over 150 mutations have been discovered in SOD1, including the point mutations G93R and G85R. Recent studies also indicate a role for SOD1 in the sporadic form of ALS and propose a prion-like function of protein misfolding. Moreover, a few of the recently identified genes involved in ALS, such as FUS and TARDBP, also demonstrate a high tendency to act similar to prions in misfolding proteins.
A recent study used zebrafish to assess overexpression of SOD1 by mRNA microinjection to study ALS etiology. In this study, vascular endothelial growth factor (VEGF) overexpression rescued the SOD1-expressing zebrafish axonopathy, while VEGF morpholino knockdown exacerbated the abnormalities. However, one of the limitations in working with ALS in vivo models is the lack of comprehensive methods to assess the presymptomatic course of the disease. The zebrafish provides advantages in the study of processes of early disease with rapid development and reach post-embryonic life about 3 days post fertilization (dpf), which is similar to neonatal mouse development.
## Zebrafish and schizophrenia
Schizophrenia is a severe neurodegenerative disorder with the etiology of hallucination, delusions, depression, agitated body movements, confused thoughts and snafu speech, anhedonia, lack of motivation, and speech problems. The defects of schizophrenia are caused by early development in the brain. About 1% of the world's population is affected by schizophrenia and is characterized by neuronal dysfunction, which results in deficiencies in various cognitive areas including visual and verbal memory, learning, and attention. Patients with schizophrenia, as well as with HD, have impaired preimpulse inhibition (PPI), a type of sensorimotor gaiting. PPI is a neurological event where the response following shocking stimulus is defeated by a weak prestimulus or prepulse and is conserved among vertebrates. The sensorimotor zebrafish gating has been described in 6 dpf larvae for PPI testing. Twin studies have a projected heritage of around 81% for schizophrenia and an environmental impact of about 11% (factors such as diet, parenting, and exposure to toxins or teratogens). A large number of cases of schizophrenia are sporadic and appear in a family without a history of the disease. Many genes have been linked to schizophrenia susceptibility. Genes with a largely robust disease connection include dystrobrevin binding protein 1 (DTNBP1), neuregulin1, disrupted in schizophrenia1 (DISC1), kinesin family member 1 (KIF1), kinesin family member 17 (KIF17), SH3, multiple ankyrin repeat domains 3 (SHANK3), and NOTCH4. Candidate genes for schizophrenia may be vital in determining neuronal migration, neurogenesis, and cell fate.
Burgess and Granatodeveloped an endophenotype of schizophrenia in zebrafish PPI. Exposure to apomorphine and ketamine influences zebrafish PPI, and therefore appears to be facilitated by similar neurotransmitters as in other animals. The same study also identified five novel mutant lines with abnormal PPI responses. One of the most intensively studied genes associated with schizophrenia is DISC1, and was first identified with a high incidence of depression, schizophrenia, and bipolar disorder in a Scottish pedigree. Furthermore, disc1 studies in zebrafish have provided new information on this gene's function.
## Zebrafish and epilepsy
Epilepsy is a common neurological disease caused by unexpected seizures that can vary from a short attention interval to severe and prolonged seizures and muscle cramps. Epilepsy has a pathological mechanism that is poorly understood and is a complex brain disorder with many fundamental causes. Zebrafish have a multifaceted nervous system with elegant behaviors, and are prone to seizure. Adult zebrafish have a wide array of established behaviors that can be studied, making them especially suitable for model development. The pentylenetetrazole (PTZ)-induced zebrafish epileptic seizure has been used to study the mechanism of epilepsy. The affordability of both larval and adult zebrafish, which allows the ontogenesis to investigate a wider range of epilepsy-related phenomena, is also useful.
Several genetically altered zebrafish are now being used to study the behavior and brain function associated with epilepsy. Zebrafish (∼5-7 dpf) are commonly placed in multiple wells and tracked using video tracking software, continuously recorded by a camera. Mutations in two family members, Potassium Voltage-Gated Channel Subfamily Q Member 2 (KCNQ2) and Potassium Voltage-Gated Channel Subfamily Q Member 3 (KCNQ3), have been correlated with inherited neonatal epilepsy, e.g., benign family neonatal convulsions. These genes are highly expressed in zebrafish, providing support for studies of epilepsy using this vertebrate model.
## Zebrafish bioassay-guided isolation of natural product drug discovery
Zebrafish was first suggested by Jones and Huffmann of the Oklahoma Research Foundation as a model for in vivo drug development in 1957, and soon thereafter zebrafish were first used to examine NP bioactivities. Zebrafish bioassay-guided identification of NPs in a number of neurological disorders can be an attractive approach for generating novel lead compounds. Over the past decade, zebrafish as a primary model for HTS in the scope of drug discovery for NPs for neurological disease has grown. Zebrafish model profits combined with robust chromatographic and spectroscopic methods are creating a path to discover and further develop HIT compounds from various plant extracts. Zebrafish has recently emerged as a strong model in a wide range of applications for rapid analysis of gene function and small molecular bioactivity. Zebrafish are well-suited to identify therapeutically potential bioactive NPs . Zebrafish were first proposed over fifty years ago as an in vivo model for the discovery of small molecules of drugs. This preliminary study examined the utility of zebrafish embryos and larvae to screen both NPs and synthetic compounds. Zebrafish offers the opportunity of in vivo swift microgram-scale bioactivity evaluation of small molecules, an attractive feature combined with high-resolution fractionation technologies and analytical methods like UHPLC-TOF-MS and NMR microflow. A recent example is the bioassayguided isolation of zebrafish with six spirostane glycosylated triterpene important for decoction and methanol extract anti-sizing activity from Solanum torvum aerial parts, which was discovered by Soura Challaland his colleagues. In addition, the recently identified flavonoid-transtephrostachin inhibitory of acetylcholinesterase has also been isolated from the leaves of Indian herb Tephrosia purpurea by a zebrafish bioassay. Zebrafish has recently emerged as a strong model in a wide range of applications for rapid analysis of gene function and small molecular bioactivity. Zebrafish are well-suited to identify therapeutically potential bioactive NPs . Zebrafish were first proposed over fifty years ago as an in vivo model for the discovery of small molecules of drugs. This preliminary study examined the utility of zebrafish embryos and larvae to screen both NPs and synthetic compounds. Zebrafish offers the opportunity of in vivo swift microgram-scale bioactivity evaluation of small molecules, an attractive feature combined with high-resolution fractionation technologies and analytical methods like UHPLC-TOF-MS and NMR microflow. A recent example is the bioassay-guided isolation of zebrafish with six spirostane glycosylated triterpene important for decoction and methanol extract anti-sizing activity from Solanum torvum aerial parts, which was discovered by Soura Challaland his colleagues. In addition, the recently identified flavonoid-trans-tephrostachin inhibitory of acetylcholinesterase has also been isolated from the leaves of Indian herb Tephrosia purpurea by a zebrafish bioassay. . Zebrafish bioassay-guided isolation and structurally characterized natural products.
## Source disease/targets molecules references
Pharbitis nil (Seeds) anti-seizure PharbitinRehmannia glutinosa (Root) angiogenesis effect NorviburtinalRhynchosia viscosa (Whole Plant) angiogenesis effect RhynchoviscinDysosma versipellis anti-angiogenesis KaempferolLigusticum sinense (Rhizoma) anti-melanogenesis 1. Lignan 2. cis-4-pentylcyclohex-3-ene-1,2-diolTephrosia purpurea (Leaves) anti-acetylcholinesterase trans-TephrostachinSolanum torvum (leaves) anti-convulsant Paniculonin A Paniculonin BSkeletonema marinoi anti-seizure Inosine
## Development of zebrafish models for neurological disorders
In order to study the genes associated with various neurodegenerative disorders, the zebrafish has proven to be a perfect system where the genetic material is directly injected into the fertilized embryo. For instant study of gene function, effective techniques for the manipulation of gene expression are available. By inserting genes into specific tissue promoters using vectors such as the Tol2 transposase system, transgenic zebrafish can be efficiently produced. The Cre/loxPand GAL4-UASgene function analysis systems can also be used at specific time points to generate conditionally expressed transgenics. The major disadvantage to induce specific mutations in the zebrafish genome was the unavailability of effective previous technologies. However, transcription activator-like effector nuclease (TALENS), zinc finger nucleases (ZFNs), and type II prokaryotic CRISPR (clustered regularly short palindromic repeats)/Cas systems for targeted gene sequences have been developed in recent times and are now being applied to create zebrafish transgenic models. Furthermore, new technologies have expanded development of adult zebrafish and cell culture-based models.
## Transgenic zebrafish models
Due to the effortless screening of genes and small molecules in zebrafish, innovative genetic pathways that enable the development stages for isolating chemical modifiers can be obtained easily.. More recently, it was suggested that many of these benefits could be applied to the study of human disease: high-content small molecular screens, genetic suppressor screens, in vivo disease progression observations, use of fluorescent reporters to identify interesting cell populations, and rapid hypothesis testing experiments in statistically robust larvae samples could provide valuable insight into disease pathogenesis or new therapeutic approaches. In 2008, ZFNs were used to describe the first targeted gene knockout in zebrafish, and morpholinos were used to show gene knockdowns in neurodegenerative diseases. For example, an appb knockdown study showed that the dramatic developmental defects in embryos and function of appb were needed for axonal outgrowth of motor neurons in zebrafish. In addition, the bace1 knockout zebrafish was generated by zinc finger nucleases. bace1 mutants in the PNS are hypomyelinated, whereas the CNS is not affected. Furthermore, study of the leucine-rich repeat kinase 2 (LRRK2) gene associated with PD is being studied in the zebrafish. Along with neuronal loss, the morpholino-mediated gene knockdown of lrrk2 zebrafish also caused developmental disturbances in the eyes, lens, and otic vesicles, including axis curvature defects, eye abnormalities, and edema. Since then, reverse genetic tools have seen unprecedented growth rates with the introduction of TALEN and CRISPR-Cas9 systems, including an apoea knockout for ADand a tardbp knockin and fus knockin for ALS. Further development of transgenic models using the recently developed CRISPR technique is set to unravel a greater potential for zebrafish in gene knockdown and knockin studies.
## Generation of a neurodegenerative model using amyloid-β42 (aβ42) in the adult zebrafish brain
The zebrafish bear extensive regenerative ability, and clinically important studies are aimed at understanding the mechanisms of zebrafish regeneration. Zebrafish are excellent tools because of their CNS regenerative capacity. Models of neurodegeneration in the adult zebrafish brain will be helpful to investigate the activation state of the neural stem/progenitor cells (NSPCs) and to identify the molecular differences between zebrafish and mammalian NSPCs to utilize them for regenerative therapies. Multifaceted inflammatory conditions in neurodegenerative diseases affect microglia, neurons, and NSPCs pleiotropically. Kizil et al. first developed a gene knockdown method based on cerebroventricular microinjection (CVMI) in vivo morpholino oligonucleotidein the adult zebrafish brain. CVMI injection in a skull incision is capable of uniformly targeting cells near the injection site, in this case the forebrain ventricular region containing neurogenic progenitor cells. The amyloid-β42 (Aβ42) induced neurotoxicity in adult zebrafish brain using CVMI of Aβ42 derivatives. One of the earliest findings in understanding the etiology of AD was the discovery of a 40-length peptide in AD brains now called Aβ, which constitute the primary component of AD-related amyloid deposits. Aβ is produced from the amyloid precursor protein (APP) with the continuous breakdown of βand γ-secretases. Importantly, the creation of Aβ through APP's proteolytic processing is heterogeneous, leading to variable Aβ lengths, especially at the peptide's carboxy terminus. 40 and 42 long residues are the two main forms of Aβ produced under normal APP processing conditions (Aβ40 and Aβ42, respectively). The shorter variety of Aβ40 is the majority of the Aβ produced in a normal individual. Approximately 5%-15% of the total Aβ pool is Aβ42, and it is possible to observe smaller amounts of other Aβs, both longer and shorter. Generally, the brain's Aβ pool has 5%-15% of Aβ42, which causes reminiscent phenotypes of amyloid pathophysiology: apoptosis, microglial activation, synaptic degeneration deficiencies, and learning. Aβ42 also results in NSPC proliferation and increased neurogenesis. This understanding can help to design regenerative therapy-based drug discovery for neurological disorders.
(Aβ40 and Aβ42, respectively). The shorter variety of Aβ40 is the majority of the Aβ produced in a normal individual. Approximately 5%-15% of the total Aβ pool is Aβ42, and it is possible to observe smaller amounts of other Aβs, both longer and shorter. Generally, the brain's Aβ pool has 5%-15% of Aβ42, which causes reminiscent phenotypes of amyloid pathophysiology: apoptosis, microglial activation, synaptic degeneration deficiencies, and learning. Aβ42 also results in NSPC proliferation and increased neurogenesis. This understanding can help to design regenerative therapy-based drug discovery for neurological disorders. (1) and targets, in this example, the forebrain that is rostral to the optic tectum. For injection, an incision is made into the skull over the optic tectum using a barbed-end canula. Through this slit, liquid is injected using a glass capillary. (1) and targets, in this example, the forebrain that is rostral to the optic tectum. For injection, an incision is made into the skull over the optic tectum using a barbed-end canula. Through this slit, liquid is injected using a glass capillary.
## Zebrafish cell culture-based neurodegenerative disease models
The developing zebrafish embryo is an excellent source for culturing cells, including neural cells. The technique to culture primary motor neuron (MN) in zebrafish has been developed for studying neurological disorders. The motor neuronal zebrafish cell culture was initiated at 24 hpf when the axonal development and outgrowth of MN started, allowing the development of MN axons in vivo in the context of the normal endogenous signs of the model organism, while also providing availability for an in vitro system. The zebrafish's primary culture techniques offer another approach to examine the neuronal population. There have been reports of primary neuron culture protocols ranging from blastula stage to 19 hpf, but these cultures are derived from MN axon pathfinding and neuromuscular development prior to normal course. Primary MN axons in zebrafish are present at 18 hpf out of the spinal cord, while the appearance of secondary MN axonal path findings range from 26 to 34 hpf. The brain explant culturesand primary cell culture of muscle fibersare possible to develop from the later development stages of zebrafish embryogenesis. The advantages of primary zebrafish cell culture provide a new foundation to develop potential therapies for neurological disorders.
A new protocoloutlines how the subcellular spreading and protein aggregation status of neurodegenerative disease-causing neurons from transgenic zebrafish embryos can be investigated. ALS and spinocerebellar ataxia type-3 (SCA3) can be studied from this protocol, as the disease-causing sarcoma-fused (FUS) and ataxin-3 proteins of the human variant gene can be expressed in the zebrafish cell culture. A combination of neuronal subtypes, including motor neurons, exhibited cultural differentiation as well as an outgrowth of neurites. The human mutant FUS mislocated from nuclei to cytosol, imitating observed in human ALS and the zebrafish FUS model. In contrast, zebrafish-grown neurons expressing human ataxin-3 with disease-associated improved polyQ repeats did not build up in nuclei as frequently reported in SCA3. Another simple and efficient protocol was recently proposed to obtain the primary cells of embryonic zebrafish. By exploiting the cell-type rich resource specific fluorescent zebrafish reporter lines, different types of differentiated cells were cultivated and monitored, proving that they continued their original morphology in culture for many days and demonstrated that before cultivation, particular types of cells could be enriched with flow cytometry. This group also successfully tested several fluorescent vital colors to facilitate subcellular imaging. This technique delivers a new tool to enhance our understanding of neurodegenerative disorders pathogenesis and help the development of mechanism-based drugs for neurological disorders.
expressed in the zebrafish cell culture. A combination of neuronal subtypes, including motor neurons, exhibited cultural differentiation as well as an outgrowth of neurites. The human mutant FUS mislocated from nuclei to cytosol, imitating observed in human ALS and the zebrafish FUS model. In contrast, zebrafish-grown neurons expressing human ataxin-3 with disease-associated improved polyQ repeats did not build up in nuclei as frequently reported in SCA3. Another simple and efficient protocol was recently proposed to obtain the primary cells of embryonic zebrafish. By exploiting the cell-type rich resource specific fluorescent zebrafish reporter lines, different types of differentiated cells were cultivated and monitored, proving that they continued their original morphology in culture for many days and demonstrated that before cultivation, particular types of cells could be enriched with flow cytometry. This group also successfully tested several fluorescent vital colors to facilitate subcellular imaging. This technique delivers a new tool to enhance our understanding of neurodegenerative disorders pathogenesis and help the development of mechanism-based drugs for neurological disorders.
# Conclusions
# Conclusions
In summary, several observational studies have shown a connection between zebrafish and human neurological disorders. Zebrafish are proving to be an ideal model for screening pharmaceutical agents prior to testing in rodents. The long-term aims of this work are to clarify the mechanisms of neurodegeneration and develop new neuroprotective compounds for the treatment of neurodegenerative diseases. In adult zebrafish, the approach of neurodegeneration using Aβ peptides can also help to design regenerative therapies in the neurodegenerative situation. The described culture of neuronal cells adds a new tool to investigate neurodegenerative diseases regarding molecular and cellular mechanisms, high-quality live cell imaging, and the discovery of new therapeutic drugs for neurological disorders. The primary embryo of zebrafish and larvae culture has the potential to provide tremendous knowledge regarding various mechanisms and treatments for human disease. Zebrafish-based assays are capable of promoting the bioassay-guided fractionation of great numbers of bioactive extracts identified in these in vivo screens, thus allowing the isolation of different novel, bioactive natural products-most of which are likely to be desirable lead compounds for the development of new, potent drugs. These initial studies support zebrafish in helping to resolve a crucial bottleneck in the discovery of NPs by allowing rapid in vivo, microgram-scale, bioassay-guided fractionation analysis, and diverse natural extract dereplication studies.
Author Contributions: The writing, review, and editing of manuscript were completed by A.P., R.K.R., and J.L.F.
## Conflicts of interest:
The authors declare no conflict of interest.
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Lytic Phages against ST11 K47 Carbapenem-Resistant Klebsiella pneumoniae and the Corresponding Phage Resistance Mechanisms
We isolated and characterized a novel phage from hospital sewage, P13, able to lyse ST11 K47 carbapenem-resistant Klebsiella pneumoniae (CRKP), a major CRKP lineage. P13 formed a large lytic plaque (3.0 to 6.0 mm in diameter) in double-layer LB agar after overnight coculture with its host bacterial strain. A translucent halo formed when the culture was prolonged to 48 h. P13 showed a narrow host range only lysing ST11 K47 CRKP with a burst size of around 167 PFU/cell and exhibited broad pH and thermal stability. Genome sequencing showed that P13 contains no virulence, lysogenic or antimicrobial resistance genes, making this lytic phage a potential agent for phage therapy. Transmission electron microscopy showed that P13 exhibited typical morphology of the family Podoviridae with an isometric head and a short noncontracted tail. Genomic analysis showed that P13 belongs to a novel species of the genus Przondovirus, subfamily Studiervirinae, family Autographiviridae. P13-resistant mutants of bacteria emerged after 4 h exposure to the phage. Interruptions of wbaP (encoding capsule polysaccharide synthesis) by insertion sequence IS903B mediated P13 resistance. The rapid emergence of resistant mutants represents a disadvantage for P13 as a therapeutic phage and highlights the need for recovery of a range of phages binding to different surface receptors of host bacteria to further extend their utility as a potential tool against CRKP. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major challenge for infection control and clinical management. Alternative therapies to antimicrobial agents are urgently needed and bacteriophages (phages) are an attractive option. However, more novel lytic phages and more studies to reveal phage-resistant mechanisms are needed to overcome phage resistance. In this study, we isolated and characterized a novel species of lytic phage active against CRKP. We found this phage exhibited delayed formation of halo, which is atypical compared to other characterized similar phages, and we provide an explanation for this phenotype based on genomic analysis. We also identified mechanisms mediating resistance to the phage.
China [bib_ref] Carbapenemresistant isolates of the Klebsiella pneumoniae complex in western China: the common..., Liu [/bib_ref] and can be further assigned to several capsule types, among which K47 and K64 are the two major capsule types of ST11 CRKP [bib_ref] Novel subclone of carbapenem-resistant Klebsiella pneumoniae sequence type 11 with enhanced virulence..., Zhou [/bib_ref].
Bacteriophages, called phages here, are viruses able to attack bacteria and are ubiquitous. Phages have been used to treat bacterial infections for many years in certain countries, such as Georgia and Poland, but have not been adopted in most countries [bib_ref] Phage therapy: a renewed approach to combat antibiotic-resistant bacteria, Kortright [/bib_ref]. This is largely due to the wide availability of antimicrobial agents and concerns around phages such as the rapid development of resistance by bacterial strains after exposure [bib_ref] Phage therapy: a renewed approach to combat antibiotic-resistant bacteria, Kortright [/bib_ref]. Due to the global problem of antimicrobial resistance, phages have regained the interest of the medical community as an alternative treatment. Novel phages are required as the prerequisite for cocktails to overcome phage resistance during therapy. Only four phages have been reported in the literature as able to lyse ST11 K47 CRKP, SH-KP152226 (1), vB_KpnP_IME205 [bib_ref] Identification of two depolymerases from phage IME205 and their antivirulent functions on..., Liu [/bib_ref] , SRD2021 [bib_ref] Bacteriophage SRD2021 recognizing capsular polysaccharide shows therapeutic potential in serotype K47 Klebsiella..., Hao [/bib_ref] , and P-KP2 [bib_ref] Combination therapy of phage vB_KpnM_P-KP2 and gentamicin combats acute pneumonia caused by..., Wang [/bib_ref]. We isolated and characterized a novel phage able to lyse ST11 K47 CRKP, expanding the repertoire of phages against CRKP. Here, we reported its morphological, physiological, genomic characteristics and the mechanisms that bacteria developed to counter phage attacks.
# Results
Recovery of a phage able to rapidly lyse ST11 K47 CRKP. We isolated a phage from sewage collected at West China Hospital, named P13 (also called 066013), able to lyse 020030, an ST11 K47 CRKP [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref]. P13 formed a clear plaque with a 3.0 to 6.0 mm diameter in 0.7% LB agar plate after overnight culture at 37°C but no obvious halo surrounding the phage plaque was present [fig_ref] FIG 1: The morphology of phage P13 [/fig_ref]. After prolonged culture for 48 h, a halo of 7 to 10 mm in diameter was observed [fig_ref] FIG 1: The morphology of phage P13 [/fig_ref] , suggesting that P13 encodes a polysaccharide depolymerase [bib_ref] Bacteriophage-encoded depolymerases: their diversity and biotechnological applications, Pires [/bib_ref]. Under the TEM, P13 displayed typical morphology of the family Podoviridae, with an approximately 50 nm isometric head and an approximately 10 nm tail [fig_ref] FIG 1: The morphology of phage P13 [/fig_ref].
The optimal multiplicity of infection (MOI) of P13 was 0.01 and at such an optimal MOI, progeny phages were produced at 2.30 6 0.23 Â 10 9 PFU/mL [fig_ref] TABLE 2: Number of progeny phages under different MOIs [/fig_ref]. A high percentage (95%) of phage P13 adsorbed cells of strain 020030 within 6 min [fig_ref] FIG 2: Growth, stability, and lytic activity of P13 [/fig_ref]. As shown in [fig_ref] FIG 2: Growth, stability, and lytic activity of P13 [/fig_ref] , the latency period for P13 was about 20 min and the estimated burst size was about 167 PFU/cell. The phage titer was stable at pH 4 to 10 but decreased by 3 and 1 log in phage titer at pH of 3 and 11, respectively [fig_ref] FIG 2: Growth, stability, and lytic activity of P13 [/fig_ref]. The titer of P13 was stable between 4 and 50°C but decreased by 1 log at 60°C and dropped to 0 after exposure to 70°C for 1 h [fig_ref] FIG 2: Growth, stability, and lytic activity of P13 [/fig_ref]. P13 was able to lyse strain 020030 within 1 h and the lysis persisted for around 4 h. After 5 h, the optical density at 600 nm (OD 600 ) value of the bacterial solution rose from 0 to 0.15 [fig_ref] FIG 2: Growth, stability, and lytic activity of P13 [/fig_ref]. The bacterial solution was streaked on an LB plate and cultured at 37°C Phages Against CRKP mSphere overnight. The colonies were P13-resistant mutants, verified using the spot assay. We found that it was only when the number of bacteria was less than 10 4 CFU (regardless of the MOI) that no resistant mutants developed. This indicated that the incidence of resistant mutants was about 10 24 . The host specificity of phage P13. An additional 18 CRKP clinical isolates belonging to six STs (ST1, ST11, ST15, ST37, ST45, and ST54) and 9 capsule types (K5, K14, K39, K38, K45, K47, K62, K64, and K106) were used to test the host range of phage P13. Our data showed that P13 has a narrow host range with the ability to only lyse ST11 K47 CRKP [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref]. Efficacy of plating (EOP) values in P13-susceptible strains tested varied from 0.005 to 1 [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref] , among which one strain (020143) exhibited a ,0.2 EOP value and was considered "low efficiency" [bib_ref] Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli, Viazis [/bib_ref].
The taxonomy of phage P13. The complete sequence of phage P13 was obtained. A total of 49 protein-coding sequences (CDS) were predicted and annotated. Gene analysis showed that P13 contained 40,967 bp with a GC content of 53.65% [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref] and did not Phages Against CRKP mSphere Phages Against CRKP mSphere contain any virulence genes, antimicrobial resistance genes, tRNA, lysogen genes, or integrase genes. The 49 CDSs of P13 encode proteins of various functions, including structure, DNA packing, lysis, metabolism, DNA replication, and transcription and [fig_ref] TABLE 2: Number of progeny phages under different MOIs [/fig_ref]. Although genes encoding the holin-endolysin system are usually in a close arrangement in the phage genome [bib_ref] Phage lysis: multiple genes for multiple barriers, Cahill [/bib_ref] , genes encoding the holin and the endolysin were spatially distant from each other in the genome of P13 . BLASTp analysis revealed P13 and SH-KP152226 and vB_KpnP_IME205 shared an identical holin sequence. However, the endolysin of P13 differed from that of SH-KP152226 in a single amino acid (Ser117Ala) but only had an 88.74% amino acid identity (100% coverage) with that of vB_KpnP_IME205. BLASTn analysis revealed that P13 exhibited the highest known similarity (96% coverage, 94.66% nucleotide identity, and 90.87% overall DNA sequence homology) with phage SH-KP152226 (accession no. MK903728.1) of the genus Przondovirus within the subfamily Studiervirinae of the family Autographiviridae (a former member of the family Podoviridae). Phylogenetic analysis of the DNA polymerase-encoding gene sequence revealed that P13 belonged to the genus Przondovirus . The main species demarcation criterion for bacterial and archaeal viruses is set at overall DNA sequence homology of 95% according to the ICTV [bib_ref] update from the ICTV Bacterial and Archaeal Viruses Subcommittee, Adriaenssens [/bib_ref]. P13 was, therefore, designated a novel species of the genus Przondovirus [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref].
The emergence of phage-resistant mutants and the mechanism of resistance. After overnight incubation of a mixture of high-titer P13 and strain 020030 at 37°C, individual colonies that grew on the plates were considered to be P13-resistant mutants. We performed genome sequencing on three randomly selected phage-resistant mutants (020030X1B1, 020030X1B2, and 020030X1B3). We did not identify any SNPs between P13-resistant Gene map of phage P13. Different colored arrows represent predicted CDSs coding different functions: red, lysis; gray, hypothetical protein (hp); orange, DNA replication, transcription, and metabolism function; green, phage structure; purple, DNA packaging. The genome map was generated using SnapGene (https://www.snapgene.com/). Phages Against CRKP mSphere mutants and the parental strain 020030 . We found that the galactose phosphotransferase-encoding wbaP gene, which is involved in the synthesis of capsule polysaccharide [bib_ref] Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types..., Pan [/bib_ref] , was interrupted by insertion sequence IS903B of the IS5 family at nucleotide position 378, 382, and 378 (numbered from the ATG start codon) in these three mutants, respectively [fig_ref] FIG 6: Genetic composition of the cps gene cluster in ST11 KL47 CRKP 020030... [/fig_ref]. By cloning the wbaP gene from the parental strain 020030 to P13-resistant mutants, the recombined transformants 020030X1B1::pwbaP and 020030X1B2::pwbaP restored susceptibility to P13 , verifying the interruption of wbaP as the P13-resistance mechanism. The findings are consistent with phage resistance due to a single nucleotide mutation in wbaP in a recent report [bib_ref] Phage resistance is associated with decreased virulence in KPC-producing Klebsiella pneumoniae of..., Henrici De Angelis [/bib_ref].
# Discussion
In this study, we recovered a novel phage, P13, of the Przondovirus genus able to rapidly lyse ST11 KL47 CRKP. P13 has a rapid adsorption time, is highly stable at a wide range of pH and temperatures, and contains no virulence, lysogenic or antimicrobial resistance genes. These characteristics make this lytic phage a promising agent for phage therapy as proposed previously (20). Phylogenetic analysis of the DNA polymerase of phages belonging to the genus Przondovirus genus. The phylogeny tree was inferred using Phylogeny.fr (http://www.phylogeny.fr/index.cgi) with "One Click mode" using MUSCLE for multiple alignments, PhyML for phylogeny, and Gblocks for eliminating poorly aligned positions and divergent regions [bib_ref] Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative, Anisimova [/bib_ref]. Accession numbers are indicated in front of phage names. P13 (066013) is marked with red color.
## Phages against crkp msphere
Only four phages have been previously described that can lyse ST11 K47 CRKP, SH-KP152226 (1), vB_KpnP_IME205 (11), SRD2021 (12), and P-KP2 [bib_ref] Combination therapy of phage vB_KpnM_P-KP2 and gentamicin combats acute pneumonia caused by..., Wang [/bib_ref]. Among these four phages, phages SRD2021 and P-KP2 belong to the family Siphoviridae and Myoviridae, respectively. In contrast, phages SH-KP152226 and vB_KpnP_IME205, like P13 in this study, morphologically belong to the family Podoviridae. Previous studies have demonstrated that the phage host range is determined by the receptor-binding proteins (RBPs) forming baseplate-attached tailspike proteins (TSPs) or tail fibers [bib_ref] Salmonella phage S16 tail fiber adhesin features a rare polyglycine rich domain..., Dunne [/bib_ref] [bib_ref] Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis, Yehl [/bib_ref]. The tail fiber protein of P13 also has a high similarity to that of SH-KP152226 with 98.36% amino acid identity and 100% coverage. This high identity is consistent with the ability of both phages to lyse ST11 K47 CRKP.
An intriguing phenomenon is the presence of a large clear center of the P13 plaque that masks the formation of a halo. The constantly increasing halo surrounding the phage plaque indicated that the phage-encoded a depolymerase [bib_ref] Bacteriophage-encoded depolymerases: their diversity and biotechnological applications, Pires [/bib_ref] , which are typically tail spike or fiber proteins in phages of families Podoviridae and Autographiviridae [bib_ref] Bacteriophage-encoded depolymerases: their diversity and biotechnological applications, Pires [/bib_ref] [bib_ref] Diversity and function of phage encoded depolymerases, Knecht [/bib_ref]. P13 encodes a tail fiber protein [fig_ref] TABLE 2: Number of progeny phages under different MOIs [/fig_ref] , which has 98.11% identity and 100% coverage with the depolymerase (a tail fiber protein, also called ORF42, accession no. ALT58497.1) of Klebsiella phage vB_KpnP_IME205. The tail fiber protein of P13 is therefore likely a phage depolymerase. Both SH-KP152226 and Multiple genome alignments among P13 (066013) and the five most closely related phages. The five phages belonged to the branch of P13 in the phylogenetic tree . The alignments were generated using Mauve [bib_ref] Mauve: multiple alignment of conserved genomic sequence with rearrangements, Darling [/bib_ref]. The height of bars reflects the similarity of regions. Fragments not aligned nor specific to a particular genome are depicted by white areas. Regions of homologous DNA shared by two or more genomes are defined as local colinear blocks, which are shown by boxes in the same color. Phages Against CRKP mSphere vB_KpnP_IME205 formed clear plaques with halos (about 7 mm in diameter) and a small clear center (about 2 to 3 mm in diameter) within the plaques. However, unlike those two phages, P13 formed a plaque (3 to 7 mm in diameter) without obvious halo in a double-layer LB plate after overnight culture. By extending culture time a halo gradually appears. It has been reported that the phage adsorption rate, lysis timing, burst size, and phage morphology all affect phage plaque properties [bib_ref] Effects of bacteriophage traits on plaque formation, Gallet [/bib_ref]. Because information on the adsorption rate, lysis timing, and burst size of SH-KP152226 and vB_KpnP_IME205 have not been reported, we were unable to untangle the reasons for this phenotypic difference in plaque formation. Nonetheless, burst size and lysis timing are governed by the holin-endolysin system [bib_ref] Phage lysis: multiple genes for multiple barriers, Cahill [/bib_ref]. and the genes encoding the holin and the endolysin are atypically distant from each other in the genome of P13. This distinct genetic architecture has been reported in other phages, such as SH-KP152226, and warrants further exploration. We compared the amino acid sequence of holin and endolysin between P13 and SH-KP152226 and vB_KpnP_IME205. The endolysin of P13 differed from that of SH-KP152226 in a single amino acid (Ser117Ala) but only had an 88.74% amino acid identity (100% coverage) with that of vB_KpnP_IME205. Therefore, the amino acid variations may also result in different phage plaque morphologies. The rapid development of resistance to this phage in target strains is a clear disadvantage for therapeutic utility. Gene wbaP encodes a membrane protein functioning as a glycosyltransferase responsible for the transfer of galactose-1-phosphate to the undecaprenyl phosphate acceptor moiety and influences the initiation of capsule polysaccharide (CPS) synthesis in the Enterobacteriaceae (25). The disruption of wbaP results in the absence of CPS and therefore abolishes phage adsorption, rendering phage resistance [bib_ref] The mutation in wbaP cps gene cluster selected by phage-borne depolymerase abolishes..., Kaszowska [/bib_ref]. Therefore, the clinical utility would require a combination of phages binding to different receptors to form a cocktail to achieve an optimal bactericidal effect. This highlights the need to recover significant numbers of new phages to create a potentially clinically useful panel of phages.
Conclusion We recovered a novel phage of the Przondovirus genus able to rapidly lyse ST11 KL47 CRKP, a major high-risk lineage of CRKP. The strict host specificity suggests that this phage is unlikely to disturb human commensal microflora like antimicrobial agents but also represents a disadvantage for a therapeutic phage. Furthermore, bacteria can escape the attack of phages through sophisticated approaches. There is still a long and complicated way to go in phage therapy.
# Materials and methods
Bacteria for phage isolation and host range analysis. We used a CRKP clinical strain, 020030, which was recovered using chromogenic ager plates (CHROMagar Orientation; CHROMagar; Paris, France) containing 2 mg/mL meropenem and 64 mg/mL linezolid from a tissue sample of a patient in West China Hospital FIG 7 Spot test assays. Phage P13 at 10 2 to 10 10 PFU/mL formed clearing zones on semisolid LB plates containing 020030 (the parental strain) or 020030X1B1::pwbaP and 020030X1B2:pwbaP (two P13 mutants with the complete wbaP-containing plasmid pwabP) but not on that containing 020030X1B1 (a P13-resistant mutant).
Phages Against CRKP mSphere [bib_ref] Carbapenemresistant isolates of the Klebsiella pneumoniae complex in western China: the common..., Liu [/bib_ref] , as the host strain for isolating phages. The genome sequence of strain 020030 has been reported previously [bib_ref] Carbapenemresistant isolates of the Klebsiella pneumoniae complex in western China: the common..., Liu [/bib_ref]. In this study, we also performed long-read sequencing on 020030 using a MinION sequencer (Nanopore; Oxford, UK). We then obtained the complete genome sequence of this strain by a hybrid assembly of both short and long reads using Unicycler [bib_ref] Unicycler: resolving bacterial genome assemblies from short and long sequencing reads, Wick [/bib_ref]. This high-quality genome assembly has been deposited in GenBank under accession no. CP028788 to CP028793. Strain 020030 belongs to ST11 and KL47 [bib_ref] Carbapenemresistant isolates of the Klebsiella pneumoniae complex in western China: the common..., Liu [/bib_ref]. We used additional other 18 CRKP clinical strains belonging to six STs (ST1, ST11, ST15, ST37, ST45, and ST54) and 9 capsular types (K5, K14, K39, K38, K45, K47, K62, K64, and K106) for phage host range analysis [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref]. Phage isolation. We collected sewage samples from the untreated influx of the wastewater processing station at West China Hospital in 2019 to isolate phages. Bacterial culture (1 mL, strain 020030) at logarithmic phase was mixed with 17 mL of filtered sewage and 2 mL 10Â LB broth (Hopebio; Qingdao, China), and then incubated at 37°C for 4 h. The coculture was centrifuged at 12,000 Â g at 4°C for 2 min and the supernatant was then filtered through a 0.22 mm membrane (Labgic Technology; Beijing, China). Tris-HCl-MgSO 4 (TM buffer) was used to dilute the supernatant. We obtained individual plaques by 10-fold dilutions of supernatant against strain 020030 using the double-layer agar method [bib_ref] Isolation and characterization of specific phages to prepare a cocktail preventing Vibrio..., Chen [/bib_ref]. We further purified isolated phage plaques at least 3 times until the formation of uniform plaques was obtained. We then obtained a lytic phage, assigned P13, able to lyse strain 020030.
Phage host range determination. As phage host range is an essential factor for phage therapy, we determined the host range of phage P13 against an additional 18 CRKP strains [fig_ref] TABLE 1: CRKP strains used in this study and the host range of phage... [/fig_ref] using the spot assay as described previously [bib_ref] Phage host range and efficiency of plating, Kutter [/bib_ref]. To avoid false-positives, we used a phage master stock at a titer of approximately 10 10 PFU/mL to perform this experiment. We recorded the strain as susceptible to P13 if a clear zone was present. We also determined the efficiency of plating (EOP) of phage P13 on susceptible strains by counting individual plaques obtained from diluted phage preparations (10 1 to 10 10 PFU/mL). EOP value is the titer of the phage on each susceptible bacterial lawn compared to the titer observed on the host bacterial strain and the category of efficiency was defined as described previously [bib_ref] Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli, Viazis [/bib_ref].
Multiplicity of infection (MOI) assay. To determine the optimal MOI in which phage lyse bacteria produce the largest amount of progeny phages, we grew the host strain 020030 to logarithmic phase, which was adjusted to 0.5 McFarland turbidity (around 10 8 CFU, CFU/mL). We mixed 1 mL of 10 6 CFU/ mL host strain with 10 mL of 10-fold diluted phages (about 10 10 , 10 9 , 10 8 , 10 7 , and 10 6 PFU/mL) as described previously [bib_ref] Two new lytic bacteriophages of the Myoviridae family against carbapenem-resistant Acinetobacter baumannii, Zhou [/bib_ref]. We counted the number of progeny phages after incubating for 3 h using the double-layer agar method (28) as described above.
Phage adsorption and one-step growth. We tested the ability of the phage to adsorb the host strain by mixing 10 mL host strain at a concentration of 10 8 CFU/mL with 100 mL of 10 8 PFU/mL phage at an MOI of 0.01. At 0, 3, 6, 9, and 12 min, samples were retrieved and filtered by 0.22 mM filters immediately to count free phage titers. We also performed a one-step growth experiment to estimate the latent period and burst size of phage P13 according to a protocol described previously with minor modifications [bib_ref] Basic phage mathematics, Abedon [/bib_ref]. Briefly, we mixed 100 mL of phage (10 8 PFU/mL) with 9.9 mL of host strain (10 8 CFU/mL) and then allowed it to adsorb for 6 min at 37°C. We retrieved 0.1 mL of the mixture to 9.9 mL prewarmed LB broth (tube A), retrieved a 1 mL aliquot from tube A to 9 mL LB broth in tube B, and retrieved a 1 mL aliquot from tube B to 9 mL LB broth (tube C), which was incubated at 37°C. At various time points, a 0.1 mL aliquot was retrieved from the corresponding tube (A, B, or C) to determine phage titers. An additional 1 mL aliquot was retrieved from tube A at the beginning for calculating the burst size. Half of the aliquot (500 mL) was filtered through a 0.22 mM membrane to remove adsorbed phages and the remaining half was not filtered. Phage-infected cells were calculated by subtracting the number of free phages in the filtered fraction from that of the unfiltered fraction. The burst size could be calculated by dividing the stationary-phase progeny counts by the number of infected cells. We performed this experiment in triplicate.
pH and thermal stability. We tested the stability of phages in different pH and temperatures using TM buffers with different pH values (2 to 13). We mixed a 100 mL aliquot of 10 9 PFU/mL phages with 900 mL TM buffer at each pH value. The phage titer was determined using the double-layer agar methodafter incubation at 25°C for 1 h. We tested the thermotolerant ability of phages at 4, 25, 37, 50, 60, and 70°C for 1 h in a water bath. We determined the phage titer at each temperature as described above. The experiments were performed in triplicate.
Transmission electron microscopy. We observed the morphology and the size of phage P13 using transmission electron microscopy (TEM) imaging. For this, we prepared phage particles as described previously. We dropped the phage particles to copper grids for 10 min and then added a drop of uranyl acetate 1.0% (wt/vol) for negative staining. We used a Hitachi Transmission Electron Microscope (Hitachi High-Tech; Tokyo, Japan) at an accelerating voltage of 80 kV for TEM.
In vitro bacteriolytic characteristics of the phages. We tested the in vitro inhibition of bacterial growth by phage by determining the bacteriolytic curve. We added 200 mL 10 8 PFU/mL of phage suspension to 20 mL of strain 020030 culture growing to the prelogarithmic phase (OD 600 = 0.20, about 10 8 CFU/mL) at an MOI of 0.01. We measured the optimal optical density (OD) values of the bacterial solution at 1 h intervals up to 8 h using a spectrophotometer (Youke; Shanghai, China). We used bacterial culture without phage and LB broth without the host strain as the positive and negative-control, respectively. We performed the assays in triplicate.
Recovery of phage-resistant mutants and its relation to MOI. We examined whether P13-resistant mutants could emerge using the double-layer agar method. We mixed 10 mL high-titer phage stocks at 10 11 PFU/mL and 100 mL culture of the host strain at logarithmic phase with 5 mL of 0.7% molten top LB agar. The mixture was gently vortexed and then poured onto 1.5% LB agar plates. After overnight incubation at 37°C Individual colonies growing on plates were considered mutants resistant to P13. We randomly picked three individual colonies (020030X1B1, 020030X1B2, and 020030X1B3) and confirmed their resistance to P13 using the spot assay [bib_ref] Isolation of four lytic phages infecting Klebsiella pneumoniae K22 clinical isolates from..., Domingo-Calap [/bib_ref]. We incubated 100 mL host strain at 10 6 to 10 2 CFU/mL with 100 mL phage P13 at 10 9 to 10 3 PFU/mL in different MOI for 18 h in a 96-well microplate to explore in which MOI P13-resistant mutants would emerge.
Whole-genome sequencing and analysis. We performed whole-genome sequencing for phage P13 to determine its taxonomy and for the three P13-resistant mutants of 020030 to investigate their resistance mechanisms. We prepared genomic DNA of phage P13 from phage particles using a Phage DNA isolation kit (Norgen Biotek; Thorold, Canada) and DNA of the three bacterial mutants from cultures at mid-logarithmic phase using a DNeasy blood and tissue kit (Qiagen; Hilden, Germany). The prepared DNA was ultrasonically sheared into 350 bp before the construction of 150-bp paired-end libraries. Genome sequencing was performed using HiSeq X10 (Illumina; San Diego, CA, USA). We trimmed the generated raw reads of phage P13 using Trimmomatic v0.38 [bib_ref] Trimmomatic: a flexible trimmer for Illumina sequence data, Bolger [/bib_ref] and then assembled them into draft genomes using SPAdes v3.13.0 [bib_ref] SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing, Bankevich [/bib_ref].
Prokka [bib_ref] Prokka: rapid prokaryotic genome annotation, Seemann [/bib_ref] and Rapid Annotations Subsystems Technology (RAST, http://rast.nmpdr.org/) were used for genome annotation. BLASTP on CDS was performed against AMRFinderPlus v3.9 (37) for antimicrobial resistance genes, and Kleborate v2.0.0 (https://github.com/katholt/Kleborate) was used for capsule typing and virulence factor detection. We constructed a phage gene map using SnapGene (https://www.snapgene.com/) and used PHACTS [bib_ref] PHACTS, a computational approach to classifying the lifestyle of phages, Mcnair [/bib_ref] to determine the probable phage lifestyle. We further determined the taxonomic position of P13 by aligning the amino acid sequence of its DNA polymerase with those of other phages belonging to the genus Przondovirus using Phylogeny.fr (http://www.phylogeny.fr/index.cgi) with "One Click mode" using MUSCLE for multiple alignments, PhyML for phylogeny, and Gblocks for eliminating poorly aligned positions and divergent regions [bib_ref] Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative, Anisimova [/bib_ref]. Overall DNA sequence homology was defined as coverage multiplied by identity according to the International Committee on Taxonomy of Viruses (ICTV) [bib_ref] update from the ICTV Bacterial and Archaeal Viruses Subcommittee, Adriaenssens [/bib_ref]. We then performed multiple genome alignments of P13 and the five phages belonging to the branch of P13 in the phylogenetic tree using Mauve [bib_ref] Mauve: multiple alignment of conserved genomic sequence with rearrangements, Darling [/bib_ref]. We used Snippy (https://github.com/tseemann/snippy) to identify single nucleotide polymorphisms (SNPs) between the genome sequence of 020030 and those of its phage P13-resistant mutants. We used Roary v3.11.2 (41) to identify the deletion and acquisition of genomic fragments in the phage-resistant mutants.
Cloning experiments to verify the phage-resistant mechanism. Cloning experiments were performed to verify whether the interruptions in the CPS synthesis gene wbaP mediate resistance to P13. The complete wbaP sequence of strain 020030 was obtained by PCR using self-designed primers wbaP-F-SacI (AACGAGCTCTTGAATGCTTACCCCA) and wbaP-R-HindIII (AACAAGCTTTGATAGATTATCCCGACTG, the restriction sites are underlined). The amplicons were digested using the corresponding restriction endonucleases and were then ligated with the cloning vector pBC SK (Stratagene; La Jolla, CA, USA) pretreated with the same endonucleases. The recombinant plasmid, assigned pwbaP here, were chemically transformedinto Escherichia coli DH5a to facilitate cloning into the P13-resistant mutants. The transformants were screened on LB agar plates containing 50 mg/mL chloramphenicol after overnight incubation at 37°C and the presence of the cloned gene was confirmed by PCR with universal primers M13 forward/reverse and subsequent Sanger sequencing. Then, pwbaP was prepared from a DH5a transformant and was chemically transformed (28) into P13-resistant mutants 020030X1B1 and 020030X1B2, respectively. 020030X1B1 and 020030X1B2 could not grow on plates containing 50 mg/mL chloramphenicol and the corresponding transformants 020030X1B1:: pwbaP and 020030X1B2::pwbaP were selected and confirmed as described above for the DH5a transformant. 020030X1B1::pwbaP and 020030X1B2::pwbaP were tested for the susceptibility to P13 using the spot assay as described above with strains 020030 and 020030X1B1 as control.
Accession numbers. The complete sequence of phage P13 and the draft genome sequences of the phage-resistant three mutants have been deposited in GenBank under accession numbers MW042787, JAHKSU000000000, JAHKST000000000, and JAHKSS000000000, respectively.
# Supplemental material
Supplemental material is available online only.
[fig] FIG 1: The morphology of phage P13. (A) P13 forms a large plaque without halo after overnight culture. (B) A halo of 2 mm wide appeared around the P13 plaque after being cultured for 48 h. (C) Negatively stained TEM images of P13. Scale bar = 100 nm. [/fig]
[fig] FIG 2: Growth, stability, and lytic activity of P13. (A) The adsorption curve of P13. (B) The one-step growth curve of phage P13. The initial number of phage-infected cells was 1.00 6 0.12 Â 10 5 CFU/mL. The stationary-phase phage progeny number was 1.67 6 0.416 Â 10 7 PFU/mL. (C) Thermal stability of P13. (D) pH stability of P13. (E) The bacteriolytic activity of P13 at MOI of 0.01 in vitro. Values represent the mean 6 standard deviations (n = 3). [/fig]
[fig] FIG 6: Genetic composition of the cps gene cluster in ST11 KL47 CRKP 020030 and the disrupted wbaP in P13-resistant mutants. ORFs are indicated as arrows. ORFs in green represent core genes of the cps gene cluster. HP, hypothetical protein; GT, glycosyltransferase. IS refers to IS903B and D represents interruption. [/fig]
[table] TABLE 1: CRKP strains used in this study and the host range of phage P13 [/table]
[table] TABLE 2: Number of progeny phages under different MOIs [/table]
[table] TABLE S1 ,: DOCX file, 0.02 MB. TABLE S2, DOCX file, 0.02 MB. [/table]
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Experiences of health management among people at high risk of stroke in China: A qualitative study
[bib_ref] Population health management: Saving lives and saving money?, Jha [/bib_ref] [bib_ref] Clinical and occupational health management of healthcare workers living with chronic hepatitis..., Dolman [/bib_ref] [bib_ref] Using Mobile apps for health management: A new health care mode in..., Lv [/bib_ref] [bib_ref] Are education, exercise and diet interventions a cost-effective treatment to manage hip..., Mazzei [/bib_ref]
## | backg rou n d
Stroke is a major public health issue. It has become the second leading cause of death and the primary cause of long-term disability globally [bib_ref] Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics..., Virani [/bib_ref]. An estimated increase of 1.5 million stroke survivors is expected in Europe by the year 2025 [bib_ref] Epidemiology of stroke in Europe and trends for the 21st century, Béjot [/bib_ref].
Stroke also continues to present a significant public health challenge, incurring high economic costs and burdens in the global world during the last 30 years .
Furthermore, the situation in low-and middle-income countries has markedly worsened [bib_ref] The global burden of stroke and need for a continuum of care, Norrving [/bib_ref]. Stroke is the leading cause of death in China, and it is estimated that there will be more than 30 million stroke survivors by 2030, including 75% with varying degrees of disability that will present a tremendous burden to themselves, their families and society . A systematic review [bib_ref] Natural history, predictors and outcomes of depression after stroke: Systematic review and..., Ayerbe [/bib_ref] of 50 studies reported that the prevalence of depression among people after stroke was 29%, and the cumulative incidence within 5 years of stroke was 39%-52%.
This negatively affects the survivors' functional outcomes, their response to rehabilitation and their quality of life. Awareness of prevention of risk factors and adherence to health behaviour management among people at high risk of stroke in China was lower than people in developed countries. In addition, the incidence of anxiety, depression and stroke was higher than that of developed countries . This situation is especially worse in rural areas [bib_ref] Stroke in China: Advances and challenges in epidemiology, prevention, and management, Wu [/bib_ref]. People at high risk of stroke and their family members were exhausted by long-term care and financial burden.
## According to the china national stroke screening and prevention
Project (CNSSPP), a person reporting three or more of eight major risk factors, or with a history of transient ischaemic attack (TIA) or with a history of stroke, is at particularly high risk of stroke [bib_ref] Prevalence and risk factors for dyslipidemia among adults in rural and urban..., Opoku [/bib_ref] [bib_ref] Carotid atherosclerosis detected by ultrasonography: A National Cross-Sectional Study, Wang [/bib_ref]. These eight major risk factors include:
(a) history of hypertension (>140/90 mmHg) or taking antihypertensive drugs; (b) atrial fibrillation and valvular disease; (c) smoking; (d) dyslipidemia; e. diabetes mellitus; (f) low physical activity, i.e. fewer than three times weekly, of 30-minute exercise sessions, for at least 1 year; (g) overweight or obesity (BMI >26 kg/m 2 ); and h. family history of stroke. Most of these eight major risk factors are modifiable and are closely related to unhealthy lifestyle behaviour. Therefore, if unhealthy lifestyle behaviour is improved in populations at high risk of stroke through planned health management, the incidence and recurrence rate of stroke may be reduced [bib_ref] A multicomponent behavioral intervention to reduce stroke risk factor behaviors: The stroke..., Brown [/bib_ref].
In the Internet age, mhealth and telecare are increasingly a new driving force supporting basic medical and health services to cover people at scale [bib_ref] Maugeri Centre for Telehealth and Telecare: A real-life integrated experience in chronic..., Scalvini [/bib_ref]. These information tools can overcome the limitations of distance and time between health care providers and recipients and shows great potential for improving health management and health care services and promoting the sharing of advantageous medical resources [bib_ref] Applying digital information delivery to convert habits of antibiotic use in primary..., Poss-Doering [/bib_ref].
People at high risk of stroke can easily obtain online health-related information about health management through various information tools or social media platform. However, it is increasingly difficult to distinguish reliable health information from misleading content in the information explosion era . In addition, Kontos [bib_ref] Predictors of eHealth usage: Insights on the digital divide from the health..., Kontos [/bib_ref] also found that compared to those aged 65 and older, those aged 18-34 were nearly 3.5 times (OR 3.51, 95% CI 1.66-7.44) more likely to use the Internet to search for health information. As age increases, the odds of watching health-related information on social network platform decreased, but they are exactly the people who need this information most .
In conclusion, research shows that people at high risk of stroke are heterogeneous in their internet-based health management experiences, with most suffering from many challenges to their survival across different situations. Therefore, this study aimed to provide an in-depth description of experiences in health management to provide a foundation for targeted health management strategies for people at high risk of stroke.
## | me thods
## | study design
An exploratory, qualitative descriptive study using semi-structured in-depth interviews was conducted to address the study aim.
## | participants
Inclusion criteria were adults: (1) with three or more of the eight major stroke risk factors (previously described in the Introduction) or with a history of stroke or transient ischaemic attack (TIA); (2) with comprehension and communication capability; (3) who have provided informed consent to participate. Exclusion criteria included adults who had medical illnesses, serious complications or co-morbidities rendering them unable to cooperate with investigators during the study period.
In a qualitative descriptive study, the general recommendation of sample size for in-depth interviews is 20-30, which is largely based on ensuring sufficient opportunity to collect relevant data until new information from study participants no longer emerges, that is, reaching data saturation [bib_ref] A review of the quality indicators of rigor in qualitative research, Johnson [/bib_ref].
## | interview guide
The interview topic guide was developed by the team of authors based on the results of the previous investigation, expert advice and a health behaviour change theory "Knowledge-Attitude-Practice (KAP) model" [bib_ref] Evaluating the effect of knowledge, attitude and practice on self-management in patients..., Karbalaeifar [/bib_ref]. First, general broad questions were asked in order to familiarize interviewees with the content of the interview and build trust, followed by more in-depth questioning to gain information about individuals' views, and finally, questions addressed information about possible future developments. The interview guide is presented in . A pilot study was carried out with five people at high risk of stroke to test the interview schedule and identify potential issues with using the guide and improve the questions where necessary.
# | ethical considerations
The Ethical Review Board of the first affiliated hospital of Zhengzhou University approved this study. All potential participants were provided with information about the study and given the opportunity to ask questions/seek clarification prior to volunteering to participate in the study. All participants provided written informed consent before the interview was undertaken. Participants had the right to change their decision and end their participation at any time. All data obtained were only used for this study, the information from participants was anonymous, and individual participant information was not identifiable. Have you been able to accept health management guidance from a health manager via an information tool (such as a smartphone, app or telemedicine system)?
## | data collection
Have you been involved?
To get as much information as possible, follow-up questions should be asked: What happened next? What were you thinking and doing then? How did that affect you? TA B L E 1 Interview guide interviewed one or two times (with 72 hr between two interviews), on each occasion for 32-129 min, and the median length of the interviews was 55 min. Among them, five participants had to suspend during the interview due to treatment or examinations, or an outburst of emotion, and were subsequently interviewed for a second time. Interviewers used interview techniques including repetition, reflection and summarization to elicit participants' most authentic and in-depth views and feelings. During each interview, attention was also paid to non-verbal communication and expressions, such as silence, crying, sighing and surprise. These were noted by interviewers in written field notes.
Data collection and data analysis were carried out simultaneously until the data were saturated and no new topics emerged [bib_ref] The extension of Colaizzi's method of phenomenological enquiry, Edward [/bib_ref]. Finally, the transcribed interviews were checked with study participants for verification of the authenticity of the content.
# | data analysis
Each interview was transcribed verbatim into text and checked for accuracy within 24 hr of completion of the interview. Colaizzi's seven-
## | re sults
## | participant recruitment and characteristics
A total of 31 participants were included, 20 (64.5%) were male and 11 (35.5%) were female, the ages were ranged from 40 to 86, with an average age of 60.71 (SD = 11.55). Their characteristics are shown in This group experienced many health management problems. As their basic health management needs were not met or supported for a long period, health management problems were increasingly aggravated.
They survived a difficult situation and sustained physical, psychological and social stress. They wanted to maintain good health management, but they were always vulnerable to internal and external influences; most of them were in a state of exhaustion, collapse and helplessness in relation to many aspects of health management.
They demonstrated limited knowledge of health management:
The acquisition of medical information was limited, especially in remote rural areas.
…It's too far for us to go to a big hospital. There is a clinic in our village, and only one doctor there.
Sometimes we can't find him…I seldom hear about stroke prevention…We elderly people are not like young people, can easily use mobile phones and computers to read health information on the Internet (with a sign and smile) (P5).
There was a relative shortage of medical resources and an unbalanced distribution of these resources between urban and rural areas, to time and geographical constraints. The participants often got information that was provided by their relatives or friends based on their own experiences, not from professional staff, which was consequently not targeted to their particular needs and lacked detail and accuracy.
…know a little about the health management of stroke, such as 'more exercise' or 'less greasy food'… However, it is not clear how and how much to do… Prevention is mostly based on our own feelings, sometimes. It would be great if a specialist doctor talked to us more and helped us more (excited)…
(P29).
In addition, this group of people were vulnerable to the temptation of interest-oriented advertising, because of their urgent need for health management knowledge about the risk of stroke. The reliability of information was not guaranteed and the potential harm of misleading medical advertising remained.
…There are also some false health information and advertising on the Internet, and the people around me have been cheated…also some campaigns in the parks or squares that sell medicine, they said it can lower blood pressure and even prevent stroke from a secret traditional Chinese medicine prescription, I bought some, but didn't feel any effect… (with grieving and anger)…
(P14).
They demonstrated a lack of confidence in health management:
The high risk of stroke experienced by these participants af- …I downloaded a 'Talk about stroke' APP, which is rich in content and can also consult experts online. It's good… (P9).
They described promoting social interaction:
Participants at high risk of stroke did their best to communicate with people around them and get help, when they encountered problems that they could not solve; they made great efforts to seek help from professional medical staff and got psychological counselling when they were troubled by negative emotions. Moreover, they expanded their social interaction by fostering interests and hobbies; they had their own "patients group" and "social groups," sharing and encouraging each other, obtaining emotional support and social support.
…I joined a walking club and we have regular activities. I can exercise, and also chat with friends, it's good for my physical and mental health… (P20).
…I have the Wechat group (Wechat, a communication software) for stroke patients, we often exchange and share information in this group…I also added professional medical staff as friends, many times I can get professional advice from them… (P4).
They reported enhancing self-health management:
Those at high risk of stroke paid more attention to self-health management once they understood their physical condition. They learned to actively control risk factors, reduce complications and prevent disease recurrence. In addition, they improved their ability to identify and deal with emergencies.
…I know Stroke is closely related to blood pressure, blood sugar and blood lipid. I take aspirin and antihypertensive tables on time every day…test my blood pressure and blood sugar every day and record them.
(P24).
…When I first suffered from stroke, I delayed the best thrombolytic therapy and regretted it because
## | theme 3: sensitivity to multiple influencing factors
The severity of disease and complexity of disease management:
Usually, the more serious the stroke and more complications a person experiences, the greater the demand for knowledge and skills for health management. Accordingly, there were more risk factors that needed to be controlled, which may have affected the person's coping strategies and were associated with an increased burden of health management. If this group of people lacked social support, their health management was unlikely to be as effective.
## | discuss ion
This is the first reported study to use a qualitative descriptive method among people at high risk of stroke to explore the experience of health management. This qualitative study is highly valuable to nursing research, supplementing quantitative research, using a phenomenology method can describe the real experiences and feelings of people at high risk of stroke in the process of health management, and explain the deep-seated views and emotional reactions.
The findings are of significance to clinical nursing practice and can assist health care professionals to develop targeted interventions to promote health management among people at high risk of stroke and further reduce the incidence rate, recurrence rate and disability rate of stroke.
The findings indicate that health management by people at high risk of stroke faces a series of challenges mainly reflected in three aspects: limited knowledge, lack of confidence and poor compliance. One related previous study reported that low income, poor education status and poor health systems are the main factors that lead to low health expectation and awareness of health management in rural regions [bib_ref] Ecological analysis of health care utilisation for China's rural population: Association with..., Chau [/bib_ref]. [bib_ref] Strengthening public health services to achieve universal health coverage in China, Yuan [/bib_ref] also pointed out that the capacity and use of primary care and community health providers are inadequate, suggesting that public health problems and challenges remain a major constraint to promoting universal coverage of chronic disease health management despite great efforts. The complexity of individuals' socioeconomic characteristics and poor health system conditions was a major constraint to the knowledge of, confidence in and compliance with health management among people at high risk of stroke, all of which contribute to the problems reported by this group of people facing a difficult situation. Health management staff should actively exchange health knowledge with people at high risk of stroke, regularly evaluate and promote coping skills [bib_ref] A retrospective cohort study on the risk of stroke in relation to..., Lai [/bib_ref]. Practitioners can also explain the purpose, importance and necessity of health management to them and their families by using various ways, and presenting successful cases to them can help patients to realize the benefits of health management to the improvement of their quality of life [bib_ref] A family involvement and patient-tailored health management program in elderly Korean stroke..., Chang [/bib_ref]. In addition, health managers should assess the psychological state of people at high risk of stroke and encourage them to express their inner feelings and negative emotions, offering professional psychological guidance to whom with mental disorders or giving medicine and other psychotherapy when necessary [bib_ref] Psychological and emotional needs, assessment, and support post-stroke: A multiperspective qualitative study, Harrison [/bib_ref]. When it comes to improving participant compliance, [bib_ref] The effects of telemonitoring on patient compliance with self-management recommendations and outcomes..., Ding [/bib_ref] showed that innovative telemonitoring can improve compliance with self-health management, and a telehealth tool is a promising method for supporting both patients and practitioners in health management.
When people at high risk of stroke are able to accumulate some valuable experiences in health management, they gradually become active learners, improve self-health management and expand social interaction. One recent study [bib_ref] Stroke-related knowledge and lifestyle behavior among stroke survivors, Faiz [/bib_ref] reported that stroke survivors improved their stroke-related knowledge through active learning over 3-12 months, and a significant proportion of patients made changes in lifestyle behaviour. Thus, knowledge acquisition and behaviour change have a positive influence on health management and can reduce the risk of new stroke events. It is interesting that self-health management was seen as significant to health management, which is consistent with a previous study by [bib_ref] Stroke self-management support improves Survivors' self-efficacy and outcome expectation of self-management behaviors, Lo [/bib_ref] who showed that self-health management can improve self-efficacy, outcome expectation and satisfaction with the performance of health management behaviours of stroke patients. Social interaction improved health practices that positively predicted health management outcomes, and negatively correlated with psychological processes, such as stress and depression [bib_ref] Social relationships and mortality risk: A meta-analytic review, Holt-Lunstad [/bib_ref]. Social interaction was also shown to have a critical role in neurogenesis and recovery after stroke, and this was confirmed in an animal experiment [bib_ref] Social interaction plays a critical role in neurogenesis and recovery after stroke, Venna [/bib_ref]. Health management staff should regard active learning, self-health management and social interaction as positively significant predictors of health management among people at risk of stroke, and encourage them to maintain these at a better level by information tools in the Internet age.
Whether health management is effective or not is closely related to the severity and complexity of the disease, family income and social support. Greater severity and complexity of the disease can threaten belief in the benefits of health management and increase the probability of ineffective coping with self-health management [bib_ref] Symptoms of angina pectoris increase the probability of disability pension and premature..., Jespersen [/bib_ref]. One explanation is that greater severity of disease leads to more rapid functional decline and more complex diseases require greater knowledge and skills for health management, thus, individuals are more likely to burn out resulting in poor health management outcomes. Family income is a very important and even a decisive factor in health management. A previous studyincluding 6,413 community residents found that high monthly income is a protective factor for better health management. Good family income is the basis of health management and the foundation to promote better education and health management awareness. Better social support significantly predicts effective health management and is positively associated with chronic disease self-management [bib_ref] Social support and chronic disease management among older adults of Mexican heritage:..., Bustamante [/bib_ref]. It is helpful for patients to get support and help from friends and family members, and to get advice and encouragement from healthcare providers. The receipt of timely, stroke-specific, targeted, social support improves stroke survivors' confidence and coping ability, and prompts them to solve their health management problems.
# | limitations
This study has been carried out using a qualitative method in a Chinese context. Therefore, it may not be generalizable to other contexts and cultures. In addition, this study only recruited participants in hospitals, and more high-risk groups for stroke exist in the community. Therefore, we should further expand the research scope to investigate people at high risk of stroke in the community.
## | con clus ions
This study explored the experiences of health management among people at high risk of stroke and identified a number of changes to effective health management thereby providing a clear theoretical basis to inform future development of intervention measures to support ongoing health management. These findings have important reference value for the primary prevention of stroke. Health administrators and health providers should respond to this study with reform to the health system financial support, social support and information tools applicable to the Internet age.
## Auth o r co ntr i b uti o n s
## Ack n owled g em ent
We express our gratitude to all the respondents. We also thank
Professor Martyn Jones at the University of Dundee and Professor Jaclene A. Zauszniewski at the Case Western Reserve University, for improving the readability.
## Co n fli c t o f i nte r e s t
The authors stated that they have no conflicts of interest.
## Data ava i l a b i l i t y s tat e m e n t
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
## O rci d
## Lina guo
https://orcid.org/0000-0001-9843-6627
Miao Wei https://orcid.org/0000-0002-0049-8289
Yu He https://orcid.org/0000-0003-4659-7124
[fig] Lina: Guo, Yuanli Guo, Jo Booth, Miao Wei, Lin Wang, Yiru Zhu, Yu He and Yanjin Liu were responsible for the conception and design; Guo, Yuanli Guo, Yiru Zhu, Miao Wei, Lin Wang and Yu He made contributions to data collection; Guo and Yuanli Guo involved in data analysis and drafting of the manuscript; Guo and Jo Booth performed the manuscript revision; Yanjin Liu was responsible for supervision and support of this study. [/fig]
[table] Table 2: Findings present illustrative quotations and use the participant number to indicate the source of the quote. [/table]
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An Epidemiological Study on the Prevalence of Atrial Fibrillation in the Chinese Population of Mainland China
Background: Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. Since only limited data on the Chinese population, which is the largest in the world, is available, we conducted an epidemiological study on the prevalence and risk factors of AF in mainland China. Methods: This population-based study conducted by cluster sampling comprised 29079 participants forming 14 cohorts from 13 provinces across China, where the population was nearly 1 billion. Every participant underwent electrocardiogram and physical examinations and responded to the interviewer-led questionnaire(s). Univariate and multiple statistical analyses were conducted to explore the relationship between AF prevalence and risk factors. Results: The age-standardized prevalence of AF in China (≥30 y) was 0.65%, and it increased with age. Men showed a higher prevalence of AF than women (0.91% [age-standardized, 0.66%] vs. 0.65% [0.63%], P = 0.013); several significant risk factors (age, hyperthyroidism, coronary heart disease, and rheumatic heart disease) were identified for AF in the general population. Stroke prevalence was much higher in AF patients than in non-AF people (12.95% vs. 2.28%, P < 0.001). AF was confirmed to be a significant independent risk factor for stroke prevalence in the studied population (OR = 2.776, [1.814, 4.248], P < 0.001). We found that AF patients received poor treatment (2.7%, warfarin; 39.7%, aspirin). Discussion: This study conducted on a large sample size demonstrates that AF prevalence in mainland China is slightly lower than that in Western countries and similar to that in Asian areas, and confirms that AF is a serious public health problem in China. We identified several potential risk factors, but their associations with AF still need to be further studied.
# Introduction
Atrial fibrillation (AF) is the most common sustained cardiac rhythm disturbance encountered in clinical practice. According to European and Northern American experiences, AF accounts for massive health care system expenses. [bib_ref] The aging of America. Impact on health care costs, Schneider [/bib_ref] [bib_ref] Cost of an emerging epidemic: an economic analysis of atrial fibrillation in..., Stewart [/bib_ref] The increased longevity is a result of improved medical care among patients with coronary artery disease, hypertension, and heart failure; these 3 chronic cardiac conditions generally predispose patients to AF. Several preliminary epidemiological studies based on US and European populations indicated that the prevalence of AF was on the increase with each passing decade. [bib_ref] Trends in the prevalence and management of atrial fibrillation in general practice..., Majeed [/bib_ref] [bib_ref] Trends in first-ever admissions for atrial fibrillation in, Stewart [/bib_ref] [bib_ref] Secular trends in the prevalence of atrial fibrillation: The Framingham Study, Wolf [/bib_ref] Epidemiological studies in Western countries have shown that in older people, the prevalence of AF increases with age from less than 1% for persons younger than 60 y of age to approximately 10% for those who are 80 or above. [bib_ref] Trends in the prevalence and management of atrial fibrillation in general practice..., Majeed [/bib_ref] [bib_ref] Atrial fibrillation as an independent risk factor for stroke: The Framingham Study, Wolf [/bib_ref] [bib_ref] Prevalence, age distribution, and gender of patients with atrial fibrillation: analysis and..., Feinberg [/bib_ref] [bib_ref] Prevalence of diagnosed atrial fibrillation in adults, Go [/bib_ref] The Framingham Heart Study reported that men showed a higher AF prevalence than women. [bib_ref] Secular trends in the prevalence of atrial fibrillation: The Framingham Study, Wolf [/bib_ref] Data from the same study cohort also revealed AF to be an independent risk factor for stroke. [bib_ref] Atrial fibrillation as an independent risk factor for stroke: The Framingham Study, Wolf [/bib_ref] Several risk factors of AF have been identified by J Epidemiol 2008; 18 [bib_ref] Secular trends in the prevalence of atrial fibrillation: The Framingham Study, Wolf [/bib_ref] 209-216 studies based on white cohorts. [bib_ref] Prevalence, incidence, prognosis, and predisposing conditions for atrial fibrillation: population-based estimates, Kannel [/bib_ref] These factors include age, congestive heart failure, valvular heart disease, male sex, a history of myocardial infarction, hypertension, and diabetes. [bib_ref] Prevalence, incidence, prognosis, and predisposing conditions for atrial fibrillation: population-based estimates, Kannel [/bib_ref] For other ethnical populations, the Epidemiology, Practice, Outcomes, and Costs of Heart Failure (EPOCH) study indicated that the African-American race was associated with a lower prevalence of AF. [bib_ref] Racial variation in the prevalence of atrial fibrillation among patients with heart..., Ruo [/bib_ref] Data from Asian countries like Japan, [bib_ref] Rapid increase in estimated number of persons with atrial fibrillation in Japan:..., Ohsawa [/bib_ref] [bib_ref] A 15.5 year follow-up study of stroke in a Japanese provincial city, Nakayama [/bib_ref] Singapore, [bib_ref] Low prevalence of atrial fibrillation in community-dwelling Chinese aged 55 years or..., Yap [/bib_ref] and Korea 14 also showed a lower prevalence of AF (0.7%-1.5%) compared to those from Western series. However, the prevalence of AF in China, which accounts for nearly one-fourth of the world population, has not been published in international journals. In order to clarify the prevalence of AF and explore its potential risk factors in China, we performed the first large-scale epidemiological study on AF in mainland China. This study provided data for analyzing the epidemiological characteristics of AF in the Chinese population, studying the association between stroke and AF, and comparing Chinese cohorts with other populations. The present study is an advanced version of the former preliminary study. [bib_ref] An epidemiological survey of atrial fibrillation in China, Zhou [/bib_ref] [fig_ref] Table 3: Baseline characteristics of the variables included in the multivariable model * [/fig_ref] [bib_ref] Epidemiology in practice: sample size calculation for eye surveys: a simple method, Minassian [/bib_ref] and the expected prevalence of AF (approximately 1%) was obtained from the preliminary data of the Western series. [bib_ref] Trends in the prevalence and management of atrial fibrillation in general practice..., Majeed [/bib_ref] [bib_ref] Atrial fibrillation as an independent risk factor for stroke: The Framingham Study, Wolf [/bib_ref] [bib_ref] Prevalence, age distribution, and gender of patients with atrial fibrillation: analysis and..., Feinberg [/bib_ref] [bib_ref] Prevalence of diagnosed atrial fibrillation in adults, Go [/bib_ref] In the first stage, 14 centers based on 14 population-based cohorts from 13 provinces across China were established, such that they represented the average standards of living, education, and health care in mainland China. The cohorts were selected such that they represented the average characteristics of each province. There was no significant heterogeneity across these sites. The provinces studied included Guangdong, Hebei, Henan, Hubei, Hunan, Inner Mongolia, Shandong, Shanxi, Sichuan, Tianjin, Yunan, Zhejiang, and Jiangxi.
# Materials and methods
## Study population and sample
Given below is a map of the location of the clusters.
In the second stage, which was based on the data collected from the local household registration system in each chosen area, subjects were selected by cluster random sampling from J Epidemiol 2008; 18(5) 209-216 each population established in the first stage. Each participating center was responsible for 2000-2500 subjects (born in or after 1972), and among the participants, a male to female ratio of 1:1 was required. The selected subjects were asked to come to the appointed center for examinations and interviews. This study was approved by the Health Ministry of the Peoples Republic of China and the local governments. A regular hospital or health agency in each area was appointed to provide the equipments and venues for this study. All the participants received a detailed introduction to this study by the local government and medical organizations. The participants' informed consent was obtained by the local medical organizations. All the examinations were conducted free of charge, and no participant was compensated.
## Screening process
We developed an interviewer-led questionnaire specifically for the survey. The first part of the questionnaire was recorded by the physicians, who conducted face-to-face interviews with the patients. The interviewers obtained information regarding the patients' demographic characteristics and recorded the family medical history; they inquired about their smoking habits; drinking habits; and a medical history of other diseases, including diabetes, hypertension, dyslipidemia, rheumatic heart disease (RHD), coronary heart disease (CHD), hyperthyroidism, stroke, and AF. Besides, participants with self-reported RHD underwent further physical examinations and echocardiography if the diagnosis was unclear. We did not routinely perform blood tests for thyroid function, blood sugar, and blood lipid. Participants with a self-reported history of stroke were all required to provide their medical records from hospital visits and/or visits to primary care practitioners, which were carefully reviewed by qualified local neurologists. The second part of the questionnaire was recorded by 2 trained technicians who obtained the blood pressure, pulse rate, height, weight, waist, and hip measurements. After asking the patient to rest for at least 10 min in the sitting position, the blood pressure in the right arm was measured in the sitting position with the use of a standardized mercury column sphygmomanometer with a standardized protocol.The third part included a copy of an electrocardiogram (ECG) with a written interpretation. Patients with AF and suspected AF were required to provide information on the use of anticoagulants.
Hypertension was defined by a systolic blood pressure of 140mmHg or higher, diastolic blood pressure of 90mmHg or higher, the use of antihypertensive agents, or a combination of these. Participants were considered to be smokers if they were smokers at the time of the investigation or if they had quit smoking less than 6 month earlier. They were considered non-drinkers if they did not consume alcohol regularly or had quit for not less than 6 months. Diagnoses of diabetes, hyperthyroidism, dyslipidemia, and CHD were classified by physicians (interviewers) as "yes" on the basis of positive self-reported histories of these diseases. Education attainment was classified as "yes" if a subject was a high school graduate or had higher education.
Several steps were used to ensure standardization of the methods: (1) Physicians and technicians from each center received unified training prior to the screening process. (2) All participants were asked to respond to the interviewer-led questionnaire. (3) Blood pressure, pulse rate, height, weight, waist, and hip measurements were recorded by trained technicians. (4) All participants underwent an ECG examination, which was performed by trained technicians and trained physicians in each center. The physicians provided a primary written interpretation for each ECG, all of which were reviewed by an appointed senior cardiologist daily. [bib_ref] Secular trends in the prevalence of atrial fibrillation: The Framingham Study, Wolf [/bib_ref] Monitors were sent to each center to supervise the standardized procedures. (6) Trained interviewers contacted those who did not come to the interview by telephone, and then conducted a family interview wherever possible to encourage response from the patient. The screening work for each center was performed within 3 months in 2003.
## Definition of af
Subjects who were diagnosed with AF during the ECG examination in the screening process were defined as AF cases. Those who were not found to have AF according to the ECG test, but had previous medical records from visits to qualified affiliated hospital(s) of medical universities or any prior ECG record(s) for AF episode(s) were also defined as having AF (possible paroxysmal AF [bib_ref] ACC/AHA/ESC guidelines for the management of patients with atrial fibrillation: executive summary...., Fuster [/bib_ref] [bib_ref] Classification of atrial fibrillation, Gallagher [/bib_ref]. AF determination based on the ECG was first conducted by a physician, who acted as the interviewer, and then verified by an appointed senior physician or cardiologist. ECGs and medical records obtained outside the study center were copied for further verification. The ECG criteria and classification for AF were in accordance with the ACC/AHA/ESC 2001 guidelines. [bib_ref] Classification of atrial fibrillation, Gallagher [/bib_ref] In order to ensure that the maximum number of AF cases were identified, suspected patients for whom AF was not detected according to the ECG but who were able to recall AF or tachycardia episode(s), which might have been diagnosed by primary care practitioners or ECG technicians, received further reviews of their medical records within 1 y from hospital visits, visits to primary care practitioners, and other potential evidences. Their health records, if present, were also fully searched in the database of local medical organizations. Then, all these suspected AF cases were further investigated by senior cardiologists to confirm or disconfirm the diagnosis.
# Statistical analysis
We used Borland Delphi 6.0 to build a database management system to handle the data obtained in this study. Data analysis J Epidemiol 2008; 18 (5) 209-216 was conducted with the SPSS statistical software, version 12.0.1. Differences between groups were tested with Pearson's χ 2 test or Fisher's exact test when appropriate. The exact confidence intervals (C.I.) of the prevalence were calculated by StatPages.net (http://statpages.org/confint.html) based on binomial distribution. We calculated the overall as well as age-and sex-stratified point prevalence of AF in the participants .The direct age standardization method 20 was chosen to calculate the age-standardized prevalence and expected number of AF patients in the 13 studied provinces and in all of China, on the basis of the standard age distribution data from China's fifth national population census in 2000.
Two main multiple logistic regression models were constructed. In one model, the dependent variable was AF (yes/no), and the backward stepwise method was used to investigate the associations between the potential risk factors and AF prevalence. For potential independents, we defined age and waist-hip ratio (WHR) as continuous variables and the following as binomial categorical independent variables: sex (male/female), diabetes (yes/no), hyperthyroidism (yes/ no), hypertension (yes/no), RHD (yes/no), CHD (yes/no), smoking (yes/no), drinking (yes/no), dyslipidemia (yes/no), and education attainments (high school graduate or not). Univariate analyses were first conducted to test the correlation between AF prevalence and each potential independent variable. In the univariate analysis, variables with P < 0.25 were chosen as independent variables to construct the model for multivariable analyses (backward stepwise logistic regression model). The other logistic model was used to explore the association between AF and the prevalence of stroke, where the dependent variable was stroke (yes/no) and the independent variables based on previous studies 21 included age, diabetes, hypertension, dyslipidemia, smoking, and drinking. In the backward stepwise logistic regression model, the criterion for inclusion of the variables was P < 0.05, and that for exclusion was P > 0. [bib_ref] Racial variation in the prevalence of atrial fibrillation among patients with heart..., Ruo [/bib_ref]. The values for odds ratios (ORs) were linked to the prevalence of AF, i.e., values greater than 1 indicated an increased risk of high AF prevalence and values less than 1 indicated a reduced risk of high AF prevalence.
# Results
The study included 29079 subjects (mean age, 52.5 ± 22.4; range, 30-100) from 14 different Chinese cohorts (male, n = 13558; female, n = 15521), among which 224 participants were identified as having AF. The overall response rate was approximately 96%. The most common reason for nonparticipation (4%) was refusal to undergo examination. Among the 224 AF patients, 204 (91.1%) were identified on the basis of the ECGs during the screening process, and the rest (20, 8.9%) were identified via previous medical records and ECGs. Prior to data analysis, we ensured that all participants were examined for AF or suspected AF. Our results showed that the crude prevalence of AF in the studied Chinese population (≥30 y) was 0.77% (95% C.I., 0.67-0.88%). On the basis of the age composition data in the 13 studied provinces and all of China (obtained from the fifth national population census in China), the age-standardized prevalence of AF in both genders in the Chinese (≥30 y) was 0.65% (0.66% for males and 0.63% for females); the estimated number of AF patients (≥30 y) was calculated to be 2456573 in all the provinces studied and 4193857 in all of China.
The age-specific prevalence of AF increased with age (see [fig_ref] Table 1: Sex-and age-specific [/fig_ref]. The crude prevalence of AF in men was higher than that in women (0.91% vs. 0.65%, P = 0.013). In each age group, the prevalence of AF in men was higher than that in women, except for the 50-59 age group (see [fig_ref] Table 1: Sex-and age-specific [/fig_ref].
In the 224 participants with AF, only 6 took warfarin (see [fig_ref] Table 2: Information on the use of medicine by Chinese AF* patients [/fig_ref] , and only 1 of these 6 underwent an internalized normal ratio examination regularly.
The baseline characteristics of the variables and the results from the univariate analyses are listed in [fig_ref] Table 3: Baseline characteristics of the variables included in the multivariable model * [/fig_ref]. Drinking and smoking were then excluded due to a very high P value. The remaining 9 variables were independent variables that were entered into the logistic regression model in the first step.
Four significant risk factors, including age, hyperthyroidism, RHD, and CHD, were located via the backward stepwise logistic regression model (see [fig_ref] Table 4: Risk factors of AF in both genders in the Chinese population in... [/fig_ref]. Among the variables that were entered in the last step of the regression, neither dyslipidemia nor sex was found to be statistically significant on the basis of the criteria (P < 0.05) (see [fig_ref] Table 4: Risk factors of AF in both genders in the Chinese population in... [/fig_ref]. WHR (per 0.1) and waist circumference (per cm) were significantly associated with the prevalence of AF when it was not adjusted for other factors (OR = 1.173, [1.041, 1.322], P = 0.009; OR = 1.020, [1.008, 1.033], P = 0.002), but they were statistically insignificant after adjustment. As another anthropometric index, body mass index (BMI) was not significantly associated with AF prevalence even when it was not adjusted with the other variables (P = 0.552). WHR (per 0.1) became a significant risk factor for AF (OR = 1.212, [1.010, 1.456], P = 0.039) when only the male population was considered. Collinearity diagnosis was routinely performed, and no independent variable was found to have a tolerance of <0.1. Analysis of variance inflation factor (VIF), Eigenvalue, and the condition index did not show significant multicollinearity.
During the backward stepwise regression, the variable(s) removed from steps 2, 3, 4, and 5 were hypertension, diabetes, education attainment, and WHR respectively. In the univariate analysis, the prevalence of stroke in AF patients was much higher than that in the non-AF population (12.95% vs. 2.28%, P < 0.001) (See [fig_ref] Table 5a: Prevalence of stroke in AF and non-AF cohortsTable 5b [/fig_ref]. Moreover, in the logistic regression model, AF was proven to be one of the leading contributors to the prevalence of stroke (OR = 2.776, .248], P < 0.001) (See .
# Discussion
This study demonstrates that the age-standardized prevalence of AF in the general Chinese population was 6.5 per 1000 people, and that it increased with age. Given China's 6.4 billion population (≥30 y), it has been estimated that at least 4 million adults suffer from AF in mainland China today, which can be a substantial burden on public health. Here, we confirm that AF is a disease that is largely prevalent in the elderly in the general Chinese population, with the great majority of cases present among people aged 60 y and above.
The age-specific prevalence of AF in China rose dramatically from 0% in the 30-39 age group to 7.5% in the 80-89 age group. Similar trends were found in nearly all the previous studies on AF prevalence. The present study showed a lower prevalence rate of AF in Chinese people, especially in the middle-aged cohort, than in Western cohorts. [bib_ref] Trends in the prevalence and management of atrial fibrillation in general practice..., Majeed [/bib_ref] [bib_ref] Secular trends in the prevalence of atrial fibrillation: The Framingham Study, Wolf [/bib_ref] [bib_ref] Atrial fibrillation as an independent risk factor for stroke: The Framingham Study, Wolf [/bib_ref] [bib_ref] Prevalence, age distribution, and gender of patients with atrial fibrillation: analysis and..., Feinberg [/bib_ref] [bib_ref] Prevalence of diagnosed atrial fibrillation in adults, Go [/bib_ref] [bib_ref] Sex-specific increase in the prevalence of atrial fibrillation (The Copenhagen City Heart..., Friberg [/bib_ref] [bib_ref] Prevalence of atrial fibrillation and eligibility for anticoagulants in the community, Sudlow [/bib_ref] [bib_ref] Prevalence of cardiovascular disease and diabetes mellitus in residents of Rochester, Minnesota, Phillips [/bib_ref] Firstly, it should be brought to notice that ascertaining the diagnosis of AF and the characteristics of the population studied would significantly affect the final result of a study. Wolf et al. [bib_ref] Secular trends in the prevalence of atrial fibrillation: The Framingham Study, Wolf [/bib_ref] included the AF cases diagnosed from ECGs that were obtained from the subjects' private physicians and during interim hospitalizations. Inclusion of such AF cases could prevent the loss of cases with acute paroxysmal AF, which may have occurred 25 in the present or Friberg's study. [bib_ref] Sex-specific increase in the prevalence of atrial fibrillation (The Copenhagen City Heart..., Friberg [/bib_ref] Majeed et al. [bib_ref] Trends in the prevalence and management of atrial fibrillation in general practice..., Majeed [/bib_ref] used a large and well-validated general practicederived database to diagnose AF, which is more sensitive than the single ECG used in the present study; moreover, they gained baseline data from clinical practices when there was a lack of completely healthy subjects, and therefore, they tended to obtain a higher prevalence of AF. AF prevalence according to a community study on middle-aged to elderly Japanese people was only 1.3%, [bib_ref] A 15.5 year follow-up study of stroke in a Japanese provincial city, Nakayama [/bib_ref] whereas that in ill, hospitalized, elderly people could reach as high as 22%. [bib_ref] Electrocardiographic abnormalities in the sick elderly, Patel [/bib_ref] As such, the lower prevalence of related diseases, such as coronary artery disease, diabetes, and dyslipidemia, in the population studied contributed to a lower AF prevalence. Secondly, the use of different data sets is also an important reason for the difference. It has recently been indicated that AF prevalence varies according to race, 27 which may partly explain why AF prevalence found in this study is much closer to the results from Asian cohorts [bib_ref] Racial variation in the prevalence of atrial fibrillation among patients with heart..., Ruo [/bib_ref] [bib_ref] Rapid increase in estimated number of persons with atrial fibrillation in Japan:..., Ohsawa [/bib_ref] [bib_ref] A 15.5 year follow-up study of stroke in a Japanese provincial city, Nakayama [/bib_ref] [bib_ref] Low prevalence of atrial fibrillation in community-dwelling Chinese aged 55 years or..., Yap [/bib_ref] [bib_ref] Prevalence of and risk factors for atrial fibrillation in Korean adults older..., Jeong [/bib_ref] outside China than to those from the Western series. Moreover, another essential reason is the different age strata among the studies, because AF increases with age. Studies based on younger populations often indicated a lower prevalence of AF, [bib_ref] The aging of America. Impact on health care costs, Schneider [/bib_ref] [bib_ref] A 15.5 year follow-up study of stroke in a Japanese provincial city, Nakayama [/bib_ref] [bib_ref] Independent risk factors for atrial fibrillation in a population-based cohort: The Framingham..., Benjamin [/bib_ref] [bib_ref] Population prevalence, incidence, and predictors of atrial fibrillation in the Renfrew/Paisley study, Stewart [/bib_ref] and those based on older participants showed a higher prevalence. [bib_ref] Atrial fibrillation as an independent risk factor for stroke: The Framingham Study, Wolf [/bib_ref] [bib_ref] Prevalence of atrial fibrillation and eligibility for anticoagulants in the community, Sudlow [/bib_ref] [bib_ref] Prevalence of cardiovascular disease and diabetes mellitus in residents of Rochester, Minnesota, Phillips [/bib_ref] [bib_ref] Prevalence of atrial fibrillation in elderly subjects (the Cardiovascular Health Study), Furberg [/bib_ref] [bib_ref] Atrial fibrillation and mortality in an elderly population, Lake [/bib_ref] In conclusion, it can be said that AF is a disease that is increasing in prevalence; 5 therefore, it should be noted that the prevalence of AF obtained in this study may actually be lower than that reported in previous studies.
We found no significant association between smoking or drinking and the prevalence of AF. Kannel WB et al. reported that smoking was a significant risk factor in women when adjusted only for age. [bib_ref] Long-term alcohol consumption and the risk of atrial fibrillation in the Framingham..., Djousse [/bib_ref] Luc Djousse et al. concluded that long-term alcohol consumption was a strong predictor of the incidence of AF adjusted for other risk factors. [bib_ref] Prevalence, age distribution, and gender of patients with atrial fibrillation: analysis and..., Feinberg [/bib_ref] The reason as to why no significant association was found between AF prevalence and these lifestyle-related factors was probably due to the cross-sectional design of the present study.
In this study, hypertension was not significantly associated with the prevalence of AF, whether it was adjusted only for age or for all risk factors, although it was by far a powerful predisposing factor for AF incidence. [bib_ref] Prevalence, age distribution, and gender of patients with atrial fibrillation: analysis and..., Feinberg [/bib_ref] [bib_ref] Population prevalence, incidence, and predictors of atrial fibrillation in the Renfrew/Paisley study, Stewart [/bib_ref] [bib_ref] Prevalence of atrial fibrillation in elderly subjects (the Cardiovascular Health Study), Furberg [/bib_ref] Hypertension is a major contributing factor to the development of AF in longitudinal studies, but it is not always a risk factor for AF in cross-sectional analyses. Patients who have developed AF usually tend to show a lowering of systolic blood pressure ("decapitated" hypertension) compared to the blood pressure levels prior to the onset of AF. Second, some drugs used for the treatment of hypertension also suppress AF, such as betablocker and certain calcium channel blockers. These factors may attenuate the relationship between elevated blood pressure and the prevalence of AF in a cross-sectional analysis.
Bailey et al. indicated that estrogen plays a role in the occurrence of AF. [bib_ref] The effects of hormones on arrhythmias in women, Bailey [/bib_ref] Considering that the proportion of menopause almost reaches 100% among women aged 60 y or above and increases with age among women aged 30-59 y (see [fig_ref] Table 1: Sex-and age-specific [/fig_ref] , we analyzed the female subgroup aged 35-62 y (this group included the youngest as well as the oldest menopausal females) but found no significant association between AF and menopause (P = 0.59). Educational attainment was not significantly associated with AF prevalence in this study. The older Chinese people are, the lower their average level of educational attainment; this may affect the result.
On the basis of the present study, we found a significantly higher prevalence of stroke in subjects with AF than in non-AF subjects. In the multiple logistic regression model, AF was also confirmed to be one of the leading contributors to the prevalence of stroke after adjustment for age and several other known risk factors. Therefore, the strong link between AF and stroke was confirmed in the Chinese population studied. Due to the much higher prevalence of stroke in AF cases, anticoagulation therapy is highly recommended in China for stroke prevention in patients with AF. Unfortunately, the number of AF patients who took anticoagulants regularly (2.7%, warfarin and 37.9%, aspirin) was rather small. Health education and public policies are urgently required for Chinese AF patients.
The present study had some limitations. Firstly, we did not find a significant association between AF and several predisposing factors. The reasons may be due to both the cross-sectional study and the relatively low number of diagnosed AF participants. Secondly, the prevalence of diabetes was very low (3.5%, [3.3%-3.7%]) compared with that of hypertension (35.9%, [35.3%-36.4%]), which may reflect the lack of biochemical examinations. Only 91 (0.3%) of 29079 participants were found to have RHD, which may explain the wide confidence intervals for RHD. Moreover, although we obtained a single ECG recording for each participant and tried to obtain complete information from those diagnosed with AF, it was still hard to discover paroxysmal AF in some cases; this limitation is also true for other studies. Therefore, the prevalence of AF revealed in this study may represent the lower boundary of the true prevalence. With regard to the Chinese, though Dr. Qi et al. and Dr. Yap et al. reported some results based on hospitalized patients 34 and community-dwelling populations, [bib_ref] Low prevalence of atrial fibrillation in community-dwelling Chinese aged 55 years or..., Yap [/bib_ref] respectively, no data on the general population has demonstrated the incidence of AF and the correlation between it and the risk factors; this area requires to be explored by a carefully designed follow-up study.
[table] Table 1: Sex-and age-specific (by 5 y) prevalence of AF in the Chinese *This included the overall population in the 13 provinces studied, where the corresponding age strata of the general Chinese population were cited from the fifth national population census conducted in November 2000 in China; † : atrial fibrillation [/table]
[table] Table 2: Information on the use of medicine by Chinese AF* patients [/table]
[table] Table 3: Baseline characteristics of the variables included in the multivariable model *: atrial fibrillation; † : coronary heart disease; ‡ : rheumatic heart disease; § : mean ± standard deviation [/table]
[table] Table 4: Risk factors of AF in both genders in the Chinese population in mainland China [/table]
[table] Table 5a: Prevalence of stroke in AF and non-AF cohortsTable 5b. AF* as an independent variable of the prevalence of stroke in the studied Chinese population *: atrial fibrillation; † : odds ratio [/table]
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Mild hyponatremia is associated with low skeletal muscle mass, physical function impairment, and depressive mood in the elderly
Background: Mild hyponatremia (serum sodium 130-135 mEq/L) is a common electrolyte disorder in the elderly. However, its association with both sarcopenia and cognitive function remains to be clarified. Therefore, here we investigated the association of mild hyponatremia with skeletal muscle mass, physical function, and cognitive function in the elderly. Methods: We enrolled 75 participants with mild hyponatremia and 2907 with normonatremia (serum sodium, 136-145 mEq/L) aged ≥70 years who visited the Memory Disorder Outpatient Center of Japan's National Center for Geriatrics and Gerontology. Skeletal muscle mass index (SMI), grip strength (GS), walking speed (WS), one-leg standing (OLS) test times, and neuropsychological test scores were determined. Results: One-way analysis of covariance showed that elderly participants with mild hyponatremia had lower SMI (7.1 ± 0.2, 7.2 ± 0.2 kg/m 2 , p = 0.04), weaker GS (19.1 ± 1.9 vs 21.4 ± 1.8 kg, p = 0.01), slower WS (0.9 ± 0.1 vs 1.1 ± 0.1 m/s, p = 0.001), and higher GDS-15 score (6.4 ± 0.9 vs 5.2 ± 0.9, p = 0.002) than those with normonatremia. Multiple logistic regression analysis indicated that mild hyponatremia was independently associated with sarcopenia (odds ratio [OR]: 2.2, p = 0.02), slower WS (OR: 5.3, p = 0.04) and shorter OLS time (OR: 2.5, p = 0.02) as well as with severe depressive mood (OR: 2.6 p = 0.006) but not with SMI (OR: 1.6, p = 0.2) or GS (OR: 1.9, p = 0.09). Conclusions: Our results suggest that elderly people with even mild hyponatremia had physical function impairment and depressive mood.
# Background
Hyponatremia is the most common electrolyte disorder in the elderly. It occurs in about 7% of all otherwise healthy people aged 55 years or more, with incidence increasing significantly with age. Hyponatremia in the elderly has many causes, including use of medication such as diuretics and antidepressants, syndrome of inappropriate antidiuretic hormone secretion, malnutrition, and comorbidities such as cardiac disease [bib_ref] Hyponatremia in the elderly: challenges and solutions, Filippatos [/bib_ref]. In most cases though, the symptoms of hyponatremia are mild and related symptoms are subtle, so it often goes unnoticed by patients and physicians.
However, recent studies suggest that asymptomatic hyponatremia is associated with poor prognosis in the elderly. Largely asymptomatic mild-to-moderate hyponatremia was reported to be associated with gait instability, falls, and fractures [bib_ref] Is asymptomatic hyponatremia really asymptomatic?, Decaux [/bib_ref] [bib_ref] Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits, Renneboog [/bib_ref] [bib_ref] Mild prolonged chronic hyponatremia and risk of hip fracture in the elderly, Ayus [/bib_ref]. Also, even after considering the many etiologies of hyponatremia that may contribute to poor prognosis among the elderly, hyponatremia itself is considered to be an independent risk factor for poor prognosis [bib_ref] Is asymptomatic hyponatremia really asymptomatic?, Decaux [/bib_ref]. Attention deficit has also been reported in both humans and rats with mild-to-moderate hyponatremia [bib_ref] Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits, Renneboog [/bib_ref] [bib_ref] Chronic Hyponatremia causes neurologic and Psychologic impairments, Fujisawa [/bib_ref]. Thus, it seems plausible that asymptomatic hyponatremia contributes to poor prognosis through, for example, falls or fractures probably as a result of impaired attention, posture, and gait.
To date, most studies on the association between physical function and hyponatremia have involved patients with moderate hyponatremia (serum sodium [SNa] < 130 mEq/L) [bib_ref] Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits, Renneboog [/bib_ref]. So, it remains unclear whether there is an association between mild hyponatremia (SNa 130-135 mEq/L), which constitutes most cases of hyponatremia, and both physical and cognitive function in the elderly. In clinical practice, physicians are often hesitant to treat asymptomatic mild hyponatremia with SNa ≥ 130 mEq/L. However, increased mortality has been reported in patients hospitalized with even mild hyponatremia (SNa 130-134 mEq/L) [bib_ref] Mortality after hospitalization with mild, moderate, and severe hyponatremia, Waikar [/bib_ref]. Thus, we hypothesized that elderly people with even mild hyponatremia, not just those with mild-to-moderate hyponatremia, have physical or cognitive dysfunction such as impaired balance or executive dysfunction. We also hypothesized that there is an association between mild hyponatremia and lower skeletal muscle mass (SMM) in this population based on a report that found an association between chronic hyponatremia and decreased SMM in rats [bib_ref] Chronic hyponatremia exacerbates multiple manifestations of senescence in male rats, Barsony [/bib_ref].
Because homeostatic responses to environmental challenges decline with aging, hyponatremia is frequently diagnosed in the elderly. We anticipate that more frail elderly patients will present with mild hyponatremia into the future as society ages. Thus, if elderly people with mild hyponatremia tend to have physical or cognitive dysfunction, then we should consider ways to prevent and manage this condition. In this study, we investigated the association of mild hyponatremia with SMM, physical function, and neuropsychological test scores in the elderly.
# Methods
## Participants
We enrolled 2962 elderly participants with normonatremia (SNa 136-145 mEq/) and 75 with hyponatremia (SNa 130-135 mEq/L) aged ≥70 years who visited the National Center for Geriatrics and Gerontology (NCGG) for assessment of memory disorder between September 2010 and November 2017. None of the participants had any severe symptoms such as nausea, vomiting, confusion, or seizures, and all were able to carry out at least 1 of the following 3 physical function tests: grip strength (GS), walking speed (WS), and the one-leg standing (OLS) test. To eliminate pseudohyponatremia and the effect of disease on physical or cognitive function, the following inclusion criteria were adopted: (1) no history of stroke; (2) no pseudohyponatremia (TP ≤ 8.0, Glu ≤ 400, TH < 500, and T-cho ≤ 280); and (3) not taking medication for Parkinson's disease (e.g., L-dopa, dopamine agonist, trihexyphenidyl, and amantadine).
The study was approved by the NCGG Ethics Committee. All participants provided informed consent before enrollment in the study.
## Clinical measures
The following self-reported and caregiver-reported measures were obtained based on previous studies [bib_ref] Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits, Renneboog [/bib_ref] [bib_ref] Mild prolonged chronic hyponatremia and risk of hip fracture in the elderly, Ayus [/bib_ref] [bib_ref] Mortality after hospitalization with mild, moderate, and severe hyponatremia, Waikar [/bib_ref] [bib_ref] Physical Function Differences Between the Stages From Normal Cognition to Moderate Alzheimer..., Fujisawa [/bib_ref] [bib_ref] A review of drug-induced hyponatremia, Liamis [/bib_ref] : age, sex, and years of education, alcohol consumption, body mass index (BMI), history of cardiac disease, diabetes, liver disease, lung disease, depression, chronic kidney disease (CKD), cancer, dementia diagnosis, joint pain, visual impairment, hearing impairment, diuretic use, antibiotics use (e.g., clarithromycin, levofloxacin, and minocycline hydrochloride), proton pump inhibitor use, non-steroidal antiinflammatory drug (NSAID) or acetaminophen use, antiepileptic use (e.g., sodium valproate, and carbamazepine), antipsychotic use (e.g., risperidone, haloperidol, and aripiprazole), noradrenergic and specific serotonergic antidepressant (NaSSA) use, selective serotonin reuptake inhibitor (SSRI) or serotonin noradrenaline reuptake inhibitor (SNRI) use, benzodiazepine use (e.g., alprazolam, brotizolam, etizolam, and flunitrazepam), and frequency of physical activity. Those who consumed > 60.0 g/day of alcohol were defined as heavy drinkers. No exercise per week was defined as inactivity. Potassium, calcium, brain natriuretic peptide, estimated glomerular filtration rate, Creactive protein (CRP), hemoglobin (Hb), thyroidstimulating hormone, and vitamin B12 were checked from their blood samples. Anemia was defined their serum Hb was < 11.0 g/dL. Hypothyroidism was defined as a serum thyroid-stimulating hormone level of 10 μIU/mL or less.
Vitamin B12 deficiency was defined as a serum vitamin B12 level of < 200 pg/mL.
## Assessment of smm, physical function, and sarcopenia
Body weight and SMM were assessed using a multifrequency body composition analyzer (MC-180 Multi-Frequency Body Composition Analyzer; Tanita, Tokyo, Japan), which is a scale that uses bioelectrical impedance analysis to determine body composition. We calculated BMI (body weight [kg]/height squared [m 2 ]) and SMM index (SMI; i.e., SMM [kg]/height squared [m 2 ]). Low SMM was defined as SMI < 7.0 kg/m 2 in men and < 5.7 kg/m 2 in women [bib_ref] Asian Working Group for Sarcopenia: 2019 Consensus Update on Sarcopenia Diagnosis and..., Chen [/bib_ref].
Physical function was assessed by measuring GS, WS, and OLS time. Muscle strength was from upper extremity strength measured with a digital force gauge (ZP 500 N; Imada, Toyohashi, Japan) [bib_ref] Association of grip strength and related indices with independence of activities of..., Matsui [/bib_ref]. The best GS (kg) was determined, with low muscle strength defined as < 28 kg in men and < 18 kg in women, in accordance with the Asian Working Group for Sarcopenia (AWGS) criteria [bib_ref] Asian Working Group for Sarcopenia: 2019 Consensus Update on Sarcopenia Diagnosis and..., Chen [/bib_ref]. Velocity was determined by measuring normal WS, with poor physical performance defined as WS < 1.0 m/s [bib_ref] Asian Working Group for Sarcopenia: 2019 Consensus Update on Sarcopenia Diagnosis and..., Chen [/bib_ref]. Postural function was assessed using the OLS test, which measures the time in seconds (up to a maximum of 60 s) that participants can stand unassisted on one leg for as long as possible with their eyes open. The best score was used for the analysis [bib_ref] Physical Function Differences Between the Stages From Normal Cognition to Moderate Alzheimer..., Fujisawa [/bib_ref]. Impaired postural function was defined as OLS time < 15 s according to the locomotive syndrome criteria [bib_ref] Threshold values of physical performance tests for locomotive syndrome, Muramoto [/bib_ref]. Sarcopenia was defined as low SMM, low muscle strength, and/or low physical function [bib_ref] Asian Working Group for Sarcopenia: 2019 Consensus Update on Sarcopenia Diagnosis and..., Chen [/bib_ref].
## Assessment of cognitive function
Cognitive function was assessed with the Mini-Mental State Examination (MMSE) [bib_ref] Mini-mental state". A practical method for grading the cognitive state of patients..., Folstein [/bib_ref] , executive function with the Frontal Assessment Battery (FAB) [bib_ref] The FAB: a frontal assessment battery at bedside, Dubois [/bib_ref] , working memory with the Digit Span subtest (forward and backward) of the Wechsler Adult Intelligence Scale, attention with the category fluency subtest of the Hasegawa Dementia Rating Scale-Revised [bib_ref] Diagnostic accuracy of mini-mental status examination and revised hasegawa dementia scale for..., Kim [/bib_ref] , and memory with the Logical Memory II subtest of the Wechsler Memory Scale-Revised (WMS-R) [bib_ref] San Antonio: Psychological Corp. : Harcourt Brace Jovanovich, Wechsler [/bib_ref].
Cognitive impairment was indicated by MMSE score ≤ 23, FAB score ≤ 11 [bib_ref] Diagnostic performance of the Chinese frontal assessment battery in early cognitive impairment..., Chong [/bib_ref] , Digit Span forward score ≤ 5, Digit Span backward score ≤ 3, category fluency test score ≤ 5, and WMS-R Logical Memory II subtest raw score ≤ 8 for > 16 years of education, ≤ 4 for 8-15 years of education, and ≤ 2 for 0-7 years of education [bib_ref] Mini mental state examination and logical memory scores for entry into Alzheimer's..., Chapman [/bib_ref]. Depressive mood was assessed using the Geriatric Depression Scale 15 (GDS-15) [bib_ref] Development and validation of a geriatric depression screening scale: a preliminary report, Yesavage [/bib_ref] , with a score ≥ 10 indicating severe depressive mood [bib_ref] Depression in Japanese community-dwelling elderly--prevalence and association with ADL and QOL, Wada [/bib_ref]. All of the cognitive test results were reviewed by neuropsychologists.
# Statistical analysis
The Chi-squared test and Mann-Whitney's U test were used to investigate differences in characteristics between mild hyponatremia and normonatremia. One-way analysis of covariance (ANCOVA) was used to identify significant differences between the 2 groups in SMI, the 3 physical function test scores, and all the neuropsychological test scores after controlling for covariates. Then, multiple logistic regression analysis was conducted to examine the correlation between mild hyponatremia and sarcopenia, SMI, physical function test scores, and all the neuropsychological test scores. Cutoff scores for these dependent variables were based on the studies mentioned above. All the variables whose p-value was < 0.2 in [fig_ref] Table 1: Participant characteristicsValues are median [/fig_ref] were selected as covariates for both ANCOVA and multiple regression analysis as shown under Tables 3 and 4. All analyses were performed using SPSS Statistics for Windows ver. 26.0 (IBM Corp., Armonk, NY). A p-value < 0.05 was considered significant.
# Results
During the study period, 7580 individuals aged ≥70 years visited the NCGG Memory Disorder Outpatient Center. Of them, 3097 met the inclusion criteria: 8 had moderate or more severe hyponatremia (SNa < 130 mEq/L), 75 had mild hyponatremia (SNa 130-135 mEq/L), 2907 had normonatremia (SNa 136-145 mEq/L), and 107 had hypernatremia (SNa > 145 mEq/L). Thus, 75 participants with mild hyponatremia and 2907 with normonatremia were included in this study.
The characteristics of hyponatremia and normonatremia are summarized for all participants in [fig_ref] Table 1: Participant characteristicsValues are median [/fig_ref] [fig_ref] Table 2: Difference in physical and cognitive function between the groups [/fig_ref] , the mild hyponatremia group also had more cases of sarcopenia, weaker GS, slower WS, shorter OLS time, and higher GDS-15 score.
ANCOVA showed that mild hyponatremia had a sig- [fig_ref] Table 3: ANCOVA results for the comparison of skeletal mass index, physical function, and... [/fig_ref].
The results of multiple logistic regression using each cutoff score are shown in [fig_ref] Table 4: Results of logistic regression analysis for predicting sarcopenia, skeletal muscle mass, and... [/fig_ref]. After entering all variables written under [fig_ref] Table 4: Results of logistic regression analysis for predicting sarcopenia, skeletal muscle mass, and... [/fig_ref]
# Discussion
## Correlation between mild hyponatremia and smm and physical function
The mild hyponatremia group had significantly lower SMI, weaker GS, and slower WS, and nearly significantly shorter OLS time compared with the normonatremia group, even after controlling for covariates. When using the AWGS criteria for sarcopenia, the mild hyponatremia group had a significantly higher risk of sarcopenia and worse physical performance and impaired balance, and a marginally significant higher risk of worse muscle strength but was not at risk of having a lower SMI.
The present study demonstrated that elderly people with even mild hyponatremia had gait disturbances and balance impairment. Previous studies demonstrated that chronic hyponatremia causes gait disturbances in both rats [bib_ref] Chronic Hyponatremia causes neurologic and Psychologic impairments, Fujisawa [/bib_ref] and humans [bib_ref] Is asymptomatic hyponatremia really asymptomatic?, Decaux [/bib_ref]. The mechanism behind these observations is yet to be determined. However, for the mechanism whereby hyponatremia contributes to gait dysfunction and balance impairment, if we look at it from the viewpoint of the central nervous system (CNS), the brain cells adapt to hyponatremia by swelling with the loss of osmolytes, such as glutamate [bib_ref] Intracellular levels of glutamate in swollen astrocytes are preserved via neurotransmitter reuptake..., Schober [/bib_ref] , which is a neurotransmitter involved in gait and balance function [bib_ref] Mild prolonged chronic hyponatremia and risk of hip fracture in the elderly, Ayus [/bib_ref]. Rerelease of glutamate from the cytosol to extracellular space contributes to excessive activation of neuronal glutamate receptors, causing excitotoxic cell damage and death in the CNS [bib_ref] Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury, Arundine [/bib_ref]. However, it remains to be elucidated what severity of hyponatremia, and particularly whether mild hyponatremia, causes these consequences, which could lead to slow WS and balance dysfunction. On the other hand, we found that muscle strength as measured by GS was significantly different between the mild hyponatremia and normonatremia groups of elderly participants, but when using the AWGS cutoff value for sarcopenia, the logistic regression model showed that the association between mild hyponatremia and GS was not significant. Although Cairns et al. observed a 10% decrease in muscle strength when the sodium concentration was decreased from 147 to 60 mmol/L in mice [bib_ref] Changes of action potentials and force at lowered [Na+]o in mouse skeletal..., Cairns [/bib_ref] , Vandergheynst et al. did not observe any significant difference in GS when the sodium concentration was increased from 127.7 ± 2.5 mmol/L to 136.1 ± 1.8 mmol/L in humans [bib_ref] Impact of hyponatremia on nerve conduction and muscle strength, Vandergheynst [/bib_ref]. Mild hyponatremia itself is correlated with muscle strength, but it's impact on GS may be insufficient to bring the GS under the cutoff point.
Our study revealed that elderly individuals with even mild hyponatremia had poor physical function. A previous study reported that elderly patients with similar severity of hyponatremia were much more sensitive to alterations in gait tests compared with younger patients [bib_ref] Attention and postural balance are much more affected in older than in..., Renneboog [/bib_ref]. These findings imply that elderly individuals with even mild hyponatremia may be at risk of physical dysfunction.
In clinical practice, it is particularly important to know that the elderly with mild hyponatremia have low physical function because, as mentioned above, mild hyponatremia often goes unnoticed by patients and physicians and physicians are often hesitant to treat asymptomatic mild hyponatremia with SNa ≥130 mEq/L. However, a previous study reported a significant improvement in activities of daily living and cognition among geriatric patients effectively treated for hyponatremia [bib_ref] Impact of resolution of Hyponatremia on neurocognitive and motor performance in geriatric..., Brinkkoetter [/bib_ref]. Mild hyponatremia in the elderly deserves more of our attention. Additional longitudinal studies are needed to strengthen the evidence on the severity of hyponatremia that affects physical function in the elderly and the underlying mechanism.
Although we included many covariates, we might have missed some unmeasured or unknown potential confounders. In addition, we did not determine the causes or chronicity of mild hyponatremia. The duration of hyponatremia or severity of comorbidities may be additional risk factors for physical dysfunction or depressive mood. Hyponatremia could be a marker of severity in these unmeasured underlying illnesses and might signal the presence of physiologic derangements or comorbidities.
Regarding low muscle mass in elderly with mild hyponatremia, hyponatremia tended to lead to progressive SMM loss in rats [bib_ref] Chronic hyponatremia exacerbates multiple manifestations of senescence in male rats, Barsony [/bib_ref]. Hyponatremia has also been often observed in patients who are malnourished and in the elderly with sarcopenia [bib_ref] Sarcopenia as a potential cause of chronic hyponatremia in the elderly, Bertini [/bib_ref]. In the present study, although there was no significant difference in BMI between the elderly with mild hyponatremia and those with normonatremia, the elderly with mild hyponatremia might have had low protein intake and/or vitamin D deficiency. However, the impact of these factors on muscle strength may be insufficient to bring the SMI score under the cutoff point for mild hyponatremia.
## Association between mild hyponatremia and depressive mood
This study also found that mild hyponatremia was associated with depressive mood, but not with other neuropsychological tests scores. To our knowledge, this study is the first to report an association between mild hyponatremia and depressive mood in the elderly. Previously, the efficacy of tolvaptan was evaluated in patients with hyponatremia. Serum sodium concentrations increased more in the tolvaptan group than in the placebo group and a significant improvement was observed in the tolvaptan group on the Mental Component Summary (for vitality, social functioning, emotionally limited accomplishment, calmness, and sadness) of the Medical Outcomes Study 12-item Short-Form General Health Survey [bib_ref] Tolvaptan, a selective oral vasopressin V2-receptor antagonist, for hyponatremia, Schrier [/bib_ref]. The mechanism has yet to be determined, but some studies have suggested a correlation between depression and glutamate, whose release from brain cells to the extracellular space may be mediated by hyponatremia [bib_ref] Reduced glutamate in the anterior cingulate cortex in depression: an in vivo..., Auer [/bib_ref] [bib_ref] Plasma and platelet amino acid concentrations in patients affected by major depression..., Mauri [/bib_ref]. Anti-glutamate agents are known to have antidepressant effects in patients with depression [bib_ref] Beneficial effects of the antiglutamatergic agent riluzole in a patient diagnosed with..., Coric [/bib_ref]. Alternatively, elderly individuals with depressive mood might not have sufficient appetite to consume an appropriate amount of dietary sodium.
## Study strengths and limitations
This study used detailed neuropsychological test results and is the first to reveal a correlation between mild hyponatremia and depressive mood. A strength of the study is its large sample, which may have minimized many possible confounders, including pseudohyponatremia, many types of medication, and past histories. However, there are also some limitations. Despite the large sample size, few individuals with hyponatremia undertook the physical function tests, especially for WS. This left us with a small sample of mild hyponatremia, which did not allow us to adapt another mild hyponatremia cutoff, such as 134 mEq/L. Next, as mentioned above, we might have missed some unmeasured or unknown potential confounders; for example, we did not obtain enough data about locomotor system disease or endocrine disease to consider their potential effects. Lastly, this is a cross-sectional study and the specific mechanisms underlying the association of mild hyponatremia with physical function and depressive mood remain to be investigated.
# Conclusions
In this study, elderly participants with mild hyponatremia had impaired lower skeletal muscle mass, physical function, and depressive mood compared with elderly participants with normonatremia. Mild hyponatremia in the elderly deserves more attention than it currently receives. Additional longitudinal studies with a larger number of participants with hyponatremia are needed to strengthen the evidence on the severity of hyponatremia that affects physical and cognitive function in the elderly.
[table] Table 1: Participant characteristicsValues are median (interquartile range) or number (percentage). Differences were assessed using the Kruskal-Wallis test for continuous variables or the Chisquared test for categorical variables Abbreviations: BNP Brain natriuretic peptide, CRP C-reactive protein, eGFR Estimated glomerular filtration rate, NSAID Non-steroidal anti-inflammatory drug, NaSSA Noradrenergic and specific serotonergic antidepressant, SNRI Serotonin noradrenaline reuptake inhibitor, SSRI Selective serotonin reuptake inhibitors. a > 60 g/day of alcohol, b Hemoglobin < 11.0 g/dL, c thyroid-stimulating hormone ≤10μIU/mL, d vitamin B12 < 200 pg/mL [/table]
[table] Table 2: Difference in physical and cognitive function between the groups [/table]
[table] Table 3: ANCOVA results for the comparison of skeletal mass index, physical function, and psychological tests between the groups [/table]
[table] Table 4: Results of logistic regression analysis for predicting sarcopenia, skeletal muscle mass, and physical and cognitive functions CI Confidence interval, CKD Chronic kidney disease, FAB Frontal Assessment Battery, GDS-15 15-item Geriatric Depression Scale, HDS-R Hasegawa Dementia Rating Scale-Revised, MMSE Mini-Mental State Examination, NaSSA Noradrenergic and specific serotonergic antidepressant, OR Odds ratio, SNRI Serotonin noradrenaline reuptake inhibitor, SSRI Selective serotonin reuptake inhibitors, WMS-R Wechsler Memory Scale-Revised a Low skeletal muscle mass was defined as skeletal muscle mass index < 7.0 kg/m 2 in men and < 5.7 kg/m 2 in women b Reduced grip strength was defined as < 28 kg for men and < 18 kg for women c Raw WMS-R II score ≤ 8/9 for > 16 years of education, ≤ 4/5 for 8-15 years of education, ≤ 2/3 for 0-7 years of education [/table]
[bib_ref] Mild prolonged chronic hyponatremia and risk of hip fracture in the elderly, Ayus [/bib_ref]
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Biology and biotechnology of Trichoderma
[bib_ref] TrichoBLAST: a multilocus database for Trichoderma and Hypocrea identifications, Kopchinskiy [/bib_ref] [bib_ref] Phenotype microarrays for high-throughput phenotypic testing and assay of gene function, Bochner [/bib_ref] [bib_ref] Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian..., Kubicek [/bib_ref] [bib_ref] European species of Hypocrea. Part I. The green-spored species, Jaklitsch [/bib_ref] [bib_ref] European species of Hypocrea. Part I. The green-spored species, Jaklitsch [/bib_ref] [bib_ref] European species of Hypocrea. Part I. The green-spored species, Jaklitsch [/bib_ref] [bib_ref] Myths and dogmas of biocontrol, Harman [/bib_ref] [fig_ref] Figure 1: Characteristic features of Trichoderma spp [/fig_ref] [bib_ref] The giant Madagascar hissing-cockroach (Gromphadorhina portentosa) as a source of antagonistic moulds:..., Yoder [/bib_ref] [bib_ref] Bioaccumulation of mycotoxins by shellfish: contamination of mussels by metabolites of a..., Sallenave [/bib_ref] [bib_ref] Toxigenic saprophytic fungi in marine shellfish farming areas, Sallenave-Namont [/bib_ref] [bib_ref] Isolation and properties of carboxylesterases of the termite gut-associated fungus, Xylaria nigripes...., Sreerama [/bib_ref] [bib_ref] Enzyme production by recombinant Trichoderma reesei strains, Tulasne [/bib_ref] [bib_ref] Sexual development in the industrial workhorse Trichoderma reesei, Seidl [/bib_ref] [bib_ref] Light regulation of metabolic pathways in fungi, Tisch [/bib_ref] [bib_ref] Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn, Martinez [/bib_ref] [bib_ref] Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn, Martinez [/bib_ref] [bib_ref] The information highways of a biotechnological workhorse-signal transduction in Hypocrea jecorina, Schmoll [/bib_ref] [bib_ref] Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using..., Crom [/bib_ref] [bib_ref] Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of..., Seidl [/bib_ref]
## Tools for genetic manipulation of trichoderma
Due to the industrial application of T. reesei, the genetic toolkit for this fungus is the most extensive of the genus, although also research with other species is not limited by technical obstacles and most tools can also be used for all species with slight modifications. Transformation of many species is possible, and different approaches such as protoplasting , Agrobacterium-mediated transformation [bib_ref] Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation, Zeilinger [/bib_ref] , or biolistic transformation [bib_ref] Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic..., Lorito [/bib_ref] were developed. The range of selectable marker cassettes, which includes hygromycin [bib_ref] Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression..., Mach [/bib_ref] and benomyl resistance [bib_ref] Effect of benomyl and benomyl resistance on cellulase formation by Trichoderma reesei..., Peterbauer [/bib_ref] [bib_ref] Impact of light on Hypocrea jecorina and the multiple cellular roles of..., Schuster [/bib_ref] , the Aspergillus nidulans amdS gene, which enables growth on acetamide as sole nitrogen source [bib_ref] A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei, Penttila [/bib_ref] as well as the auxotrophic markers, pyr4 , arg2 [bib_ref] The arg2 gene of Trichoderma virens: cloning and development of a homologous..., Baek [/bib_ref] , and hxk1 [bib_ref] A novel carbon source-dependent genetic transformation system for the versatile cell factory..., Guangtao [/bib_ref] allows for construction of multiple mutants, which is now facilitated by the availability of a T. reesei strain with perturbed nonhomologous endjoining pathway . Sequential deletions despite a limited number of selection markers became possible by the use of a blaster cassette comprising direct repeats for homologous recombination and excision of the marker gene [bib_ref] Sequential gene deletions in Hypocrea jecorina using a single blaster cassette, Hartl [/bib_ref]. Besides knockout strategies for functional analysis of genes, also expression of antisense constructs for knockdown [bib_ref] Trichoderma atroviride G-protein alpha-subunit gene tga1 is involved in mycoparasitic coiling and..., Rocha-Ramirez [/bib_ref] [bib_ref] pH and Pac1 control development and antifungal activity in Trichoderma harzianum, Moreno-Mateos [/bib_ref] [bib_ref] The Galpha protein GNA3 of Hypocrea jecorina (anamorph Trichoderma reesei) regulates cellulase..., Schmoll [/bib_ref] was reported for Trichoderma, and RNAi has been shown to function in T. reesei [bib_ref] RNAi-mediated gene silencing of highly expressed genes in the industrial fungi Trichoderma..., Brody [/bib_ref]. Last but not least, the recent discovery of a sexual cycle in T. reesei [bib_ref] Sexual development in the industrial workhorse Trichoderma reesei, Seidl [/bib_ref] further boosts the versatility of this fungus for research and industry.
## Defense mechanisms and their exploitation
Successful colonization of a given habitat by any organism is crucially dependent on its potential to defend its ecological niche and to thrive and prosper despite competition for nutrients, space, and light. Many fungi and especially those of the genus Trichoderma are masters of this game [bib_ref] The biological control agent Trichoderma-from fundamentals to applications, Herrera-Estrella [/bib_ref] [bib_ref] Overview of mechanisms and uses of Trichoderma spp, Harman [/bib_ref] [bib_ref] Trichoderma-plant-pathogen interactions, Vinale [/bib_ref]. Their defense mechanisms comprise both enzymatic and chemical weapons, which make Trichoderma spp. efficient mycoparasites, antagonists, and biocontrol agents-characteristics that can be exploited by using Trichoderma spp. or the metabolites secreted by these fungi as biological fungicides to fight plant diseases caused by pathogenic fungi [bib_ref] Evaluation of Trichoderma spp. as a biocontrol agent against soilborne fungi and..., Spiegel [/bib_ref] [bib_ref] Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens..., Marra [/bib_ref] [bib_ref] Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures..., Navazio [/bib_ref]. Thereby Trichoderma spp. play an important role in the three-way interaction with the plant and the pathogen [bib_ref] In vivo study of Trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent..., Lu [/bib_ref] [bib_ref] The molecular biology of the interactions between Trichoderma spp., phytopathogenic fungi, and..., Woo [/bib_ref].
## Trichoderma's strategies for combat
After publication of Trichoderma lignorum (later found to be T. atroviride) acting as a parasite on other fungi in 1932 [bib_ref] Trichoderma lignorum as a parasite of other soil fungi, Weindling [/bib_ref] , research on antagonistic properties of Trichoderma spp. progressed rapidly. Nowadays, the most important species in this field are T. atroviride (in earlier reports sometimes misidentified as T. harzianum), T. harzianum, T. virens, and Trichoderma asperellum [bib_ref] Biocontrol mechanisms of Trichoderma strains, Benitez [/bib_ref] , while T. reesei, the biotechnological workhorse, can rather be seen as a model organism used because of the established molecular biological methods and available recombinant strains [bib_ref] Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require..., Seidl [/bib_ref]. Trichoderma spp. are able to control ascomycetes, basidiomycetes, and oomycetes [bib_ref] Understanding Trichoderma: between biotechnology and microbial ecology, Monte [/bib_ref] [bib_ref] Biocontrol mechanisms of Trichoderma strains, Benitez [/bib_ref] , and recently, also their effect on nematodes was reported [bib_ref] Biological control effects of Pochonia chlamysdosporia and Trichoderma isolates from Benin (West-Africa)..., Kyalo [/bib_ref] In their defensive actions, Trichoderma spp. apply lytic enzymes [bib_ref] Significance of lytic enzymes from Trichoderma spp. in the biocontrol of fungal..., Viterbo [/bib_ref] , proteolytic enzymes [bib_ref] Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum..., Suarez [/bib_ref] , ABC transporter membrane pumps [bib_ref] Identification of a new biocontrol gene in Trichoderma atroviride: the role of..., Ruocco [/bib_ref] , diffusible or volatile metabolites , and other secondary metabolites [bib_ref] Secondary metabolites from species of the biocontrol agent Trichoderma, Reino [/bib_ref] as active measures against their hosts or they succeed by their impairing growth conditions of pathogens [bib_ref] Biocontrol mechanisms of Trichoderma strains, Benitez [/bib_ref]. Interestingly, the success of these actions is not independent of the surrounding temperature [bib_ref] Effect of temperature on antagonistic and biocontrol potential of Trichoderma sp. on..., Mukherjee [/bib_ref] , which can be crucial for the use as a biocontrol agent in different climates. Large-scale studies on gene expression during biocontrol at least in part reflect these findings [bib_ref] Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens..., Marra [/bib_ref] [bib_ref] Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence..., Samolski [/bib_ref] and reveal additional components with potential effectivity such as a superoxide dismutase and amino acid oxidase [bib_ref] Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response..., Tseng [/bib_ref] to be secreted under these conditions. Moreover, the response of Trichoderma to its host has also been shown to involve stress response, response to nitrogen shortage, cross pathway control, lipid metabolism, and signaling processes [bib_ref] Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of..., Seidl [/bib_ref].
## Regulatory mechanisms triggering the defense of trichoderma
Signal transduction pathways triggering the genes involved in biocontrol and mycoparasitism have been studied in considerable depth and include heterotrimeric G-protein signaling, mitogen-activated protein kinase (MAPK) cascades, and the cAMP pathway [bib_ref] Trichoderma biocontrol: signal transduction pathways involved in host sensing and mycoparasitism, Zeilinger [/bib_ref]. Especially the MAP-kinase TVK1, characterized in T. virens [bib_ref] Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase, Mendoza-Mendoza [/bib_ref] [bib_ref] TmkA, a mitogen-activated protein kinase of Trichoderma virens, is involved in biocontrol..., Mukherjee [/bib_ref] [bib_ref] The MAP kinase TVK1 regulates conidiation, hydrophobicity and the expression of genes..., Mendoza-Mendoza [/bib_ref] as well as its orthologs in T. asperellum (TmkA; [bib_ref] Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic..., Viterbo [/bib_ref] and T. atroviride (TMK1; [bib_ref] Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk 1 differentially affects..., Reithner [/bib_ref] , is important in regulation of signaling mechanisms targeting output pathways relevant for efficient biocontrol. Transcript levels of the respective genes increased upon interaction with plant roots in T. virens and T. asperellum [bib_ref] Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic..., Viterbo [/bib_ref]. Deletion of T. atroviride tmk1 causes higher antifungal activity and improved protection against Rhizoctonia solani but reduced mycoparasitic activity [bib_ref] Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk 1 differentially affects..., Reithner [/bib_ref]. In agreement with this study, lack of T. virens TVK1 considerably increases biocontrol effectivity of this fungus [bib_ref] Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase, Mendoza-Mendoza [/bib_ref]. Hence, although deletion of the respective genes causes reduced mycoparasitic efficiency, the biocontrol abilities of the mutant strains are enhanced.
As for the action of the pathway of heterotrimeric Gprotein signaling, two genes have been studied so far with respect to biocontrol related mechanisms in Trichoderma spp.: the class I (adenylate cyclase inhibiting) G-alpha subunits TGA1 of T. atroviride and TgaA of T. virens as well as the class III (adenylate cyclase activating) G-alpha subunits TGA3 of T. atroviride and GNA3 of T. reesei. TGA1 plays an important role in regulation of coiling around host hyphae and regulates production of antifungal metabolites. Lack of TGA1 results in enhanced growth inhibition of host fungi [bib_ref] Trichoderma atroviride G-protein alpha-subunit gene tga1 is involved in mycoparasitic coiling and..., Rocha-Ramirez [/bib_ref] [bib_ref] The G protein alpha subunit Tga1 of Trichoderma atroviride is involved in..., Reithner [/bib_ref]. For TgaA, a host specific involvement as shown in case of the action of MAP-kinases has been reported [bib_ref] Role of two Gprotein alpha subunits, TgaA and TgaB, in the antagonism..., Mukherjee [/bib_ref]. TGA3 on the other hand is crucial for biocontrol since deletion of the corresponding gene resulted in avirulent strains . Since constitutive activation of GNA3 in T. reesei is suggested to positively influence mycoparasitism, a similar mechanism, may be at work in this fungus ). These results are in agreement with analysis of cAMP signaling components, which indicate a positive role of cAMP in biocontrol [bib_ref] cAMP signalling is involved in growth, germination, mycoparasitism and secondary metabolism in..., Mukherjee [/bib_ref]. Recently, also an important role in biocontrol of T. virens has been reported for the homolog of the VELVET protein, so far mainly known as light-dependent regulator protein [bib_ref] Regulation of morphogenesis and biocontrol properties in Trichoderma virens by a VELVET..., Mukherjee [/bib_ref].
Attempts were made to identify characteristics among all these genes and enzymes regulated upon interaction of Trichoderma with a pathogen, which could be used to distinguish efficient from nonefficient biocontrol strains isolated from nature [bib_ref] Identification of potential marker genes for Trichoderma harzianum strains with high antagonistic..., Scherm [/bib_ref]. However, only further, extensive studies will reveal the reliability of standardized marker gene assays for evaluation of potential biocontrol strains.
## Trichoderma as a protector of plant health
The beneficial action of Trichoderma spp. is not limited to fighting pathogens; they have also been shown to be opportunistic plant symbionts, enhancing systemic resistance of plants [bib_ref] Induction of defense responses in cucumber plants (Cucumis sativus L. ) By..., Yedidia [/bib_ref] [bib_ref] Induced systemic resistance and plant responses to fungal biocontrol agents, Shoresh [/bib_ref] , a response which is improved by ceratoplatanin family proteins [bib_ref] Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of..., Seidl [/bib_ref]. Perception of the signals transmitted by Trichoderma in the plant requires the function of a MAPK [bib_ref] Characterization of a mitogen-activated protein kinase gene from cucumber required for Trichoderma-conferred..., Shoresh [/bib_ref] , and also in the fungus itself, a MAPK signaling is crucial for full induction of systemic response in the plant [bib_ref] Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic..., Viterbo [/bib_ref]. By colonizing plant roots, which is significantly enhanced by swollenin [bib_ref] Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization, Brotman [/bib_ref] or invading them, they are also carried through soil and occupy new niches. This interaction with plants as well as their rhizosphere competence leads to enhanced root proliferation, better growth, and protection of the plants against toxic chemicals, against which Trichoderma spp. themselves show a remarkable resistance. Hence, these fungi are promising agents that can be applied for remediation of polluted soil and water by treatment of appropriate plants with spores [bib_ref] Uses of Trichoderma spp. to alleviate or remediate soil and water pollution, Harman [/bib_ref].
## Secondary metabolites
In order to survive and compete in their ecological niche, fungi apply not only enzymatic weapons but also have a potent arsenal for chemical warfare at their disposal [bib_ref] Trichoderma-plant-pathogen interactions, Vinale [/bib_ref]. Thereby, not only potential antibiotics (for example the peptaibols) but also mycotoxins and more than 100 metabolites with antibiotic activity including polyketides, pyrones, terpenes, metabolites derived from amino acids, and polypeptideswere detected in Trichoderma spp. and have been suggested to be used for chemotaxonomy of these species. However, the evolution of peptaibol formation seems to be too complex to allow for prediction of peptaibol production profiles form phylogenetic relationships [bib_ref] Facts and challenges in the understanding of the biosynthesis of peptaibols by..., Kubicek [/bib_ref] [bib_ref] Intact-cell MALDI-TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea:..., Neuhof [/bib_ref]. One of the first characterized secondary metabolites of Trichoderma spp. was the peptide antibiotic paracelsin . A wide variety of peptaibols was identified in [bib_ref] Characterisation of the peptaibiome of the biocontrol fungus Trichoderma atroviride by liquid..., Stoppacher [/bib_ref]. Interestingly, the four trichothecene mycotoxin-producing species (Trichoderma brevicompactum, Trichoderma arundinaceum, Trichoderma turrialbense, and Trichoderma protrudens) are not closely related to those species used in biocontrol, which not only means that the application of biocontrol in agriculture does not pose a risk in this respect but also indicates that these mycotoxins do not play a major role in the defense mechanisms of these fungi [bib_ref] Trichothecene production by Trichoderma brevicompactum, Nielsen [/bib_ref]. As many other fungi, also Trichoderma spp. have been shown to produce a broad array of volatile organic compounds, which recently have received closer attention [bib_ref] Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride..., Stoppacher [/bib_ref]. With respect to regulation of peptaibol biosynthesis in Trichoderma spp., several factors are known to be relevant. Environmental cues such as light, pH, nutrients, starvation, or mechanical injury impact this process. Efficient production of peptaibols predominantly occurs in solid cultivation and correlates with conidiation [bib_ref] Light regulation of metabolic pathways in fungi, Tisch [/bib_ref]. Signaling molecules involved range from the blue light photoreceptors BLR1 and BLR2 to the G-alpha subunits GNA3 (TGA3) and GNA1 as well as protein kinase A [bib_ref] Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated..., Komon-Zelazowska [/bib_ref]. Thereby, GNA3 is essential for peptaibol formation, but on the other hand, stimulation of peptaibol formation in the absence of BLR1 and BLR2 is still possible [bib_ref] Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated..., Komon-Zelazowska [/bib_ref].
## Trichoderma spp. as industrial workhorses
Shortly after the discovery of T. viride QM6a by the US army during World War II [bib_ref] History of the cellulase program at the U. S. Army Natick Development..., Reese [/bib_ref] , the outstanding efficiency of its cellulases led to extensive research toward industrial applications of these enzymes. Later on, this species was renamed T. reesei in honor of Elwyn T. Reeseand became the most important cellulase producer worldwide. Until now, this species is the most important one of the genus for industrial purposes.
## Cellulases and plant cell wall-degrading enzymes
Rising energy costs and the imminent climate change led to an increased attention to biofuel production [bib_ref] Genomics of cellulosic biofuels, Rubin [/bib_ref]. As a potent cellulase producer, research with T. reesei is nowadays particularly focused on improvement of efficiency of the enzyme cocktail produced in order to decrease overall costs of production of bioethanol from cellulosic waste material [bib_ref] Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives, Kumar [/bib_ref] , although applications in the pulp and paper industry and textile industry are also important. After the early mutation programs and strain improvement, the protein secretion capacity of industrial strains now reaches 100 g/l, with up to 60% of the major cellulase Cel7a (CBHI) and 20% of Cel6a (CBHII). High levels of cellulase and hemicellulase gene expression can be achieved upon cultivation on cellulose, xylan, or a mixture of plant polymers [bib_ref] Regulation of gene expression in industrial fungi: Trichoderma, Mach [/bib_ref] as well as on lactose [bib_ref] The Dxylose reductase of Hypocrea jecorina is the major aldose reductase in..., Seiboth [/bib_ref] , all of which are agricultural or industrial byproducts. The natural inducer of at least a subset of these enzymes is believed (yet not definitely proven) to be sophorose, a transglycosylation product of cellobiose [bib_ref] Induction of cellulolytic enzymes in Trichoderma reesei by sophorose, Sternberg [/bib_ref] [bib_ref] Transglycosylation products of cellulase system of Trichoderma reesei, Vaheri [/bib_ref]. Targeted strategies to further enhance the efficiency of the enzymes secreted include elucidation of regulatory mechanisms both at the promotor level [bib_ref] Regulation of gene expression in industrial fungi: Trichoderma, Mach [/bib_ref] [bib_ref] Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial..., Schmoll [/bib_ref] as well as with respect to signal transduction ). However, auxiliary components acting on the substrate could also enhance efficiency of its degradation [bib_ref] Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins,..., Saloheimo [/bib_ref].
Metabolic engineering in recent years provided intriguing insights into these processes ), and exploration of the genome sequence of T. reesei revealed that this industrial workhorse possesses the smallest amount of genes within Sordariomycetes encoding the enzymes which made it so popular-plant cell wall-degrading enzymes [bib_ref] Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn, Martinez [/bib_ref]. Availability of the genome sequence also spurred genome wide analysis of early mutant strains and identification of putatively beneficial mutations, which caused their high efficiency (Le [bib_ref] Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using..., Crom [/bib_ref]. Interestingly, it seems that even early mutants such as RutC-30 bear considerable alterations of their genome [bib_ref] The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85..., Seidl [/bib_ref]. These novel tools also facilitated characterization of the enzyme cocktails secreted by these strains [bib_ref] Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains, Herpoel-Gimbert [/bib_ref]. In addition to these efforts enzyme engineering approaches [bib_ref] Modeling cellulase kinetics on lignocellulosic substrates, Bansal [/bib_ref] , improvement of the secretion machinery [bib_ref] Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in..., Kruszewska [/bib_ref] as well as screening of the enormous variety of plant cell wall-degrading enzymes from nature isolates or other organisms secreting cellulases and directed evolution [bib_ref] Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei, Nakazawa [/bib_ref] complement the optimization of the regulatory mechanism of available production strains. Hence, with the aid of Trichoderma, economically reasonable production of second generation biofuels from waste products is on the way.
## Heterologous protein production
Filamentous fungi are versatile cell factories and frequently used for heterologous protein expression [bib_ref] Fungal biotechnology, Adrio [/bib_ref] , especially if they have generally regarded as safe status [bib_ref] Heterologous protein expression in filamentous fungi, Nevalainen [/bib_ref] , as has T. reesei [bib_ref] On the safety of Trichoderma reesei, Nevalainen [/bib_ref]. The industrial use of T. reesei as a producer of heterologous proteins started more than 20 years ago with the production of calf chymosin [bib_ref] A novel fungal expression system: secretion of active calf chymosin from the..., Harkki [/bib_ref] [bib_ref] Enzyme production by recombinant Trichoderma reesei strains, Tulasne [/bib_ref]. Shortly thereafter, even expression of immunologically active antibody fragments [bib_ref] Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei, Nyyssonen [/bib_ref] in T. reesei was achieved and numerous enzymes and performance proteins followed. Nowadays, T. reesei is one of the most commonly used filamentous fungi for heterologous protein production [bib_ref] Heterologous protein expression in filamentous fungi, Nevalainen [/bib_ref].
Based on the efficient expression as well as the considerable knowledge on regulation of cellulase genes, their promotors are routinely used for heterologous protein production [bib_ref] Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial..., Schmoll [/bib_ref]. Consequently, improvements in cellulase transcription are also beneficial for these applications. In many cases, the signal peptide of Cel7a (CBHI) is used to facilitate efficient secretion of the product into the culture medium. Nevertheless, also alternative promotors were also shown to be useful for certain applications [bib_ref] Production of recombinant proteins in the filamentous fungus Trichoderma reesei, Keränen [/bib_ref]. In general, the high efficiency and the inducibility of the cellulase promotors have proven beneficial in many applications. Using the cellulase promotors, also relatively cheap carbon sources such as cellulose or lactose can be used for production. Nevertheless, it must be considered that the large amount of enzymes secreted into the culture medium can be an issue in specific purification of the heterologous protein, and the complex substrates used could induce extracellular proteases, which are deleterious for the yield of the process [bib_ref] Production of recombinant proteins in the filamentous fungus Trichoderma reesei, Keränen [/bib_ref]. For further improvement, promotor modifications, for example with the chb1-promotor , can increase yields of the protein to be expressed.
## Food industry
With their long history of safe industrial scale enzyme production, Trichoderma spp. have also been extensively applied for production of food additives and related products [bib_ref] On the safety of Trichoderma reesei, Nevalainen [/bib_ref] [bib_ref] Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei:..., Blumenthal [/bib_ref]. Currently, various Trichoderma enzymes are applied to improve the brewing process (β-glucanases), as macerating enzymes in fruit juice production (pectinases, cellulases, hemicellulases), as feed additive in livestock farming (xylanases) and for pet food. Cellulases are mainly applied in baking, malting, and grain alcohol production . However, not only enzymes but also metabolites of Trichoderma spp. are used as additives. One of the first products isolated from T. viride was a chemical with characteristic coconut-like aroma, a 6-pentyl-α-pyrone with antibiotic properties, the production of which was constantly improved to reach concentrations of more than 7 g/L in extractive fermentation cultures in T. atroviride nowadays [bib_ref] Production of 6-pentyl-[alpha]-pyrone with Trichoderma atroviride and its mutant in a novel..., Oda [/bib_ref]. An interesting idea is the application of cell wall-degrading enzymes, for example of T. harzianum, as food preservatives because of their antifungal effect , but so far this suggestion has not found broad application. With a similar aim, T. harzianum mutanase can be used in toothpaste to prevent accumulation of mutan in dental plaque [bib_ref] Optimization of conditions for the efficient production of mutan in streptococcal cultures..., Wiater [/bib_ref].
## Black sheep in the genus trichoderma
In addition to the highly beneficial and frequently used species, the genus Trichoderma also comprises opportunis-tic human pathogens, which show efficient growth at body temperature and mycoparasitic species, which are a significant threat to mushroom farms.
## Human pathogenic species
Besides such long-known and well-studied pathogenic fungi as Candida, Aspergillus, or Crypotcoccus, also the genus Trichoderma comprises opportunistic human pathogens, which pose a serious and often lethal threatespecially to HIV-infected persons and other immunocompromised patients. Belonging to the emerging fungal pathogens, these fungi are often not recognized or diagnosed in a stadium when efficient treatment is problematic [bib_ref] Infections due to emerging and uncommon medically important fungal pathogens, Walsh [/bib_ref]. Trichoderma species have been reported to cause respiratory problems due to volatile organic compounds they produce [bib_ref] Volatile organic compounds from the indoor mould Trichoderma viride cause histamine release..., Larsen [/bib_ref] , but more importantly, they can infect immunocompromised patients (Trichoderma citrinoviride, T. harzianum, and Trichoderma longibrachiatum and Hypocrea orientalis) after transplantations or suffering from leukemia or HIV [bib_ref] Clinical importance of the genus Trichoderma. A review, Kredics [/bib_ref]. The typically poor prognosis of such infections is (besides delayed diagnosis) predominantly due to the low susceptibility of these fungi to commonly used antifungal agents [bib_ref] In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials..., Kratzer [/bib_ref] , which often necessitates combined treatment with different drugs [bib_ref] In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials..., Kratzer [/bib_ref] [bib_ref] Invasive pulmonary infection due to Trichoderma longibrachiatum mimicking invasive Aspergillosis in a..., Alanio [/bib_ref]. Nevertheless, few data on investigation of virulence factors of these fungi are available [bib_ref] Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains, Kredics [/bib_ref]. Among the clinical isolates, T. longibrachiatum and H. orientalis are the most common ones. Interestingly, no specific phylogenetic characteristics of the clinical isolates as compared to environmental isolates could be found, and no correlation between virulence or pathogenicity and genomic structure was detected [bib_ref] Intraspecific mitochondrial DNA polymorphism within the emerging filamentous fungal pathogen Trichoderma longibrachiatum, Antal [/bib_ref]. However, most intriguingly, T. longibrachiatum not only causes disease; at the same time, it seems to be a source for potential antifungal drugs efficient against Candida and Aspergillus species [bib_ref] Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum, Vicente [/bib_ref].
## Green mold disease
Cultivation of the edible mushrooms Agaricus bisporus (champignon) and Pleurotus ostreatus (oyster mushrooms) on mushroom farms all over the world is of considerable economic importance. In the 1980s, a mixture of strains at first identified as T. harzianum was found to cause deleterious infections in these farms [bib_ref] Infection of mushroom compost by Trichoderma species, Seaby [/bib_ref] with losses between 30% and 100%. Actually, these strains represented two new species, Trichoderma aggressivum fsp. europeae and T. aggressivum fsp. aggressivum . Since then, this "green mold disease" has spread all over the world [bib_ref] Genetically closely related but phenotypically divergent Trichoderma species cause green mold disease..., Komon-Zelazowska [/bib_ref] and was shown to be mainly caused by Trichoderma pleurotum, Trichoderma pleuroticola in P. ostreatus [bib_ref] Two new species of Trichoderma associated with green mold of oyster mushroom..., Park [/bib_ref] , and T. aggressivum in A. bisporus. Nevertheless, also T. harzianum, T. longibrachiatum, Trichoderma ghanense, T. asperellum, and T. atroviride have been detected in Agaricus compost and Pleurotus substrates [bib_ref] Green mold diseases of Agaricus and Pleurotus spp. are caused by related..., Hatvani [/bib_ref] , but aggressive colonization of the substrate has not been proven for these species. This threat to commercial mushroom production has recently also led to the development of methods for rapid and specific detection of these fungi in cultivation substrates [bib_ref] Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates..., Kredics [/bib_ref]. A similar objective led to the development of a key for identification of Trichoderma species commonly associated with commercially grown mushrooms [bib_ref] Genetic comparison of the aggressive weed mould strains of Trichoderma harzianum from..., Muthumeenakshi [/bib_ref].
## Future prospects
More than ever before sustainable economy and protection of our environment are dominant topics in our everyday life and one alarming report about contaminated landscapes or catastrophes caused by climate change follows another. Today, 87% of energy used in the world comes from nonrenewable sources like natural gas, oil, and coal [bib_ref] Progress and challenges in enzyme development for biomass utilization, Merino [/bib_ref]. Although biofuel production is now being pushed in order to decrease the requirement for fossil fuels, the raw materials therefore originate from commodities and land also needed for food. In this respect, production of the so-called second generation biofuels from agricultural waste products by the aid of cellulases and hemicellulases produced for example by T. reesei and fermentation of the resulting oligosaccharides by yeast provides an alternative strategy. However, for an economically competitive process an increase in efficiency of more than 40-fold would be necessary, which is a formidable challenge for research with Trichoderma.
Sustainability is also the major driving force for investigation of biocontrol with Trichoderma. As opportunistic plant symbionts and effective mycoparasites, numerous species of this genus have the potential to become commercial biofungicides. The challenge in this field of research will be the development of reliable screening techniques, which allow for prediction of the biocontrol efficiency of a given isolate by determination of the key factors for this process. Nevertheless, also the ecological effects of widespread application of a single (or few) fungal species in agriculture remain to be investigated in order to ensure a truly beneficial effect for the environment.
Besides these major applications of Trichoderma spp., also the fields of green and white biotechnology become increasingly important for environmentally safe production of enzymes and antibiotics. These industrial applications will also benefit from studies on molecular physiology and regulatory processes, which continuously reveal novel and valuable metabolites and enzymes as well as components to be modified or adjusted for cost effective high yield production.
Last but not least, the extensive studies on diverse physiological traits available and still progressing for Trichoderma make these fungi versatile model organisms for research on both industrial fermentations as well as natural phenomena. (2006)
[fig] Figure 1: Characteristic features of Trichoderma spp. a T. reesei and b T. atroviride growing on plates, c T. reesei or H. jecorina growing in daylight and showing light responsive conidiation, d fruiting body formation of T. reesei upon crossing with a nature isolate of H. jecorina, (e, f) T. longibrachiatum germinating and growing on human cells, g, i T. reesei (left) during confrontation with Pythium ultimum (right), h T. atroviride (left) during confrontation with R. solani (right) [/fig]
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Long‐term safety and tolerability of oral tofacitinib in patients with Crohn’s disease: results from a phase 2, open‐label, 48‐week extension study
Background:Tofacitinib is an oral, small molecule Janus kinase inhibitor that is being investigated for inflammatory bowel disease.Aims: This 48-week open-label extension study primarily investigated long-term safety of tofacitinib 5 and 10 mg b.d. and secondarily investigated efficacy as maintenance therapy in patients with Crohn's disease.Methods: Patients who had completed the phase 2b maintenance study, or withdrawn due to treatment failure, were enrolled. Patients in remission (Crohn's disease activity index <150) at baseline received tofacitinib 5 mg b.d.; all others received 10 mg b.d. A single dose adjustment was allowed after 8 weeks' fixed, open-label treatment. Results: Sixty-two patients received tofacitinib 5 mg b.d.; 88 received 10 mg b.d. Both groups had similar rates of adverse events and serious infections. Crohn's disease worsening was the most frequent adverse event for tofacitinib 5 (33.9%) and 10 mg b.d. (19.3%). Patients not in remission at baseline, receiving 10 mg b.d., had higher rates of serious adverse events (19.3%) and discontinuation attributed to insufficient clinical response (30.7%) vs 5 mg b.d. (8.1% and 9.7%, respectively). At week 48, of patients with baseline remission receiving 5 mg b.d., 87.9% maintained remission and 75.0% sustained remission as observed (46.8% and 38.7%, respectively, by non-responder imputation). Study design prevented between-dose efficacy comparisons.Conclusions:No new safety signals emerged. Although both doses showed generally similar safety outcomes for overall adverse events, serious adverse events were more frequent for tofacitinib 10 than 5 mg b.d. Discontinuation due to insufficient clinical response was lower among patients in remission at baseline. ClinicalTrials.gov:NCT01470599.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
# | introduction
Crohn's disease is a chronic, progressive inflammatory disease of the gastrointestinal tract 1 that has a significant impact on patients' quality of life. [bib_ref] Quality of life in inflammatory bowel disease, Love [/bib_ref] Current therapies for Crohn's disease include corticosteroids, thiopurines, methotrexate, anti-tumour necrosis factor α antibodies, anti-integrin antibodies and anti-p40 antibodies. [bib_ref] American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in..., Lichtenstein [/bib_ref] However, not all patients respond to these medications, leaving an unmet need for novel therapies. [bib_ref] Characterizing unmet medical need and the potential role of new biologic treatment..., Gordon [/bib_ref] Tofacitinib is an oral, small molecule Janus kinase inhibitor approved in several countries for the treatment of ulcerative colitis.
It has also been investigated for Crohn's disease. The efficacy and safety of tofacitinib for inducing and maintaining clinical remission (defined as Crohn's disease activity index score <150) in patients with moderate-to-severe Crohn's disease have previously been investigated in two phase 2b studies. [bib_ref] Tofacitinib for induction and maintenance therapy of Crohn's disease: results of two..., Panés [/bib_ref] showed clinical response-100 (defined as a CDAI score reduction of at least 100 points from baseline) or remission vs placebo, although these differences were also not statistically significant. [bib_ref] Tofacitinib for induction and maintenance therapy of Crohn's disease: results of two..., Panés [/bib_ref] Here, we present results of a phase 2b open-label extension study that evaluated the safety and tolerability of tofacitinib, and exploratory efficacy over 48 weeks of treatment and 4 weeks of follow-up. Guidelines. All patients provided written informed consent.
## | materials and methods
## | study design
## | study patients
To be eligible for this study, patients with Crohn's disease had either completed the 26-week, double-blind, maintenance study or had withdrawn from the maintenance study after meeting pre-specified treatment failure criteria. Maintenance study treatment failure was defined as meeting both of the following criteria on two consecutive visits, at least 2 weeks apart, in patients who had completed ≥4 weeks of treatment in the maintenance study: an increase in CDAI ≥ 100 points from maintenance study baseline value and an absolute CDAI score ≥175 points. Patients with active (draining) fistulae, intra-abdominal or perineal collection, or abscess were excluded. Full details of eligibility criteria and prohibited concomitant medications are included in Files S3 and S4.
All patients who withdrew early or completed the open-label extension study received a safety follow-up visit approximately 4 weeks after the last study medication dose. For patients who discontinued, study procedures were completed as per end of treatment/early termination.
## | primary objective: safety outcomes
The primary objective was to investigate the long-term safety and tolerability of tofacitinib 5 and 10 mg b.d. in patients with Crohn's disease during the 48-week study and the 4-week follow-up. Safety outcomes included incidence and severity of adverse events, classified by Medical Dictionary for Regulatory Activities version 19.0 coding.
Abnormalities and changes from baseline were monitored for clinical laboratory values, vital sign measurements, electrocardiogram measurements and clinically significant changes in physical examination.
Safety endpoints of special interest were adjudicated, including major adverse cardiovascular events, hepatic injury, opportunistic infection, malignancy, gastrointestinal perforation and interstitial lung disease.
There was a histopathological central over-read of specimens from potential malignancies, and the malignancy adjudication committee reviewed clinical and histopathological results. Safety outcomes were assessed at baseline, weeks 8, 16, 24, 36, 48 and 52. The 4-week safety follow-up visit at week 52 represented the end of the study.
## | secondary objective and secondary efficacy outcomes
The secondary objective was to investigate the effects of tofacitinib maintenance therapy on clinical remission, patient quality of life, and biomarkers measured by C-reactive protein and faecal calprotectin, through 48 weeks.
Secondary binary efficacy outcomes included clinical remission, sustained clinical remission (achieved clinical remission at both weeks 24 and 48) and steroid-free clinical remission at week 48 among patients receiving steroids at baseline. Other endpoints included CDAI scores over time, time to relapse, serum high-sensitivity C-reactive protein and faecal calprotectin over time, changes from baseline, and corticosteroid use. Relapse was defined as an increase in CDAI of >100 points from baseline, and an absolute CDAI score of >220 points. The proportion of patients who relapsed, and the time to relapse, was analysed for patients in clinical remission at baseline who initially received tofacitinib 5 mg b.d. Also evaluated was the proportion of patients switching (at the investigator's discretion) from tofacitinib 5 to 10 mg b.d., or vice versa. Efficacy outcomes were monitored through 48 weeks, to end of treatment, or to early termination for those who discontinued the study.
## | patient-reported outcomes
The following patient-reported outcomes were collected during this open-label extension study: the Inflammatory Bowel Disease Questionnaire (IBDQ), [bib_ref] A new measure of health status for clinical trials in inflammatory bowel..., Guyatt [/bib_ref]
## | statistical analyses
Descriptive summary statistics, including two-sided 95% confidence intervals for efficacy, biomarkers and patient-reported outcome endpoints, were calculated based on observed data (no imputation of missing data) by initially assigned dose group from the full analysis . A greater proportion of patients assigned 10 mg b.d. were taking corticosteroids and had higher mean CDAI scores and higher median C-reactive protein and faecal calprotectin concentrations, compared with those receiving 5 mg b.d. . Ileocolonic disease location was recorded in a numerically higher proportion of patients receiving tofacitinib 10 mg b.d. than 5 mg b.d.
# | results
## | patients
## | safety outcomes
The proportion of patients with adverse events was 79.0% and 76.1% for tofacitinib 5 and 10 mg b.d., respectively ( Discontinuation due to insufficient clinical response was more frequent for patients who entered the study not in remission, receiving 10 mg b.d. (30.7%), compared with 5 mg b.d. (9.7%). By investigator decision, some cases of discontinuation due to insufficient clinical response did not have an associated adverse event of worsening Crohn's disease recorded, and one patient discontinued due to an adverse event of worsening Crohn's disease but did not have this specified as a reason for insufficient clinical response. Laboratory values meeting predetermined laboratory criteria for re-test or discontinuation were similarly low for both tofacitinib groups.
During this study, there were no deaths, adjudicated cases of cardiovascular events, interstitial lung disease or hepatic injury, or malignancy (excluding non-melanoma skin cancer [NMSC]).
One case of basal cell carcinoma of the skin was reported in the F I G U R E 2 Patient disposition diagram. Safety analyses patient population. Includes enrolled patients who received ≥1 dose of study medication. N represents the total number of patients in each group; n represents the number of patients with events. † Dose assignment was determined based on last available haematocrit result prior to open-label extension study entry. Baseline CDAI scores were recalculated using baseline haematocrit results received after treatment assignment was determined. Therefore, one patient in the tofacitinib 5 mg b.d. group and four patients in the 10 mg b.d. group had remission status that did not correspond to their initial dose assignment. b.d., twice daily; CDAI, Crohn's disease activity index
## | exploratory efficacy outcomes
This study had no primary efficacy endpoints; all efficacy endpoints, patient-reported outcomes and biomarkers were exploratory end- The adverse event of worsening Crohn's disease leading to discontinuation was termed insufficient clinical response. Only patients who were not in remission were assigned tofacitinib 10 mg b.d. Infections that were reported as a serious adverse event are described in the
## | patient-reported outcomes
For patients who entered the study in remission receiving tofacitinib 5 mg b.d., the majority (range: 69.5%-81.0%) showed high (≥170) IBDQ total scores at baseline, weeks 8, 24 and 48 . At week 48, mean change from baseline IBDQ total score (−2.8) and
individual IBDQ domains showed a general maintenance of values over time, with change from baseline range −3.1-0.1 (see .
At week 48, small mean changes from baseline were observed for SF-36v2 domains (−0.2 and −1.2 for PCS and MCS, respectively), and for EQ-5D/VAS score (1.2). For PRTI assessment, 66.7% and 33.3% of patients reported being "extremely satisfied" or "satisfied," respectively, with their study drug, 88.1% of patients reported they preferred their study drug, and 83.3% reported they would use the same drug again.
In the tofacitinib [bib_ref] Direct and indirect effects of tofacitinib on treatment satisfaction in patients with..., Panés [/bib_ref] Sustained clinical remission was defined as remission at both weeks 24 and 48. Relapse was defined as an increase in CDAI of >100 points from baseline, and an absolute CDAI score of >220 points. Patients who discontinued due to lack of efficacy were treated as a relapse event, with an event time at the time of drop out. All data represent full analysis set. N represents the total number of patients analysed, n represents the number of patients achieving clinical remission or sustained clinical remission. . Substantial improvements (37. [bib_ref] Evaluation of the meaningfulness of health-related quality of life improvements as assessed..., Coteur [/bib_ref] For the PRTI assessment, 32.6% and 41.3% of patients reported being "extremely satisfied" or "satisfied" with their study drug, respectively. 56.5% of patients reported they preferred their study drug, and 60.9% reported they would use the same drug again.
# | discussion
No new or unexpected safety signals were observed during this study. Rates of adverse events were generally similar for patients receiving tofacitinib 5 and 10 mg b.d. and were consistent with the previous 26-week maintenance study. [bib_ref] Tofacitinib for induction and maintenance therapy of Crohn's disease: results of two..., Panés [/bib_ref] In the maintenance study, Crohn's disease worsening was also the most frequently occurring adverse event, 5 although rates were lower than those reported here.
Patient-years of tofacitinib exposure in the maintenance study were In this open-label extension study, rates of serious adverse events were higher with tofacitinib 10 mg b.d. than 5 mg b.d., and compared with rates observed in the maintenance study. [bib_ref] Tofacitinib for induction and maintenance therapy of Crohn's disease: results of two..., Panés [/bib_ref] The majority of patients receiving tofacitinib reported positive outcomes of "extremely satisfied" and "satisfied" for the PRTI assessments of patient satisfaction.
Proportions of patients responding positively were slightly higher for 5 mg b.d. than 10 mg b.d.
This study had several limitations. Only patients not in remission were assigned tofacitinib 10 mg b.d.; therefore, as the two patient populations had different disease activity at the outset, this precluded definitive comparison of efficacy outcomes between doses.
Patients were permitted to switch doses after 8 weeks of receiving their initially assigned study dose and, as such, efficacy and safety outcomes were analysed by initial dose assignment, not taking dose changes into account. Additionally, this was an open-label study, without a control group.
In summary, no new safety signals were observed through
week 52 of this open-label extension study of tofacitinib in patients with Crohn's disease. Rates of adverse events were generally similar among both groups, and were consistent with rates observed in the previous tofacitinib Crohn's disease maintenance study. There were higher rates of serious adverse events and discontinuation due to adverse events in the tofacitinib 10 mg b.d. group. However, as patients were only assigned tofacitinib 10 mg b.d. if they were not in remission at baseline and therefore had more active disease than those receiving 5 mg b.d., and because dose switching was permitted, direct comparisons between dose groups were not conducted.
Patients in remission at open-label extension study entry had a lower rate of discontinuation due to insufficient clinical response than those not in remission at study entry. Exploratory efficacy analyses at week 48 appeared to show that many patients receiving tofaci-
## | data sharing statement
Upon request, and subject to certain criteria, conditions and exceptions (see https://www.pfizer.com/science/clinical-trials/trial-dataand-results for more information), Pfizer will provide access to individual de-identified participant data from Pfizer-sponsored global interventional clinical studies conducted for medicines, vaccines and medical devices (a) for indications that have been approved in the US and/or EU or (b) in programmes that have been terminated (ie, development for all indications has been discontinued). Pfizer will also consider requests for the protocol, data dictionary and statistical analysis plan. Data may be requested from Pfizer trials 24 months after study completion. The de-identified participant data will be made available to researchers whose proposals meet the research criteria and other conditions, and for which an exception does not apply, via a secure portal. To gain access, data requestors must enter into a data access agreement with Pfizer.
## Acknowledg ements
Declaration of personal interests: The authors would like to thank the patients, investigators and study teams who were involved in this study. J. Panés has received consulting fees from AbbVie, Amgen, Boehringer Ingelheim, Celgene, Ferring, Genentech/Roche, GSK, Janssen, MSD, Oppilan, Pfizer Inc, Robarts, Shire, Takeda, Theravance and TiGenix. G. R. D'Haens has served as an advisor for
[fig] F: I G U R E 4 A, Mean CDAI score through week 48, and mean changes from baseline at week 48 for B, CDAI, C, C-reactive protein and D, faecal calprotectin, for tofacitinib 5 mg b.d. All data represent full analysis set (observed). N represents the total number of patients analysed at each time point, n represents the number of patients with data at week 48 or at baseline and week 48. b.d., twice daily; CDAI, Crohn's disease activity index; SD, [/fig]
|
The Prevailing Catalytic Role of Meteorites in Formamide Prebiotic Processes
Meteorites are consensually considered to be involved in the origin of life on this Planet for several functions and at different levels: (i) as providers of impact energy during their passage through the atmosphere; (ii) as agents of geodynamics, intended both as starters of the Earth's tectonics and as activators of local hydrothermal systems upon their fall; (iii) as sources of organic materials, at varying levels of limited complexity; and (iv) as catalysts. The consensus about the relevance of these functions differs. We focus on the catalytic activities of the various types of meteorites in reactions relevant for prebiotic chemistry. Formamide was selected as the chemical precursor and various sources of energy were analyzed. The results show that all the meteorites and all the different energy sources tested actively afford complex mixtures of biologically-relevant compounds, indicating the robustness of the formamide-based prebiotic chemistry involved. Although in some cases the yields of products are quite small, the diversity of the detected compounds of biochemical significance underlines the prebiotic importance of meteorite-catalyzed condensation of formamide.
## The numerous biogenic functions of meteorites
Upon their arrival on the surface of this planet, meteorites deliver part of their energy into the atmosphere, with the rest being released at the impact event. The materials provided to the impact site are both a source of substrates for further reactions endowed with possible biogenic relevance, and a potential source of catalytic activity, or both. The fact that meteorites are as a whole a very complex system, prevents generalization of this list of properties and means that different meteorites are considered differently. For example, an iron-nickel meteorite can hardly be a source of organic material but it can be a source of energy and catalysis, while a chondrite could represent, in the first place, a source of catalysis and organics.
We focus here on the catalytic properties exerted by meteoric materials in the prebiotic chemistry of formamide. However, meteorite-dependent catalysis cannot be treated separately from the other functions exerted by meteorites. Hence, we firstly summarize these other functions whilst trying to sketch a general frame.
## Meteorites are providers of impact energy during their passage through the atmosphere
The first modelling of this phenomenon from a prebiotic perspective was conducted by Chyba and Sagan [bib_ref] Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules-An inventory for..., Chyba [/bib_ref]. Part of their model has recently found theoretical and experimental support in the work of Ferus et al. [bib_ref] High-energy chemistry of formamide: A unified mechanism of nucleobase formation, Ferus [/bib_ref] [bib_ref] Formation of nucleobases in a Miller-Urey reducing atmosphere, Ferus [/bib_ref] [bib_ref] High energy radical chemistry formation of HCN-rich atmospheres on early, Ferus [/bib_ref]. and Sagan calculated that the organic syntheses driven by impact shocks were important sources of organic molecules on the early Earth, in addition to the direct delivery by extraterrestrial bodies and to the organic syntheses by energy sources such as UV or electrical discharges. The quantities of organics produced by the Heavy Bombardment (around 3.8 Gy ago) were calculated to correspond to the quantities of organics produced by other energy sources. Particularly relevant, among these organics, was HCN. On this basis, shown [bib_ref] High energy radical chemistry formation of HCN-rich atmospheres on early, Ferus [/bib_ref] that HCN can be produced during impact events by the reprocessing of carbonaceous and nitrogenous materials from both the impactor and the atmosphere. The investigation of high-energy events on a range of model starting mixtures shows the production of highly reactive cyano radicals and excited CO as the most abundant products initially. The subsequent reactions of these first formed excited species lead to the production of HCN and of its hydration product, formamide NH 2 CHO. In addition, it was shown [bib_ref] High-energy chemistry of formamide: A unified mechanism of nucleobase formation, Ferus [/bib_ref] that the high-energy synthesis of nucleic bases from formamide can occur during the impact of an extraterrestrial body through the initial dissociation of the formamide molecule producing large amounts of CN and NH radicals. These radicals can further react with formamide to produce nucleic bases. Chyba and Sagan's idea that the highly reactive triple-bonded HCN has a propensity to form when organic matter is challenged with sources of high energy [bib_ref] Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules-An inventory for..., Chyba [/bib_ref] , and that this energy can be provided by impactors as meteorites [bib_ref] Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules-An inventory for..., Chyba [/bib_ref] , has thus found experimental evidence [bib_ref] High-energy chemistry of formamide: A unified mechanism of nucleobase formation, Ferus [/bib_ref] [bib_ref] Formation of nucleobases in a Miller-Urey reducing atmosphere, Ferus [/bib_ref] [bib_ref] High energy radical chemistry formation of HCN-rich atmospheres on early, Ferus [/bib_ref].
## Meteorites as agents for geodynamism
Starters of Earth's tectonics. Plate tectonics allows chemicals essential to life to flow from the Earth's interior to the surface. Analysis of the characteristic titanium isotopes of shales dating to 3.5 Gy has shown that more than half of the continental crust was made of felsic rocks originated from subduction. Felsic rocks like granite form in the presence of heat and water in subduction events, and are indicative of plate tectonics. This datation is half a billion years earlier than expected from the temperature of the young Earth, too hot at that time to drive plate tectonics. Simulation modelling [bib_ref] Impact-driven subduction on the Hadean Earth, O'neill [/bib_ref] shows that a large single impact could trigger a subduction zone that remains active for 10 million years, providing a solution to this apparent Hadean-times contradiction. Given the known strong incidence of impacts between 4 and 3.5 Gy ago, intermittent and frequent meteorite-induced early subduction events are explained. A planet characterized by high geochemical activity was probably a more favorable scenario for life than a steady setting.
Activators of local hydrothermal systems. This role has been pointed out by G. Osinski and collaborators [bib_ref] Impact-generated hydrothermal systems on Earth and Mars, Osinski [/bib_ref] and combines well with the geothermal waters scenario envisaged by A. Y. Mulkidjanian et al. [bib_ref] Origin of first cells at terrestrial, anoxic geothermal fields, Mulkidjanian [/bib_ref] as a shrine for life, for which evidence has been provided [bib_ref] Earliest signs of life on land preserved in ca. 3.5 Ga hot..., Djokic [/bib_ref]. Osinski first reported evidence that most impact events resulting in the formation of complex impact craters (i.e., >2-4 and >5-10 km diameter on Earth and Mars, respectively) were potentially capable of generating a hydrothermal system. The longevity and size of the hydrothermal systems can be evaluated based on the impact crater record and on the impact-generated hydrothermal deposits. These show the strong chemical activity generated at impact sites. It was suggested that hydrothermally altered and precipitated rocks can provide nutrients and habitats for a life-long period of time after hydrothermal activity has ceased. Mulkidjanian et al. presented a comprehensive discussion and data on the advantages of hydrothermal systems as sites where prebiotic phenomena could have occurred [bib_ref] Origin of first cells at terrestrial, anoxic geothermal fields, Mulkidjanian [/bib_ref] , essentially favored by the physical-chemical conditions characterizing these type of environments.
A key property of geothermal systems is their long-term stability and the possibility of generating proficient energy cycles [bib_ref] Earliest signs of life on land preserved in ca. 3.5 Ga hot..., Djokic [/bib_ref].
## As providers of simple organic biogenic materials, the role of meteorites varies enormously
The biogenic materials can be at first an approximation of two types: the simpler forms of atom aggregations, devoid of elaborated chemical information, like CO, CO 2 , H 2 O, NH 3 , and CH 4 , and the products of the chemical reactions occurred during the history of each meteorite starting from these simple compounds, like nucleobases, amino acids, sugars, and hydrocarbons, representing the next step of chemical complexity. Both types of materials profoundly differ among meteorites for their composition, for their relative abundances, and for the average complexity, as expected from the fact that meteorites are of the most diverse origin, composition, and history [bib_ref] High molecular diversity of extraterrestrial organic matter in Murchison meteorite revealed 40..., Schimitt-Kopplin [/bib_ref] [bib_ref] Understanding prebiotic chemistry through the analysis of extraterrestrial amino acids and nucleobases..., Burton [/bib_ref].
## As catalysts
The starting material selected for testing the catalytic properties of different types of meteorites was formamide. In prebiotic chemistry, formamide can be considered a substrate, a solvent, and a catalyst itself. Summarizing the reasons for the prebiotic interest of formamide:
Simplicity. Formamide is a central compound in the one-carbon atom chemistry domain. HCN is one of the most abundant one-carbon atom compounds in space [bib_ref] Molecular and atomic line surveys of galaxies. I: The dense, star-forming gas..., Geach [/bib_ref] , where its stability is due to the low temperature. The reaction of HCN with water leads to formamide NH 2 CHO. Formamide is also the product of the reaction between formic acid and ammonia at temperatures lower than 200-150 - C [bib_ref] A model gas study of ammonium formate, methanamide and guanidinium formate as..., Krocher [/bib_ref]. Noteworthy, formic acid was the major species observed in the Miller-Urey type experiments [bib_ref] Organic compounds synthesis on the primitive Earth, Miller [/bib_ref]. The formation of formamide in a Miller-Urey-type experimental set-up [bib_ref] Formation of nucleobases in a Miller-Urey reducing atmosphere, Ferus [/bib_ref] [bib_ref] Miller experiments in atomistic simulation, Saitta [/bib_ref] and in atmosphere/meteorite impact chemistry [bib_ref] Formation of nucleobases in a Miller-Urey reducing atmosphere, Ferus [/bib_ref] has been shown.
Formamide is a logical starting point for reconstituting prebiotic syntheses because of its availability, stability, and solvent properties.
Availability. Formamide is largely available in the Universe, both far and close to us. It has been detected in interstellar space [bib_ref] Observations of interstellar formamide: Availability of a prebiotic precursor in the galactic..., Adande [/bib_ref] [bib_ref] Shedding light on the formation of the pre-biotic molecule formamide with ASAI, Lopez-Sepulcre [/bib_ref] [bib_ref] Microwave detection of interstellar space, Rubin [/bib_ref] , in star-forming regions, and in comets [bib_ref] Complex organic molecules in comets C/2012 F6 (Lemmon) and C/2013 R1 (Lovejoy):..., Biver [/bib_ref]. The concentration of formamide in the Hadean Earth has the problem of a difficult solution, because it largely depends on the pathway of its formation, on the environment considered, and on the scenario selected; arbitrariness in this matter is high. However, the relevance of this problem greatly decreases considering the possibility of natural methods for its concentration, as thermovection [bib_ref] Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases, Niether [/bib_ref] , and considering the fact that geothermal volcanic scenarios are more likely [bib_ref] Origin of first cells at terrestrial, anoxic geothermal fields, Mulkidjanian [/bib_ref] [bib_ref] Earliest signs of life on land preserved in ca. 3.5 Ga hot..., Djokic [/bib_ref] than ocean-based scenarios. The absence of oceans in the earliest times decreases the severity of the formamide-related "concentration" problem. It has been shown that formamide-based condensation reactions may occur in the presence of substantial amounts of water [bib_ref] The key role of meteorites in the formation of relevant prebiotic molecules..., Rotelli [/bib_ref] [bib_ref] Formamide-based prebiotic chemistry in the Phlegrean fields, Botta [/bib_ref]. Data for the formation pathway of formamide under a space condition are reported in reference [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref].
Stability. Formamide is stable as a liquid between 4 and 210 - C [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref] [bib_ref] Genetics first or metabolism first? The formamide clue, Saladino [/bib_ref] with limited azeotropic effects. The stability in water is high (t1 /2 = 199 years at 25 - C and 7.3 days at 120 under neutral pH) compared to other HCN derivatives [bib_ref] Aspects of the hydrolysis of formamide: Revisitation of the water reaction and..., Slebocka-Tilk [/bib_ref].
Properties as a solvent. In this respect, formamide is particularly interesting as a possible prebiotic solvent alternative to water. Formamide is a polar solvent that may promote the synthesis of all types of prebiotic precursors [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref] [bib_ref] Genetics first or metabolism first? The formamide clue, Saladino [/bib_ref] , the prebiotically relevant solubilization of phosphates [bib_ref] Nucleoside phosphorylation by phosphate minerals, Costanzo [/bib_ref] , and the phosphorylation [bib_ref] Nucleoside phosphorylation by phosphate minerals, Costanzo [/bib_ref] [bib_ref] Nucleosides and deoxynucleoside phosphorylation in formamide solutions, Schoffstall [/bib_ref] and the polymerization of activated monomers [bib_ref] Generation of RNA molecules by base catalyzed click-like reaction, Costanzo [/bib_ref]. The earliest Earth had little or no free water [bib_ref] Dehydration melting at the top of the lower mantle, Schmandt [/bib_ref]. Upon accumulation following its release and/or delivery, water revealed itself as mostly deleterious to unprotected biogenic precursors. The "Water Paradox" [bib_ref] Is there a common chemical model for life in the universe?, Benner [/bib_ref] [bib_ref] Paradoxes in the origin of life, Benner [/bib_ref] is reasonably only solved by "Non-aqueous-Solvents first". When it entered the scene, water most probably acted as an effector of selection, pipelining evolution based on the preferential survival of water-resistant phenotypes (as are nucleic acids polymers relative to nucleic acids monomers [bib_ref] Origin of informational polymers: Differential stability of 3 -and 5 -phosphoester bonds..., Saladino [/bib_ref] [bib_ref] Origin of Informational Polymers: Differential Stability of Phosphoester Bonds in Ribo Monomers..., Saladino [/bib_ref] on the organization of semi-permeable membranes-contained systems.
## State of the art about the synthetic capacity of formamide with terrestrial catalysts
A large body of previous studies has established that in the presence of catalysts of terrestrial origin, formamide affords nucleic bases (the five biologically extant ones, and others), numerous carboxylic acids, several aminoacids, condensing agents, long-chained aliphatic compounds, and a large body of miscellanea [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref] [bib_ref] Genetics first or metabolism first? The formamide clue, Saladino [/bib_ref].
The catalysts analyzed were calcium carbonate, alumina, silica, zeolite (Y type), titanium dioxide, clays of the montmorillonite family, cosmic dust analogues (CDAs) of terrestrial olivines (from fayalite to forsterite), mineral phosphates, iron-sulphur and iron-copper-sulphur minerals, zirconium minerals, and borate minerals. Some of these compounds are also components of meteorites. The results showed that all the catalytic systems analyzed were active, yielding mixtures of nucleobases, aminoacids, carboxylic acids, sugars, and condensing agents, varying according to the catalyst. Results revealing the robustness of formamide chemistry are reviewed in [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref] [bib_ref] Genetics first or metabolism first? The formamide clue, Saladino [/bib_ref]. Given that the purpose of these analyses was aimed at terrestrial scenarios, the condensation reactions of formamide with terrestrial minerals were routinely performed with thermal energy, and a variety of possible geochemical scenarios were modelled [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref] [bib_ref] Genetics first or metabolism first? The formamide clue, Saladino [/bib_ref] , including serpentinization-related chemical gardens [bib_ref] A global scale scenario for prebiotic chemistry: Silica-based self-assembled mineral structures and..., Saladino [/bib_ref].
## The catalytic activity of meteorites
Following a preliminary study on the chondrite Murchison [bib_ref] Catalytic effects of Murchison material: Prebiotic synthesis and degradation of RNA precursors, Saladino [/bib_ref] , we considered the catalytic role of several types of meteorites in the chemistry of formamide using different sources of energy. The purpose of analyzing differently-powered reactions was to start exploring which scenario(s), among the plausible ones, could be fruitfully evaluated experimentally. The energies tested were: thermal [bib_ref] The key role of meteorites in the formation of relevant prebiotic molecules..., Rotelli [/bib_ref] [bib_ref] Formamide-based prebiotic chemistry in the Phlegrean fields, Botta [/bib_ref] [bib_ref] Meteorites as Catalysts for Prebiotic Chemistry, Saladino [/bib_ref] , proton irradiation from accelerated Helium [bib_ref] Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under..., Saladino [/bib_ref] , and high-energy radiation from accelerated Boron [bib_ref] First evidence on the role of heavy ion irradiation of meteorites and..., Saladino [/bib_ref].
The meteorites were used as powered fractions of the original sample (generally, 0-125 micro meter, with or without a pre-treatment aimed to remove eventually present endogenous organics) and selected as representative of the four major classes (iron, stony-iron, chondrites, achondrites), keeping the number of relevant sub-classes reasonably limited. Thus, comprehensive series of syntheses were performed analyzing the differently-powered catalytic activities of large panels of meteorites. Noteworthy, the meteorites performed in a very similar way with or without pre-treatments, suggesting that the mineralogical modifications eventually occurring during the pre-treatments do not significantly modify the selectivity and specificity of the formamide condensation [bib_ref] Meteorites as Catalysts for Prebiotic Chemistry, Saladino [/bib_ref].
The synthetic reactions under thermal energy were typically performed in closed vessels at 160 - C (or lower, down to 80 - C), as described in the numerous publications reviewed in [bib_ref] Current advances in prebiotic chemistry under space conditions, Carota [/bib_ref] [bib_ref] Genetics first or metabolism first? The formamide clue, Saladino [/bib_ref]. The irradiation experiments were performed at the Accelerators of the Joint Institute for Nuclear Research (Dubna, Russia), as described in [bib_ref] Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under..., Saladino [/bib_ref] [bib_ref] First evidence on the role of heavy ion irradiation of meteorites and..., Saladino [/bib_ref].
As a general conclusion, the syntheses catalyzed by meteorites in formamide are more complex and higher-yield than the corresponding ones obtained with terrestrial catalysts. However, given the complexity of the systems analyzed, exceptions are numerous and this conclusion can only be validated case-by-case (see below). Summarizing the various systems analyzed.
## Thermal energy-triggered condensations catalyzed by iron, stony-iron, chondrites, and achondrites
The treatment of powdered materials from 12 different types of meteorites of the four classes, preliminarily deprived of the possibly present organics, was performed at low (60 - C) and high (140 - C) temperatures [bib_ref] Meteorites as Catalysts for Prebiotic Chemistry, Saladino [/bib_ref]. Canyon-Diablo, Campo-del-Cielo, and Sikhote-Alin were the three types of Iron meteorites analyzed; Seymchan and NWA 4482 were the two Stony-Iron; NWA 2828, Gold Basin, Dhofar 959, Murchison, and NWA 1465 the five Chondrites; and NWA 5357 and Al-Haggounia the two Achondrites. References, composition, historical and terrestrial provenience, and cosmo-origin data of these meteorites are available in [bib_ref] Meteorites as Catalysts for Prebiotic Chemistry, Saladino [/bib_ref].
The results showed the one-pot synthesis of an astonishingly reach and composite panel of compounds of prebiotic interest, encompassing heterocycles (nucleic bases), carboxylic acids, amino acids, and a rich miscellanea of low-molecular-weight compounds including potential condensing agents like carbodiimide and urea. Remarkably, among the recovered nucleic bases, uracil, cytosine, adenine, guanine, and the closely related species isocytosine, dihydrouracil, and hypoxanthine, were observed. Among the amino acids we detected glycine, alanine, valine, leucine, and phenylalanine. Carboxylic acids were observed up to C-9 (nonanoic acid). In the conditions tested, the only recovered product in the absence of meteorites was purine; for a detailed analysis and discussion of the chemical aspects involved and of the meteorite-type selectivity observed, see [bib_ref] Meteorites as Catalysts for Prebiotic Chemistry, Saladino [/bib_ref]. The general order of reactivity was iron and stony-iron > achondrite and chondrite. The same condensation reactions were performed with terrestrial catalysts as the reference (troilite, pyrrothite, pyrite, chalcopyrite, volcanic basalt, olivine, hydrotalcite). The comparison showed that meteorites were more efficient catalysts than simple terrestrial catalysts, highlighting the presence of synergistic effects between the different mineralogical components of the meteorite. The yield and the composition of the products varied as a function of the meteorite, but the panel of compounds obtained was in each case a composite and representative of a potential "prebiotic soup". This may be taken as an indication that meteorite-catalyzed syntheses could have occurred in several of the early Earth environments for which prebiotic scenarios have been proposed, as: hydrothermal alkaline vents [bib_ref] The origin of membrane bioenergetics, Lane [/bib_ref] , anoxic geothermal fields [bib_ref] Origin of first cells at terrestrial, anoxic geothermal fields, Mulkidjanian [/bib_ref] , or less defined "warm little ponds" scenarios. In addition to the interest per se, this set of data provides a basis of comparison with the data obtained when exposing the same synthetic system to high-energy proton irradiation.
## Proton irradiation-triggered condensations catalyzed by iron, stony-iron, chondrites, and achondrites
A set of meteorites similar to that analyzed for thermal energy-driven reactions (including in this new study Chelyabinsk, which had fallen in the meantime) was analyzed for reactions occurring at a low temperature under irradiation with accelerated protons, thus mimicking the solar wind/flare radiation [bib_ref] Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under..., Saladino [/bib_ref]. Note that a recent study suggests that solar flares could play a remarkable role in shaping the composition of the atmosphere of the primordial Earth [bib_ref] Prebiotic chemistry and atmospheric warming of early Earth by an active young..., Airapetian [/bib_ref]. As for thermal energy-driven reactions, the synthesis of a large panel of molecules was observed. The products isolated from the reaction mixtures were carboxylic acids as long as C20 (arachidic acid); amino acids (glycine, alanine, proline); nucleic bases (uracil, thymine, cytosine, adenine and guanine, among others); and, notably, sugars (ribose, 2 -deoxyribose, glucose, 2 -deoxyglucose, among others).
Most notably, four nucleosides, namely cytidine, uridine, adenosine, and thymidine, were synthesized. The detailed mechanism involved in the selective formation of the β-glycosidic bond has been reported for the reaction catalyzed by the chondrite NWA 1465. In summary, sugars and nucleic bases are synthesized separately from formamide by a radical cyanide (- CN) based mechanism [bib_ref] Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under..., Saladino [/bib_ref] , and they can then be regio-and stereo-selectively connected through the formation of a C1 sugar-centred radical intermediate. Formation of the β-glycosidic bond, that so far required complex laboratory procedures and external interventions, may thus occur in a one-pot reaction.
These results show that, as an alternative to heat, radiation is a plausible and powerful energy source for prebiotic processes [bib_ref] Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under..., Saladino [/bib_ref] [bib_ref] First evidence on the role of heavy ion irradiation of meteorites and..., Saladino [/bib_ref] [bib_ref] Irradiation of pyrimidine in pure H 2 O ice with high-energy ultraviolet..., Nuevo [/bib_ref]. As a general trend, stony-iron, chondrite (with the only exception of Chelyabinsk), and achondrite meteorites were more active than iron meteorites. The major conclusion from this analysis is that meteorites perform as catalysts under proton irradiation conditions better than terrestrial minerals, as exemplified by the high yields obtained, and by the one-pot synthesis of molecules as complex as ribo-and 2 -deoxy ribonucleosides.
## Heavy ion-triggered condensations catalyzed by stony-iron and chondrites
The energy source of 11 B-boron beams was studied because of the interstellar abundance of boron in our Galaxy, and because of its presence in cosmic rays near the Earth [bib_ref] Galactic cosmic ray Origin of Li, Be and B in stars, Reeves [/bib_ref] [bib_ref] Cosmic abundance of Boron, Cameron [/bib_ref] [bib_ref] Nucleosynthesis of 11 B-rich boron in the pre-solar cloud recorded in meteoritic..., Chausidon [/bib_ref]. As for the reactions with Heat and Protons as energy sources, a large panel of compounds was also observed in this case, including purine and pyrimidine nucleic bases (uracil, cytosine, adenine, and guanine), nucleic base analogues, heterocycles, and carboxylic acids involved in metabolic pathways [bib_ref] First evidence on the role of heavy ion irradiation of meteorites and..., Saladino [/bib_ref]. The presence of amino imidazole carbonitrile (AICN), 4,6-diamino purine (4,6-DAP), and 2,4-diamino pyrimidine (2,4-DAPy) among the observed products suggested the occurrence of unified mechanisms based on the generation of radical cyanide species (- CN). The case of differential purine versus pyrimidine nucleobase synthesis depending on the type of meteorite and of radiation used [fig_ref] Figure 1: The 11 B and Proton irradiation-powered synthesis of nucleobases from formamide in... [/fig_ref] is noteworthy. With 11 B beams, a lower variety of products was detected relative to heat and protons, amino acids, and nucleosides not being observed in the reaction mixtures, probably due to the different penetration depth of the ions. On the other hand, Chelyabinsk and Dhofar 959 produced higher amounts of nucleic bases, probably due to the higher linear energy transfer (LET) value, as discussed in. The differential synthesis of carboxylic acids as a function of the meteorite used is shown in [fig_ref] Figure 2: The 11 B and Proton irradiation-powered synthesis of carboxylic acids from formamide... [/fig_ref]. Taken together, these data suggest that the Cosmic Radiation high-energy particles can act as an efficient energy source for meteorite-catalyzed prebiotic syntheses. used is shown in [fig_ref] Figure 2: The 11 B and Proton irradiation-powered synthesis of carboxylic acids from formamide... [/fig_ref]. Taken together, these data suggest that the Cosmic Radiation high-energy particles can act as an efficient energy source for meteorite-catalyzed prebiotic syntheses. used is shown in [fig_ref] Figure 2: The 11 B and Proton irradiation-powered synthesis of carboxylic acids from formamide... [/fig_ref]. Taken together, these data suggest that the Cosmic Radiation high-energy particles can act as an efficient energy source for meteorite-catalyzed prebiotic syntheses.
## Thermal energy-triggered condensations catalyzed by chondrites in formamide/water mixtures
General considerations [bib_ref] Origin of first cells at terrestrial, anoxic geothermal fields, Mulkidjanian [/bib_ref] and geopaleontological findings [bib_ref] Earliest signs of life on land preserved in ca. 3.5 Ga hot..., Djokic [/bib_ref] lead us to select a model based on geothermal fields onto whose waters, we may imagine, meteorites abundantly impacted for several hundred million years, carrying their load of endogenous and impact-related organics, providing energy and catalytic capacity locally. We have thus analyzed the catalytic effects of six different chondrites on the synthetic capacity of formamide and of formamide/water mixtures [bib_ref] The key role of meteorites in the formation of relevant prebiotic molecules..., Rotelli [/bib_ref] [bib_ref] Formamide-based prebiotic chemistry in the Phlegrean fields, Botta [/bib_ref]. The chondrites were ALH 84028, EET 92042, MIL 05024, LAR 04318, GRO 95551, and GRO 95566. The waters tested were pure water, seawater, and thermal waters from the Bagnaccio volcanic area (Viterbo, Italy) and from the Phlegrean Fields (Naples, Italy). In this last instance, the meteorite NWA 4482 was analyzed. The results showed abundant syntheses, in spite of the fact that the role of water in prebiotic chemistry is controversial due to its nucleophilic character inducing solvolysis reactions and to the fact that both HCN and formamide are degraded in water. Mixtures of water and formamide afforded amino acids, nucleic bases, and carboxylic acids, as did the other systems described above. Volcanic thermal waters were the most active system for the catalytic activity of meteorites. These data hint to the important role that chondrites could have played in the early Earth subjected to the Heavy Bombardment, detailing that the mineral components that eventually reached the ground could have promoted the catalysis of organics in the hydrous and oxidizing environment that they encountered.
## Impact-triggered synthesis of nucleobases and their precursors in the presence of meteorites
Ferus et al. have studied the catalytic effect of meteorites on the formation of various nucleic bases from formamide in an extraterrestrial impact simulated with laser-zapping experiments [bib_ref] High-energy chemistry of formamide: A simpler way for nucleobase formation, Ferus [/bib_ref]. In contrast to iron-nickel meteorites, chondritic meteorites (i.e., NWA 6472) have shown a good catalytic performance, enabling the formation of all four RNA nucleobases. When the irradiation was performed in the presence of an iron-nickel meteorite (Campo del Cielo), only cytosine, uracil, and guanine were detected among the reaction products. The lower performance of iron-nickel meteorites in the impact experiments was interpreted by the radical scavenging ability of iron. reports the general overview of the products obtained from formamide depending on the type of energy source and meteorite. . Products obtained from formamide in the presence of different energy sources and types of meteorites.
## Products
Ref.
## Reaction type meteorite type
Nucleobases and their analogs: uracil, cytosine, adenine, guanine, isocytosine, dihydrouracil and hypoxanthine. Aminoacids: glycine, alanine, valine, leucine, phenylalanine. Carboxylic acids: from oxalic acid (C-2) up to nonanoic acid (C-9). Condensing agents: carbodiimide and urea.
[38]
Condensation process under thermal energy conditions in formamide
# Conclusions
The overall result of the meteorite-catalyzed condensations of formamide [bib_ref] The key role of meteorites in the formation of relevant prebiotic molecules..., Rotelli [/bib_ref] [bib_ref] Formamide-based prebiotic chemistry in the Phlegrean fields, Botta [/bib_ref] [bib_ref] Catalytic effects of Murchison material: Prebiotic synthesis and degradation of RNA precursors, Saladino [/bib_ref] [bib_ref] Meteorites as Catalysts for Prebiotic Chemistry, Saladino [/bib_ref] [bib_ref] Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under..., Saladino [/bib_ref] [bib_ref] First evidence on the role of heavy ion irradiation of meteorites and..., Saladino [/bib_ref] is that in each condition tested, independent of the type of meteorite involved, the panel of compounds obtained is variegate and abundant. In addition, formamide in the presence of meteorites condenses into a plethora of prebiotic compounds, independent of the type of energy driving the reactions. A detailed analysis of the results is only possible analyzing the data reported in the specific studies on a case-by-case basis. However, the results tell us that the robustness of the formamide-meteorite chemistry is so marked that it makes it impossible to prefer one single scenario over the others. The conclusion is that looking for the components of the shrine of life, one has to look not for a single exclusive scenario, but for the one that was more prone to facilitate the next steps into complexity.
The key lesson that we learned from this ensemble of analyses is that, from a prebiotic perspective, the chemistry of formamide is very robust. From a "chance versus necessity" perspective, these first steps are thermodynamically necessary. Given the variety of energies tested, the disparate provenience of the catalysts analyzed, and the fact that the reactions may occur in pure formamide and in formamide/water mixtures (water being pure or variously mineralized), these results show that the same reactions are prone to occur in a very large set of environments. This reinforces the vision that from the point of view of prebiotic chemistry, this planet is not particularly special. The steps that follow these initial prebiotic stages, the steps leading towards complex life, are a different matter.
The key players in this logic are meteorites. Their provenience is by definition exogenous, which entails a heuristically relevant consideration: meteorites provide (in addition to minerals which are common components of this planet) catalysts which are not present on Earth. Elementally equal minerals may have very different crystal structures, explaining the observed different catalytic activities. Also relevant is that the combination of the components of the meteorites depend upon their non-terrestrial geological history.
Meteorites have formed in a number of different conditions that never occurred on Earth, enriching the catalogue of chemical possibilities beyond our terrestrial local and historically determined possibilities. This explains the interest in the observation that the large majority of products obtained through their catalysis is the same that can be obtained with terrestrial catalysts. This highlights the fact that the chemistry of HCN and of its accumulation-prone derivative formamide is likely to be met everywhere.
[fig] Figure 1: The 11 B and Proton irradiation-powered synthesis of nucleobases from formamide in the presence of meteorites. The amount of recovered nucleobase is given as μg obtained from 1.0 mL of formamide. The reactions were carried out in the presence of the indicated meteorite (A). (B-D) Show that the different type of radiations yield different compounds. Data from[39,40]. [/fig]
[fig] Figure 2: The 11 B and Proton irradiation-powered synthesis of carboxylic acids from formamide in the presence of meteorites. Conditions as inFigure 1. Data from[39,40]. [/fig]
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IRIS: a method for reverse engineering of regulatory relations in gene networks
Background:The ultimate aim of systems biology is to understand and describe how molecular components interact to manifest collective behaviour that is the sum of the single parts. Building a network of molecular interactions is the basic step in modelling a complex entity such as the cell. Even if gene-gene interactions only partially describe real networks because of post-transcriptional modifications and protein regulation, using microarray technology it is possible to combine measurements for thousands of genes into a single analysis step that provides a picture of the cell's gene expression. Several databases provide information about known molecular interactions and various methods have been developed to infer gene networks from expression data. However, network topology alone is not enough to perform simulations and predictions of how a molecular system will respond to perturbations. Rules for interactions among the single parts are needed for a complete definition of the network behaviour. Another interesting question is how to integrate information carried by the network topology, which can be derived from the literature, with largescale experimental data.Results:Here we propose an algorithm, called inference of regulatory interaction schema (IRIS), that uses an iterative approach to map gene expression profile values (both steady-state and timecourse) into discrete states and a simple probabilistic method to infer the regulatory functions of the network. These interaction rules are integrated into a factor graph model. We test IRIS on two synthetic networks to determine its accuracy and compare it to other methods. We also apply IRIS to gene expression microarray data for the Saccharomyces cerevisiae cell cycle and for human B-cells and compare the results to literature findings.Conclusions: IRIS is a rapid and efficient tool for the inference of regulatory relations in gene networks. A topological description of the network and a matrix of gene expression profiles are required as input to the algorithm. IRIS maps gene expression data onto discrete values and then computes regulatory functions as conditional probability tables. The suitability of the method is demonstrated for synthetic data and microarray data. The resulting network can also be embedded in a factor graph model.
# Background
Although all cells of an organism contain the same DNA, each cell only transcribes and translates a fraction of that DNA. Each cell has a particular interaction pattern that involves genes, proteins and molecules; these complex schema are known as gene regulatory networks. Full understanding of gene interactions can be used to identify methods to control the behaviour of genes directly involved in disease processes. Methods used to infer patterns of interaction among molecular components from observed data are called reverse engineering algorithms. Even if gene-gene interactions only partially describe real networks because of post-transcriptional modifications and protein regulation, using microarray technology it is possible to combine measurements for thousands of genes into a single analysis step that provides a picture of cell gene expression. Therefore, many reverse engineering algorithms rely on gene expression data [bib_ref] Using Bayesian Networks to Analyze Expression Data, Friedman [/bib_ref]. These methods differ in the type of expression profiles used, so there are algorithms for time-series data [bib_ref] D: Inference of Gene Regulatory Networks and Compound Mode of Action from..., Bansal [/bib_ref] , algorithms for steady-state data [bib_ref] Reverse Engineering of Regulatory Networks in Human B Cells, Basso [/bib_ref] [bib_ref] ARACNE: an Algorithm for the Reconstruction of Gene Regulatory Networks in a..., Margolin [/bib_ref] and algorithms that work on both types of data [bib_ref] Advances to Bayesian Network Inference for Generating Causal Networks from Observational Biological..., Yu [/bib_ref]. A graphical representation of the gene regulatory network is often used, with Bayesian networks probably the most popular graphical models used in this scenario [bib_ref] Using Bayesian Networks to Analyze Expression Data, Friedman [/bib_ref] [bib_ref] Advances to Bayesian Network Inference for Generating Causal Networks from Observational Biological..., Yu [/bib_ref] [bib_ref] Estimation of Genetic Networks and Functional Structures Between Genes by Using Bayesian..., Imoto [/bib_ref] [bib_ref] Dynamic Bayesian Network and Nonparametric Regression for Nonlinear Modeling of Gene Networks..., Kim [/bib_ref] [bib_ref] Inferring Subnetworks from Perturbed Expression Profiles, Pe'er [/bib_ref]. The major limitation of Bayesian networks is that they cannot represent cyclic structures. To overcome this limitation, methods based on dynamic Bayesian networks have been proposed [bib_ref] Dynamic Bayesian Multinets, Blimes [/bib_ref] [bib_ref] Learning the Structure of Dynamic Probabilistic Networks, Friedman [/bib_ref] [bib_ref] Linear Modeling of Genetic Networks from Experimental Data, Someren [/bib_ref] [bib_ref] Applying Dynamic Bayesian Networks to Perturbed Gene Expression Data, Dojer [/bib_ref].
However, the network topology alone is not enough to perform simulations and predictions of how a molecular system will respond to perturbations. The rules for interactions among the single parts are needed for a complete definition of the network behaviour. Another interesting question is how to integrate the information carried by the network topology, which can be derived from the literature, with large-scale experimental data. Much attention has recently been focused on the modelling [bib_ref] MetaReg: a Platform for Modeling, Analysis and Visualization of Biological Systems Using..., Ulitsky [/bib_ref] and inference [bib_ref] Structural Systems Identification of Genetic Regulatory Networks, Xiong [/bib_ref] of activation rules between molecular components in the cell. Although this can be considered a simpler problem than inference of the network topology, it is important to point out the following:
- Many interaction patterns between molecular components can be obtained from the literature (using, for example, databases such as Ingenuity Pathway Analysis) and integrated in the inference algorithm.
- Owing to the limited amount of experimental data, it can be convenient to solve a simplified problem and exploit as much as possible prior knowledge about the phenomenon being investigated.
- Modelling of the interaction pattern between molecular components can be useful when performing large-scale simulations and deriving hypotheses about the behaviour of biological systems under different conditions [bib_ref] MetaReg: a Platform for Modeling, Analysis and Visualization of Biological Systems Using..., Ulitsky [/bib_ref].
In the present study we follow this research direction. However, instead of considering continuous expression levels, as in [bib_ref] Structural Systems Identification of Genetic Regulatory Networks, Xiong [/bib_ref] , we use a discrete representation of gene activation. Indeed, methods based on graphical models often work on discrete data obtained from real-valued gene expression profiles. The discretisation step is of fundamental importance for good accuracy of subsequent computational steps. Ślęzak and Wróblewski proposed a discretisation approach based on rough set theory in which quality functions are used for roughly discretised data, with inexact dependence between attribute rankings [bib_ref] Rough Discretization of Gene Expression Data, Ślęzak [/bib_ref]. Friedman et al. considered two different approaches to discretise real-valued data [bib_ref] Using Bayesian Networks to Analyze Expression Data, Friedman [/bib_ref]. In the first approach they discretised expression levels to several discrete states according to a fixed discretisation rule and demonstrated that this approach is sensitive to the discretisation procedure. In the second method they combined a linear regression model with the model dependence and measurements, but this approach was strongly affected by the linear dependence. Pe'er et al. proposed a new discretisation procedure for each gene in which gene-specific variation is used to estimate the normal distribution mixture by standard k-means clustering [bib_ref] Inferring Subnetworks from Perturbed Expression Profiles, Pe'er [/bib_ref]. A probabilistic approach is used to identify interactions between genes, such as activation and inhibition, so this method provides a description of the gene network with the interaction features, but many of these interactions are undirected. Moreover, the discretisation step is sensitive to the choice of the number of states that a gene may attain. In this paper we propose a new discretisation approach that depends on the expression profile data (both time-course and steady-state values) for each gene, so that different genes with different expression profiles lead to different discretisation rules. Indeed, we also use this approach to reduce the effect of noise.
After discretisation, the problem faced is how to infer the rules, and not just the pattern, by which the various molecular components interact with each other. We call this problem the inference of regulatory relations given a well-specified gene regulatory network. Gat-Viks et al. proposed an approach to learn improved regulatory functions from high-throughput data using a discrepancy score in which discretisation is carried out as a preprocessing step [bib_ref] Modeling and Analysis of Heterogeneous Regulation in Biological Networks, Gat-Viks [/bib_ref] , but the discretisation rules must be determined and tuned rather arbitrarily and each variable is discretised using the same rule. Gat-Viks et al. also proposed a more flexible approach to learn the regulatory functions in a gene network [bib_ref] A Probabilistic Methodology for Integrating Knowledge and Experiments on Biological Networks, Gat-Viks [/bib_ref] , which is represented as a factor graph [bib_ref] Factor Graphs and the Sum-Product Algorithm, Kschischang [/bib_ref] to model cyclic structures. In this approach discretisation is carried out according to an expectation maximisation (EM) algorithm that, combined with the factor graph model, provides a very flexible discretisation scheme. However, in practice this flexibility can lead to over fitting and may decrease learnability, so the authors suggested to use the same or a few discretisation schemes for all the variables. Chuang et al. proposed an approach to infer gene relations from time-course expression profiles in which first and second derivatives are used to detect time-lagged correlated gene pairs. The basic assumption is that pairs of correlated genes exhibit either a complementary pattern (that represents a repressor relation) or a similar pattern (that represents an activator relation). In our approach we propose a simple regulatory function inference that is particularly fast and yields rather good accuracy, even if the network is cyclic. In this step we use an observation similar to that of Chuang et al.for expression profile patterns, but here we use discrete data. Finally, we merge the inferred regulatory functions into a factor graph representation as reported by Gat-Viks et al. [bib_ref] A Probabilistic Methodology for Integrating Knowledge and Experiments on Biological Networks, Gat-Viks [/bib_ref].
## Implementation
## Biological model
We first define a simple model for biological networks [bib_ref] Modeling and Analysis of Heterogeneous Regulation in Biological Networks, Gat-Viks [/bib_ref]. A biological network can be modelled through a direct graph G(V, E), where each node v ∈ V represents a gene that can be in a discrete state D = {0,1}, representing an inactive and an active state, respectively. If a gene v ∈ V has at least one parent then it is called a regulated gene and we define as R v the set of parents (regulators) of v. If a gene v ∈ V has no parents, then it is a stimulator and we define as V s the set all stimulators of the network. In addition, we represent the expression data using an n × m matrix M, where n is the number of genes in the network and m is the number of experiments performed or samples. For each 1 ≤ i ≤ n and 1 ≤ j ≤ m the value of M [i, j] is the expression level of gene i in sample j.
## Iris algorithm
In this section we describe our approach to infer the regulatory relations in gene networks from high-throughput data. IRIS needs an input network topology N, an expression profile data matrix M. The method consists of two main steps: (i) Dicretisation and (ii) Regulation Functions Learning. The details are reported in the following subsections.
## Discretisation
This steps is aimed at computing a binary matrix from the observed gene activation levels. We compute the first discretisation step using the rule:
with 2 ≤ j ≤ m. And for j = 1 we use: shows an example of matrix S for three genes A, B and C. The values of S computed as above are used to compute neighbouring values. In particular, the uncertain values (NaN) are recovered as follows:
[formula] ′ M i j s [ , ]1 1 = ∈ −∞ ∈ +∞ if if Otherw τ τ i ise ⎧ ⎨ ⎪ ⎪ ⎩ ⎪ ⎪ (2) S i j S i j S i j M i j x S i i [ , ] [ , ] [ , ] [ , ] [ = − = + = ≤ 0 1 0 1 0 1 if AND AND if , , ] [, ] [, ] j S ij M ij x i − = + = ≥ ⎧ ⎨ ⎩ 1 1 1 1 AND AND (3) [/formula]
Example of the discrete state matrix S Example of the discrete state matrix S.
with 2 ≤ j <m -1 and where is the median value for the expression of gene i. To apply these rules we use an iterative approach that runs until either all values are assigned to a valid active/inactive state or no recovery action is performed in the last iteration. [fig_ref] Figure 2: Recovery step example [/fig_ref] shows an example of recovery computation.
## Regulation functions learning
In this step we use the matrix S to compute the PTs and TTs. To infer the PTs we use relative frequencies. Consider a gene v and the set of its regulators R v . Then the matrix S contains several state assignments for the genes in R v and v itself. Let Γ v be the set of all possible state assignments of the variables in R v .
Then we have:
[formula] where |{r v , v = s}| are the occurrence numbers of state assignment {r v ∪ v = s} in S. Let [/formula]
PT({v = s}|{r v }) be the conditional probability that gene v is in state s given the state assignment r v ∈ Γ v . Then we compute the conditional probabilities for v as follows:
Using (6) we compute the TT for each gene as:
[formula] ∀ r v ∈ Γ v and where TT({r v })[ = − = + = ′ ≤ 1 1 1 1 0 1 if AND AND if i i j NaN S i j M i j S i j , ] [, ] [/formula]
[, ]
[ , ]
[formula] − = + = ′ ≥ − = 1 1 1 0 0 1 0 AND AND if AND AND if AND S i j NaN M i j S i j NaN S i j [ , ] [ , ] [/formula]
[ , ]
[ , ] v to the state assignment {r v }. This situation is indicated as -1 in the TTs.
[formula] + = ′ ≤ − = + = 1 0 0 1 1 0 0 0 AND ′ ≤ ⎧ ⎨ ⎪ ⎪ ⎩ ⎪ ⎪ M i j [ , ](4)x i freq r v r v r freq r v r v v v v v v v ({ , }) |{ , }| ({ , }) |{ , = = = ∈ = = = 0 0 1 1 with Γ } }| (5) PT ({ }|{ }) ({ , }) ({ , }) ({ , } v r freq r v v freq r v v f r e q r v v v = = = = + = 0 0 0 1 ) ) ({ }|{ }) ({ , }) ({ , }) ∀ ∈ = = = = in PT r S v r freq r v v freq r v v v v v Γ 1 1 0 + + = freq r v v ({ , }) 1 (6) TT if PT PT if PT ({ }) ({ }|{ }) ({ }|{ }) ({ }| r v r v r v v v v = = > = = 0 0 1 1 1 { { }) ({ }|{ }) r v r v v > = − ⎧ ⎨ ⎪ ⎩ ⎪ PT Otherwise 0 1 (7) P v r P v r v v ({ }|{ }) ({ }|{ }) . = = = = 0 1 0 5 0 [/formula]
## Recovery step example
## Integration with factor graph
A factor graph is a class of probabilistic models that were originally applied to coding/decoding problems. Using a factor graph we can model complex domain knowledge in which feedback loops play a fundamental role. One of the important advantages of factor graphs is their combination with the sum-product algorithm [bib_ref] Factor Graphs and the Sum-Product Algorithm, Kschischang [/bib_ref] , which is a message-passing algorithm for efficiently computing marginal distributions, even in the presence of cycles.
In our approach the factor graph model is used to combine structural information (network topology) with the inferred regulatory functions. A factor graph contains two types of nodes: factor and variable nodes. We have a variable node for each gene and a factor node linking two variable nodes if and only if a relationship between these two nodes exists. Consider the gene regulatory network depicted in containing four genes: A and B are regulators of C, C is a regulator of D, and D is regulator of B. Note that gene A is a stimulator of the network. Since there are three regulated genes, we have three PTs representing P(C|A, B), P(D|C) and P(B|D). To translate this gene network into an equivalent factor graph network, we perform the following steps:
- Each gene node becomes a variable node of the factor graph;
- For each regulatory function a factor node must be inserted to link the genes involved;
- Each stimulator must be linked to a specific factor node.
Applying these rules, we obtain the factor graph in . This model can answer questions such as: "What is the probability that gene C is active given that genes A and B are inactive?" and "What is the likelihood that genes B and A are inactive given that gene C is active?". For this purpose, we set the state of the observed genes and use the sum-product algorithm to compute the posterior distribution of hidden genes. Here we follow the propagation of belief in directed graphs for Forney-style factor graphs. If a stimulator is not fixed, then its factor node will be set with a uniform distribution. rithms have been proposed for this taskfrom Monte Carlo methods to variational methods, mean field methods and belief propagation (BP). The sum-product algorithm, adopted in this paper, is a special case of belief propagation. It is well-known that belief propagation yields exact results if the graphical model does not contain cycles. If the graphical model contains loops, the sumproduct algorithm can still yield accurate results using little computational effort. However, if the influence of loops is large, the approximate marginals calculated by BP can have large errors and the quality of the BP results may not be satisfactory. Many recent research efforts in statistical machine learning are devoted to the development of efficient approximate inference algorithms for cyclic graphical models (see for example [bib_ref] Loop corrections for approximate inference on factor graphs, Mooij [/bib_ref]. For the purposes of this paper we could have to deal with cycles in the case of inference, when we use PTs, and computation of steady states, when using TTs. We adopt the belief propagation algorithm for the former and the algorithm of Gat-Viks et al. [bib_ref] Modeling and Analysis of Heterogeneous Regulation in Biological Networks, Gat-Viks [/bib_ref] for the latter. In any case the IRIS method, proposed here, is by no way influenced by the presence of cycles. In particular IRIS takes as input the description of the network and the expression profiles giving in output a map of the regulatory relations between sets of regulators and regulated genes, this means that all the information used by IRIS are based on "local" relationships between a gene and the set of its regulators. The influence of cycles appears in the successive phases for the use of these relationships in inference tasks. However, the inference in cyclic PGMs is still a very important research question in the field of statistical machine learning and its solution is, of course, outside the scopes of this paper.
# Results
In this section we report IRIS results for both synthetic networks and microarray expression profiles. IRIS needs a well-defined gene network as input. We say that a gene network is well defined if each of its interactions allows us to distinguish between regulator genes and regulated genes. Given a well-defined network, we have genes with zero regulators (called stimulators representing environmental conditions), genes with one regulator, genes with two regulators, and so on. If a gene has at least one regulator then it has a regulatory function that describes its response to a particular stimulus by its regulator(s). In our approach we suppose, without loss of generality, that a gene can be in one of two states: inactive and active, represented as 0 and 1, respectively. This assumption is commonly used in the literature to distinguish the response of a gene to a given experimental condition.
Given a well-defined gene regulatory network, IRIS computes the regulatory functions, providing two different descriptions: a description in which each interaction is described as a conditional probability table, which we refer to as a potential . These two different descriptions allow different analyses.
Using the PTs we can execute an inference step to compute the a posteriori probability of hidden genes given observed genes, so that, for example, it is possible to understand how to control a gene using particular environmental conditions. Using the TTs we can compute the steady states of a gene regulatory network. In this scenario, we deal with the problem of the cyclic structure of gene networks, so we use an approach based on the factor graph model [bib_ref] Factor Graphs and the Sum-Product Algorithm, Kschischang [/bib_ref] as an inference engine and the idea of feedback sets [bib_ref] Modeling and Analysis of Heterogeneous Regulation in Biological Networks, Gat-Viks [/bib_ref] to compute the steady states of the networks.
## Results for synthetic networks
The synthetic networks used to test IRIS were generated using SynTReN [bib_ref] SynTReN: a Generator of Synthetic Gene Expression Data for Design and Analysis..., Bulcke K Van Den [/bib_ref] , it creates synthetic transcriptional regulatory networks and produces simulated gene expression data that approximate experimental data. Network topologies are generated by selecting sub-networks from previously described regulatory networks. Several parameters can be used to adjust the complexity of the data set generated. All gene expression values are normalized between 0 (no transcription) and 1 (maximum level of transcription). In addition, the data generated can be altered by a specified level of biological and experimental noise. Using SynTReN, we generated two synthetic networks representing a sub-pathway of the E. coli regulatorynetwork [fig_ref] Figure 4: Synthetic network for E [/fig_ref] Most of the rules governing the activation of genes in these networks have already been investigated in several studies. In particular, for the E. coli network we use conclusions from references [bib_ref] Cross-induction of glc and ace Operons of Escherichia coli Attributable to Pathway..., Peelicer [/bib_ref] [bib_ref] Transcriptional Activation by FNR and CRP: Reciprocity of Binding-site Recognition, Sawers [/bib_ref] [bib_ref] The Art and Design of genetic Screens: Escerichia Coli, Shuman [/bib_ref] [bib_ref] Gene Replacement without Selection: Regulated Suppression of Amber Mutations in Escherichia Coli, Herring [/bib_ref] to obtain the regulatory relations for glcD, focA and lacZ. For S. cerevisiae we use the results of Wilcox et al. [bib_ref] Transcriptional Profiling Identifies two Members of the ATP-binding Cassette Transporter Superfamily Required..., Wilcox [/bib_ref] to obtain the regulatory rela-Synthetic network for E. coli [fig_ref] Table 1: Percentage of correct entries in the inferred truth tables for synthetic networks... [/fig_ref] for a maximum biological 0.5. The size of each regulation table, which depends on the number of regulator genes, is also reported. If |R i | is the number of regulators of the ith gene, then the regulation TT will have a size of , assuming that a gene can be in two states. The results show that all but one activation rule were correctly inferred by IRIS.
Both networks generated by SynTReN have an acyclic structure, so we can use the EM-MAP algorithmto compare the performance of IRIS. We used an EM-MAP algorithm implementation of the Bayes net toolbox (BNT) [bib_ref] The Bayes Net Toolbox for Matlab, Murphy [/bib_ref] to validate the IRIS discretisation scheme. shows results for different discretisation methods such as equal frequency (diamonds), global width (squares) and equal width (circles). For these methods we used discretisation into two bins. The EM-MAP results for IRIS discretisation are reported in [fig_ref] Table 2: Execution time for IRIS and EM-MAP [/fig_ref] lists the execution times (taken using a Linux PC with Intel Core Duo CPU at 1.8 Ghz) for IRIS and EM-MAP. We report the running times not for an absolute evaluation, but for a relative comparison, indeed the proposed method yields similar or slightly better results than EM-MAP and requires less computational resources. Actually the running time of IRIS depends linearly on the number of genes, and the number of samples, however it depends exponentially from the maximum in-degree of the node network.
Since the IRIS discretisation scheme can be influenced by the order of the values for expression of each gene in the data set, it is interesting to investigate how the performance changes on randomly changing the order of the data. This is useful for a steady-state data set, for which the order of the expression profile has no biological meaning. Actually the difference between the expression levels at successive points of the profile has been often used to characterise the behaviour of a gene in different conditions, with particular reference to time course experiments (see for example, as it can be used as an indicator of increase/decrease of expression profiles correlated in time. IRIS makes use of this difference in the discretisation procedure to fix the values which are not significantly low or high with respect to the mean expression level in the profile. Actually we found useful to compare differences in the expression levels over different conditions even for non time course data. The intuitive reason for the use of the information carried by the difference of expression levels is based of the fact that in this way we can observe if two or more genes have a common or an opposite pattern, which are indicator of activation and inhibition respectively. For example, suppose that we have a network where the gene A is a regulator of another gene B and let E 1 and E 2 be two different experimental conditions of a steady state dataset. Suppose also that a 1 (b 1 ) and a 2 (b 2 ) are the expression levels of A(B) under E 1 and E 2 respectively. Now we can distinguish two different situations: a) a 1 <a 2 and b 1 <b 2 (or a 1 >a2 and b 1 >b 2 ): here we can state that in the experiment E 2 both genes have an expression level greater (lower) then in E 1 , in other words, the two genes have a similar behaviour.
## | |
## R i
Synthetic network for S. cerevisiae Synthetic network for S. cerevisiae. Truth tables (TTs) were computed using the data set with the maximum biological noise level (0.50). "TT Size" reports the number of possible state assignments for the regulator genes. Note that the value for the i-th regulated gene is , where |Ri| is the number of regulators and each gene can be in two states. We distinguish the number of correct/incorrect/undefined inferred states for each regulatory relation and compute these as percentages of the total number of states. Each value was obtained as the mean for 10 runs. For EM-MAP we also report the number of iterations to reach convergence.
[formula] CLN2 SSL1 2 2 0 0 CDC28 SSL1 2 2 0 0 NOT3 SSL1 2 2 0 0 FIT2 SSL1 PDR11 4 4 0 0 CDC6 PDR11 2 2 0 0 CEF1 PDR11 2 1 1 0 LEU2 PDR11 2 2 0 0 CLB6 PDR11 2 2 0 0 DAL80_GZF3 PDR11 2 2 0 0 A2 PDR11 IPT1 4 4 0 0 [/formula]
## | |
b) a 1 >a 2 and b 1 <b 2 (or a 1 <a 2 and b 1 >b 2 ): here we can state that in E 1 the gene A has an expression level greater (lower) then in E 2 , whereas, the gene B has an expression level in E 1 lower (greater) than in E 2 , in other words, the two genes have an opposite behaviour which can be observed form an opposite sign of the expression derivative for the genes in the experimental condition E 2 .
If two genes show the behaviour of the case a) than we can suppose that these two genes have a relationship of activation, whereas, in the second case they have a relationship of inhibition. The fact that for steady state data we have uncorrelated values, suggests to choose any set of points to extract second order information. In order to maintain, a coherence with the case of time course data, without loss of generality, we use the previous point in the expression profile matrix. In order to demonstrate that the choice of the previous point does not significantly affect the results, we evaluated the results in terms of Kullback-Leibler divergence with the known solution on a steady state dataset performing random permutations on the expression profile of each gene. These results reported in show that for different permutations the Kullback-Leibler divergence is nearly constant, in other words, the choice of the previous point has the same impact on the results that could have the choice of any other point for the computation of the difference. Finally, the use of multiple points instead of just the previous one did not produce significant improvements, at the expenses of an increased computational time.
## Results for microarray expression profiles
We also applied the IRIS algorithm to two real data sets comprising microarray expression profiles for the yeast mitotic cell cycle and human B-cells. shows the network topology for the yeast mitotic cell cycle extracted from the study by Noman and Iba [bib_ref] Inferring Gene Regulatory Networks Using Differential Evolution with Local Search Heuristics, Noman [/bib_ref] , where we consider only known interactions reported in the literature. In this network the transcriptional factors SWI4, SWI6 and MBP1 are directly linked to the cyclins CLN1, CLN2, CLN3, CLB5 and CLB6, which bind to the cyclin-dependent kinase protein CDC28, whereas SIC1 is an inhibitor of the cyclin CDC28 complex. Using the literaturewe obtain a description of the regulatory relations for this network (for more details see Additional
## Yeast mitotic cell cycle
Kullback-Leibler divergence for IRIS using randomised data sets Kullback-Leibler divergence for IRIS using randomised data sets. The S. cerevisiae synthetic network and the data set with biological noise of 0.5 were used in this test. The x-axis represents the percentage of columns swapped randomly. The D KL values reported are the means for 100 runs.
Network for the S. cerevisiae cell cycle Network for the S. cerevisiae cell cycle.
file 1: regulation_true_descriptions.pdf). These relationships can be considered the truth tables for this pathway.
To infer the regulatory functions we use the microarray data from Spellman et al. [bib_ref] Comprehensive Identification of Cell Cycle-regulated Genes of the Yeast Saccharomyces cerevisiae by..., Spellinan [/bib_ref]. Finally, the inferred TTs can be used to compute the steady states of the network using an appropriate algorithm [bib_ref] Modeling and Analysis of Heterogeneous Regulation in Biological Networks, Gat-Viks [/bib_ref]. In this case three steady states are identified [fig_ref] Table 5: Steady states for the yeast mitotic cell-cycle network obtained using IRIS [/fig_ref]. Among them the first and the third one have been described in literature.
## Human b-cells
Recent studies have demonstrated that the organisation of a gene regulatory network often follows a scale-free nature [bib_ref] Scale-free networks in cell biology, Albert [/bib_ref]. A scale-free network is characterised by an inverse relationship between the number of nodes and their connectivity. Another feature of gene networks is the presence of highly connected genes (called hubs). These networks typically contain short feedback loops. To test the suitability of IRIS for scale-free gene regulatory networks, we inferred the regulatory relations from human B-cell data, for which we considered the MYC gene as a major hub. MYC codes for a protein that binds to the DNA sequence of other genes. When MYC is mutated or over expressed, the protein does not bind correctly and often causes cancer. Both the gene expression profiles and network topology were extracted from the results of Basso et al. [bib_ref] Reverse Engineering of Regulatory Networks in Human B Cells, Basso [/bib_ref]. The network topology represents the MYC gene and 55 genes directly connected to it. To infer the regulatory rules of this network, we used a subset of 100 expression profiles (in [bib_ref] Reverse Engineering of Regulatory Networks in Human B Cells, Basso [/bib_ref] 336 samples are used). Here we use the MYC target gene database (MYC-DB).
Because MYC-subnetwork has an acyclic structure (differently to S. cerevisiae cell cycle) we can use EM-MAP algorithm to infer the PTs for this network. In order to evaluated the method in terms of scalability, reports the Kullback-Leibler divergences obtained by IRIS (red asterisk line) and EM-MAP (blue crossed line) for different values of m/n (m is the number of samples and n is the number of genes). As can be seen from the as the ratio m/n increases the accuracy gets better. This is also evident from where the percentage of correct (blue bars), undefined (green bars) and incorrect (red bars) evaluations of IRIS algorithm are reported.
# Conclusions
This paper described a method to infer regulatory relations in gene networks from expression data. The basic features of IRIS are a simple discretisation method to translate real-valued measurements into two discrete states (active and inactive) and a regulatory inference rule.
To compare the proposed approach with other methods, we reported results for synthetic networks. The main conclusion is that the proposed method yields similar or slightly better results than other well-known approaches, but requires much less computational resources.
## Biological findings observed genes hidden genes inference results
Strong relationship between cyclins CLB5 and CLB6 [bib_ref] CLB5 and CLB6, a new Pair of B Cyclins Involved in DNA..., Schwob [/bib_ref] and between CLN1 and CLN2To infer the conditional probability reported in the "Inference Results" column, the sum-product algorithm was used, and the state of the "Observed Genes" is fixed to derive the potential behaviour of "Hidden Genes". We also tested IRIS on two real data sets to infer interaction rules for the yeast mitotic cell cycle and the human MYC sub-network. IRIS exhibited good accuracy for these networks compared to literature-derived rules. IRIS relies on knowledge of the network topology, which can be extracted from on-line databases (e.g. KEGG) or can be obtained from network reverse engineering algorithms. In other words regulatory network parameter estimation and model selection are treated and performed as two different tasks. This approach could be useful in studying gene regulatory networks with hundreds of genes as a set of smaller sub-networks, as reported for MYC expression profiles.
IRIS is useful for extracting the main rules within a gene network with a well-defined topology. This information can then be used in subsequent analysis steps, such as probabilistic inference or as a preliminary step for building models of complex biological systems [bib_ref] MetaReg: a Platform for Modeling, Analysis and Visualization of Biological Systems Using..., Ulitsky [/bib_ref].
## Availability and requirements
[fig] Figure 2: Recovery step example. (a) Data obtained by the discretisation rule defined in (1). (b) Data obtained after one iteration of the rules defined in (3) and (4). (c) Final data for which all uncertain values were recovered. [/fig]
[fig] Figure 4: Synthetic network for E. coli. tion for FIT2. Since all the regulators of A2 have an inhibitory function, the gene will be in an active state if and only if both regulators are inactive. For more details we inferred the regulatory functions for both synthetic gene networks. To evaluate the accuracy of the PTs computed by IRIS, we used the Kullback-Leibler divergence (D KL ) [30], which is a measure of the difference between two probability distributions P and Q, with D KL (P||Q) = 0 if and only if P = Q. Figure 6 shows mean D KL (P true ||P IRIS ) values as a function of the noise level (asterisks). The results in terms of correct TT entries inferred by the algorithm are reported in [/fig]
[fig] Comparison: Kullback-Leibler divergence for (a) E. coli and (b) S. cerevisiae synthetic networks Figure 6 Kullback-Leibler divergence for (a) E. coli and (b) S. cerevisiae synthetic networks. The x-axis shows the biological noise levels used to generate the data sets. The y-axis represents the D KL values obtained as the mean for all tables of a network for the corresponding biological noise level. The Figure reports the D KL values of EM-MAP obtained using different discretisation approaches: IRIS (crosses), equal frequency (diamonds), global width (squares) and equal width (circles) and the D KL values obtained using IRIS algorithm both in discretisation step and in regulation function learning process (asterisks). [/fig]
[fig] •: Project name: Inference of Regulatory Interaction Schema (IRIS) Project home page: http://bioinformatics.bio gem.it:8081/BioPlone/downloadfolder/iris-downage Operating system(s): Platform independent Programming language: MATLAB Other requirements: Graphviz is required if the user wants to create a file containing the gene regulatory network topology License: GNU GPL Any restrictions to use by non-academics: None [/fig]
[table] Table 1: Percentage of correct entries in the inferred truth tables for synthetic networks for E. coli and S. cerevisiae.E. ColiTrueTable vs IRIS Inferred TT [/table]
[table] Table 2: Execution time for IRIS and EM-MAP. [/table]
[table] Table 3: Percentage of correct entries in inferred truth tables for the S. cerevisiae mitotic cell-cycle network.TrueTable vs IRIS Inferred TT This table follows the same schema as for Table 1. [/table]
[table] Table 6: shows the results obtained by IRIS on the complete data set composed by 100 samples. Among the genes in MYC-DB, we obtained 93% of correct evaluations. Genes with no specified regulation in MYC-DB such as EEF1E1, TRAP1 and PAICS resulted up-regulated in IRIS. In particular for PAICS this up-regulation was confirmed in[39]. In addition there are 31 genes in the Basso et al. network with no MYC-DB entries, IRIS identifies 24 regulatory relationships that deserve further biological investigation. [/table]
[table] Table 4: Inference results. Column "Biological Findings" lists a short description of the features of interest and references. [/table]
[table] Table 5: Steady states for the yeast mitotic cell-cycle network obtained using IRIS [/table]
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Correlation holes and slow dynamics induced by fractional statistics in gapped quantum spin liquids
SUPPLEMENTARY NOTE 1: DERIVATION OF THE MICROSCOPIC MODELWe focus our attention on a classical Z 2 lattice gauge theory perturbed by a small, transverse magnetic field h. The model is composed of spin-1/2 degrees of freedom, σ i , which live on the bonds (labelled by the index i) of a square lattice with N = L × L sites (labelled by the index s) wrapped around a cylinderHere i ∈ s denotes the four spins that reside on the bonds surrounding the lattice site s. The coupling constant J ( h) is positive by convention. Treating the magnetic field h perturbatively, we arrive at the following ring-exchange Hamiltonian in the ground state sector:up to a constant energy shift that arises due to the virtual creation and annihilation of excitations. The plaquette operator B p is defined as B p = i ∈p σ z i , where i ∈ p denotes the four spins surrounding the plaquette p. The toric code Hamiltonian [1] is generated perturbatively and lifts the macroscopic degeneracy of the classical Z 2 theory.The ground state of the effective model, Supplementary Eq. (3), is characterised by eigenvalues +1 for all (commuting) operators A s and B p (and has a topological degeneracy that is immaterial for the purpose of the present work). Excitations correspond to states in which plaquette operators B p and/or star operators A s have negative eigenvalues. We will refer to the energetically costly star defects as spinons, and to the lower-energy plaquette defects as visons (h 4 /J 3 J, since J h by construction). Let us then consider the two spinon sector, relevant for the intermediate temperatures of interest, T h, J. The magnetic field h makes the spinons dynamicalwhere ss denotes neighbouring sites on the square lattice, and σ ss is the spin on the bond connecting sites s and s .Since the magnetic field is applied parallel to the z axis, the vison configuration remains precisely static. The operators b s , b † s are hardcore bosons representing the spinon excitations, which live on the sites of the lattice. Note that the spins σ x ss and the operators b s are not independent: A s = e iπb † s b s . Crucially, each spinon hopping event is accompanied by a spin flip in the σ x basis. In order to derive an effective tightbinding model, we make a gauge choice by fixing the string, S(γ i ) = k ∈γ i σ z k , used to defined the state corresponding to a spinon residing on site i; the path γ i ends on site i. Choosing a different string S(γ i ) may lead to an additional phase: e iφ = S(γ i )S(γ i ) , φ = 0 or π. Having made this choice, the hopping matrix element between adjacent sites i, j is given bywhere γ i ∪ γ j and the bond i j form a closed loop. Moving a spinon around a plaquette p, whose bonds are indexed by i j ∈ p, the spinon acquires a phaseThe string operators do not appear in Supplementary Eq. (5) since S(γ i ) 2 = 1. Hence, for a given vison configuration, and when considering gauge invariant quantities, we can map Supplementary Eq. (4) onto a nearest neighbour tight-binding modelwhere the Peierls phases φ ss = −φ s s are, according to Supplementary Eq. (5), determined by the positions of the visonseach vison contributing a π-flux threading the plaquette on which it resides. With a cylindrical geometry, it is possible to choose a gauge in which the hopping amplitudes are real and uniform in one direction and acquire an appropriate minus sign in the orthogonal direction, according to the specific vison realisation[2]. Imposing periodic boundary conditions, the total flux threading all plaquettes must be an integer multiple of 2π, i.e., p (∇ × A) p = 0 (mod 2π).
## Supplementary note 1: derivation of the microscopic model
We focus our attention on a classical Z 2 lattice gauge theory perturbed by a small, transverse magnetic field h. The model is composed of spin-1/2 degrees of freedom, σ i , which live on the bonds (labelled by the index i) of a square lattice with N = L × L sites (labelled by the index s) wrapped around a cylinder
[formula] H = −J s A s − h i σ z i , A s = i ∈s σ x i .(1) [/formula]
Here i ∈ s denotes the four spins that reside on the bonds surrounding the lattice site s. The coupling constant J ( h) is positive by convention. Treating the magnetic field h perturbatively, we arrive at the following ring-exchange Hamiltonian in the ground state sector:
[formula] H (0) eff = −J s A s − 5 16 h 4 J 3 p B p (2) ≡ − ∆ s 2 s A s − ∆ v 2 p B p ,(3) [/formula]
up to a constant energy shift that arises due to the virtual creation and annihilation of excitations. The plaquette operator B p is defined as B p = i ∈p σ z i , where i ∈ p denotes the four spins surrounding the plaquette p. The toric code Hamiltonianis generated perturbatively and lifts the macroscopic degeneracy of the classical Z 2 theory.
The ground state of the effective model, Supplementary Eq. (3), is characterised by eigenvalues +1 for all (commuting) operators A s and B p (and has a topological degeneracy that is immaterial for the purpose of the present work). Excitations correspond to states in which plaquette operators B p and/or star operators A s have negative eigenvalues. We will refer to the energetically costly star defects as spinons, and to the lower-energy plaquette defects as visons (h 4 /J 3 J, since J h by construction). Let us then consider the two spinon sector, relevant for the intermediate temperatures of interest, T h, J. The magnetic field h makes the spinons dynamical
[formula] H (2) eff = 4J − h ss b † s σ z ss b s + h.c. ,(4) [/formula]
where ss denotes neighbouring sites on the square lattice, and σ ss is the spin on the bond connecting sites s and s .
Since the magnetic field is applied parallel to the z axis, the vison configuration remains precisely static. The operators b s , b † s are hardcore bosons representing the spinon excitations, which live on the sites of the lattice. Note that the spins σ x ss and the operators b s are not independent: A s = e iπb † s b s . Crucially, each spinon hopping event is accompanied by a spin flip in the σ x basis. In order to derive an effective tightbinding model, we make a gauge choice by fixing the string, S(γ i ) = k ∈γ i σ z k , used to defined the state corresponding to a spinon residing on site i; the path γ i ends on site i. Choosing a different string S(γ i ) may lead to an additional phase: e iφ = S(γ i )S(γ i ) , φ = 0 or π. Having made this choice, the hopping matrix element between adjacent sites i, j is given by
[formula] t i j = −h S(γ i )S(γ j )σ z i j , [/formula]
where γ i ∪ γ j and the bond i j form a closed loop. Moving a spinon around a plaquette p, whose bonds are indexed by i j ∈ p, the spinon acquires a phase
[formula] h −4 i j ∈p t i j = i j ∈p σ z i j = 1 − 2n p .(5) [/formula]
The string operators do not appear in Supplementary Eq. (5) since S(γ i ) 2 = 1. Hence, for a given vison configuration, and when considering gauge invariant quantities, we can map Supplementary Eq. (4) onto a nearest neighbour tight-binding model
[formula] H (2) eff ({φ ss }) = 4J − h ss b † s e iφ s s b s + h.c. (6) ≡ ∆ s s b † s b s − t s ss b † s e i A s s b s + h.c. ,(7) [/formula]
where the Peierls phases φ ss = −φ s s are, according to Supplementary Eq. (5), determined by the positions of the visonseach vison contributing a π-flux threading the plaquette on which it resides. With a cylindrical geometry, it is possible to choose a gauge in which the hopping amplitudes are real and uniform in one direction and acquire an appropriate minus sign in the orthogonal direction, according to the specific vison realisation. Imposing periodic boundary conditions, the total flux threading all plaquettes must be an integer multiple of 2π, i.e., p (∇ × A) p = 0 (mod 2π).
## Supplementary note 2: competing strip configuration
As presented in the main text, the free energy of a spinon confined to a disc of radius ξ surrounded by disordered visons is given by
[formula] F d (ξ) = j 2 0 t s (ξ + ξ 0 ) 2 + πT ξ 2 ln 1 + e −β∆ v .(8) [/formula]
Neglecting the effects of nonzero ξ 0 , and for temperatures satisfying T ∆ v , one arrives at the following expression for the radius ξ * which minimises the disc free energy:
[formula] ξ d * = j 2 0 t s πT ln 2 1/4 .(9) [/formula]
For ξ ξ 0 , the disc model then predicts that the system will become completely free of visons at a temperature
[formula] T d * 16 j 2 0 π ln 2 t s L 4 .(10) [/formula]
In a square system of finite size with periodic boundary conditions, we observe an instability in our MC simulations whereby the shape of the depleted patch changes upon approaching T d * from a disc to a strip wrapping around the system. This is a finite size effect where the spinon wave function overlaps with itself across the periodic boundary conditions. It can be readily understood in light of the competition between the disc free energy and the free energy of a strip of width 2ξ in a system of size L × L, with the corresponding vison strip free energy:
[formula] F s (ξ) = π 2 t s 4(ξ +ξ 0 ) 2 + 2T ξ L ln 1 + e −β∆ v .(11) [/formula]
Notice that the strip width that minimises this free energy is, for ξ ξ 0 and T ∆ v ,
[formula] ξ s * = π 2 t s 4T L ln 2 1/3 ,(12) [/formula]
and the temperature at which the system becomes entirely free of visons is given by
[formula] T s * 2π 2 ln 2 t s L 4 .(13) [/formula]
In order to determine whether or not the system makes a transition from the disc state at high temperature to the strip state at low temperature, we compare the two free energies F d and F s . Solving for F d (T) = F s (T), we find that the two free energies coincide at a temperature In the absence of mutual statistics between the particles, the spinon only endows the visons with an effective chemical potential, but does not induce significant correlations between their positions. The data in both panels are for a system of size L = 16, averaged over 2 6 histories.
[formula] T ds = 1 4 4 3 6 j 6 0 π ln 2 t s L 4 ,(14) [/formula]
and that the disc free energy is lower than the strip one for T > T ds . All of T d * , T s * , and T ds scale with system size as ∝ L −4 , and therefore the O(1) prefactors determine whether or not an instability between the two vison configurations occurs. We find that T d * , T s * < T ds , which implies that at the level of the saddle point approximation one expects a transition from the disc to the strip state at a temperature given by [fig_ref] Figure 1 |: Effect of density-density interactions between spinons and visons [/fig_ref] , before the visons are eventually expelled from the system altogether below T s * . Incidentally, the L −4 scaling with system size is indeed confirmed by the numerics in the inset of [fig_ref] Figure 2 |: Evolution of the vison-depleted patch diffusion constant with its radius [/fig_ref] in the main text, where the data for the vison density n p are shown to collapse for various system sizes L when plotted as a function of T L 4 /t s .
## Supplementary note 3: spinon-vison interactions
Here, we discuss the possibility that the Hamiltonian includes an explicit short-range interaction term between the spinons and visons. In particular, suppose that the spinons and visons are coupled by a density-density term of the form
[formula] H int = H eff + U s 1 4 p ∈s n p b † s b s .(15) [/formula]
That is, each spinon interacts with the four adjacent plaquettes p surrounding each site s (denoted by p ∈ s). The noninteracting Hamiltonain H eff is given by . We expect on rather general grounds that the interactions are repulsive U > 0 and weak |U| < t s . If the mutual statistics between the species is removed, i.e., A ss = 0, then a typical configuration of visons gives rise to a diagonal (on-site) disordered potential term in the spinon tight binding Hamiltonian which also localises the spinons, as is generally expected in two spatial dimensions. However, since interactions are weak (|U| < t s ) the localisation length significantly exceeds the lattice spacing and we do not observe the formation of well-defined depleted patches. On the contrary, the vison density behaves smoothly, as if responding to a slowly-varying chemical potential with weak correlations between their positions. This behaviour can be seen in Supplementary [fig_ref] Figure 1 |: Effect of density-density interactions between spinons and visons [/fig_ref] , where the strength of the interactions is set to U = t s /4. In these simulations, the localisation length exceeds the system size, and presence of a spinon translates into a uniform chemical potential µ U/L 2 that controls the vison density as a function of temperature. If the mutual statistics is reinstated, we observe the revival of the nontrivial correlations between the visons, implying the existence of a well-defined vison-depleted patch around the spinon. The linchpin of the formation of the vison-depleted patches and the implied nontrivial vison correlations is an effective disorder that gives rise to a localisation length (i.e., penetration depth) significantly smaller than the radius of the patch ξ loc ξ, which eminently comes about as a result of mutual statistics and not of short-range spinon-vison interactions |U| < t s .
## Supplementary note 4: diffusive patch motion
In this section we study the dynamics of the vison-depleted patches as a function of temperature. Since there is no preferred direction for the motion of the patches, one generally expects them to perform an unbiased random walk across the system. Notice however that the motion of a patch over one lattice Let us define the centre of the patch, r p , using the maximum of the spinon ground state wavefunction: |ψ 0 (r p )| 2 = max r |ψ 0 (r)| 2 . Asymptotically, one expects that r 2 p (t) 2Dt. This behaviour is indeed observed in the MC simulations, and the resulting values of D as a function of patch size ξ are shown in [fig_ref] Figure 2 |: Evolution of the vison-depleted patch diffusion constant with its radius [/fig_ref].
Treating the patches as classical particles that satisfy the reaction-diffusion equation A + A ∅, one may write down an equation for the evolution of the spinon density in the longwavelength limit∂ ρ s ∂t
[formula] + ∇ · J = −K ρ 2 s + η(r, t) ,(16) [/formula]
where the current J = −D∇ρ s , the annihilation constant K ∝ D, and η(r, t) is a temperature-dependent source term that represents pairwise creation of the spinons at nonzero temperatures (with appropriate spectral properties). The rate of spinon annihilation in equilibrium is given by K ρ 2 s . In order for the system to fall out of equilibrium as discussed in the main text, the cooling rate must be greater than the rate at which spinons can annihilate in order to remain in equilibrium, i.e., dρ s (T(t))/dt K ρ 2 s . At the mean field level (i.e., neglecting spatial fluctuations of the spinon density ρ s ), one may estimate the required cooling rate:
[formula] dT dt DT 2 ∆ s e −∆ s /T .(17) [/formula]
Once again we see that, thanks to the exponentially small density of spinons in equilibrium at low temperature, this condition is likely to be easily (if not unavoidably) accessible experimentally in the study of quantum spin liquid materials.
## Supplementary note 5: effective patch growth model
Here we present an effective model of the growth of randomly distributed vison-depleted patches. The model represents a caricature of the dynamics of the system between tem- in the main text, capturing both the plateau in the density of spinons between T b → T c and the kinematically-locked regime between T c → T d .
[formula] peratures T b → T c → T d in [/formula]
Suppose that the initial density of patches is ρ 0 (equal to the equilibrium spinon density), and that their positions are random and uncorrelated. We assume that the centres of the patches remain stationary, whereas their radii are monodispersed at the typical equilibrium value, ξ, at temperature T. Then, the patches will grow as temperature is reduced until any two patches overlap, at which point the corresponding spinons annihilate. This process is described by the following mean field theory in the dilute limit, ρξ1. When changing ξ → ξ + dξ one can infer from geometric arguments that the reduction in the patch density is dρ = −8πρ 2 ξdξ. One can then solve for the resulting density
[formula] ρ(ξ) ρ 0 = 1 1 + 4πρ 0 ξ 2 .(18) [/formula]
In the limit ρ 0 ξ 2 1, the density remains essentially constant and unresponsive. This regime corresponds to the plateau between T b → T c in . The density begins to decay appreciably once a significant number of the patches start to touch, i.e., ρ 0 ξ 2 ∼ 1. This condition defines the temperature T c , which represents the crossover between the plateau and the kinematically-locked regime. In the opposite limit, ρ 0 ξ 2 1, the density of patches decays as ρ ∼ ξ −2 , corresponding to the kinematically-locked regime between temperatures T c → T d in . Supplementary Eq. (18) implies however that πξ 2 ρ = 1/4 in this regime, which no longer satisfies the condition of diluteness that underpinned our simple modelling. Numerical simulations of the above effective model of patch growth [fig_ref] Supplementary, Figure 3 |: Evolution of vison-depleted patch density with patch radius [/fig_ref] show that the relationship ρ ∝ ξ −2 predicted by Supplementary Eq. (18) does indeed hold in the regime ρ 0 ξ 2 1, but with a modified prefactor, πξ 2 ρ 1/6.
[fig] Figure 1 |: Effect of density-density interactions between spinons and visons. Left panel: vison density as a function of temperature in the absence (blue circles) and presence (red squares) of mutual statistics between the spinons and visons. The dashed blue line shows excellent agreement with the density of noninteracting visons in the presence of a uniform chemical potential µ, as discussed in the text. Right panel: corresponding vison correlations at the temperature T 1 indicated by the vertical dotted line on the left panel. [/fig]
[fig] Figure 2 |: Evolution of the vison-depleted patch diffusion constant with its radius.. Dependence of the diffusion constant, D, on the vison-depleted patch radius ξ in equilibrium at temperature T, obtained from MC simulations, where time is measured in units of MC sweeps. The MC data inFig. 2of the main text are used to map between temperature and patch radius ξ. The solid line through the MC data is a guide to the eye. The inset shows that D(ξ) is consistent with power law behaviour for sufficiently large ξ: D ∼ ξ [/fig]
[fig] Supplementary, Figure 3 |: Evolution of vison-depleted patch density with patch radius. Numerical simulations of the effective patch model: as the radius ξ is increased, all neighbouring pairs of patches separated by a distance 2ξ or less are removed from the system. The centres of the patches remain fixed. The dashed line corresponds to the analytical result in Supplementary Eq. (18), valid in the dilute limit ρξ2 1, and the solid line corresponds to the numerical results. The data correspond to 25 000 randomly distributed patches, averaged over 20 histories. The Voronoi diagrams show representative configurations of patches at densities ρ 0 , ρ 0 /10, and ρ 0 /10 2 (left to right). spacing requires the coordinated rearrangement of a number of visons which scales with the size of the patch. Hence, one expects the diffusion constant, D, (equivalently, the characteristic time scale) of the patch to decrease (grow) with decreasing temperature, as it involves the rearrangement of a larger number of visons. [/fig]
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Rhodobacter capsulatus AnfA is essential for production of Fe‐nitrogenase proteins but dispensable for cofactor biosynthesis and electron supply
## | material s and me thods
## | strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study are listed in in Appendix 1. Rhodobacter capsulatus minimal medium V (RCV) was prepared as previously described. In this medium, a fixed nitrogen source and molybdate (Mo) have been omitted. Traces of Mo arising from impurities of the chemicals used support residual Mo-nitrogenase activity but are low enough to permit the production of Fe-nitrogenase.
To examine diazotrophic growth, cultures were inoculated in 3 ml RCV medium in screw-capped 17-ml Hungate tubes before the exchange of headspace air for pure N 2 gas and incubation in the light. When required, 10 mM serine was added as a fixed nitrogen source, which (in contrast to ammonium) does not inhibit nitrogen fixation.
## | construction of rhodobacter capsulatus anfh-lacz reporter strains and β-galactosidase assays
The anfH promoter was narrowed down by nested deletions. For this, appropriate primer pairs were used to PCR-amplify promoter variants F1 to F6 , thereby adding BamHI and HindIII sites. Corresponding BamHI-HindIII fragments were cloned into the broad-host-range vector pBBR1MCSbefore insertion of a lacTeT cassette (carrying a promoterless lacZ gene, a tetracycline resistance gene, and an oriT transfer origin) from plasmid pYP35into the HindIII site. The resulting reporter plasmids carrying transcriptional lacZ fusions were designated pBBR_F1-lacZ to pBBR_F6-lacZ.
To generate site-directed substitution mutations in the anfH promoter , plasmid pBBR_F1-lacZ served as a template.
Base-pair substitutions were introduced using the QuikChange protocol (Stratagene). The resulting pBBR_F1-lacZ derivatives carrying transcriptional lacZ fusions to different promoter variants were designated pBBR_Mut1-lacZ to pBBR_Mut7-lacZ, pBBR_Mut2/6-lacZ, and pBBR_Mut2/7-lacZ.
The reporter plasmids were conjugationally transferred into the R. capsulatus wild-type strain B10S. Following phototrophic growth of the R. capsulatus reporter strains in RCV medium with 10 mM serine (no Mo added) until the late logarithmic phase, LacZ (β-galactosidase) activity was determined. In the absence of ammonium (-NH 4 + ), the superior regulator NtrC activates transcription of nifA and anfA in concert with the housekeeping sigma factor RpoD. MopA and MopB independently repress anfA in the presence of molybdate (+MoO 4 2-;. NifA and AnfA activate their target genes by partnering with the alternative sigma factor RpoN. Noteworthy, NifA indirectly controls AnfA-mediated anfHDGK expression by controlling RpoN production. Involvement of NifA-activated genes in biosynthesis of the iron-molybdenum cofactor (FeMoco) of Mo-nitrogenase and the iron-only cofactor (FeFeco) of Fe-nitrogenase and electron transfer to both nitrogenases is indicated. (b) Production of active Fe-nitrogenase in a strain lacking AnfA. In this study, we constructed strain YP515-BS85 containing mutations in the anfA and nifD genes (marked by red crosses) and a chromosomal substitution of the anfH promoter (P anfH ) by the nifH promoter (P nifH ) thereby putting anfHDGK expression under NifA control. This strain grew under N 2 -fixing conditions suggesting that AnfA is dispensable for FeFeco biosynthesis and electron supply to Fe-nitrogenase. For further details, see text
## | examination of anfa binding to the anfh promoter
In vitro binding of the DNA-binding domain of AnfA (AnfA_DBD)
to the anfH promoter was examined by electrophoretic mobility shift assays (EMSA) as previously described .
To overexpress AnfA_DBD, appropriate primers were used to PCRamplify a DNA fragment coding for the C-terminal 72 amino acid residues of AnfA in Appendix 2) thereby adding SacII and NcoI sites. The corresponding SacII-NcoI fragment was cloned into the expression vector pASK_IBA45+ (IBA GmbH Göttingen) resulting in hybrid plasmid pYP409. For purification of the Streptagged AnfA_DBD, Escherichia coli BL21 (DE) carrying pYP409 was cultivated with AHT induction before cell disruption and Strep-Tactin affinity chromatography as previously described .
The F1 fragment (carrying the wild-type anfH promoter) and its variants were labeled with γ-32 P-ATP, and free γ-32 P-ATP was removed by gel filtration using Illustra ProbeQuant G-50 Micro
Columns (GE-Healthcare). After 20 min incubation of labeled promoter variants with increasing amounts of the purified AnfA_DBD protein, bound and free DNAs were separated in 6% polyacrylamide gels. Radioactive bands were detected by phosphor screen exposure.
## | substitution of the rhodobacter capsulatus anfh promoter by the nifh promoter
To replace the anfH promoter (P anfH ) by the nifH promoter (P nifH ), we exchanged the 266 bp anfA-anfH intergenic region by the 267 bp fdxD-nifH intergenic region. For this purpose, we constructed mutagenesis plasmid pYP516 containing the 3′ end of anfA (including the translation stop codon, TGA), a gentamicin (Gm) resistance cassette, P nifH , and the 5′ end of anfH (starting with the translation start codon, ATG). To replace the anfH promoter and to delete the anfA gene in a single step, we constructed mutagenesis plasmid pYP515
containing an anfA upstream fragment (but lacking the anfA coding region), a Gm cassette, P nifH , and the 5′ end of anfH. Plasmids pYP516 and pYP515 were conjugationally introduced into the R. capsulatus strain BS85 (ΔnifD), in which the nifD gene is disrupted by a spectinomycin (Sp) cassette. BS85 does not exhibit Mo-nitrogenase activity, and hence, any nitrogenase activity observed in this background can be assigned to Fe-nitrogenase.
Promoter replacement mutants were identified by selection for Gm resistance and screening for loss of vector-encoded tetracycline resistance indicating marker rescue by double cross-over events.
The resulting mutants were called YP516-BS85 (P anfH → P nifH , anfA + , ΔnifD) and YP515-BS85 (P anfH → P nifH , ΔanfA, ΔnifD).
In contrast to strain YP515-BS85 (P anfH → P nifH , ΔanfA, ΔnifD), strain KS94A-YP415 (ΔanfA, ΔnifD) contains the wild-type anfH promoter upstream of the anfHDGKOR3 operon. In this strain, the anfA and nifD genes are disrupted by Sp and Gm cassettes, respectively.
## | re sults
## | localization of the rhodobacter capsulatus anfh promoter by nested deletions
Rhodobacter capsulatus AnfA is essential for the expression of the anfHDGKOR3 operon, but the anfH promoter has not been investigated. The coding regions of anfH and its upstream gene, anfA, are separated by 266 bp . This intergenic region includes three conspicuous sequences, namely (a) a GC-rich inverted repeat sequence followed by a T-rich stretch likely acting as Rho-independent terminator of anfA transcription, (b) two 17 bp direct repeats each encompassing inverted repeat sequences, which are promising candidates as
AnfA-binding sites, and (c) a highly conserved RpoN-binding site.
For clarity, the 17 bp sequences will from now on be called distal and proximal AnfA-binding sites.
To localize the anfH promoter (P anfH ), we analyzed the effects of nested promoter deletions on anfH expression. For this purpose, we generated transcriptional fusions between P anfH fragments,
## | effects of anfa-binding site mutations on anfh expression
To dissect the function of the distal and proximal AnfA-binding sites in P anfH , we generated pBBR_F1-lacZ variants carrying site-directed
## | effects of anfa-binding site mutations on promoter binding by anfa
The AnfA protein encompasses three domains, namely a GAF, an AAA+, and a HTH domain, involved in environmental sensing, activation of RNA polymerase, and promoter binding, respectively.
To test the direct binding of AnfA to P anfH , we performed electrophoretic mobility shift assays (EMSA). For this, the radiolabeled F1 fragment (carrying the wild-type anfH promoter) and its variants
## | nifa-driven anfhdgkor expression restores fe-nitrogenase activity in a strain lacking anfa
Productive nitrogen fixation by the Fe-nitrogenase requires more than the expression of the anfHDGKOR3 operon. Current knowledge suggests that AnfA is required only for anfHDGKOR3 expression and has little impact on the expression of NifA-activated genes like nifB and rnfA, which are essential for FeFeco biosynthesis and electron transfer to Fe-nitrogenase, respectively. We, therefore, speculated that AnfA might be dispensable for the production of active (N 2 -fixing)
## F i g u r e 2
Effect of nested deletions in the R. capsulatus anfH promoter on anfH-lacZ expression. (a) Cis-regulatory elements in the anfA-anfH intergenic region. The DNA sequence encompasses the AnfA translation stop codon (TGA), the Rho-independent anfA transcription terminator, two AnfA-binding sites (AnfA_BS), the RpoN-binding site (RpoN_BS), and the AnfH translation start codon (ATG). Arrowheads mark inverted repeat sequences. The start sites of anfH promoter deletion variants F1 to F6 are indicated. (b) Reporter fusions between anfH promoter deletion variants and lacZ. Promoter variants F1 to F6 were cloned into a broad-host-range vector, before insertion of a lacZ cassette (designed for transcriptional fusions) immediately downstream of the anfH start codon resulting in reporter plasmids pBBR_F1-lacZ to pBBR_F6-lacZ (Materials and Methods). (c) Expression of anfH-lacZ fusions. R. capsulatus reporter strains carrying pBBR_F1-lacZ to pBBR_ F6-lacZ were phototrophically grown in RCV minimal medium with 10 mM serine but without Mo addition, conditions allowing anfHDGKOR3 expression. LacZ (β-galactosidase) activity is given in Miller units. The results represent the means and standard deviations of five independent experiments R. capsulatus reporter strains carrying pBBR_F1-lacZ (WT) and its variants (Mut1 to Mut7, Mut2/6, and Mut2/7) were phototrophically grown in RCV minimal medium (no Mo added) with 10 mM serine. LacZ (β-galactosidase) activity is given in Miller units. Data for WT control are the same as in . operon is the only member of the AnfA regulon, nitrogen fixation by Fe-nitrogenase should become entirely NifA-dependent in the P anfH → P nifH strain .
To determine the effect of P anfH → P nifH substitution on anfH-DGKOR3 expression, we generated two R. capsulatus strains, which carry the same P anfH → P nifH promoter substitution, but either in the wild-type (anfA + ) or ΔanfA background. Subsequently, we introduced transcriptional anfH-lacZ reporter fusions in these strains and, as a control, in the wild-type strain B10S by chromosomal integration of plasmid pMH187 as previously described. The resulting reporter strains were grown with either serine or ammonium as a nitrogen source before LacZ activity was determined.
The wild-type control (containing the native AnfA-dependent anfH promoter) showed the expected high anfH-lacZ expression in serine cultures, while no expression was observed in ammonium cultures ;. Both P anfH → P nifH strains expressed anfH-lacZ to comparable levels as the wild-type control showing that P anfH → P nifH substitution mediated effective anfHDGKOR3 expression independent of AnfA.
To test whether nitrogen fixation by Fe-nitrogenase was entirely NifA-dependent in the P anfH → P nifH strains, we introduced a polar nifD mutation (ΔnifD) in these strains, before examination of diazotrophic growth. Regardless of the nifD mutation, these strains were expected to express all the other NifA-dependent nitrogen fixation genes involved in cofactor biosynthesis and electron transport . Since ΔnifD strains lack Mo-nitrogenase, diazotrophic growth of these strains depends entirely on Fe-nitrogenase. As controls, we included the wild type, the parental ΔnifD strain, and a ΔanfA-ΔnifD strain lacking both nitrogenases.
The wild-type and ΔnifD strains grew well with N 2 as the sole nitrogen source, while the ΔanfA-ΔnifD strain failed to grow diazotrophically consistent with earlier studies. Both P anfH → P nifH strains grew almost as well as the parental ΔnifD strain suggesting that P anfH → P nifH substitution indeed decouples production of functional Fe-nitrogenase from AnfA.
Taken together, our findings show that substitution of the AnfAdependent anfH promoter by the NifA-activated nifH promoter restores anfHDGKOR expression and Fe-nitrogenase activity in a strain lacking AnfA. In other words, AnfA appears to be dispensable for FeFeco biosynthesis and electron delivery to Fe-nitrogenase.
## | d iscuss i on
Despite the wide distribution of Fe-nitrogenases, our knowledge of Fe-nitrogenase-related promoters was so far limited to one species, the γ-proteobacterium Azotobacter vinelandii. Here, we Binding sites of dyad symmetry are typically bound by dimeric F I G U R E 4 Effect of P anfH → P nifH substitution on anfH-lacZ expression and diazotrophic growth. (a) Effect of P anfH → P nifH substitution on anfH-lacZ expression. R. capsulatus strains carrying a chromosomally integrated transcriptional anfH-lacZ fusion based on plasmid pMH187were phototrophically grown in RCV minimal medium (no Mo added) with either 10 mM serine or 10 mM ammonium. The strains used were as follows: B10S:pMH187 (anfA + , anfH-lacZ), YP516:pMH187 (P anfH → P nifH , anfA + , anfH-lacZ), and YP515:pMH187 (P anfH → P nifH , ΔanfA, anfH-lacZ). LacZ (β-galactosidase) activity is given in Miller units. The results represent the means and standard deviations of five independent experiments. (b) Effect of P anfH → P nifH substitution on Fe-nitrogenase activity in R. capsulatus strains lacking AnfA. R. capsulatus strains were phototrophically grown in RCV minimal medium (no Mo added) with N 2 as the sole nitrogen source. The strains used were as follows: B10S (wild type), BS85 (anfA + , ΔnifD), YP516-BS85 (P anfH → P nifH , anfA + , ΔnifD), YP515-BS85 (P anfH → P nifH , ΔanfA, ΔnifD), and KS94A-YP415 (ΔanfA-ΔnifD). The results represent the means and standard deviations of three independent measurements (a) (b)
regulatorssuggesting that AnfA proteins also bind target promoters as dimers. The AnfA-binding site consensus previously defined for A.
vinelandii, C-N-GG-N 3 -GGTA, and our consensus share the strictly conserved GTA motif . The . Together these findings suggest that AnfA proteins in different bacteria require the GTA motif, but differ in their dependence on the TAC motif to activate their target promoters.
## A. vinelandii
Rhodobacter capsulatus AnfA is essential for anfHDGKOR3 expression and consequently, for nitrogen fixation by Fe-nitrogenase. Remarkably, a strain lacking AnfA but driving anfHDGKOR3 expression by NifA regained the capacity to grow diazotrophically via Fe-nitrogenase . This means that AnfA in the wild type is required for AnfHDGKOR3 production, but dispensable for FeFeco biosynthesis and electron delivery to Fenitrogenase. For a regulatory model, see . Our findings do not necessarily exclude other AnfA targets than the anfH promoter.
Indeed, AnfA affects the expression of different nitrogen fixation genes including iscN, nifE, fprA, and nifB. None of these genes, however, is preceded by an obvious AnfA-binding site (or just a GTA motif) suggesting that AnfA control of these genes is indirect.
In contrast to the R. capsulatus nifB promoter, we found potential AnfA-binding sites in the nifB promoters of Rhodospirillum rubrum (TAC-N 6 -GTA) and Rhodomicrobium vannielii (CAC-N 6 -GTA) suggesting direct nifB activation by AnfA in these strains. In line with the requirement of NifB for the activity of all three nitrogenases in A.
vinelandii, the nifB promoter can be activated by NifA, VnfA, or AnfA, which bind to overlapping sites in the nifB promoter.
Our finding that only a single Fe-nitrogenase-related target, the anfH promoter, strictly requires activation by AnfA in R. capsulatus, raises the question of why this diazotroph needs AnfA. One explanation is that AnfA contributes (indirectly) to fine regulation of NifA-dependent genes. Also, Mo repression of anfA introduces a regulatory level to prevent the production of Fe-nitrogenase under Mo-replete conditions. This guarantees the exclusive activity of Mo-nitrogenase, which exhibits higher N 2 -reducing activity than Fe-nitrogenase .
## Ack n owled g em ents
We thank F. Narberhaus (Bochum, Germany) for helpful discussions and continuous support. We thank J. Eisfeld (Bochum, Germany)
## F i g u r e 5
AnfA-binding sites in proteobacterial anfH promoters. (a) Comparison of AnfA-binding sites. Binding of AnfA to distal and proximal sites has been experimentally shown for R. capsulatus (this study) and A. vinelandii. Affiliation of bacterial strains to the α-and γ-proteobacteria, and the numbers of nucleotides (N) between cis-regulatory elements are indicated. Known and presumed AnfA-binding sites encompass strictly conserved GTA and partially conserved TAC motifs (highlighted in red). Lower and upper case lettering in the consensus sequences indicates conservation in at least four or five of the respective sequences, respectively. (b) AnfAbinding site logo. The AnfA-binding site consensus based on all distal and proximal sites shown in (a) was generated using the weblo go.berke ley.edu program (a)
## (b)
Bits for help with electrophoretic mobility shift assays. This work was financed by a grant from the Deutsche Forschungsgemeinschaft (DFG) (Ma 1814/4-2) to BM.
## Co n fli c t o f i nte r e s t s
None declared.
## Auth o r co ntr i b uti
## E th i c s s tatem ent
None required.
## Data ava i l a b i l i t y s tat e m e n t
All data are provided in full in the Section 3.
## O rci d
Bernd Masepohl https://orcid.org/0000-0002-1747-7177
A PPE N D I X 2 F I G U R E A 1 Comparison of AnfA proteins
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Complications Among Adults Hospitalized With Influenza: A Comparison of Seasonal Influenza and the 2009 H1N1 Pandemic
Background. Persons with influenza can develop complications that result in hospitalization and death. These are most commonly respiratory related, but cardiovascular or neurologic complications or exacerbations of underlying chronic medical conditions may also occur. Patterns of complications observed during pandemics may differ from typical influenza seasons, and characterizing variations in influenza-related complications can provide a better understanding of the impact of pandemics and guide appropriate clinical management and planning for the future.Methods. Using a population-based surveillance system, we compared clinical complications using International Classification of Diseases, Ninth Revision (ICD-9) discharge diagnosis codes in adults hospitalized with seasonal influenza (n = 5270) or 2009 pandemic influenza A(H1N1) (H1N1pdm09; n = 4962).Results. Adults hospitalized with H1N1pdm09 were younger (median age, 47 years) than those with seasonal influenza (median age, 68 years; P < .01), and differed in the frequency of certain underlying medical conditions. Whereas there was similar risk for many influenza-associated complications, after controlling for age and type of underlying medical condition, adults hospitalized with H1N1pdm09 were more likely to have lower respiratory tract complications, shock/sepsis, and organ failure than those with seasonal influenza. They were also more likely to be admitted to the intensive care unit, require mechanical ventilation, or die. Young adults, in particular, had 2-4 times the risk of severe outcomes from H1N1pdm09 than persons of the same ages with seasonal influenza.Conclusions. Although H1N1pdm09 was thought of as a relatively mild pandemic, these data highlight the impact of the 2009 pandemic on the risk of severe influenza, especially among younger adults, and the impact this virus may continue to have.
Influenza causes considerable morbidity during each annual influenza season. Although influenza is most often a self-limited respiratory illness, severe illness including hospitalization and death can occur as a result of complications stemming from the influenza virus infection. In the United States, an estimated 100 000-300 000 hospitalizations [bib_ref] Hospitalizations associated with influenza and respiratory syncytial virus in the United States, Zhou [/bib_ref] and 3300-48 000 deaths are attributable to influenza each year. Since the 1960s, several factors have been recognized to increase the risk for complications of influenza, including extremes of age, certain underlying chronic diseases, and pregnancy [bib_ref] Influenza immunization: statement, Burney [/bib_ref] [bib_ref] Prevention and control of seasonal influenza with vaccines: recommendations of the Advisory..., Fiore [/bib_ref].
The most common influenza-associated complications are pulmonary, especially pneumonia, but a number of organ systems can be affected [bib_ref] Complications of viral influenza, Rothberg [/bib_ref]. Cardiovascular complications, such as myocardial infarction, have been associated with antecedent respiratory infection, including influenza [bib_ref] Influenza as a trigger for acute myocardial infarction or death from cardiovascular..., Warren-Gash [/bib_ref]. Neurologic complications including seizures and encephalopathy have also been documented among persons with influenza [bib_ref] Influenza virus and CNS manifestations, Studahl [/bib_ref] , more often observed in children [bib_ref] A population-based study of neurologic manifestations of severe influenza A(H1N1)pdm09 in California, Glaser [/bib_ref]. Complications may also result from the exacerbation of underlying chronic medical conditions following infection, such as asthma, chronic obstructive pulmonary disease (COPD), or heart disease [bib_ref] Respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a..., Rohde [/bib_ref] [bib_ref] Asthma and pneumonia, Browne [/bib_ref].
In April 2009, the influenza A(H1N1)pdm09 virus (H1N1pdm09) caused the first influenza pandemic in >40 years [bib_ref] Emergence of a novel swine-origin influenza A (H1N1) virus in humans, Dawood [/bib_ref]. Patterns of complications among persons with influenza may differ between influenza pandemics and annual seasons due to either differences in age groups most affected or virulence of circulating strains. An appreciation of particular patterns of complications and how they may differ during an influenza pandemic can help in understanding the clinical impact of the 2009 pandemic, guide clinicians toward the most effective diagnosis and management of patients with influenza, and assist in future resource planning.
Several studies have described patients hospitalized with H1N1pdm09, including those with influenza-related complications, in case series or at single-hospital sites or geographic areas [bib_ref] A population-based study of neurologic manifestations of severe influenza A(H1N1)pdm09 in California, Glaser [/bib_ref] [bib_ref] Pandemic Influenza A (H1N1) Virus Hospitalizations Investigation Team. Influenza-associated pneumonia among hospitalized..., Jain [/bib_ref] [bib_ref] Hospitalized patients with 2009 H1N1 influenza in the United States, Jain [/bib_ref] [bib_ref] Complications and outcomes of pandemic 2009 influenza A (H1N1) virus infection in..., Lee [/bib_ref] [bib_ref] Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1)..., Louie [/bib_ref] [bib_ref] Risk factors for hospitalisation and poor outcome with pandemic A/H1N1 influenza: United..., Nguyen-Van-Tam [/bib_ref] [bib_ref] Influenza pneumonia: a comparison between seasonal influenza virus and the H1N1 pandemic, Riquelme [/bib_ref]. However, there has been limited evaluation of influenza-related complications in a large multicenter population of adults hospitalized with influenza during multiple influenza seasons, with the ability to directly compare complications from seasonal and pandemic influenza and with a large enough sample size to assess more rare complications. In this analysis, we describe data on >9000 adult patients during 2005-2010 from the Emerging Infections Program (EIP) Influenza Surveillance Network, a population-based surveillance system in the United States for patients hospitalized with laboratoryconfirmed influenza. The purpose of this analysis was to describe and compare influenza-associated complications among adults hospitalized with H1N1pdm09 and seasonal influenza. Complications among children in this network have been previously described [bib_ref] Complications and associated bacterial coinfections among children hospitalized with seasonal or pandemic..., Dawood [/bib_ref].
# Methods
## Study population and case definitions
Since 2005, the EIP influenza surveillance network has conducted population-based surveillance of adults hospitalized with laboratory-confirmed influenza virus infection, using a standardized surveillance protocol in 240 hospitals of 10 geographically diverse surveillance areas across the United States. Included in this analysis are adult patients hospitalized with laboratory-confirmed influenza identified in EIP during An adult is included in EIP influenza surveillance if he/she is ≥18 years of age, resides in the surveillance area, and is hospitalized with laboratory confirmation of influenza virus infection. Laboratory testing for influenza is ordered at the discretion of clinicians providing medical care, and confirmation may include a positive result from viral culture, direct or indirect fluorescent antibody staining, rapid antigen test, reverse transcription polymerase chain reaction (PCR), or documentation of a positive test result in a patient's medical record. Patients are identified through hospital laboratory and admission databases, infection control logs, and hospital discharge data for patients with a documented positive influenza test. Through medical record review, data are collected for each patient regarding demographic characteristics, medical history, clinical course, and outcomes (eg, admission to intensive care unit, mechanical ventilation, or death), as well as the first 9 discharge codes using the International Classification of Diseases, Ninth Revision (ICD-9).
Patients identified through surveillance were classified as having seasonal influenza (hospitalized 1 October 2005-14 April 2009) or H1N1pdm09 (hospitalized 15 April 2009-30 April 2010). Patients hospitalized during the pandemic period but known to be infected with influenza B viruses or seasonal influenza A subtypes were excluded.
Complications were classified based on ICD-9 discharge codes and included from the following categories: pulmonary, cardiovascular, neurologic, metabolic, musculoskeletal, cerebrovascular, and endocrine. A complete list of included complications and associated ICD-9 codes are included in Supplementary [fig_ref] Table 1: Characteristics of Adults Hospitalized With Laboratory-Confirmed Seasonal or Pandemic InfluenzaAbbreviations [/fig_ref].
# Data analysis
Clinical characteristics of patients and frequencies of complications were compared using χ 2 tests for categorical variables, and medians and distributions of continuous variables were compared using the Wilcoxon-Mann-Whitney test. To control for the contribution of age and underlying medical condition to the risk of complications, we calculated adjusted relative risks of complications in patients with H1N1pdm09 compared with patients with seasonal influenza using multivariable log-binomial regression. All analyses were performed using SAS software version 9.2 (Cary, North Carolina).
# Results
A total of 5959 adult patients with influenza were identified through EIP surveillance during the seasonal influenza periods from 1 October 2005 through 14 April 2009, and 5446 adult patients were identified during the pandemic period 15 April 2009-30 April 2010. Among adults identified during the pandemic period, 66 (1.2%) were excluded because they were infected with influenza B viruses (n = 52) or seasonal influenza A virus subtypes (n = 14 for H3N2). Unknown subtypes were assumed to be the predominant H1N1pdm09. In addition, a total of 689 (11.6%) patients with seasonal and 418 (7.8%) patients with pandemic influenza were excluded from the analysis because they were missing ICD-9 code data. Patients without ICD-9 data did not differ in risk of intensive care unit (ICU) admission, mechanical ventilation, or death. The data collection instrument captures the first 9 ICD-9 codes; among included patients, 5388 (52.7%) had all 9 ICD-9 code fields completed.
Adults hospitalized with H1N1pdm09 were younger (median age, 47 years) than those with seasonal influenza (median age, 68 years) (P < .01; [fig_ref] Table 1: Characteristics of Adults Hospitalized With Laboratory-Confirmed Seasonal or Pandemic InfluenzaAbbreviations [/fig_ref]. [fig_ref] Figure 1: Age distribution of adults hospitalized with seasonal or pandemic influenza, 2005-2010, United... [/fig_ref] shows the substantial differences in the age distribution of patients during the seasonal and pandemic periods. During the seasonal influenza period, the largest proportional age group of patients was ≥80 years of age (29%), whereas this age group represented the smallest fraction of patients with pandemic influenza (4%), and only 21% of patients hospitalized with H1N1pdm09 were ≥60 years of age. Fewer than half of patients had documented influenza vaccination in either group, although significantly more during prepandemic seasons (46%) than during the pandemic season (23%; P < .001), when vaccine was not available until after the peak of the fall wave. Antiviral treatment was higher during the pandemic, with 83% of patients receiving antiviral medications compared with 54% during prepandemic seasons (P < .001).
Patients hospitalized during the pandemic were only slightly less likely to have a documented underlying medical condition than during the seasonal influenza period (80% vs 84%; P < .01); however, the types of medical conditions reported varied between the 2 groups [fig_ref] Table 1: Characteristics of Adults Hospitalized With Laboratory-Confirmed Seasonal or Pandemic InfluenzaAbbreviations [/fig_ref]. Patients hospitalized with H1N1pdm09 were more likely than patients with seasonal influenza to have asthma or be pregnant; they were substantially less likely to have COPD or other chronic lung diseases, chronic cardiovascular disease, or chronic metabolic diseases [fig_ref] Table 1: Characteristics of Adults Hospitalized With Laboratory-Confirmed Seasonal or Pandemic InfluenzaAbbreviations [/fig_ref]. A greater percentage of adults hospitalized with H1N1pdm09 were admitted to the ICU (23%) or required mechanical ventilation (13%) than adults hospitalized with seasonal influenza (15% ICU, P < .001; 9% mechanical ventilation, P < .001). Mortality was similar in both groups (4%) [fig_ref] Table 1: Characteristics of Adults Hospitalized With Laboratory-Confirmed Seasonal or Pandemic InfluenzaAbbreviations [/fig_ref].
We examined several complications affecting a number of organ systems [fig_ref] Table 2: Frequency of Complications Among Persons Hospitalized With Laboratory-Confirmed Seasonal or Pandemic Influenza,... [/fig_ref]. Among seasonal patients, 64% had at least 1 of the included complications recorded, compared with 71% of patients with H1N1pdm09 (P < .01). Overall, patients with 1 of the included complications had 5.5% risk of mortality compared with 0.3% mortality in patients without any of the included complications (P < .001; relative risk = 15.4 after controlling for age and underlying condition). Whereas pulmonary complications were the most commonly recorded during both periods [fig_ref] Table 2: Frequency of Complications Among Persons Hospitalized With Laboratory-Confirmed Seasonal or Pandemic Influenza,... [/fig_ref] , there was a significant difference between the 2 periods, with 55% of seasonal influenza patients having at least 1 recorded pulmonary complication, compared with 64% of patients with H1N1pdm09 (P < .001). Of pulmonary complications, pneumonia was the most commonly recorded (seasonal: 35%, pandemic: 43%, P < .01). Other common complications included renal failure (12% among both seasonal and pandemic patients, P = .99), cardiovascular complications (seasonal, 6%; pandemic, 5%; P = .02), and shock/sepsis (seasonal, 6%; pandemic, 10%; P < .01). Neurologic, musculoskeletal, cerebrovascular, and endocrine complications were also noted, but less frequently (<5% of seasonal or pandemic patients).
Although the frequency of many complications differed between the seasonal and pandemic period, for some the associations were no longer significant after we controlled for patients' ages and underlying medical condition(s), indicating that the risk of those complications did not differ during the pandemic for persons of similar age and medical history. These complications included exacerbations of asthma and COPD, most cardiovascular and neurologic complications, and musculoskeletal, cerebrovascular, and endocrine complications.
There were several complications, however, that were significantly more likely to occur among adults hospitalized with H1N1pdm09 than adults with seasonal influenza, even after controlling for age and underlying medical conditions. These were most commonly respiratory complications such as pneumonia (adjusted relative risk [aRR] = 1.3) and respiratory failure (aRR = 1.8), as well as nonrespiratory complications such as renal failure (aRR = 1.3) and shock/sepsis (aRR = 1.7). Some other, less frequent complications were also more likely among patients hospitalized with H1N1pdm09 including acute respiratory distress syndrome (aRR = 3.0), acute heart failure (aRR = 3.4), hemoptysis (aRR = 1.8), and encephalopathy (aRR = 1.5). We further found that adults hospitalized with H1N1pdm09 were approximately 1.5 times more likely to experience clinical outcomes such as ICU admission, mechanical ventilation, or death than those hospitalized with seasonal influenza, after controlling for age and underlying conditions.
To examine whether these associations were modified by age, we repeated the analysis in 3 age strata: 18-39 years, 40-64 years, and ≥65 years [fig_ref] Figure 2: The relative risk of selected complications [/fig_ref]. Young adults <40 years of age hospitalized with H1N1pdm09 were particularly affected, with approximately 2.5 times the risk of respiratory failure, twice the risk of ICU admission or mechanical ventilation, and 4 times the risk of death compared with adults <40 years hospitalized with seasonal influenza.
# Discussion
Although the 2009 influenza pandemic is considered to have been a relatively mild pandemic, especially when compared with the previous 3 documented influenza pandemics, many serious complications did occur. We found that compared with adults hospitalized during 4 previous influenza seasons, those hospitalized with pandemic influenza were younger and more likely to have lower respiratory tract complications (including pneumonia) and corresponding indicators of severe illness (including shock/sepsis and acute organ failure), even after controlling for differences in age and comorbidities. Furthermore, adults hospitalized with influenza during the pandemic were also more likely to be admitted to the ICU, require mechanical ventilation, or die during hospitalization.
These findings are consistent with observations from previous influenza pandemics, which have noted an increased risk of lower respiratory tract complications, including viral pneumonia with or without bacterial coinfection [bib_ref] Studies on influenza in the pandemic of 1957-1958. II. Pulmonary complications of..., Louria [/bib_ref] [bib_ref] Pulmonary complications of interpandemic influenza A in hospitalized adults, Murata [/bib_ref]. Likewise, the findings from this large case series are similar to a few smaller recent studies that were able to directly compare adults with H1N1pdm09 or seasonal influenza. In Hong Kong, adults hospitalized with H1N1pdm09 had a significantly greater frequency of complications and mortality despite being younger than patients hospitalized with influenza in 2007-2008 [bib_ref] Complications and outcomes of pandemic 2009 influenza A (H1N1) virus infection in..., Lee [/bib_ref].
A small study of patients hospitalized with influenza in Australia in 2009 found an increased risk of admission to intensive care among persons infected with H1N1pdm09 vs cocirculating seasonal influenza strains [bib_ref] Comparison of adult patients hospitalised with pandemic (H1N1) 2009 influenza and seasonal..., Dalton [/bib_ref] , whereas patients with outpatient medically attended H1N1pdm09 in the United States experienced a greater risk of pneumonia than patients with seasonal influenza H1N1 or H3N2 in previous seasons [bib_ref] Clinical characteristics and 30-day outcomes for influenza A 2009 (H1N1), Belongia [/bib_ref]. Postpandemic, 2 studies have also found an increased risk of pneumonia [bib_ref] Influenza in the immediate post-pandemic era: a comparison with Seasonal and Pandemic..., Rahamat-Langendoen [/bib_ref] or ICU admission/death [bib_ref] Patients hospitalized with laboratory-confirmed influenza during the 2010-2011 influenza season: exploring disease..., Chaves [/bib_ref] among patients with H1N1pdm09 compared with those with other seasonal influenza strains. During a pandemic, the spread of an antigenically novel influenza virus against which there is limited preexisting immunity may be expected to result in an increased risk of severe respiratory complications compared with annually circulating strains of seasonal influenza. One notable hallmark of the 2009 influenza pandemic was the increased risk of influenza among younger persons, which was reflected in the substantial differences in the age distribution we observed between adults hospitalized with pandemic or seasonal influenza. Whereas this trend is often seen during influenza pandemics [bib_ref] Pandemic versus epidemic influenza mortality: a pattern of changing age distribution, Simonsen [/bib_ref] , some degree of cross-reactive immunity among persons of older ages may have contributed to relatively low levels of infection with H1N1pdm09 in older adults [bib_ref] Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus, Hancock [/bib_ref]. Although we did find that all adults had a higher risk of respiratory complications and severe outcomes with H1N1pdm09 than seasonal influenza, we further stratified by age and found that younger adults aged <40 years were particularly affected, including a 4 times greater risk of death during the pandemic compared with adults of similar age hospitalized with seasonal influenza.
This trend toward more severe illness, especially in younger persons, has important implications for clinical management during pandemics, as younger persons are also likely to have different comorbidities that need to be managed in parallel. As we found, patients hospitalized during the pandemic were more likely to have asthma or be pregnant, medical histories associated with younger age. While we did not have data on obesity for hospitalized patients prior to the pandemic, other studies have indicated that morbid obesity was associated with hospitalization during the pandemic even in the absence of other medical conditions [bib_ref] Morbid obesity as a risk factor for hospitalization and death due to..., Morgan [/bib_ref] , and thus may be a frequent comorbidity seen among younger adults with severe influenza. Conversely, adults hospitalized during the pandemic were less likely to have conditions such as COPD, chronic heart disease, or chronic metabolic diseases, which are more prevalent among older adults.
Consistent with these differences in age and medical history, the overall pattern of other complications observed among patients hospitalized with influenza during the 2009 pandemic differed from that observed during previous seasonal epidemics. During the pandemic, for example, there were fewer exacerbations of COPD or acute myocardial infarctions, but more hospitalizations with acute complications of asthma. Much of these differences in frequency were attributable to differences in age and corresponding underlying comorbidities, as the risk of such complications did not differ during the pandemic for individuals of similar ages and medical history. Nonetheless, there was a different distribution of complications during the pandemic and as clinicians may expect to encounter a younger demographic of patients with severe influenza during pandemics, the approach to resource planning for patients and their associated medical complications may need to reflect these differences.
Finally, an increased incidence of influenza in the population during the pandemic resulted in a corresponding increased incidence of hospitalization compared with previous influenza seasons [bib_ref] Estimating the burden of 2009 pandemic influenza A (H1N1) in the United..., Shrestha [/bib_ref]. In 1 year during the pandemic, we identified 5446 patients with H1N1pdm09 in the same surveillance areas, similar in number to 4 previous influenza seasons combined (n = 5959). An increase in the number of hospitalizations during a pandemic not only affects demands on hospital capacity, but also means an increased number of patients with complications that may require additional resources, staffing, and equipment (eg, supplemental oxygen, mechanical ventilation, dialysis) to effectively manage them.
This analysis is subject to some limitations. First, complications were classified based on ICD-9 discharge coding and were not confirmed with medical record review; however, our frequencies were similar to other smaller series of hospitalized patients during the 2009 pandemic that used chart review and/or radiology for confirmation of complications [bib_ref] Pandemic Influenza A (H1N1) Virus Hospitalizations Investigation Team. Influenza-associated pneumonia among hospitalized..., Jain [/bib_ref] [bib_ref] Hospitalized patients with 2009 H1N1 influenza in the United States, Jain [/bib_ref] [bib_ref] Complications and outcomes of pandemic 2009 influenza A (H1N1) virus infection in..., Lee [/bib_ref]. While we also cannot exclude the possibility of changes in ICD-9 coding practices during the pandemic, this surveillance system has been operating using standardized methods and in the same sites across all of the included years and when we compared the distributions of other recorded ICD-9 codes between patients before and during the pandemic [fig_ref] Table 2: Frequency of Complications Among Persons Hospitalized With Laboratory-Confirmed Seasonal or Pandemic Influenza,... [/fig_ref] , there were no differences in the proportion coded for pneumonia and influenza, any respiratory disease, or respiratory and circulatory diseases. Second, only the first 9 discharge ICD-9 codes were captured during data collection. Approximately half of the included patients had 9 codes recorded; thus, we were unable to determine if they had additional codes in which one of the included complications were recorded. However, we considered acute and often severe complications, which may have been more likely to be listed higher among discharge codes.
Finally, patients included in this surveillance system are tested for influenza at the discretion of the treating clinicians; thus, some patients who truly had influenza but were not tested would not be represented in our analysis. This may be especially true of patients who do not present with a typical influenza-like illness, but with complications of underlying medical conditions or other nonrespiratory complications. In this study population, approximately 5% of patients did not have any respiratory ICD-9 code [fig_ref] Table 2: Frequency of Complications Among Persons Hospitalized With Laboratory-Confirmed Seasonal or Pandemic Influenza,... [/fig_ref] , and about 12% of patients with 1 of the included complications did not have a respiratory complication [fig_ref] Table 2: Frequency of Complications Among Persons Hospitalized With Laboratory-Confirmed Seasonal or Pandemic Influenza,... [/fig_ref]. These complications may be underrepresented if physicians are less likely to test such patients for influenza. Likewise, an increased use of the more sensitive PCR to diagnose influenza during the pandemic could have increased the detection of influenza during 2009.
In the largest case series describing clinical complications of influenza, we observed a greater proportion of lower respiratory complications and severe outcomes among persons hospitalized with influenza during the 2009 pandemic compared to previous influenza seasons. In addition, although the risk of many other complications did not differ for persons of similar age and medical history, these were often less likely overall because of the younger age of patients with severe influenza during the pandemic. Understanding the patterns of influenza-associated complications and how those may differ during influenza pandemics can help direct clinicians toward the most effective management of ill patients and assist in resource planning for future seasons and pandemics. Although the 2009 pandemic was thought of as relatively mild, these data highlight the impact of this pandemic on increasing the risk of severe illness from influenza, especially among younger adults, and the impact this virus may continue to have in the future.
## Supplementary data
Supplementary materials are available at Clinical Infectious Diseases online (http://cid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
## Notes
[fig] Figure 1: Age distribution of adults hospitalized with seasonal or pandemic influenza, 2005-2010, United States. [/fig]
[fig] Figure 2: The relative risk of selected complications (with 95% confidence intervals) among patients hospitalized with pandemic influenza vs seasonal influenza, stratified by age. Abbreviations: ARDS, acute respiratory distress syndrome; ICU, intensive care unit. [/fig]
[table] Table 1: Characteristics of Adults Hospitalized With Laboratory-Confirmed Seasonal or Pandemic InfluenzaAbbreviations: COPD, chronic obstructive pulmonary disease; IQR, interquartile range. [/table]
[table] Table 2: Frequency of Complications Among Persons Hospitalized With Laboratory-Confirmed Seasonal or Pandemic Influenza, as Categorized by ICD-9 Code [/table]
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Unusual mechanical failures of intrathecal baclofen pump systems: symptoms, signs, and trouble shooting
Introduction Although intrathecal baclofen (ITB) therapy is an effective treatment for spasticity, it has several disadvantages and a risk of complications. Methods We present six pediatric patients who suffered from unusual mechanical failures of intrathecal baclofen pump systems. Results With these case-vignettes, we provide a systematic approach on how to interpret the symptoms of ITB complications and an advice which further diagnostic and therapeutic steps to follow. We underline the seriousness of baclofen overdose, underdosing or withdrawal.
# Introduction
Reducing spasticity makes care easier, prevents secondary orthopedic problems and relieves pain. This improves the patient's quality of life. Baclofen has an anti-spastic effect, but cerebral side effects often occur before therapeutic anti-spastic effects of oral administration are observed. To increase efficacy, intrathecal baclofen therapy (ITB) has been used to treat spasticity since the 1980s. ITB yields better reduction in spasticity at doses 1000 times lower than oral baclofen and minimizes adverse effects [bib_ref] Intrathecal baclofen withdrawal syndrome-a life-threatening complication of baclofen pump: a case report, Mohammed [/bib_ref].
The indication for ITB treatment arises from the severity of spasticity and dystonia. Children with a gross motor function classification system (GMFCS) level IV or V benefit most. The treatment is palliative and constitutes a challenge with respect to the multimorbidity of the patients.
The baclofen pump is inserted subcutaneously in the lower abdomen and connected to a catheter as part of a surgical procedure. This catheter is placed under the skin and inserted into the intrathecal space at a lumbar level and then moved upwards. Placement of the tip of the intrathecal catheter has been suggested at the T1-T2 level for spastic quadriplegia, the T6-T10 level for spastic diplegia and in the midcervical region for dystonia [bib_ref] Intrathecal baclofen for generalized dystonia, Albright [/bib_ref]. The dose is adjusted individually, since in children there are no clear age-related or body weight-correlated recommendations [bib_ref] Intrathekale Baclofentherapie: Palliativmaßnahme bei spastischen und dystonen Bewegungsstörungen, Voss [/bib_ref]. Clinically, anti-spastic effects of continuous intrathecal dose changes occur with a 2-4-h delay [bib_ref] Intrathecal baclofen therapy in Germany: proceedings of the IAB-interdisciplinary working group for..., Dressler [/bib_ref]. A loss of efficacy in the long-term course in patients with a stable underlying disease (e.g. cerebral palsy) is seldomly observed [bib_ref] Intrathekale Baclofentherapie: Palliativmaßnahme bei spastischen und dystonen Bewegungsstörungen, Voss [/bib_ref].
Although ITB is an effective treatment of spasticity, it suffers from several disadvantages. The pump reservoir must be refilled with baclofen regularly through an injection. In addition, the pump must be surgically exchanged about every 5-7 years for battery exhaustion. Furthermore physicians must be aware of potentially serious complications [fig_ref] Table 1: Symptoms and therapy of baclofen-underdosing/withdrawal and overdosing [/fig_ref]. A withdrawal syndrome is the most common cause of a life-threatening event, but toxicity from overdose due to mechanical system malfunction has also to be beared in mind [bib_ref] Intrathecal baclofen toxicity and deep coma in minutes, Tunali [/bib_ref]. In children, ITB complications occur in about 24-30% of cases [bib_ref] Intrathecal baclofen therapy-practical approach: clinical benefits and complication management, Winter [/bib_ref] [bib_ref] Analysis of complications in 430 consecutive pediatric patients treated with intrathecal baclofen..., Motta [/bib_ref] [bib_ref] Complications of intrathecal baclofen pumps in children, Gooch [/bib_ref] , and the complications can present challenges for clinicians, so a high index of suspicion is needed to make the correct diagnosis [bib_ref] Baclofen pumps: uses and complications, Woolf [/bib_ref].
# Patients and methods
We here describe six unusual mechanical ITB complications [fig_ref] Table 2: Overview of cases with mechanical ITB complication [/fig_ref] , occurring in most of the patients after a long and successful treatment period, which obscured some of the hints to diagnosis and treatment of the failure [fig_ref] Table 1: Symptoms and therapy of baclofen-underdosing/withdrawal and overdosing [/fig_ref]. We use SynchroMed II pumps from Medtronic with corresponding Ascenda catheters. We applied a variable flow for patient case 6 and a fixed flow for all other patients.
## Case 1
At the age of 11 years, the boy suffered from a traffic accident with considerable brain trauma. He developed a severe residual syndrome with spasticity GMFCS V and apallic syndrome. With 12 years a baclofen pump was implanted with good effect on muscle tone. The pump was refilled regularly.
Five years after baclofen pump implantation, for the first time a pump alarm arose. A few hours later, he presented at the emergency room because of high muscle tension, sweating and trembling. Creatine kinase was increased to 15.02 μkat/l (reference < 4.1). The suspected baclofen withdrawal symptoms were treated with oral baclofen, diazepam, continuous administration of analgesics and high-volume infusion therapy.
In search for the cause, an X-ray was taken, and because this was unremarkable, contrast medium was applied via the side port of the baclofen pump to test the catheter system. No contrast medium flow into the catheter system could be Repositioning the catheter for high cervical baclofen delivery with high flow did improve the situation Step-by-step procedure for suspected baclofen pump problems Clinical symptoms (see [fig_ref] Table 1: Symptoms and therapy of baclofen-underdosing/withdrawal and overdosing [/fig_ref] shown. In a subsequent computed tomography (CT), no leakage was demonstrated, neither. Disconnection of the catheter was not detectable, so pump replacement was indicated. During intraoperative evaluation, a corroded and thus no longer functional pump was found [fig_ref] Figure 1: Case 1, photo of the corroded pump [/fig_ref] , a very rare complication [bib_ref] Intrathecal pump: an abrupt intermittent pump failure, Riordan [/bib_ref] [bib_ref] Intrathecal baclofen in spinal spasticity: frequency and severity of withdrawal syndrome, Stetkarova [/bib_ref]. A new pump was implanted and the symptoms resolved.
## Case 2
The girl had fell victim to carbon monoxide poisoning at the age of 2 years. As a result, she developed spasticity GMFCS V and an apallic syndrome. A baclofen pump was implanted 1 month after the accident, and intrathecal baclofen therapy showed good effect on muscle tension. Five years later the patient was presented with a soft, indolent abdominal . Spasticity had worsened in the previous weeks, so the baclofen dosage had been increased step by step. The swelling on the abdomen was punctured and serous fluid was collected.
The fluid showed a high protein content (total protein 8.4 g/l) and no inflammatory cells, so it was interpreted as leakage out of the catheter. Surgical revision was performed, and the connector from the catheter to the pump was found to be loose. Baclofen was leaking from the proximal and cerebrospinal fluid from the distal part. The pump at the end of battery lifetime and the connector were replaced. Subsequently, a lower baclofen dosage was required to obtain sufficient treatment effects.
## Case 3
At the age of three, the boy suffered from hypoxic brain damage and subsequent bilateral spasticity GMFCS V as result of a drowning accident. Four months later, a baclofen pump was implanted with good effect. During the longer course, the patient developed inactivity osteoporosis and experienced several bone fractures. With 12 years, treatment with bisphosophonates was therefore initiated.
Overall, ITB was carried out in the patient for 13 years without any problems. Shortly before the planned explantation of the pump because of low battery charge, the patient's spasticity worsened. A CT scan was performed under suspicion of catheter dysfunction which showed subdural contrast depots at two points and a subcutaneous catheter rupture .
For that reason, both pump and catheter were explanted. Intraoperatively, it was recognized that the tissue around the pump was hardly movable, calcareous coating of the pump was noticed, and the entry point of the catheter into the spinal canal was found to be surrounded by chalky, strongly adherent tissue. The calcification was very unusual and was interpreted as an adverse side effect of the bisphosphonate therapy .
Six days after replacement, wound dehiscence developed with purulent secretion. The patient suffered from fever and laboratory results showed leukocytosis and increased serum CRP values. Staphylococcus aureus was identified in local wound specimen and blood culture. Antibiotic treatment with cefotaxime and clindamycin was started. The new pump and the entire catheter system had to be completely removed.
The patient then developed pronounced withdrawal symptoms after the pump explantation: shakiness, sweating, agitation and increase in spasticity. He received high doses of oral baclofen, midazolam, clonidine and dronabinol to alleviate the withdrawal symptoms. The patient's condition slowly improved, and a new pump system was reimplanted several months later uneventfully.
## Case 4
Following peripartal varicella encephalitis associated with intracerebral haemorrhage, the newborn girl developed bilateral spastic cerebral palsy GMFCS V. At seven years ITB was started with an initially good response. The first complication occurred as soon as 1 month after pump implantation. An increase in spasticity and a higher frequency of crying periods were reported. By increasing the baclofen dose from 150 to 320 μg/d, no improvement of the symptoms could be achieved. X-ray demonstrated a dislocation of the intrathecal catheter . The catheter was rolled up at the level of L3 and could be repositioned into the intrathecal space neurosurgically.
A long-term complication occurred 4 years later when spasticity and restlessness increased again. An X-ray was inconspicuous. Two months later, a discrepancy during pump filling was found. While 2.1 ml were supposed to be in the pump reservoir, 11 ml could be extracted. This showed a reduced extrusion of baclofen without programmed lowering of the flow rate. The catheter access port was punctured. Neither cerebrospinal fluid could be aspirated nor contrast agent injected. Surgical pump explantation revealed a coiled spinal catheter which had led to a functional closure of the catheter. Retrospectively, the detection of the once more subcutaneously twisted catheter on the X-ray images remains challenging .
## Case 5
The patient presented with an Addison's crisis and severe hypoglycemia as initial manifestation of X-linked adrenoleukodystrophy at the age of 4 years. He developed progressive leukodystrophy as typical feature of the underlying disease and bilateral basal ganglion necrosis which we interpreted as consequence of the severe Addison crisis. This resulted in a mixed picture of spastic and dystonic-dyskinetic movement disorder, initial GMFCS lay at III, decreasing to IV. One month after the neurological deterioration, baclofen pump was implanted.
Difficulties with the intrathecal baclofen therapy started nearly immediately. The symptoms varied widely with intermittent weakness in the legs. This was interpreted as a sign of overdose. These symptoms alternated with phases of Case 2, photo of the abdomen shows subcutaneous bulging by dislocated catheter Case 3, CT shows catheter rupture: spiral CT of the spine after injection of the baclofen pump with 15 ml solutrast: evidence of a subcutaneous contrast medium extravasation as a sign of a tube rupture dorsal to L4 (long white arrow). In addition, most likely misalignment of the catheter tip with two subdural contrast agent deposits (short white arrows). Only minute amounts of contrast medium are displayed intrathecally Case 3, histological workup displays steps of heterotopic bone formation with fibrous mesenchymal tissue lying adjacent to calcifying areas and immature woven bone (Scalebar corresponds to 100 μM) increased spasticity suggesting underdosing. The fluctuating symptoms led to frequent reprogramming of the baclofen pump rate. The dose varied between 25 and 510 μg/d. CT was performed because of the unstable treatment results and revealed an epidural position of the dislocated intrathecal catheter . This incorrect positioning most likely resulted in the delivery of changing amounts of intrathecal baclofen which explained the fluctuating symptoms. After revision of the catheter, baclofen was effective at a dose of 350 μg/d.
During follow-up the dose had to be increased only as the disease progressed.
## Case 6
Periventricular leukomalacia because of preterm birth was the cause of bilateral spastic cerebral palsy GMFCS V in this patient. At seven years, the boy received a baclofen pump. Case 4, lateral spine X-ray: the catheter is dislocated and a large part lies rolled up subcutaneously, so that it ends in the soft tissues between the lumbar spinous processes Case 4, X-ray fluoroscopy: after puncturing the access port of the pump, with correct needle position documented on the image, neither CSF can be aspirated nor contrast medium applied. The part of the tube system that is not covered by the pump is shown correctly, the tip of the catheter is projected at the level of the 6th thoracal vertebra (long white arrow). The short white arrows disclose the rolling up of the catheter Case 5, spiral CT of the skull base and the upper cervical spine after contrast medium application via the side port of the baclofen pump:
The tube system was intact up to the intraspinal entry of the tube at the level of L4/L5, without evidence of leakage. Contrast medium distribution presents epidurally dorsal to the CSF space at C2 (long arrow), pushing between the dural sheets of the tentorium (short arrow). There is no evidence of intrathecal contrast medium ITB showed a good response until the baclofen pump was changed after 7 years when the battery was low. Three months after changing the pump, the patient developed a spastic crisis. X-ray was inconspicuous without signs of catheter interruption or leakage. A pump malfunction was assumed, and a replacement of the pump was planned. For short-term treatment of the withdrawal symptoms, an additional, external CSF catheter was placed to administer baclofen intrathecally which led to a rapid improvement. A new pump was implanted shortly afterwards and was connected to the remaining catheter system. However, during the following 5 months, problems persisted with increased intrathecal baclofen demand, interrupted by phases with signs of overdose as increased fatigue and somnolence. When analysing the difference between internal and external baclofen administration were twofold: the external catheter was positioned very high, and baclofen was more diluted leading doubled flow velocity. A rostral baclofen distribution problem has been discussed. For that reason a new spinal catheter was positioned to the level of C0/C1, and baclofen was thinned down to 1 mg/ml (compared to the standard dilution of 2 mg/ml) to allow for a reasonable intrathecal flow velocity delivered by the pump. This finally resulted in improvement and stabilisation of the previously unfavourable situation. Employing higher flow and extremely high catheter placement intraventricular placement could be avoided, what had been discussed as alternative solution.
# Discussion
Any change in the patient's clinical state could be a warning sign of baclofen pump dysfunction.
Problems with ITB can be divided into three categories: first, there are application errors. These include incorrect programming or filling of the pump with the wrong drug dilution or failure to recognize a pending battery change. Second, there are mechanical flow problems. Here it is important to identify the localization: Is it the pump itself that has a defect and either delivers too much, too little or no baclofen at all, is it the connection point pump to abdominal catheter or abdominal catheter to spinal catheter part, is it in the course of the catheter or is it the intrathecal positioning of the catheter? Third, there are infections, here beyond the scope of this manuscript.
Depending on the dynamics of the onset and severity of the under-or overdosing symptoms, diagnostic and therapeutic steps should be initiated in a targeted manner. Symptomrelated treatment should be initiated concomitant to the search for the underlying causes of malfunction [fig_ref] Table 1: Symptoms and therapy of baclofen-underdosing/withdrawal and overdosing [/fig_ref].
An increase in spasticity may be a relative underdosing due to a therapeutic change in pump rate or because of an increased need with progressive worsening of the underlying disease. However, it is a warning sign, when spasticity suddenly deteriorates or when the dosage has been increased and the spasticity continues to worsen. At any level of the pump and catheter system, there might be the problem causing underdosing or withdrawal; therefore, each level must be checked step by step. Incorrect filling of the pump with low concentration of baclofen, lower dosage, a mechanical problem of the pump or failure to transport the drug out of the pump through the catheter into the intrathecal space due to dislocation, kinking, obstruction or malposition of the catheter tip, for example, can lead to a lack of baclofen at the site of action.
Overdosing occurs less frequently and may occur as result of an iatrogenic mistake when filling of the pump was incorrect (high baclofen concentration) or the pump rate too high. Another reason might be that the pump itself has a mechanical problem and is transporting more baclofen solution than programmed. Catheter problems mostly result in too little baclofen arriving intrathecally; therefore this usually does not lead to overdosing but eventually to fluctuating treatment responses.
If there are fluctuating symptoms with an alternation of over-or underdosing, this may be due to pump dysfunction. However, a malpositioning of the catheter tip, similar to case 5, should also be considered, when varying amounts of baclofen arrive intrathecally. Changes in intrathecal baclofen distribution may also lead to fluctuating symptoms, even though the catheter tip is correctly located intrathecally. We report this in case 6. Other causes of pump or catheter complications were made unlikely in this case by extensive diagnostic workup. Without proof of a mechanical dysfunction, improving the situation can be tried in these cases by placing the catheter tip to the C0/C1 level, as an alternative to intraventricular placement in case of efficacy decline.
Children who are immobilized by chronic disease are at high risk of developing secondary osteoporosis. The probability of receiving ITB and concomitant treatment with bisphosphonates is therefore increased. The intraoperative findings in case 3 with a combination of ITB and bisphosphonates with pronounced calcifications were impressive and so far not observed by us in other patients. We assume this to be a side effect of the bisphosphonates. We also suspect that these calcifications of the tissue contributed to the subsequent infection of the pump pocket. We could not find any other similar case report in the literature.
# Conclusion
We describe different causes of ITB failure, the resulting symptoms and signs, as well as an approach to trouble shooting.
Patients with spasticity GMFCS level IV and V may benefit significantly from ITB. However, the benefit should also be balanced against the risk of potentially life-threatening complications.
[fig] Figure 1: Case 1, photo of the corroded pump [/fig]
[table] Table 1: Symptoms and therapy of baclofen-underdosing/withdrawal and overdosing [/table]
[table] Table 2: Overview of cases with mechanical ITB complication [/table]
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Protocol for the development of the Wales Multimorbidity e-Cohort (WMC): data sources and methods to construct a population-based research platform to investigate multimorbidity
# Abstract
Introduction Multimorbidity is widely recognised as the presence of two or more concurrent long-term conditions, yet remains a poorly understood global issue despite increasing in prevalence. We have created the Wales Multimorbidity e-Cohort (WMC) to provide an accessible research ready data asset to further the understanding of multimorbidity. Our objectives are to create a platform to support research which would help to understand prevalence, trajectories and determinants in multimorbidity, characterise clusters that lead to highest burden on individuals and healthcare services, and evaluate and provide new multimorbidity phenotypes and algorithms to the National Health Service and research communities to support prevention, healthcare planning and the management of individuals with multimorbidity. Methods and analysis The WMC has been created and derived from multisourced demographic, administrative and electronic health record data relating to the Welsh population in the Secure Anonymised Information Linkage (SAIL) Databank. The WMC consists of 2.9 million people alive and living in Wales on the 1 January 2000 with follow-up until 31 December 2019, Welsh residency break or death. Published comorbidity indices and phenotype code lists will be used to measure and conceptualise multimorbidity. Study outcomes will include: (1) a description of multimorbidity using published data phenotype algorithms/ ontologies,investigation of the associations between baseline demographic factors and multimorbidity, [bib_ref] Epidemiology of multimorbidity and implications for health care, research, and medical education:..., Barnett [/bib_ref] identification of temporal trajectories of clusters of conditions and multimorbidity and (4) investigation of multimorbidity clusters with poor outcomes such as mortality and high healthcare service utilisation. Ethics and dissemination The SAIL Databank independent Information Governance Review Panel has
# Introduction
Multimorbidity is defined by the UK's Academy of Medical Sciences (AMS) and World Health Organization (WHO) as the presence of two or more concurrent long-term conditions, which is a global and growing phenomenon. Multimorbidity is more prevalent in older individuals and associated with high healthcare utilisation and mortality, but with large numbers of patients of all age suffering from multimorbidity. [bib_ref] Epidemiology of multimorbidity and implications for health care, research, and medical education:..., Barnett [/bib_ref] [bib_ref] The epidemiology of multimorbidity in primary care: a retrospective cohort study, Cassell [/bib_ref] [bib_ref] Multimorbidity and mortality in older adults: a systematic review and meta-analysis, Nunes [/bib_ref] [bib_ref] Multimorbidity and survival for patients with acute myocardial infarction in England and..., Hall [/bib_ref] With an ageing population, it is estimated that two in Strengths and limitations of this study ► Creation and access to a multisourced population based, deeply phenotyped e-cohort. ► Future use of this resource removes the need for data management and cleaning of source data, accelerating research and which could also support efforts for reproducibility of results. ► Variety of individual and household level data available on demography, health status, healthcare utilisation, both primary and secondary healthcare, and mortality to support a wide range of analytical approaches to addressing scientific questions. ► Input from multiple disciplines and institutions from across all four nations of the UK to help understand, measure and address multimorbidity. ► Routine data do not capture data on some important aspects, such as quality of life. Open access three people in England aged 65 years or over will experience multimorbidity by 2035 and nearly one fifth will have complex multimorbidity (four or more conditions). [bib_ref] Projections of multimorbidity in the older population in England to 2035: estimates..., Kingston [/bib_ref] Much of what is known about multimorbidity is based on a limited and fragmented knowledge base, largely derived from studies of older people in high-income countries or hospital populations. The 2018 AMS report concluded that multimorbidity is an unhelpful term implying random assortment of disease when it often refers to clusters of specific diseases. Once identified, these disease clusters can be addressed specifically through research, healthcare policy development and service delivery. The identification of previously unrecognised disease clusters may also provide biological and clinical insights into their aetiology, prevention and treatment. The AMS report identified specific research gaps and proposed a list of priorities (box 1). Several can be addressed through a combination of health data science, epidemiology and statistics and by exploiting the potential from creating deeply phenotyped cohorts from population and clinical data sources.
Responding to this agenda, we created a privacy protecting total population electronic cohort-the Wales Multimorbidity e-Cohort (WMC)-as a platform to study these issues in depth, collaborating with scientists from many different institutions and disciplines, clinicians, and members of the public from across the UK to create a broader team science approach.
The objectives of this work are to understand prevalence, trajectories and determinants of multimorbidity, and identify clusters causing the greatest healthcare burden. The WMC will also contribute data on incidence, prevalence and burden to the Global Burden of Diseases (GBD) Study, and provide new multimorbidity phenotypes to e-cohorts with local participants, and phenotyping algorithms to many e-cohorts that use routine data. [bib_ref] The dementias platform UK (DPUK) data portal, Bauermeister [/bib_ref] We expect that findings from these analyses will provide evidence to health policy leads in order to support prevention and the complex healthcare planning and management of multimorbid individuals. Members of the public are embedded in the research team to ensure the resource focuses on issues of concern to the public. This paper describes the creation of the WMC and the statistical approaches that will be developed to support the diverse research objectives.
# Methods
The WMC was developed by linking multiple routinely collected population and clinical data sources on the population of Wales from 2000 to 2019. We used the privacy-protecting Secure Anonymised Information Linkage (SAIL) Databank, to contribute to the Health Data Research UK National Implementation Multimorbidity Resource (HNIMR) project and extended to 2020 for the Medical Research Council (MRC) funded Welsh Multimorbidity Machine Learning project . SAIL is one of the most comprehensive, privacy protecting, linked data Trusted Research Environments in the UK. SAIL uses data from many different sources and provides linkage at individual and household level. [bib_ref] Use of data linkage to measure the population health effect of non-health-care..., Lyons [/bib_ref] It has supported many different study designs, including large-scale community-based or clinical condition-based observational studies, disease surveillance, evaluation of natural experiments of environmental interventions, embedded trials and the Dementias Platform UK. [bib_ref] All Wales injury surveillance system revised: development of a population-based system to..., Lyons [/bib_ref] [bib_ref] Support and assessment for fall emergency referrals (safer) 2: a cluster randomised..., Snooks [/bib_ref] [bib_ref] Study protocol for investigating the impact of community home modification services on..., Hollinghurst [/bib_ref] [bib_ref] Longitudinal access and exposure to green-blue spaces and individual-level mental health and..., Mizen [/bib_ref] [bib_ref] Risk factors for 1-year mortality and hospital utilization patterns in critical care..., Szakmany [/bib_ref] [bib_ref] Emergency hospital admissions associated with a non-randomised housing intervention meeting national housing..., Rodgers [/bib_ref] [bib_ref] Risk of emergency hospital admission in children associated with mental disorders and..., Paranjothy [/bib_ref] [bib_ref] The secure Anonymised information linkage databank dementia e-cohort (SAIL-DeC), Schnier [/bib_ref] Cohort design and characteristics The WMC is a clearly defined complete population cohort. Cohort entry includes all residents in Wales, alive and living on 1 January 2000. Cohort censorship was defined by the first date of migration out of Wales/ residency break, death, or the study endpoint on 31 December 2019 (figure 1). Within these constraints, the cohort is designed to be without selection bias and to achieve complete follow-up. WMC also provides a fully generalisable population sample against which findings from more selected samples may be compared.
The WMC contains 2 902 101 individuals aged 0-99 at cohort start date with 46 million person years of follow-up available [fig_ref] Table 1: WMC baseline demographics [/fig_ref]. Individuals have a minimum of 1-day follow-up (cohort end date = 2 January 2000) and maximum of 20 years of follow-up (cohort end date = 31 December 2019).
The Heatmap in [fig_ref] Figure 3: Heatmap of person years of WMC follow-up, by age group, sex and... [/fig_ref] visualises the person years of follow-up by age, sex and area level deprivation. The more years of follow-up available the darker the colour. Age is calculated at the cohort start, therefore, younger individuals will have more years of available follow-up compared with older individuals. On average, there are less person years of follow-up available for the least deprived 15-24 years old compared with their respective age group in other areas of Wales.
## Data sources
The WMC has used and combined anonymised health, social and environmental data held within the SAIL Databank ( www. saildatabank. com).
The baseline characteristics for the WMC have been created using the Welsh Demographic Service Dataset Box 1 The Academy of Medical Sciences identified research gaps ► The scale and nature of multimorbidity and how it is changing over time. ► Which clusters of conditions cause the biggest problems for patients. ► The causes of the most common clusters including links with sex, ethnicity, income and lifestyle. ► The best ways to prevent the patients developing multimorbidity, and whether this requires different approaches to just preventing individual conditions. ► How doctors can increase the benefits and reduce the risks of treatment for patients with multimorbidity. ► How to organise healthcare systems to deal with multimorbidity more effectively and how best to use digital technology in caring for patients. Anonymised linkage fields Linkage fields are used to anonymously link between data sources in the SAIL Databank and have been previously described elsewhere. [bib_ref] The Sail databank: linking multiple health and social care datasets, Lyons [/bib_ref] SAIL uses a multiple encryption system in which a trusted third party, the NHS Wales Informatics Service, uniquely matches identities (NHS number, name, date of birth and residential address/ Unique Property Reference Number (UPRN)) and replaces these with unique identifiers. For individuals this is called an Anonymised Linkage Field (ALF) and
## Open access
Residential Anonymised Linkage Field (RALF) for pseudonymised residences before uploading data to SAIL.
## Demographic data
The cohort includes the following variables: ALF, age in years, sex, date of death, date of movement out of Wales, RALF at both cohort inception and cohort end and Care Home Anonymised Linkage Fields (CHALFs) at cohort end date. The CHALF was derived from a data extract from Care Inspectorate Wales in 2020 for all adult care home settings. [bib_ref] Study protocol for investigating the impact of community home modification services on..., Hollinghurst [/bib_ref] Geographical variables associated with the RALF and CHALF include Lower layer Super Output Area (LSOA) 2001 at cohort inception and LSOA 2011 at cohort end. These have been mapped to the Welsh Index of Multiple Deprivation version 2011 and 2019, respectively, to derive socioeconomic deprivation quintiles and urban/rurality categories. Health data All admissions to hospital (inclusive of critical care admissions), outpatient, emergency department attendances treated in NHS hospitals as well as disease registries and laboratory test results data are available for cohort participants, GP data for diagnoses and treatments from SAIL providing practices are data for approximately 80% of the population. [bib_ref] Measuring follow-up time in routinely-collected health datasets: challenges and solutions, Thayer [/bib_ref] All relevant health events recorded in clinical data sources will be joined onto the WMC to identify diagnosis [fig_ref] Figure 2: WMC pyramid for age [/fig_ref].
The Upset plot in [fig_ref] Figure 4: Number of WMC individuals utilising healthcare services recorded in multisource data sources,... [/fig_ref] demonstrates the number of WMC participants that have interacted with the various healthcare settings from 1 January 2000 to their cohort censorship end date.For example, 780 830 (26.9%) individuals have used GP, inpatient, outpatient and emergency department services as well as had at least one laboratory test within their WMC coverage.
Phenotyping the e-cohort Published comorbidity indices and phenotype code lists (International Classification of Diseases 10th revision (ICD-10), OPCS Classification of Interventions and Procedures version 4 (OPCS4) and primary care Read Codes version 2) will be used to measure and conceptualise multimorbidity. These include those created by: CALIBER initiative; Charlson Comorbidity Index; Common Mental Disorders (CMD); Elixhauser Comorbidity Index; GBD Study and the NHS Quality and Outcomes Framework (QOF). [bib_ref] Case-Finding for common mental disorders of anxiety and depression in primary care:..., John [/bib_ref] [bib_ref] A chronological map of 308 physical and mental health conditions from 4..., Kuan [/bib_ref] [bib_ref] Comorbidity scores for administrative data benefited from adaptation to local coding and..., Bottle [/bib_ref] [bib_ref] Coding algorithms for defining Charlson and Elixhauser co-morbidities in Read-coded databases, Metcalfe [/bib_ref] [bib_ref] Coding algorithms for defining comorbidities in ICD-9-CM and ICD-10 administrative data, Quan [/bib_ref] [bib_ref] A new method of classifying prognostic comorbidity in longitudinal studies: development and..., Charlson [/bib_ref] [bib_ref] Comorbidity measures for use with administrative data, Elixhauser [/bib_ref] [bib_ref] Uk phenomics platform for developing and validating electronic health record phenotypes: caliber, Denaxas [/bib_ref] [bib_ref] Contacts with primary and secondary healthcare prior to suicide: case-control wholepopulation-based study..., John [/bib_ref] [bib_ref] Overview of the SF-36 health survey and the International quality of life..., Ware [/bib_ref] Diagnostic codes relating to HIV will not be included in any outputs to conform with SAIL policies. They are part of the list of redacted codes not allowed to be used for research using the data.All ICD-10 and OPCS4 codes provided at the three character level were expanded to include all children terms.
## Caliber
Phenotyping algorithms created from the CALIBER resource using ICD-10, OPCS4 and Read Codes will be used to identify 300 physical and mental health conditions recorded in both primary and secondary healthcare. There are 1645 distinct ICD-10 codes (at three and four-character level) for 300 conditions, however, when capturing all ICD-10 codes to include variation in coding entry (eg, C796-instead of C796) and expanding the code list to the four-character level (F200 instead of F20), there are 3702 distinct ICD-10 codes (at the four-character level) recorded in the inpatient data. This is important to note as to link solely on standardised codes would result in loss of information and potential reporting of false negatives.
There are 587 distinct OPCS4 codes (at three and fourcharacter level) for 28 conditions and 8588 distinct Read Codes (at the five-character level) for 275 conditions.
## Charlson comorbidity index
The Aylin and Bottle Charlson amended ICD-10 code list will be used for inpatient diagnosis and the Metcalfe et Open access al [bib_ref] Coding algorithms for defining Charlson and Elixhauser co-morbidities in Read-coded databases, Metcalfe [/bib_ref] Charlson Read Code list will be utilised for primary care recorded diagnosis. The ICD-10 codes have been taken from the pool of diagnosis codes recorded within hospital admissions data, containing 1024 distinct codes (at the four-character level) for 16 conditions. The GP data contains 4545 distinct Read Codes at the five-character level.
## Common mental disorders
The John et al validated algorithm will be used to identify CMD in GP data. [bib_ref] Case-Finding for common mental disorders of anxiety and depression in primary care:..., John [/bib_ref] The algorithm has used a combination of diagnosis, treatment and symptoms Read Codes in identifying CMD. Individuals with CMD are identified as either having a historical diagnosis code, currently treated or, having a current diagnosis/current symptom code. There are 89 distinct diagnosis codes, 15 symptom codes and 601 treatment codes.
## Elixhauser comorbidity index
The Quan et al (2005) Elixhauser ICD-10 code list will be utilised for inpatient diagnosis and the Metcalfe et al [bib_ref] Coding algorithms for defining Charlson and Elixhauser co-morbidities in Read-coded databases, Metcalfe [/bib_ref] Elixhauser Read Code list will be utilised for primary care recorded diagnosis. The ICD-10 codes have been taken from the pool of diagnosis codes recorded within hospital admissions data and contains 1423 distinct codes (at the four-character level) for 30 conditions. The general practice data contains 6074 distinct Read codes at the five-character level.
## Gbd study
The GBD 2019 ICD-10 codes will be used to identify 130 health conditions in secondary healthcare data. There are 3497 distinct ICD-10 codes at the three and fourcharacter level.
## Quality outcome framework
The QOF conditions business rule V.38 will be used to identify 18 health conditions in primary care data.The 18 conditions are asthma, atrial fibrillation, obesity, coronary heart disease, chronic obstructive pulmonary disease, cancer, chronic kidney disease, dementia, depression, diabetes, epilepsy, heart failure, hypertension, learning difficulties, peripheral arterial disease, rheumatoid arthritis, serious mental illness and stroke. There are 2275 distinct Read Codes available at the five-character level for the 18 QOF conditions.
# Statistical analysis
The WMC provides an accessible research ready data asset to further understanding of multimorbidity through the use of biostatistical and machine learning approaches. Our collaborative team will work across a number of projects to develop and evaluate statistical and machine learning algorithms to address the following broad analytical challenges: ► What is the prevalence of multimorbidity in the WMC, and how does prevalence of multimorbidity change over time?
► What are common clusters of multimorbidity in the WMC, and how do they correspond to or differ from, common clusters of multimorbidity identified in other datasets? ► Which clusters of multimorbidity occur less frequently than one would expect based on the prevalence of their constituent conditions? ► How does multimorbidity develop across the life course (ie, trajectories)? ► What are the biological, psychological and social determinants of different clusters and trajectories of multimorbidity? ► Which clusters and trajectories of multimorbidity are associated with poor health outcomes? ► Which clusters and trajectories of multimorbidity are associated with high service utilisation? ► Does multimorbidity in specific groups (eg, patients with musculoskeletal conditions) differ from multimorbidity in general? The overarching aim is to evaluate and provide new multimorbidity phenotypes and algorithms to the NHS and research communities to support prevention, healthcare planning and the management of individuals with multimorbidity.
We will draw on both methods from statistics (eg, regression analysis, longitudinal mixed models, multiple correspondence analysis, factor analysis, 43 multistate models and latent class analysis) and machine learning (eg, k-means clustering, semantic similarity clustering, market basket analysis, network models 44 and deep learning). We will use resampling methods to assess the stability of identified multimorbidity clusters and develop visualisation techniques to summarise multimorbidity clusters and their associations with risk factors and outcomes.
Analyses will be coded in R, WinBUGS, and Python and made available to WMC users via a Git library to maximise transparency and reproducibility. [bib_ref] WinBUGS -a Bayesian modelling framework: concepts, structure, and extensibility, Lunn [/bib_ref] Patient and public involvement The proposal to develop WMC was submitted to the independent Information Governance Review Panel (IGRP) that includes members of the public (IGRP Project: 0911). We worked with this group to refine the study protocol. The scientific steering group includes two members of the public who have contributed to this paper. The HNIMR has a work package on patient and public involvement with a panel drawn from across the UK which meets to discuss the research work and feed into the research and dissemination plans.
## Ethics and dissemination
The use of deidentified data in SAIL complies with National Research Ethics Service (NRES) guidance. [bib_ref] A case study of the secure anonymous information linkage (Sail) gateway: a..., Jones [/bib_ref] Applications to use data held within the SAIL Databank, an ISO: 27001 and UK Statistics Authority (UKSA) Digital Economy Act (DEA) accredited Trusted Research Environment, must first be approved by the independent Findings from this study will be disseminated widely through a variety of routes, including to health policy and NHS leads across UK, the AMS and the Royal Colleges, as well as traditional scientific outlets. The team includes NHS clinicians and informaticians to allow for early NHS adoption of useful findings. Members of the public embedded in the team will create plain English summaries and lead at public facing meetings.
[fig] Figure 1: WMC flow diagram, based on inclusion criteria. LSOA; lower layer super output area, WDSD; Welsh Demographic Service Dataset, WMC; Wales Multimorbidity e-Cohort, WMML; Welsh Multimorbidity Machine Learning. [/fig]
[fig] Figure 2: WMC pyramid for age (years) at cohort inception. WMC, Wales Multimorbidity e-Cohort. [/fig]
[fig] Figure 3: Heatmap of person years of WMC follow-up, by age group, sex and area-level deprivation at cohort inception. WMC, Wales Multimorbidity e-Cohort. [/fig]
[fig] Figure 4: Number of WMC individuals utilising healthcare services recorded in multisource data sources, 20 most common combinations presented. WMC, Wales Multimorbidity e-Cohort. copyright. on February 15, 2021 at Swansea University. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2020-047101 on 19 January 2021. Downloaded from [/fig]
[table] copyright: on February 15, 2021 at Swansea University. Protected by http://bmjopen.bmj.com/ Open access (WDSD) and the Annual District Death Extract (ADDE) mortality registry data from the Office for National Statistics. The WDSD contains administrative information concerning the resident population of Wales that are registered to a Welsh General Practice, a free to use National Health Service (NHS) system at the point of primary care registration in the UK. The ADDE data contains information about the dates and causes of all deaths relating to residents in Wales, including those that died outside of Wales. SAIL holds general practitioner (GP) data for approximately 80% of the population with coverage extending to all local authorities in Wales. The Welsh Longitudinal General Practice data will be used to identify the subpopulation of individuals who are registered to a practice providing data to SAIL to identify which individuals have GP data present and avoid underestimation of conditions or severity of conditions not managed through hospital admission.The Welsh Health Survey Dataset and the National Survey for Wales Dataset with data on well-being measures, social class, education, housing and wealth are available for 9905 and 33 295 cohort participants respectively.24 [/table]
[table] Table 1: WMC baseline demographics [/table]
[table] Table 2: Clinical data sources available for the WMC [/table]
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Increased Fibrinolysis as a Specific Marker of Poor Outcome After Cardiac Arrest
# Background
The process of fibrinolysis is inevitable to regain microvessel patency and restore vital organ perfusion after intravascular clotting. Endothelial tissue-type plasminogen activator (t-PA) ensures clot dissolution by converting plasminogen into plasmin at the site of primary vascular damage averting permanent circulatory compromise and subsequent thrombotic organ failure. In contrast, primary hyperfibrinolysis occurs without preceding intravascular clotting and is associated with poor outcome in several critical conditions including trauma and sepsis [bib_ref] Consecutive thrombelastography clot strength profiles in patients with severe sepsis and their..., Ostrowski [/bib_ref]. In particular in traumatic coagulopathy, fibrinolysis has been investigated intensively and identified as an early and independent predictor of mortality [bib_ref] Hyperfibrinolysis after major trauma: Differential diagnosis of lysis patterns and prognostic value..., Schöchl [/bib_ref].
Although underlying mechanisms likely differ from that in trauma-associated fibrinolysis, which involves tissue injury and significant crystalloid hemodilution [bib_ref] Hyperfibrinolysis at admission is an uncommon but highly lethal event associated with..., Cotton [/bib_ref] , previous studies likewise reported an association between the presence of fibrinolysis and unfavorable prognosis in cardiac arrest [bib_ref] Asphyxia by drowning induces massive bleeding due to hyperfibrinolytic disseminated intravascular coagulation, Schwameis [/bib_ref] , with the highest fibrinolytic activity found in patients with early death.
Hypoperfusion resulting in release of endothelial t-PA (6) is considered one possible mechanism of fibrinolysis occurring during cardiopulmonary resuscitation (CPR) [bib_ref] Hyperfibrinolysis in out of hospital cardiac arrest is associated with markers of..., Viersen [/bib_ref]. The importance of hypoxia as a causal factor is best seen in the severe hyperfibrinolysis of young and previously healthy drowning patients where preceding activation of coagulation can be excluded [bib_ref] Asphyxia by drowning induces massive bleeding due to hyperfibrinolytic disseminated intravascular coagulation, Schwameis [/bib_ref]. Hence, the extent of fibrinolysis at admission reflects prolonged or insufficient resuscitation efforts and may therefore provide reliable prediction of poor outcome [bib_ref] Asphyxia by drowning induces massive bleeding due to hyperfibrinolytic disseminated intravascular coagulation, Schwameis [/bib_ref].
In this context, previous studies have already identified fibrin degradation products, such as d-dimer, as an early indicator of unfavorable outcome in cardiac arrest [bib_ref] Usefulness of the D-dimer concentration as a predictor of mortality in patients..., Szymanski [/bib_ref]. D-dimer is a well-known marker of fibrinolysis and can be routinely assessed, but however lacks specificity, which makes the definition of a reliable predictive threshold unlikely.
Yet, thrombelastometry has proven useful for point-of-care detection of increased fibrinolysis in cardiac arrest [bib_ref] Hyperfibrinolysis in out of hospital cardiac arrest is associated with markers of..., Viersen [/bib_ref] [bib_ref] Hyperfibrinolysis is common in out-of-hospital cardiac arrest: Results from a prospective observational..., Schöchl [/bib_ref].
The current study aimed to assess whether there is an optimal fibrinolysis cutoff value assessed by thrombelastometry to predict poor outcome in a preselected cohort of successfully resuscitated adult patients with out-of-hospital cardiac arrest.
# Methods
## Study population
Eligible for inclusion were adults (≥ 18 yr) with out-of-hospital cardiac arrest of presumed cardiac origin, subjected to targeted temperature management, who had achieved return of spontaneous circulation (ROSC) at admission to the ICU section of the emergency department at the Medical University of Vienna. Exclusion criteria comprised thrombolytic therapy and application of intravascular cooling or extracorporeal bypass device, as both may affect fibrinolytic activity. All patients were treated with therapeutic hypothermia at a target temperature range of 33°C ± 1°C. Patients were cooled using cooling pads (EMCOOLS Flex.Pad, Emcools AG, Pfaffstaetten, Austria) [bib_ref] Surface cooling for induction of mild hypothermia in conscious healthy volunteers -a..., Testori [/bib_ref] or water-circulating gel-coated pads (Arctic Sun 5000 Temperature Management System; Medivance, Louisville, CO) with or without cold fluids. Target temperature between 32°C and 34°C was maintained for 24 hours after first achievement of less than 34°C. Rewarming was performed at a rate of 0.25-0.5°C per hour. Body temperature was recorded with an esophageal and bladder probe.
Resuscitation-related variables were analyzed and reported according to Utstein recommendations as described previously [bib_ref] European Resuscitation Council; Australian Resuscitation Council; New Zealand Resuscitation Council; Heart and..., Jacobs [/bib_ref]. The primary endpoint was a composite of poor neurologic function or death, defined as a Cerebral Performance Category (CPC) of 3-5 (severe cerebral disability; coma or vegetative state; or brain death, respectively) at day 30 post resuscitation [bib_ref] Assessment of neurological prognosis in comatose survivors of cardiac arrest. BRCT I..., Edgren [/bib_ref]. Neurologic function at 30 days was assessed by study fellows through structured face-to-face interview with the patient or by means of structured telephone interview with the patient, the relatives, treating physicians, or nursing home members. Study fellows assessing outcome were all blinded to results obtained by rotational thrombelastometry (ROTEM).
Sustained ROSC was defined as recovery of spontaneous circulation for more than 20 minutes. No-and low-flow intervals were defined as the time from collapse to initiation of CPR and the time from CPR initiation to sustained ROSC, respectively.
No-and low-flow intervals were determined through immediate structured telephone interviews with the dispatch center, the emergency physicians, and paramedics at the scene and the bystander who performed the emergency call. The International Society on Thrombosis and Haemostasis disseminated intravascular coagulation (DIC) eight-point score was applied for DIC calculation [bib_ref] Towards definition, clinical and laboratory criteria, and a scoring system for disseminated..., Taylor [/bib_ref]. A score greater than or equal to 5 is compatible with overt DIC (Supplemental, Supplemental Digital Content 1, http://links.lww.com/CCM/D848).
For sample size calculation, we used the methods described by Buderer (15) based on specificity. We anticipated a specificity level of 90% with 95% CI (width of 10%) and a given prevalence of hyperfibrinolysis of 40% based on previously published results [bib_ref] Hyperfibrinolysis is common in out-of-hospital cardiac arrest: Results from a prospective observational..., Schöchl [/bib_ref]. The high level of anticipated specificity was estimated based on our own results from drowning patients, where a very high predictive value for death was demonstrated, if signs of hyperfibrinolysis were present [bib_ref] Asphyxia by drowning induces massive bleeding due to hyperfibrinolytic disseminated intravascular coagulation, Schwameis [/bib_ref].
The study was approved by the Ethics Committee of the Medical University of Vienna and carried out in accordance with the Declaration of Helsinki. A waiver was obtained for informed consent at admission, and patients were informed of their study participation on regaining consciousness.
# Laboratory methods
Blood sampling for thrombelastometry and laboratory studies were performed as soon as vascular access was available. Analysis of whole blood viscoelastic properties was done in 3.8% sodium-citrated whole blood samples using ROTEM (TEM International GmBH, Munich, Germany) as described previously [bib_ref] Validation of rotation thrombelastography in a model of systemic activation of fibrinolysis..., Spiel [/bib_ref]. The following ROTEM tests were applied: EXTEM, which tests the extrinsic pathway of coagulation, and APTEM, which corresponds to EXTEM but additionally comprises the antifibrinolytic aprotinin to definitively reveal the presence of increased fibrinolysis. Fibrinolysis is given as maximum lysis (ML) (%), which represents the percentile difference between the highest and lowest clot amplitude (Supplemental [fig_ref] Figure 1: Sensitivity and specificity [/fig_ref] , Supplemental Digital Content 1, http://links.lww.com/CCM/ D848; reference range < 15%, as specified by the manufacturer).
For enzyme-linked immunoassay (ELISA) analysis, blood was collected into tubes containing EDTA or 3.8% citrated plasma. Obtained samples were centrifuged for 10 minutes at 2,000g. Plasma was stored at -80°C until being tested. Tissue plasminogen activator antigen (TECHNOZYM t-PA Combi Actibind; Technoclone, Vienna, Austria), plasminogen activator inhibitor (PAI)-1 (TECHNOZYM PAI-1 Actibind; Technoclone) [bib_ref] Validation of rotation thrombelastography in a model of systemic activation of fibrinolysis..., Spiel [/bib_ref] , and prothrombin fragments F1 + 2 (EnzygnostF 1 + 2; Siemens, Marburg, Germany) (17) assays were performed using commercially available ELISA kits. The lower limits of detection are 0.01 ng/mL (t-PA antigen), 20 pmol/L (F1 + 2), and 0.49 IU/mL (PAI-1), respectively. All assays were performed according to manufacturer's instructions.
# Statistical analysis
Variables are presented as absolute values (n), relative frequencies (%), and median (interquartile ranges [IQRs]). Prevalence of increased fibrinolysis and overt DIC is given as a proportion www.ccmjournal.org e997 with a 95% CI. Potentially missing data for demographic variables were not imputated. Between-group comparisons were performed using the Mann-Whitney U test for continuous variables or the chi-square test/Fisher exact test for nominal variables. We performed exact bivariable logistic regression to estimate the effect on poor neurologic function or death at day 30 of ML and remaining candidate predictors, which were judged to be clinically plausible, including age, no-flow time, low-flow time, pH, lactate, d-dimer levels, sex, CPC prior to cardiac arrest, arrest site, witness status, presence or absence of bystander resuscitation, initial rhythm, epinephrine dose administered during resuscitation, and the rate of sustained ROSC at admission. Results are given as odds ratios/median unbiased estimates with 95% CI and are available in Supplemental (Supplemental Digital Content 1, http://links. lww.com/CCM/D848).
The optimal cutoff for ML to predict poor 30-day outcome was assessed by computing a receiver operating characteristic curve. Specificity and sensitivity were calculated with 95% CI. The Kaplan-Meier method was used to describe survival according to the optimal cutoff for ML.
Generally, a two-sided p value of less than 0.05 was considered statistically significant. We used IBM SPSS Statistical Software, Version 22.0 (IBM Corp., Armonk, NY) and Stata Statistical Software: Release 15 (StataCorp., College Station, TX) for statistical analysis and GraphPad Prism Version 7.00 for Windows (GraphPad Software, La Jolla, CA) to draw figures.
# Results
Seventy-eight patients (median age 59 yr; 47-69; 78% male) were included in the study. Results from thrombelastometry at admission and all data for the primary outcome were available.
Median core temperature at admission was 35.3°C . Fibrinolysis exceeding the normal reference value of 15% was present in 36% of patients (28/78; 95% CI, 25-48%). The rate of overt DIC was 4% overall (3/78; 95% CI, 1-11%). In total, 54% of patients (42/78) had a poor 30-day outcome including 23 nonsurvivors (30%). Kaplan-Meier estimates of survival to day 30 according to ML cutoff of greater than or equal to 20% are available with the supplement (Supplemental , Supplemental Digital Content 1, http://links.lww.com/ CCM/D848).
While nine patients died due to multiple organ failure, in 14 patients a decision to withdraw life-sustaining therapy was made by treating physicians after determination of unfavorable neurologic prognosis. Characteristics of the study patients according to 30-day outcome (CPC) are shown in.
The admission ML cutoff predicting poor outcome with 100% specificity (95% CI, 90-100) was greater than or equal to 20% (sensitivity 41%; 95% CI, 26-58) [fig_ref] Figure 1: Sensitivity and specificity [/fig_ref]. The corresponding positive and negative predictive values were 100% and 59%, respectively. The receiver operating characteristic curve of ML for prediction of poor 30-day outcome as well as specificity, sensitivity, and cumulative frequency distributions of covariables are given with the supplement (Supplemental The study included only patients with presumed cardiac cause of cardiac arrest who had achieved return of spontaneous circulation at hospital admission. Laboratory variables and core temperature were assessed immediately after admission. Body temperature was recorded with an esophageal and bladder probe. Missing data were as follows: no flow time, n = 1; initial rhythm, n = 2; prothrombin time, n = 3; lactate levels, n = 2; epinephrine, n = 2; pH, n = 3; temperature at admission, n = 2.
# Discussion
The current study prospectively investigated the value of fibrinolysis to predict 30-day outcome early after cardiac arrest.
The study was built on previous studies identifying hypoperfusion and hypoxia as triggers of primary fibrinolysis [bib_ref] Hyperfibrinolysis in out of hospital cardiac arrest is associated with markers of..., Viersen [/bib_ref]. Although prolonged or poor resuscitation efforts may cause hyperfibrinolysis, the prognostic relevance of fibrinolysis occurring during CPR, however, remained unclear. This study specifically tested the hypothesis that increased fibrinolysis in thrombelastometry may predict poor outcome.
The prevalence of increased fibrinolysis (ML > 15%) in our study is in good agreement with previous findings [bib_ref] Hyperfibrinolysis is common in out-of-hospital cardiac arrest: Results from a prospective observational..., Schöchl [/bib_ref]. However, we were interested in the optimal cutoff for fibrinolysis to specifically predict poor outcome. The greater than or equal to 20% cutoff value that predicted poor outcome with 100% specificity found in the current study corresponds well to the cutoff for increased fibrinolysis recently observed in healthy volunteers [bib_ref] Evaluation of between-, within-and day-to-day variation of coagulation measured by rotational thrombelastometry..., Jilma-Stohlawetz [/bib_ref].
From previous research, we interpret fibrinolysis as a marker of tissue hypoperfusion due to prolonged resuscitation efforts or poor-quality CPR performance and consecutive accumulation of t-PA [bib_ref] Asphyxia by drowning induces massive bleeding due to hyperfibrinolytic disseminated intravascular coagulation, Schwameis [/bib_ref]. This hypothesis is mainly based on data obtained from patients with drowning-related out-of-hospital cardiac arrest (OHCA), who are characterized by severely prolonged no-and low-flow intervals along with massive bleeding at hospital admission [bib_ref] Asphyxia by drowning induces massive bleeding due to hyperfibrinolytic disseminated intravascular coagulation, Schwameis [/bib_ref]. Their bleeding phenotype was accompanied by increased t-PA levels and absent clotting signature in thrombelastometry, which were reversed by adding aprotinin in vitro. A subsequent forearm-ischemia model conducted in healthy volunteer confirmed the increase of t-PA levels following interruption of arterial blood flow. Further previous studies likewise reported high plasma fibrinolytic activity in cardiac arrest patients with early death (5) and a good correlation between t-PA levels and markers of hypoperfusion [bib_ref] Hyperfibrinolysis in out of hospital cardiac arrest is associated with markers of..., Viersen [/bib_ref]. In accordance, patients with ML greater than or equal to 20% had significantly higher t-PA levels at admission. Levels of the t-PA inhibiting protein PAI-1 were comparatively low in patients with and without increased fibrinolysis, which is in agreement with current literature [bib_ref] Massive fibrin formation with consecutive impairment of fibrinolysis in patients with out-of-hospital..., Gando [/bib_ref]. This relates to the lack of a storage compartment for PAI-1-in contrast to t-PA stored in endothelial cells-which forbids a readily available release upon hypoxic conditions. Upon acute t-PA release, PAI-1 is consumed [bib_ref] Clearance of tissue plasminogen activator (TPA) and TPA/plasminogen activator inhibitor type 1..., Chandler [/bib_ref] [bib_ref] Von Willebrand factor and fibrinolytic parameters during the desmopressin test in patients..., Giraldi [/bib_ref] , and therefore it seems plausible that its activity is low in the very early phase after arrest. PAI-1 levels have been found to likewise increase only within 6 hours after acute hypoxia, contributing to the fibrinolytic shutdown in the subacute phase of the postcardiac arrest syndrome [bib_ref] Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator..., Pinsky [/bib_ref].
Low-flow times and lactate levels in the current study were significantly higher in patients with a ML greater than or equal to 20%, which supports the above findings. Likewise, the rate of nonshockable rhythm tended to be higher, which may suggest longer preceding no-flow intervals before initiation of CPR in these patients. No-flow times did not differ significantly between groups, but are, however, usually only estimates with low reliability.
We suggest that fibrinolysis is mainly triggered by hypoperfusion which could also explain the low negative predictive value of ML found in the current study. It is conceivable that patients with prolonged no-flow times but high-quality CPR performance-and thus, hypothetically, rapid clearance of t-PA-suffer poor neurologic recovery despite lack of fibrinolysis at admission. The quality of CPR performance is, however, difficult to assess routinely.
An alternative or possibly contributing mechanism of increased fibrinolysis in cardiac arrest may be thrombinrelated plasmin activation following blood exposure to interstitial tissue factor through hypoxic endothelial damage. Along these lines, Adrie et al [bib_ref] Coagulopathy after successful cardiopulmonary resuscitation following cardiac arrest: Implication of the protein..., Adrie [/bib_ref] found a significant association between thrombin levels and organ dysfunction after successful resuscitation. Thrombin may also contribute to delayed t-PA (23) and/or urokinase-type plasminogen activator [bib_ref] Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells, Miles [/bib_ref] release from endothelial cells.
In our study, patients' prothrombin fragments were markedly elevated in both, patients with and without increased fibrinolysis, suggesting early thrombin generation in cardiac arrest. Yet, although thrombin-mediated coagulopathy likely becomes important hours or days after successful resuscitation, possibly as part of the postcardiac arrest syndrome, its contribution to increased fibrinolysis detected by thrombelastometry very early after successful resuscitation is questionable. Furthermore, in tissue factor/thrombin-driven coagulopathy, we would expect substantial consumption of clotting factors and platelets resulting in what is referred as DIC with fibrinolytic phenotype [bib_ref] Disseminated intravascular coagulation at an early phase of trauma is associated with..., Hayakawa [/bib_ref].
Consumptive coagulopathy, however, was very rare in our study patients, and the rate of overt DIC at admission did not differ between patients with and those without increased fibrinolysis. The low prevalence of overt DIC in our study patients, however, is in striking contrast to data from a retrospective Asian study, which reported overt DIC rates of 33% in resuscitated cardiac arrest with greater than 90% hospital mortality [bib_ref] Prognostic implication of initial coagulopathy in out-of-hospital cardiac arrest, Kim [/bib_ref]. As we analyzed a highly preselected subset of patients, the results of the two studies are difficult to compare. Yet, it would be of interest whether these differences result from different resuscitation policies (including termination-of-care rules) and whether overt DIC simply represents a premortem sign.
Another possible contributor to the impairment of coagulation may be hypothermia, but, however, a substantial impact on increased fibrinolysis in our patients seems unlikely. Median body temperature at admission was above 35°C, which is in agreement with recent literature [bib_ref] TTM Trial Investigators: Targeted temperature management at 33°C versus 36°C after cardiac..., Nielsen [/bib_ref] , and did not differ significantly between patients with and without ML greater than or equal to 20%. Furthermore, previous studies did not report patterns of increased fibrinolysis under intentional hypothermia [bib_ref] Hypothermia and acidosis synergistically impair coagulation in human whole blood, Dirkmann [/bib_ref] [bib_ref] Recombinant tissue-type plasminogen activator-evoked hyperfibrinolysis is enhanced by acidosis and inhibited by..., Dirkmann [/bib_ref] [bib_ref] Hypothermic anticoagulation: Testing individual responses to graded severe hypothermia with thromboelastography, Ruzicka [/bib_ref] [bib_ref] A thromboelastometric evaluation of the effects of hypothermia on the coagulation system, Rundgren [/bib_ref].
We were further interested in whether exogenous epinephrine may contribute to the extent of increased fibrinolysis in cardiac arrest. Catecholamines are known to promote release of t-PA from the endothelium [bib_ref] Effects of sympathetic activation by adrenergic infusions on hemostasis in vivo, Von Känel [/bib_ref] , and the amount of epinephrine given during resuscitation has previously been linked to poor outcome regardless of the length of resuscitation [bib_ref] Is epinephrine during cardiac arrest associated with worse outcomes in resuscitated patients?, Dumas [/bib_ref]. In the current study, there was an insignificant trend of higher cumulative epinephrine doses in patients who presented with ML greater than or equal to 20%. In regression analysis, however, the dosage of epinephrine administered had no effect on mortality after adjustment for ML greater than or equal to 20, which may suggest no causative relationship. Whether exogenous epinephrine directly contributes to fibrinolysis or simply reflects longer resuscitation efforts, and thus prolonged preceding hypoperfusion remains speculative and might be answered in a further study. Furthermore, yet, no data are available on the interaction between endogenous catecholamine levels and fibrinolysis in cardiac arrest. Our own recent data derived from a subset of 1,188 cardiac arrest patients underline the outcome-predictive role of sympathoadrenergic activation following resuscitation indicated by both, increased short-and long-term mortality in cardiac arrest along with the amount of immature peripheral neutrophils at admission [bib_ref] Mortality in patients resuscitated from out-of-hospital cardiac arrest based on automated blood..., Weiser [/bib_ref]. It is unclear whether this simply reflects the gradual extent of stress to the body following resuscitation or whether endogenous catecholamines may have a role in subsequent alterations of the coagulation and fibrinolytic system, as it has been described in trauma and septic patients [bib_ref] Sympathoadrenal activation and endotheliopathy are drivers of hypocoagulability and hyperfibrinolysis in trauma:..., Ostrowski [/bib_ref] [bib_ref] Association between sympathoadrenal activation, fibrinolysis, and endothelial damage in septic patients: A..., Johansson [/bib_ref]. In addition to increased fibrinolysis caused by stress-mediated sympathoadrenergic catecholamine release, neutrophil elastase has been linked as trigger for fibrinolysis in trauma [bib_ref] Disseminated intravascular coagulation at an early phase of trauma is associated with..., Hayakawa [/bib_ref]. However, to date, no comparable data are available on the contribution of neutrophil elastase to promote fibrinolysis in cardiac arrest.
Some limitations need to be considered while interpreting the results. The current prospective observational study analyzed a strictly preselected cohort of OHCA patients including only those with presumed cardiac cause of cardiac arrest, who had achieved ROSC at admission, and were subjected to targeted temperature management. The possibly associated selection bias has to be considered, and study results need to be interpreted with appropriate caution.
Furthermore, it has to be mentioned that there is currently no widely accepted "gold standard" assay available for detection of systemic fibrinolysis. A possible risk of bias relating to the lack of a reference standard must be taken into account. Future studies determining thrombelastometry thresholds for increased fibrinolysis in OHCA need to validate their findings against standardized methods for detection of fibrinolysis, which may become available in the future [bib_ref] Measuring fibrinolysis: From research to routine diagnostic assays, Longstaff [/bib_ref]. Further investigations also need to confirm the predictive performance of a greater than or equal to 20% ML cutoff found in this study.
# Conclusions
Increased fibrinolysis at admission may be interpreted as cumulative surrogate marker for hypoperfusion and hypoxia, that is, the duration of no-flow time and resuscitation quality. The current study provides a predictive cutoff value for a readily available bedside marker with 100% positive predictive value for poor outcome, which could be of interest for both treating physicians and relatives.
[fig] Figure 1: Sensitivity and specificity (%) of increasing fibrinolysis values (maximum lysis [ML], %) to predict poor neurologic function or death at day 30 after admission (n = 78 [/fig]
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Evaluation of the effectiveness of a quality improvement intervention to support integration of maternal, child and HIV care in primary health care facilities in South Africa
## = yes 2 = no
Interviewer: I am now going to ask you some questions about the father of this child: A6 Is the father of this child alive? Ngabe usaphila ubaba wontwana? More than one response allowed 1 = More than one mother/child carer present 2 = More than one health worker present 3 = Other
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Cardiac tamponade and coronary artery pseudoaneurysm after brachial arterial embolectomy, possible role for an aberrant origin of the right coronary artery
A patient developed hemopericardium shortly after left brachial arterial embolectomy using an embolectomy catheter. Evaluation disclosed evolving pseudoaneurysm of the right coronary artery that was successfully managed by stenting. Misplacement of the embolectomy catheter within the coronary vessel was facilitated by an anomalous origin of the right coronary artery. This complication highlights the importance of correct insertion of the embolectomy catheter using the markers to avoid maladvancement and damage to central vessels. (J Vasc Surg Cases and Innovative Techniques 2018;4:27-30.)
Remote embolectomy catheters introduced by Thomas Fogarty 1 in 1961 have revolutionized surgical embolectomy procedures, and this is now the principal method applied for peripheral arterial embolectomies. Initial advancement of the catheter is usually carried out without fluoroscopy unless specific anatomic pitfalls are identified or suspected. Herein we report a case of a patient who developed a right coronary artery (RCA) pseudoaneurysm and hemopericardium after upper limb embolectomy without the use of fluoroscopy. This complication conceivably reflects maladvancement of the catheter into the coronary artery, facilitated by its aberrant anatomic origin. The patient gave consent for the publication of her case. Aortic dissection was also excluded. After pericardiocentesis, the patient remained stable; the pericardial catheter was removed with no evidence of recurrence of pericardial effusion. During the next 5 weeks, the patient developed weakness associated with mild fever, cough, and shortness of breath. She was hospitalized twice with presumed (although not clinically confirmed) diagnoses of congestive heart failure and respiratory infection. C-reactive protein concentration was elevated.
## Case report
Chest CT without contrast medium revealed bilateral pleural effusions more prominent on the left, cardiomegaly with mild pericardial effusion, and a new mass in the right atrioventricular groove, compatible with a pseudoaneurysm of the RCA [fig_ref] Fig 1. a ,: Computed tomography [/fig_ref]. This finding was confirmed by CT angiography and c). In light of these findings, the patient underwent coronary angiography, performed through the right common femoral artery. Repeated attempts to cannulate the RCA with several catheters including Judkins right (JR4), Williams, and Amplatz left (AL1) failed because of an anomalous origin of the RCA. After injection of contrast material in the right coronary sinus of Valsalva, the aberrant origin was located at a posterior position with an ostium directed upward [fig_ref] Fig 2: Aberrant origin of the right coronary artery [/fig_ref].
Finally, intubation of the RCA ostium was achieved using a multipurpose (MP) catheter, a catheter with a blunt angle that is directed downward and is used in similar cases of [fig_ref] Fig 1. a ,: Computed tomography [/fig_ref]. An aneurysm in the midsegment of the RCA, 3.5 cm in diameter, was demonstrated and successfully excluded by a pericardial covered stent (3.5/27 mm [ITGI Medical, Nir 'Aqiva, Israel]; [fig_ref] Fig 3: Coronary angiography of the right coronary artery [/fig_ref]. The pericardial covered stents are highly deliverable, fully covered stents that are used to treat and to seal coronary perforations and aneurysms. [bib_ref] Pericardial covered stent for coronary perforations, Chen [/bib_ref] The coronary arteries were otherwise normal. The patient was treated with aspirin, clopidogrel, and low-dose apixaban with an unremarkable clinical course until hospital discharge. She was clinically stable for 5 months of follow-up and then died suddenly of unknown cause.
# Discussion
The balloon embolectomy catheter, introduced by Fogarty, 1 is widely used for the removal of arterial emboli and thrombi. It is a convenient, minimally invasive, and safe method, although complications related to balloon inflation, such as arterial wall dissection, rupture, vessel obstruction, embolic events including stroke, and formation of arteriovenous fistulas, have been reported. [bib_ref] Complications encountered during arterial embolectomy with the Fogarty balloon catheter, Schweitzer [/bib_ref] [bib_ref] Complications secondary to thrombectomy with the Fogarty balloon catheter, Albrechtsson [/bib_ref] [bib_ref] Mechanisms and prevention of arterial injuries caused by balloon embolectomy, Dobrin [/bib_ref] Our patient developed hemopericardium and tamponade within 30 minutes after transbrachial embolectomy that was performed without fluoroscopy. The time of onset of this complication and the absence of pericardial effusion before the procedure as seen on the chest CT scan [fig_ref] Fig 4: Computed tomography [/fig_ref] strongly suggested an association with the embolectomy procedure; nevertheless, the mechanism of this connection remained unclear because it was not explored at that time. The rarity of this complication contributed to the insufficient workup performed in looking for the cause.
Based on the clinical course during the next month (as described above), we postulated that the hemopericardium was a result of a small perforation of the RCA induced by the embolectomy catheter that was pushed blindly from the brachial artery to the ascending aorta and then into the RCA down to its midsegment. The anomalous anatomy of the RCA ostium directed upward at the base of the right coronary sinus [fig_ref] Fig 2: Aberrant origin of the right coronary artery [/fig_ref] enabled the undesirable insertion of the catheter into the RCA. In the same way, it was easily targeted by the multipurpose catheter during the coronary intervention. The multipurpose catheter is a relatively straight catheter with a shape that fairly resembles the embolectomy catheter. This resulted in a small perforation with bleeding into the pericardial space and consequent cardiac tamponade. The elevated pericardial pressure potentially contributed to formation of a clot that was organized around the middle segment of the RCA and prevented ongoing bleeding. However, this process eventually resulted in the subsequent development of the RCA pseudoaneurysm. This hypothesis is also strengthened by the absence of any RCA disease or pericardial effusion on CT that was performed before the embolectomy [fig_ref] Fig 4: Computed tomography [/fig_ref]. The systemic inflammatory reaction that was observed during the period after the tamponade could be attributed to a Dressler phenomenon caused by the presence of blood within the pericardium.
This adverse outcome may have been prevented by attention paid to avoid advancement of the catheter far beyond the estimated length of the thrombosed arterial segment using the catheter markers. Another option is the use of fluoroscopy during the embolectomy procedure to guide the catheter. Nevertheless, the chance for such complication seems exceptionally slim as it requires two improbable scenarios: maladvancement of the embolectomy catheter as well as an anomalous origin of the coronary artery, facilitating advancement of the catheter into the coronary vessel. The operators should strictly assess the anticipated length of catheter insertion, be aware of this possible complication, and be careful during the advancement of the embolectomy catheter to avoid damage to other vessels.
# Conclusions
We report a very rare complication of a presumed perforation of a coronary artery and hemopericardium, with subsequent formation of a coronary pseudoaneurysm, attributed to brachial arterial catheter embolectomy. We believe that the anomalous origin of the RCA contributed to this complication.
[fig] Fig 1. a ,: Computed tomography (CT) scan performed 5 weeks after the management of acute tamponade; a new rounded mass is observed in the territory of the right coronary artery (RCA; arrow). b and c, CT angiography reveals RCA pseudoaneurysm. [/fig]
[fig] Fig 2: Aberrant origin of the right coronary artery (RCA) directed upward (arrow) demonstrated during injection of contrast material at the right coronary sinus by AL1 catheter. [/fig]
[fig] Fig 3: Coronary angiography of the right coronary artery (RCA) with multipurpose catheter. a, A large pseudoaneurysm in the midsegment of the artery (arrow). b, The RCA after occlusion of the pseudoaneurysm with pericardial covered stent (3.5/27 mm; ITGI Medical). [/fig]
[fig] Fig 4: Computed tomography (CT) scan performed before the embolectomy procedure showing no evidence of pericardial effusion or right coronary artery (RCA) disease. [/fig]
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Realizing and characterizing chiral photon flow in a circuit quantum electrodynamics necklace
Gauge theory plays the central role in modern physics. Here we propose a scheme of implementing artificial Abelian gauge fields via the parametric conversion method in a necklace of superconducting transmission line resonators (TLRs) coupled by superconducting quantum interference devices (SQUIDs). The motivation is to synthesize an extremely strong effective magnetic field for charge-neutral bosons which can hardly be achieved in conventional solid-state systems. The dynamic modulations of the SQUIDs can induce effective magnetic fields for the microwave photons in the TLR necklace through the generation of the nontrivial hopping phases of the photon hopping between neighboring TLRs. To demonstrate the synthetic magnetic field, we study the realization and detection of the chiral photon flow dynamics in this architecture under the influence of decoherence. Taking the advantages of its simplicity and flexibility, this parametric scheme is feasible with state-of-the-art technology and may pave an alternative way for investigating the gauge theories with superconducting quantum circuits. We further propose a quantitative measure for the chiral property of the photon flow. Beyond the level of qualitative description, the dependence of the chiral flow on external pumping parameters and cavity decay is characterized. C ircuit quantum electrodynamics (QED) 1-4 is the realization of cavity QED 5,6 in superconducting quantum circuits. It employs the superconducting coplanar transmission line resonators (TLRs) 1,2 to substitute the standing-wave optical cavities and the superconducting qubits 7-10 to replace the atoms. Due to its flexibility and scalability, this on-chip architecture has been regarded as a promising platform for quantum computation 4 and quantum simulation 15,16 . Recently, several theoretical schemes have been proposed to generate artificial gauge fields for microwave photons and polaritons 11-14 in circuit QED lattices15,16. While the idea of synthesizing gauge fields was first proposed and realized in the context of ultracold atoms 17-19 and magnetooptical systems 20 , circuit QED takes the advantages of individual addressing and in situ tunability of circuit parameters 4,15,16 . Moreover, the effective strong photon-photon repulsion can be induced through a variety of mechanisms, including electromagnetically induced transparency (EIT) 21-23 , Jaynes-Cummings-Hubbard (JCH) nonlinearity 24,25 , and nonlinear Josephson coupling[26][27][28]. Combining the strong photon correlation with the synthetic gauge fields, the circuit QED system is showing prospective potential in the investigation of bosonic fractional quantum Hall liquids 29,30 and nontrivial topological edge states for microwave photons 31 .In the pioneering work 13 and its generalization 14 , a method of generating effective magnetic fields for polaritons in a two dimensional cavity lattice has been proposed. For each site on the lattice, the mixing phase between the atomic and photonic components of the polariton is controlled by an EIT type modulation of the atom trapped in the cavity, and the inter-site polariton hopping is induced by the untunable evanescent coupling between neighboring cavities. When hopping from one particular site to its neighbor, the polariton acquires a hopping phase which is the difference of the mixing phases subjected to the two neighboring sites. To obtain nontrivial gauge fields, the hopping along the horizontal and vertical directions should be controlled independently, and two-mode cavities (TMCs) and double EIT processes are consequently required. The construction and pumping of the complicated multi-level artificial atoms are still challenging in current experimental setup. There is also another scheme proposed in which a passive circulator is used to induce nontrivial hopping phases between TLRs OPEN SUBJECT AREAS: QUANTUM SIMULATION SINGLE PHOTONS AND QUANTUM EFFECTS QUANTUM OPTICS
Gauge theory plays the central role in modern physics. Here we propose a scheme of implementing artificial Abelian gauge fields via the parametric conversion method in a necklace of superconducting transmission line resonators (TLRs) coupled by superconducting quantum interference devices (SQUIDs). The motivation is to synthesize an extremely strong effective magnetic field for charge-neutral bosons which can hardly be achieved in conventional solid-state systems. The dynamic modulations of the SQUIDs can induce effective magnetic fields for the microwave photons in the TLR necklace through the generation of the nontrivial hopping phases of the photon hopping between neighboring TLRs. To demonstrate the synthetic magnetic field, we study the realization and detection of the chiral photon flow dynamics in this architecture under the influence of decoherence. Taking the advantages of its simplicity and flexibility, this parametric scheme is feasible with state-of-the-art technology and may pave an alternative way for investigating the gauge theories with superconducting quantum circuits. We further propose a quantitative measure for the chiral property of the photon flow. Beyond the level of qualitative description, the dependence of the chiral flow on external pumping parameters and cavity decay is characterized. C ircuit quantum electrodynamics (QED) [bib_ref] Cavity quantum electrodynamics for superconducting electrical circuits: An architecture for quantum computation, Blais [/bib_ref] [bib_ref] Strong coupling of a single photon to a superconducting qubit using circuit..., Wallraff [/bib_ref] [bib_ref] Coherent dynamics of a flux qubit coupled to a harmonic oscillator, Chiorescu [/bib_ref] [bib_ref] Atomic physics and quantum optics using superconducting circuits, You [/bib_ref] is the realization of cavity QED [bib_ref] Manipulating quantum entanglement with atoms and photons in a cavity, Raimond [/bib_ref] [bib_ref] Algebraic method for solving a class of coupled-channel cavity QED models, Wu [/bib_ref] in superconducting quantum circuits. It employs the superconducting coplanar transmission line resonators (TLRs) 1,2 to substitute the standing-wave optical cavities and the superconducting qubits 7-10 to replace the atoms. Due to its flexibility and scalability, this on-chip architecture has been regarded as a promising platform for quantum computation [bib_ref] Atomic physics and quantum optics using superconducting circuits, You [/bib_ref] and quantum simulation [bib_ref] Circuit QED lattices: Towards quantum simulation with superconducting circuits, Schmidt [/bib_ref] [bib_ref] On-chip quantum simulation with superconducting circuits, Houck [/bib_ref]. Recently, several theoretical schemes have been proposed to generate artificial gauge fields for microwave photons and polaritons [bib_ref] Synthetic gauge fields and homodyne transmission in Jaynes-Cummings lattices, Nunnenkamp [/bib_ref] [bib_ref] Time-reversal-symmetry breaking in circuit-QED-based photon lattices, Koch [/bib_ref] [bib_ref] Fractional Quantum Hall State in Coupled Cavities, Cho [/bib_ref] [bib_ref] Quantum simulation of an artificial Abelian gauge field using nitrogen-vacancy-center ensembles coupled..., Yang [/bib_ref] in circuit QED lattices [bib_ref] Circuit QED lattices: Towards quantum simulation with superconducting circuits, Schmidt [/bib_ref] [bib_ref] On-chip quantum simulation with superconducting circuits, Houck [/bib_ref]. While the idea of synthesizing gauge fields was first proposed and realized in the context of ultracold atoms [bib_ref] Creation of effective magnetic fields in optical lattices: the Hofstadter butterfly for..., Jaksch [/bib_ref] [bib_ref] Spin-orbit coupling in quantum gases, Galitski [/bib_ref] [bib_ref] Artificial gauge potentials for neutral atoms, Dalibard [/bib_ref] and magnetooptical systems [bib_ref] Observation of unidirectional backscattering-immune topological electromagnetic states, Wang [/bib_ref] , circuit QED takes the advantages of individual addressing and in situ tunability of circuit parameters [bib_ref] Atomic physics and quantum optics using superconducting circuits, You [/bib_ref] [bib_ref] Circuit QED lattices: Towards quantum simulation with superconducting circuits, Schmidt [/bib_ref] [bib_ref] On-chip quantum simulation with superconducting circuits, Houck [/bib_ref]. Moreover, the effective strong photon-photon repulsion can be induced through a variety of mechanisms, including electromagnetically induced transparency (EIT) [bib_ref] Strongly interacting polaritons in coupled arrays of cavities, Hartmann [/bib_ref] [bib_ref] Cross-Kerr-effect induced by coupled Josephson qubits in circuit quantum electrodynamics, Hu [/bib_ref] [bib_ref] Giant Kerr Nonlinearities in Circuit Quantum Electrodynamics, Rebić [/bib_ref] , Jaynes-Cummings-Hubbard (JCH) nonlinearity [bib_ref] Observation of Resonant Photon Blockade at Microwave Frequencies Using Correlation Function Measurements, Lang [/bib_ref] [bib_ref] Quantum phase transitions of light, Greentree [/bib_ref] , and nonlinear Josephson coupling [bib_ref] Networks of nonlinear superconducting transmission line resonators, Leib [/bib_ref] [bib_ref] Photon Solid Phases in Driven Arrays of Nonlinearly Coupled Cavities, Jin [/bib_ref] [bib_ref] Josephson-junctionembedded transmission-line resonators: From Kerr medium to in-line transmon, Bourassa [/bib_ref]. Combining the strong photon correlation with the synthetic gauge fields, the circuit QED system is showing prospective potential in the investigation of bosonic fractional quantum Hall liquids [bib_ref] Fractional Quantum Hall States of Photons in an Array of Dissipative Coupled..., Umucalilar [/bib_ref] [bib_ref] Fractional Quantum Hall Physics in Jaynes-Cummings-Hubbard Lattices, Hayward [/bib_ref] and nontrivial topological edge states for microwave photons [bib_ref] Majorana-like Modes of Light in a One-Dimensional Array of Nonlinear Cavities, Bardyn [/bib_ref].
In the pioneering work 13 and its generalization 14 , a method of generating effective magnetic fields for polaritons in a two dimensional cavity lattice has been proposed. For each site on the lattice, the mixing phase between the atomic and photonic components of the polariton is controlled by an EIT type modulation of the atom trapped in the cavity, and the inter-site polariton hopping is induced by the untunable evanescent coupling between neighboring cavities. When hopping from one particular site to its neighbor, the polariton acquires a hopping phase which is the difference of the mixing phases subjected to the two neighboring sites. To obtain nontrivial gauge fields, the hopping along the horizontal and vertical directions should be controlled independently, and two-mode cavities (TMCs) and double EIT processes are consequently required. The construction and pumping of the complicated multi-level artificial atoms are still challenging in current experimental setup. There is also another scheme proposed in which a passive circulator is used to induce nontrivial hopping phases between TLRs capacitively connected to it 12 through a virtual-resonant process. Due to its dispersive nature, this scheme is suitable to construct Kagomé and honeycomb lattices with coordination number three because the circulator induces effective photon hopping between any two of the TLRs connected to it. When applied to other lattice configurations with coordination number larger than three (e. g. square lattice with coordination number four), this scheme will result in unwanted cross-talk.
In this manuscript, we consider an alternative mechanism of implementing artificial Abelian gauge fields and propose a minimum circuit to demonstrate this method. Our work is inspired by the laser assisted tunneling technique used in ultracold atoms [bib_ref] Creation of effective magnetic fields in optical lattices: the Hofstadter butterfly for..., Jaksch [/bib_ref] and recent works of Josephson-embedded circuit QED systems 28,33-35 . We consider a necklace consisting of three TLRs coupled by superconducting quantum interference devices (SQUIDs) [bib_ref] Networks of nonlinear superconducting transmission line resonators, Leib [/bib_ref] [bib_ref] Josephson-junctionembedded transmission-line resonators: From Kerr medium to in-line transmon, Bourassa [/bib_ref] [bib_ref] Many body physics with coupled transmission line resonators, Leib [/bib_ref] [bib_ref] Tunable coupling engineering between superconducting resonators: From sidebands to effective gauge fields, Peropadre [/bib_ref] which can be harmonically modulated. With appropriate modulating pulses, effective parametric photon conversion between eigenmodes of the necklace can be induced [bib_ref] Quantum superposition of a single microwave photon in two different 'colour' states, Z-Bajjani [/bib_ref] , which manifests itself as photon hopping between neighboring TLR sites. This modulation in turn leads to an accumulated hopping phase during the hopping process, which can be regarded as an effective magnetic field imposed on the photons. As our method endows hopping phases directly to the links between TLRs, we expect that the experimental setup of our scheme is much simpler because the complicated double EIT pumping is not necessary. In addition, since our scheme does not rely on the dispersive mechanism, the above-mentioned cross-talk difficulty can also be circumvented. Moreover, the effective hopping strength in our scheme can be controlled by the modulating pulses. Such advantage may offer potential facilities in the future study of the competition between synthetic gauge fields, photon hopping, and Hubbard repulsion.
We further study the chiral photon flow dynamics of the necklace, which is a direct evidence of the breaking of time reversal symmetry (TRS) in the proposed circuit. We numerically simulate the photon flow dynamics in the presence of decoherence based on reported experimental data [bib_ref] Quantum superposition of a single microwave photon in two different 'colour' states, Z-Bajjani [/bib_ref] [bib_ref] Fabrication and characterization of superconducting circuit QED devices for quantum computation, Frunzio [/bib_ref] [bib_ref] Coherent quantum state storage and transfer between two phase qubits via a..., Sillanpää [/bib_ref]. Our results imply that the unidirectional character of the photon flow survives in the cavity decay. The feasibility of detecting such phenomena with the recently-developed photon detection technique [bib_ref] Quantum superposition of a single microwave photon in two different 'colour' states, Z-Bajjani [/bib_ref] [bib_ref] Deterministic Entanglement of Photons in Two Superconducting Microwave Resonators, Wang [/bib_ref] [bib_ref] Planck Spectroscopy and Quantum Noise of Microwave Beam Splitters, Mariantoni [/bib_ref] is also discussed. Moreover, to quantitatively describe the chiral flow, we introduce the concepts of photon position vector and chiral area. We show that the direction and the strength of the chiral photon flow can be represented by the chiral area which is the directed area swept by the photon position vector in a given time. With the proposed quantitative measure, we quantify the chiral flow character in general cases and investigate its detailed dependence on external pumping and decoherence processes.
# Results
Implementing artificial gauge field with dynamic modulation method: the theoretical model. We start from a photon hopping process between three cavities described by the following Hamiltonian
[formula] H~g 12 a { 1 a 2 e ih12 zg 23 a { 2 a 3 e ih23 zg 31 a { 3 a 1 e ih31 zh:c,ð1Þ [/formula]
where a i . a { i are the annihilation/creation operators of the ith site for i 5 1, 2, 3, g ij are the i « j hopping rates, and h ij are the corresponding hopping phases. We can imagine that there is a photon initially prepared in the cavity 1 and hopping on the cavity necklace. When finishing the 1 R 3 R 2 R 1 circulation, the photon accumulates a phase h S 5 h 12 1 h 23 1 h 31 , which is similar to the Aharonov-Bohm phase of an electron circulating in an external magnetic field. Consequently, h S can be regarded as an artificial magnetic field imposed on the charge-neutral photon, and the TRS of this circuit keeps intact if and only if h S [ pZ [bib_ref] Synthetic gauge fields and homodyne transmission in Jaynes-Cummings lattices, Nunnenkamp [/bib_ref] [bib_ref] Time-reversal-symmetry breaking in circuit-QED-based photon lattices, Koch [/bib_ref]. To break the TRS, we synthesize non-trivial h 12 , h 23 and h 31 by the dynamic modulation method [bib_ref] Realizing effective magnetic field for photons by controlling the phase of dynamic..., Fang [/bib_ref] : we consider a three-cavity Hamiltonian
[formula] H TC~H0 zS ivj H ij ,ð2Þ [/formula]
with
[formula] H 0~S 3 i~1 v i a { i a i ,ð3ÞH ij~Vij t ð Þ a { i za i {a { j {a j h i 2 ,ð4Þ [/formula]
where v i is the eigenfrequency of the ith cavity and V ij (t) is the coupling constant between the ith and jth cavities. Here we assume that V ij (t) can be tuned harmonically and in situ (we will discuss the physical realization in the next subsection). Moreover, we assume that the parameters in H TC satisfy the far off-resonance condition:
[formula] v 3 {v 2 ,v 2 {v 1 f g ? V 12 t ð Þ j j, V 23 t ð Þ j j, V 31 t ð Þ j j f g :ð5Þ [/formula]
In the first step we consider the 1 « 2 hopping. If V 12 (t) is static, the 1 « 2 photon hopping can hardly be induced because the two cavities are far off-resonant. Meanwhile, we can implement the effective 1 « 2 photon hopping by modulating V 12 (t) dynamically as
[formula] V 12 t ð Þ~{g 12 cos v 1 {v 2 ð Þ t{h 12 ½ :ð6Þ [/formula]
Physically, V 12 (t) carries energy quanta filling the gap between the two cavity modes. For a photon initially placed in the 1st cavity, it can absorb an energy quantum v 2 2 v 1 from the 1 « 2 link, convert its frequency to v 2 , and hop finally into the 2nd cavity. We can further describe this process in a more rigorous way: in the rotating frame with respect to H 0 , H 12 becomes
[formula] H I12~e iH0t H 12 e {iH0t <g 12 a { 1 a 2 e ih12 zh:c,ð7Þ [/formula]
because the other terms are fast oscillating and thus are safely neglected. From Eq. (7), we notice that both the effective hopping strength and the hopping phase can be controlled by the modulating pulse V 12 (t). Similarly, we can induce the effective 2 « 3 and 3 « 1 hopping process by modulating V 23 (t) and V 31 (t) as
[formula] V 23 t ð Þ~{g 23 cos v 2 {v 3 ð Þ t{h 23 ½ ,ð8ÞV 31 t ð Þ~{g 31 cos v 3 {v 1 ð Þ t{h 31 ½ :ð9Þ [/formula]
Summarizing the three pulses up, we get
[formula] H I~HI12 zH I23 zH I31~g12 a { 1 a 2 e ih12 zg 23 a { 2 a 3 e ih23 zg 31 a { 3 a 1 e ih31 zh:c,ð10Þ [/formula]
which directly reproduces the model (1).
The superconducting circuit implementation: a SQUID-coupled three-TLR necklace. Here we show explicitly the implementation of Eq. (1) in a circuit QED necklace. We propose a circuit consisting of three TLRs with different lengths L n but the same capacitance c and inductance l per unit length, coupled by three grounding SQUIDs with capacitance C Ja and maximal Josephson coupling energy E a for n 5 1, 2, 3 and a 5 a, b, c, as shown in(this architecture has also been exploited to study the entanglement generation through dynamic Casimir effect recently [bib_ref] Dynamical Casimir Effect Entangles Artificial Atoms, Felicetti [/bib_ref]. For each of the SQUID loops, an external static flux biasW a is added to modulate the effective Josephson coupling energy as E Ja~Ea cos pW a W 0 À Á with W 0 5 h/ 2e the flux quantum. We assume that the inductance/capacitance of the resonator is much bigger than the inductance/capacitance of the SQUIDs such that the following inequalities hold:
[formula] Ll, DL j jl?L Ja ,ð11Þ [/formula]
Lc, DL
[formula] j jc?C Ja ,ð12Þ [/formula]
where L Ja~W 2 0 4p 2 E Ja is the effective inductance of the ath SQUID for a 5 a, b, c, L 5 L 1 1 L 2 1 L 3 is the total length of the TLR necklace, and DL 5 min{jL i 2 L j j, i ? j} characterizes the length difference between the TLRs. Focusing only on the lowest three modes, this necklace can be described by the Hamiltonian
[formula] H 0~S 3 m~1 v m a { m a m ,ð13Þ [/formula]
where v m is the eigenfrequency of the mth eigenmode for m 5 1, 2, 3, and a m a { m are the corresponding annihilation/creation operators. While Eq. (13) is derived in detail in Methods, we can explain the mode structure of the necklace in an intuitive way. The presence of the grounding SQUIDs can modify the eigenmodes of the individual TLRs and induce the TLR-TLR coupling. From the point of view of TLR 1, the SQUID a plays the role of a shortcut of TLR 2, because the In addition, we set E Ja /E Ca 5 100 with E Ca 5 2e 2 /C Ja . With the chosen parameters, we get v 1 /2p 5 11.5 GHz, v 2 /2p 5 9.5 GHz and v 3 /2p 5 8.2 GHz for I S 5 3 mA. In (b), we quantify the localization property of the mth eigenmode by E m /v m where E m is the energy stored in the mth TLR for m 5 1, 2, 3. The critical current I S of the SQUIDs varies from 0.5 mA to 4 mA, and the other parameters are chosen as the same as incurrent coming from TLR 2 will largely flow through SQUID a directly to the ground, without crossing TLR 1. This allows us to define separated and localized modes for the TLR necklace: due to the small inductances of the grounding SQUIDs, the edges of the TLRs can be regarded as grounding nodes, and the lowest three eigenmodes can be approximated by the three individual l/2 modes of the TLRs. For the mth eigenmode, we calculate its normalized node flux distribution function f n,m (x) in the nth TLR based on data from recent experiments [bib_ref] Quantum superposition of a single microwave photon in two different 'colour' states, Z-Bajjani [/bib_ref] [bib_ref] Fabrication and characterization of superconducting circuit QED devices for quantum computation, Frunzio [/bib_ref] [bib_ref] Coherent quantum state storage and transfer between two phase qubits via a..., Sillanpää [/bib_ref] and study its localization property versus the Josephson coupling energies of the grounding SQUIDs. For the TLRs, the circuit parameters are chosen as c 5 1.6 3 10 210 F ? m 21 , l 5 4.08 3 10 27 H?m 21 , L 1 5 6 mm, L 2 5 7 mm and L 3 5 8 mm. For the grounding SQUIDs, we choose the effective critical currents I a 5 2pE Ja /W 0 of the three grounding SQUIDs on the level of I a g [0.5 mA, 4 mA] for a 5 a, b, c. As shown in, larger critical currents lead to better localization, this is consistent with our previous description of the roles played by the grounding SQUIDs. With proper choice of the parameters, the eigenmode amplitudes jf n,m (x)j 2 become sufficiently large only for n 5 m while negligible for n ? m.
Moreover, since the currents of two neighboring TLRs flow to the ground through the same grounding SQUID, by the modulation of the grounding SQUIDs we can establish the effective inter-TLR parametric hopping. For the 1 « 2 coupling, we add an extra a. c. flux driving dW a (t) 5 DW a cos[(v 1 2 v 2 )t 2 h a ] to the staticW a which induces the parametric coupling Hamiltonian (see Methods)
[formula] H int,a~2 g a cos v 1 {v 2 ð Þ t{h a ½ a { 1 a 2 zh:c,ð14Þ [/formula]
with g a the coupling strength between modes 1 and 2. In the rotating frame with respect to H 0 in Eq. (13), such modulation results in the effective 1 « 2 hopping which can be described by
[formula] H int,a~ga e iha a { 1 a 2 zh:c:ð15Þ [/formula]
In addition, we can add similar pumping pulses on the SQUIDs b and c to induce the 2 « 3 and 3 « 1 hoppings, respectively. Summing up the three modulations, we get the effective Hamiltonian
[formula] H I~ga e iha a { 1 a 2 zg b e ih b a { 2 a 3 zg c e ihc a { 3 a 1 zh:c,ð16Þ [/formula]
which is identical with Eq. (1) through the mappings g a R g 12 , g b R g 23 and g c R g 31 . We should emphasize that g a , g b , and g c can be modulated independently by the amplitudes of the a. c. pulses, and the three phases h a , h b , and h c are determined by the initial phases of the corresponding pulses. The range of the effective coupling strength g a can be estimated base on reported experimental data: we set I a g [1, 4] mA,W a W 0 [ 0:4, 0:6 ½ , and DW a /W 0 g [0.01, 0.02] for a 5 a, b, c. The resulted coupling strengths are in the range g a /2p g [bib_ref] Suppressing charge noise decoherence in superconducting charge qubits, Schreier [/bib_ref] [bib_ref] Fractional Quantum Hall Physics in Jaynes-Cummings-Hubbard Lattices, Hayward [/bib_ref] MHz. For simplicity, in the following we consider the homogenous hopping situation g T 5 g a 5 g b 5 g c .
The realization and detection of the chiral photon flow. To demonstrate the presence of the synthetic gauge field we study the chiral photon flow in this necklace which is the analog of electron circulation in an external magnetic field. We initialize the necklace such that there is initially a photon in the 1st mode and numerically simulate its subsequent time evolution in the presence of decoherence using the master equation
[formula] dr dt~{ i H I ,r ½ z 1 2 X 3 j~1 k j 2a j ra { j {a { j a j r{ra { j a j ,ð17Þ [/formula]
where r is the density matrix of the necklace and k j is the decay rate of the jth eigenmode. The circuit parameters are chosen as the same as those used in the calculation of Figs. 1(b) and 1(c), with the pumping strength g T /2p 5 20 MHz and the homogenous decay rate k/2p 5 250 kHz. In the three situations h S 5 p/2, p and 3p/2, we calculate the energy stored in the TLRs and plot our results in [fig_ref] Figure 2 |: Chiral photon flow in the TLR necklace in the presence of decay [/fig_ref]. As shown in the first panel which corresponds to h S 5 p/2, the energy population in the three TLRs exhibits clear temporal phase delay which implies that the photon is flowing unidirectionally, first from TLR 1 to TLR 2 and then from TLR 2 to TLR 3. Such chiral character is a significant demonstration of the breaking of TRS in this system. Similarly, the third panel which corresponds to h S 5 3p/2 describes the chiral photon flow with the opposite direction. Meanwhile, the second panel implies that, in the trivial case h S 5 p, the energy initially stored in TLR 1 transfers symmetrically to its left and right. It should be emphasized that, although the cavity decay rate k we choose is stronger compared with reported experimental data [bib_ref] Deterministic Entanglement of Photons in Two Superconducting Microwave Resonators, Wang [/bib_ref] , the chiral character of the photon flow in the first and third panel still survives. The environment causes severe photon damping but influences little on the unidirectional character of the photon dynamics. Therefore, we expect that the chiral photon flow pattern in this necklace can be realized and measured by the photon number detection technique developed in recent experiments [bib_ref] Quantum superposition of a single microwave photon in two different 'colour' states, Z-Bajjani [/bib_ref] [bib_ref] Deterministic Entanglement of Photons in Two Superconducting Microwave Resonators, Wang [/bib_ref] [bib_ref] Planck Spectroscopy and Quantum Noise of Microwave Beam Splitters, Mariantoni [/bib_ref]. For the three measurement devices shown in, we can use three phase qubits capacitively coupled to the corresponding TLRs [bib_ref] Quantum superposition of a single microwave photon in two different 'colour' states, Z-Bajjani [/bib_ref] [bib_ref] Deterministic Entanglement of Photons in Two Superconducting Microwave Resonators, Wang [/bib_ref]. Since the frequencies of the qubits can be adjusted by their d. c. bias currents, the initialization and the measurement of the chiral flow dynamics can be proceeded by the following steps: in the first step, we tune the frequencies of the qubits to be large off-resonance with the TLRs, and prepare the qubit 1 in its excited state. In this step, the coupling between the qubits and TLRs are effectively turned off. In the second step, we tune on the qubit-TLR coupling by adiabatically tuning the qubit 1 in resonance with the TLR 1 for a duration T 1 5 p/2g q1 such that the excited qubit 1 emit a photon to the TLR 1 (here g qn denote the coupling strengths between TLR n and qubit n for n 5 1, 2, 3). Through this manipulation, the single-photon initial state is prepared. After the initialization, we turn off the qubit-TLR coupling and turn on the external a. c. flux pumping on the grounding SQUIDs for a duration T 0 during which the TLR necklace experiences the chiral photon flow. To measure the photon flow dynamics, we could prepare the three qubits all in their ground state, turn off the external a. c. flux pumping, and then turn on the TLR-qubit coupling by tuning the frequencies of the qubits in resonance with the corresponding TLR modes. In this way we can load the TLR photons into the corresponding qubits and extract the evolution of photon distribution on the necklace through the measurement of the three qubits [bib_ref] Rabi Oscillations in a Large Josephson-Junction Qubit, Martinis [/bib_ref].
A quantitative measure of chiral photon flow. We further consider how to describe the chiral pattern of the photon flow. In [fig_ref] Figure 2 |: Chiral photon flow in the TLR necklace in the presence of decay [/fig_ref] as well as in Refs. 11 and 12, the dynamics of perfect chiral flow and perfect non-chiral flow have been investigated. Meanwhile, in more general cases the chiral character which is not perfect but does exist becomes fogged. To characterize how ''chiral'' the photon flow is, in the following we introduce a quantitative method. As shown in [fig_ref] Figure 3 |: Photon position vectorṼ and the chiral area S [/fig_ref] , we assign three unit vectors to the three TLRs,
[formula] V 1 !~1 ,0 ð Þ for the TLR 1, V 2 !~{ 1=2, ffiffi ffi 3 p . 2 for the TLR 2, and V 3 !{ 1=2,{ ffiffi ffi 3 p 2 À [/formula]
Á for the TLR 3. We then represent the photon distribution on the necklace by the photon position vector
[formula] V ! t ð Þ~n 1 t ð ÞV ! 1 zn 2 t ð ÞV ! 2 zn 3 t ð ÞV ! 3 where n j t ð Þ~Tr r t ð Þa { j a j [/formula]
is the photon number population in the TLR j for j 5 1, 2, 3. The initial condition used in [fig_ref] Figure 2 |: Chiral photon flow in the TLR necklace in the presence of decay [/fig_ref] corresponds to the initial position V ! 0 ð Þ~V ! 1 , and the state evolution can be expressed by the motion of V ! t ð Þ on the two dimensional plane. The traces of V ! t ð Þ for some typical values of h S and k are shown in Figs. 4(a) and 4(b). While the 6th panel ofindicates the perfect chiral flow in the situation h S 5 p/2 and k 5 0, the other traces become chaotic and irrational as h S departs from p/2 and the influence of decoherence is taken into account.
To grasp the chiral character from the complicated traces of the photon position vector, our idea is to sum up the area swept by V ! t ð Þ in a given time, as shown in [fig_ref] Figure 3 |: Photon position vectorṼ and the chiral area S [/fig_ref]. We define the chiral area as
## S~1
2T
[formula] ð T 0 V ! t ð Þ|d V ! ,ð18Þ [/formula]
where T is a time scale sufficiently longer than 1/g T but significantly smaller than 1/k. S has the following properties which make it a suitable measure of the chiral character:
1. S is directed. A clockwise trace and its counterclockwise correspondence (e. g. the h S 5 p/2 and 3p/2 situations shown in [fig_ref] Figure 2 |: Chiral photon flow in the TLR necklace in the presence of decay [/fig_ref] result in chiral areas with opposite signs. Therefore, the sign of S can be used to represent the direction of the chiral flow. 2. In the perfect non-chiral case h S 5 p shown in the middle panel of [fig_ref] Figure 2 |: Chiral photon flow in the TLR necklace in the presence of decay [/fig_ref] , the photon transfers symmetrically to the TLR 2 and TLR 3. Therefore, the trace of V ! t ð Þ is always along the direction of V ! 1 and zero chiral area is obtained, i. e. S 5 0 for h S 5 p. 3. Obviously, S achieves its maximal value when the photon flow is perfect chiral. Moreover, a faster rotation of V ! t ð Þ (which means larger g T ) and a longer V ! t ð Þ (which means more photons involved) lead certainly to a bigger S. From this point of view, S can also be used to demonstrate the influence of driving and dissipation on the chiral photon flow.
The chiral area concept can help us to go beyond the qualitative description of the state evolution in special cases and move into a quantitative and general level of investigation. Despite the complicated evolution V ! t ð Þ might undergo, the chiral area presents an intuitive and physical description of the chiral character. With this definition we calculate the chiral area versus the h S and k, and show our results in. It can be seen that the chiral area S decreases rapidly as the total phase h S departs from p/2. This is in agreement with our observation of the vector traces shown in Figs. 4(a) and 4(b). Our calculation indicates that the h S window suitable for the observation of chiral photon flow is not wide. Meanwhile, the width of this window is barely influenced by the decay rate k.
# Discussion
The implementation of the Abelian gauge field in the three-TLR necklace can be regarded as a minimal model. Through a variety of generalizations, the proposed dynamic approach can be used to construct a scalable and flexible quantum simulator of gauge theories. First of all, we consider the scalability of this method, i. e. how to synthesize a gauge field on a circuit QED lattice using this dynamic approach. We take the construction of a square lattice as an example. As shown in, we can build a two-dimensional square TLR lattice by four kinds of TLRs with eigenfrequencies v 1 /2p 5 8 GHz, v 2 /2p 5 9 GHz, v 3 /2p 5 10 GHz and v 4 /2p 5 11 GHz placed in an interlaced form and connected to the ground by the grounding SQUIDs. We assume that the small capacitance/inductance conditions in Eqs. [bib_ref] Synthetic gauge fields and homodyne transmission in Jaynes-Cummings lattices, Nunnenkamp [/bib_ref] and (12) still hold. To introduce effective passive photon hopping with nontrivial hopping phases, we add extra twotone a. c. flux driving with frequencies 1 GHz and 3 GHz and appropriate initial phases to the loops of the grounding SQUIDs. In this situation, the cross-talk between next-nearest-neighbor TLRs can be neglected because the corresponding frequency (2 GHz) is far offresonant with the a. c. flux pumping. Moreover, we can introduce offdiagonal disorder [bib_ref] Field theory of the random flux model, Altland [/bib_ref] [bib_ref] Kosterlitz-Thouless-Type Metal-Insulator Transition of a 2D Electron Gas in a Random Magnetic..., Xie [/bib_ref] (i. e. random magnetic fields) into the lattice through randomization of the initial phases of the a. c. driving pulses. While the simulation of diagonal disorder has been realized in ultracold atoms [bib_ref] Disordered quantum gases under control, Sanchez-Palencia [/bib_ref] and in optical fiber systems [bib_ref] Anderson Localization and Nonlinearity in One-Dimensional Disordered Photonic Lattices, Lahini [/bib_ref] , the simulation of offdiagonal disorder of bosonic particles has also attracted research interest in recent years [bib_ref] From Anderson to anomalous localization in cold atomic gases with effective spin-orbit..., Edmonds [/bib_ref]. We can also introduce the effective photon-photon interaction to the non-interacting system described in this manuscript by the JCH method, i. e. we couple the TLRs to superconducting qubits resonantly. Though the resonant coupling, the photons are dressed by the qubits and inherit the nonlinearity of the qubits [bib_ref] Observation of Resonant Photon Blockade at Microwave Frequencies Using Correlation Function Measurements, Lang [/bib_ref] [bib_ref] Quantum phase transitions of light, Greentree [/bib_ref]. From the above points of view, we can expect that this dynamic modulation approach may pave a new way of investigating the novel physics of the competition between artificial gauge fields, diagonal and off-diagonal disorder, and Hubbard repulsion in the circuit QED system.
[formula] V(t) V(t dt) dV 1 V (TLR1) 2 V (TLR2) 3 V (TLR3) O(0,0) A B [/formula]
In summary, we have investigated the implementation of artificial gauge fields in the circuit QED system by the method of dynamic modulation. We have numerically simulated the photon chiral flow in a three-TLR necklace, discussed the feasibility of observing this phenomenon, and proposed a quantitative measure of the chiral character. Our work may offer new perspectives to future studies of quantum simulation and parametric quantum optical physics in SQUID-embedded circuit QED systems.
# Methods
Quantization of the TLR necklace. The Lagrangian of the system can be written as:
[formula] L~X 3 n~1 1 2 ð Ln dx cV 2 n x,t ð Þ{ 1 l L Lx Q n x,t ð Þ 2 " # z X a~a,b,c C Ja 2 V 2 a { 1 2L Ja Q 2 a ,ð19Þ [/formula]
where V n (x, t) describes the voltage distribution on the nth TLR for n 5 1, 2, 3,
[formula] Q n x,t ð Þ~ð t {? [/formula]
V n x,t ð Þdt is the corresponding node flux distribution, and V a /Q a are the voltage/node flux at locations of the SQUID a for a 5 a, b, c. In deriving Eq. (19), we have linearized the grounding SQUIDs as {E Ja cos 2pQ a =W 0 ½ < Q a ð Þ 2 2L Ja . This assumption is valid because Q a (t) < 0. Following the Euler-Lagrangian equation, we get the equation of motion of the node flux in the bulk of the TLRs
[formula] v 2 L 2 x Q n~L 2 t Q n ,ð20Þ [/formula]
with v~1 . ffiffiffi lc p
. At the edges of TLRs, based on Kirchhoff's law we get the following boundary conditions
[formula] Q 1 x c ð Þ~Q 3 x' c ð Þ~Q c ,ð21Þ [/formula]
Q 1 x a ð Þ~Q 2 x a ð Þ~Q a , ð22Þ ð26Þ where x a 5 L 1 , x b 5 L 1 1 L 2 , x c 5 0 and x' c~L1 zL 2 zL 3 are the locations of the grounding SQUIDs (the cyclic boundary condition is applied as shown in Eq. (21)).
[formula] Q 2 x b ð Þ~Q 3 x b ð Þ~Q b ,ð23Þ [/formula]
Here without loss of generality we assume the three SQUIDs have the same effective inductance L J and capacitance C J . The eigenmodes of the necklace can be obtained by the method of separation of variables. We set Q n (x, t) 5 f n (x)g(t) with f n x ð Þ~A n cos kxzh n ð Þ , ð27Þ
where A n /h n are the normalized amplitude/phase of the eigenmode in the nth TLR, and k is the wave vector. Substituting Eq. [bib_ref] Photon Solid Phases in Driven Arrays of Nonlinearly Coupled Cavities, Jin [/bib_ref] to Eqs. (21)-(26) we get the following transcendental equations
[formula] { tan kx' c zh 3 ð Þ z tanh 1~1 L J k { C J c k,ð28Þ [/formula]
{ tan kx a zh 3 ð Þ z tan kx a zh 2 ð Þ 1
[formula] L J k { C J c k,ð29Þ{ tan kx b zh 2 ð Þ z tan kx b zh 3 ð Þ 1 L J k { C J c k,ð30Þ [/formula]
cos h 1 cos kx a zh 2 ð Þcos kx b zh 3 ð Þ cos kx a zh 1 ð Þcos kx b zh 2 ð Þcos kx' c zh 3 ð Þ :
When the phases and the wave vector are obtained, the amplitude distribution of the eigenmode in the three TLRs can be further determined by v 1 2 v 2 is comparable with the SQUID plasma frequency v pa~1 . ffiffiffiffiffiffiffiffiffi C J L J p , the device can not be considered as a passive element because complex quasi-particle excitation behavior will emerge. Meanwhile, with parameters chosen in this manuscript, we can verify that v pa ? v 1 {v 2 j jis satisfied.
[fig] Figure 1 |: (a) Schematic plot of the SQUID-coupled three-TLR necklace. This circuit is constructed by three TLRs connected through the grounding SQUIDs. For each SQUID a, the SQUID loop is penetrated by a static bias fluxW a and a dynamic modulation pulse dW a with a 5 a, b, c. Moreover, each TLR is coupled to a measurement device which can detect its photon number. The lowest three eigenfrequencies of the circuit are labeled by v 1 , v 2 , and v 3 , respectively. (b), (c) The localization property of the eigenmodes. (c) depicts the normalized mode functions of the three lowest eigenmodes of the circuit QED necklace versus critical currents of the SQUIDs. The three panels of (c) describe the eigenmode functions corresponding to the eigenmode 1, 2, and 3 respectively. The blue solid line, green dash line and red dot-dashed line correspond to the situations of I S 5 I a 5 I b 5 I c 5 1, 2, 3 mA respectively. [/fig]
[fig] Figure 2 |: Chiral photon flow in the TLR necklace in the presence of decay. The three panels from top to bottom represent the three situations h S 5 p/2, p, and 3p/2, respectively. The solid (blue), dashed (green), and dot-dashed (red) lines represent the energies stored in the TLR 1, 2 and 3, respectively. For the three grounding SQUIDs we assume that they have identical maximal critical current 5 mA and identical static flux bias W a~Wb~Wc~0 :3W 0 . The amplitudes of the pumping pulses are chosen as DW a/b/c /W 0 5 1.4/2.0/1.4% such that the homogeneous coupling strength g T /2p 5 g a /2p 5 g b /2p 5 g c /2p 5 20 MHz is induced. The decay rate is chosen as k/2p 5 250 kHz. The other circuit parameters are chosen the same as those used in the calculation of Figs. 1(b) and 1(c). [/fig]
[fig] Figure 3 |: Photon position vectorṼ and the chiral area S. The unit vectorsV 1~1 ,0 ð Þ,Ṽ 2~{ 1=2, ffiffi ffi 3 p . 2 , andṼ 3~{ 1=2,{ ffiffi ffi 3 p .2 are assigned to the modes 1, 2, and 3 of the necklace, respectively. The photon position vector is defined asṼ t ð Þ~n 1 t ð ÞṼ 1 zn 2 t ð ÞṼ 2 zn 3 t ð ÞṼ 3 with n j t ð Þ~Tr r t ð Þa { j a j , and its evolution trace is represented by the dashedline. The chiral area S~1 2T ð T 0Ṽ t ð Þ | dṼ is the directed area swept bỹV t ð Þ and can be used to characterize the chiral property of the photon flow. [/fig]
[fig] Figure 4 |: (a),(b) Evolution traces ofṼ t ð Þ at different h S with/without the presence of decoherence. (a) depicts the traces in the dissipationless situation k/2p 5 0, and (b) corresponds to k/2p 5 150 kHz. The panels 1-6 in (a) and (b) correspond to h S 5 p/6, p/4, p/3, 5p/12, 17p/36, and p/2, respectively. (c) Chiral area S versus h S in the presence of decoherence. The total time T is set as T 5 1 ms, and the solid, dotted, dot-dashed, and dashed lines correspond to the situations with decay rates k/2p 5 0, 100, 200, and 500 kHz, respectively. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8352 | DOI: 10.1038/srep08352 [/fig]
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Delta Variant with P681R Critical Mutation Revealed by Ultra-Large Atomic-Scale Ab Initio Simulation: Implications for the Fundamentals of Biomolecular Interactions
# Introduction
The COVID-19 pandemic started two years ago and continues with unabated intensity, with no clear end in sight, despite many repeated attempts to contain it. The recent emergence of various variants of concern (VOC) in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [bib_ref] Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's..., Zhong [/bib_ref] , such as Alpha, Beta, Delta [bib_ref] SARS-CoV-2 variants of concern are emerging in India, Singh [/bib_ref] , and Gamma, and variants of interest (VOI), such as Eta, Iota, Kappa [bib_ref] Reduced neutralization of SARS-CoV-2 B. 1.617 by vaccine and convalescent serum, Liu [/bib_ref] , Lambda, and Mu, instigates new anxieties. Among the new VOC, the Delta variant causes a more severe infection and spreads faster than previous variants of the SARS-CoV-2 virus, emerging as the dominant strain in the world [bib_ref] How the Delta variant achieves its ultrafast spread, Reardon [/bib_ref] , causing worries among the general population and solidifying the belief that the battle against the pandemic will be a long one. In a broader resolved mutations provides many fundamental insights that could lead to a new level of understanding in the development of therapeutic drug design against the SARS-CoV-2 virus and its variants.
## Model design and construction
Large biomolecular systems, such as proteins, have complex structures and contain many amino acids linked together in a specific order. Currently, we are capable of conducting ab initio simulations with up to 5000 atoms for a single calculation by adopting a divide and conquer strategy to investigate the S-protein. We briefly describe the model used in this study.
The SD2 to FP region marked as region 3 in [fig_ref] Figure 1: The illustration of SD2-FP model construction [/fig_ref] is our SD2-FP model, which is used in the actual atomic-scale calculation in the present work. The other broader structural regions consist of different structural domains in the S-protein of the SARS-CoV-2, which shows the specific mutation sites in the Delta variants. This is fully illustrated in [fig_ref] Figure 1: The illustration of SD2-FP model construction [/fig_ref] in the Supplementary Materials (SM). The SD2-FP models involved in the present work are: (a) the wild type (WT), (b) the mutated model P681R, (c) the mutated model D614G, and (d) the double mutation with both D614G and P681R.
The initial structure for the regions shown in [fig_ref] Figure 1: The illustration of SD2-FP model construction [/fig_ref] was obtained from Woo et al. from [PDB ID 6VSB] [bib_ref] Developing a fully glycosylated full-length SARS-CoV-2 spike protein model in a viral..., Woo [/bib_ref] , which originated from the study by Wrapp et al. [bib_ref] Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Wrapp [/bib_ref]. Here, it should be mentioned that the 6VSB Cryo-EM structure from Wrapp et al. has many missing amino acids (AAs) due to the limitation in their technique. Different prediction methods to model the missing AAs of 6VSB were used and the details of obtaining the full-length SARS-CoV-2 spike (S) protein structure can be found in Woo et al. [bib_ref] Developing a fully glycosylated full-length SARS-CoV-2 spike protein model in a viral..., Woo [/bib_ref]. We chose Chain A in its up conformation. Sequence numbers 592-834 in S-protein were used for SD2-FP model (6VSB_1_2_1). We used our procedure to construct a manageable size model and summarize it as follows. First, we selected all residues of the SD2 and FP region to create the SD2-FP model (residue 592 to 834). Next, we removed the glycans and the associated hydrogen (H) atoms from the SD2-FP model and later added the H atoms using the Leap module with ff14SB force field in the AMBER package [bib_ref] The Amber biomolecular simulation programs, Case [/bib_ref] , which then yields the WT model used as a template to generate the mutated models. To prepare the mutated models with a single mutation (P681R or D614G) or a double mutation (P681R and D614G), we used Dunbrack backbone-dependent rotamer library [bib_ref] Rotamer libraries in the 21st century, Dunbrack [/bib_ref] , implemented by the UCSF Chimera package [bib_ref] UCSF Chimera-A visualization system for exploratory research and analysis, Pettersen [/bib_ref]. In total, we created four SD2-FP models, including the WT model, mutated with P681R and D614G mutations, and double mutation consisting of both D614G and P681R. These models were minimized with 100 steepest descent steps and 10 conjugate gradient steps using UCSF Chimera to avoid bad clashes. These models (see Figures 1 and S1) were then further optimized using Vienna ab initio simulation package (VASP), as described in Section S1.1 in SM.
## Amino-acid-amino-acid bond pair (aabp)
The VASP-optimized structures were used as input in the orthogonalized linear combination of atomic orbitals (OLCAO) methodto calculate the electronic structure and interatomic interactions in biomolecules. The details of the OLCAO method are described in Section S1.2 of SM. Using OLCAO, we calculated the bond order (BO), which quantifies the strength of the bond between two atoms and usually scales with the bond length (BL). The sum of all BO values within a single structure unit gives the total bond order (TBO). The relatively new concept of BO and TBO in biomolecules quantifies the cohesion of the system. Before we extend our formulation and analysis of BO and TBO to the amino-acidamino-acid bond pair (AABP), we will first discuss briefly the 20 canonical amino acids with distinct residues listed in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] and illustrated through their functional groups in .
Amino acids are the basic structural units of proteins, sharing three common structural elements: an amine group, a carboxyl group, and a side chain residue. Different functional groups comprising the side chain consign to each of the 20 canonical amino acids distinct physical properties that influence the protein structure and function. The peptide bond links two adjacent AAs with a covalent bond between C1 (carbon number one) of one AA and N2 (nitrogen number two) of another, creating a linear chain connecting AAs with chemically distinct side chain residues into different linear sequences that can form long polypeptide chains that are able to fold upon themselves, thereby giving rise to diverse, functionally distinct proteins. Any discussion of the structure, properties, and functionalities of proteins must therefore originate from the unique structures and properties of the 20 canonical AAs [bib_ref] PubChem substance and compound databases, Kim [/bib_ref] [bib_ref] Peptides and Proteins, Ryadnov [/bib_ref].
Various physical properties characterize and differentiate the canonical AAs in bathing aqueous solutions, such as the number of atoms, size, molecular weight, hydropathy, polarity, charge, protonation/deprotonation dissociation constants, etc. In view of our aspiration to embark on a detailed ab initio investigation, unprecedented in size and scope, of the nature and effects of point mutations on interatomic interactions in the spike protein, most of the listed quantifiers do not seem to be appropriate and some appear to be distinctly missing. Based upon what the ultra-large scale ab initio methodology can provide, we focus on two structural quantifiers: the well-known partial charge (PC) parameter and a modification of the BO parameter referred to as the amino-acid-amino-acid bond pair (AABP). The first one addresses the polarity and charge structure of the molecule and certainly responds to all of the alterations wrought by the mutation in the protein sequence. The second one is a generalization of the BO and TBO and is specifically tailored to proteins by defining the amino-acid-amino-acid bond pair (AABP) as where the summations are over all atoms α in AA u and all atoms β in AA v. AABP embodies all possible bonding between two amino acids, including both covalent and hydrogen bonding (HB). AABP is a single parameter that quantifies the amino-acid-amino-acid interaction, so that the stronger the interaction, the larger the AABP, and vice versa.
[formula] AABP(u, v) = ∑ α u ∑ β v ρ αi,βj(1) [/formula]
We stress that the use of this novel AABP concept is not the same as using the conventional description of the amino-acid-amino-acid interaction in biomolecules, since the distance of separation and the atomic interactions between two AAs are difficult to quantify accurately. The AABP values are calculated from quantum mechanical wave functions to study different types of interactions in biomolecules, such as nearest neighbor (NN) and non-NN, also designated as off-diagonal or non-local (NL) interactions between AAs. Non-NN AAs are not vicinal in the 1D primary sequence space but are vicinal in the 3D embedding folding space. The AABP concept can help to foment a better understanding of the overall 3D interactions, not only of proteins, but of complex biomolecular systems in general.
The AABP defined above is a unique feature in the present study. Simulation methodologies that have been routinely and extensively used by the biomolecular research community [bib_ref] Targeting SARS-CoV-2: A systematic drug repurposing approach to identify promising inhibitors against..., Khan [/bib_ref] [bib_ref] Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions, Wang [/bib_ref] , such as classical molecular dynamics (MD) with its onerous energy or enthalpy calculations, are based on different types of presumably transferable a priori force field specifications. As such, they cannot reveal the atomic details of the real interatomic interactions and mostly rely on the atomic potential parametrizations and assumed geometric structures, which are both inherently limited. On the other hand, the ab initio molecular dynamics methodology, intended for a more realistic simulation of complex biomolecular systems and processes from first principles, is, at present, hampered by the excessive computational times and resources, making it inapplicable to the analysis of even modest-sized proteins. The use of the concept of bond order, as implemented in this work, can quantitatively characterize the AA-AA interaction in 3D folding space and can also be applied to larger scale protein-protein interactions, or the interaction of different segments of the same protein, thus providing a promising and valuable alternative (see Section 4.2 for details).
Our approach here will be based on the characterization of the wild type and mutated protein by the PC and AA-AA bond pair parameters. By judiciously labeling each mutation as a data point, with specific details for the different components of the partial charge and AA-AA bond pair parameters, it will furthermore facilitate its application in a machine learning (ML) protocol when many mutation data become available.
# Results
This section is conveniently divided into four sections, but the key section is Section 4.3 AABP data for mutations in the structural domain of SD2-FP containing the furin cleavage site [fig_ref] Figure 1: The illustration of SD2-FP model construction [/fig_ref]. The AABP data are based on the results of the model structures using VASP and the OLCAO for the electronic interactions. [fig_ref] Table 2: Data representation for ML [/fig_ref] lists the structure information from VASP optimization for the four SD2-FP models in the Delta variant: (a) wild type (WT), (b) mutated P681R (R681), (c) mutated D614G (G614), and (d) double mutation (DM) labeled as G614-R681. In addition, [fig_ref] Table 2: Data representation for ML [/fig_ref] also lists two HR1-CH models in the Delta variant: (e) wild type (WT) D950 and (f) mutated D950N (N950). As can be seen in [fig_ref] Table 2: Data representation for ML [/fig_ref] , the energy is sufficiently converged to the level of 0.03 to 0.04 eV, which is less than 10 −5 eV per atom, including H atoms, but the entailed computational resources consumed are humongous. The VASP-optimized structure is used as the input for the DFT calculation using OLCAO.
The results are divided into the following four sections. Sections 4.1 and 4.2 are standard electronic structures routinely present for the analysis in biomolecular systems [bib_ref] Intra-and intermolecular atomic-scale interactions in the receptor binding domain of SARS-CoV-2 spike..., Adhikari [/bib_ref] [bib_ref] Ultra-Large-Scale Ab Initio Quantum Chemical Computation of Bio-Molecular Systems: The Case of..., Ching [/bib_ref] [bib_ref] Key interacting residues between RBD of SARS-CoV-2 and ACE2 receptor: Combination of..., Jawad [/bib_ref] [bib_ref] First-Principles Simulation of Dielectric Function in Biomolecules, Adhikari [/bib_ref] [bib_ref] Solvent Effect on the Structure and Properties of RGD Peptide (1FUV) at..., Baral [/bib_ref]. Section 4.2 describes the key data on interatomic interactions between all atoms whose results are used for the main Section 4.3 on the AABP data for the four SD2-FP models (a) to (d) listed in [fig_ref] Table 2: Data representation for ML [/fig_ref]. Section S2 in SM provides the additional results from the two HR1-CH models (e) and (f), listed in [fig_ref] Table 2: Data representation for ML [/fig_ref] , that support the observation in Section 4.3.
## Electronic structure
In an ab initio calculation of any materials, the focus is on the density of states (DOS) or its components, the partial DOS (PDOS). In small molecules, researchers tend to use the list of energy levels separated by HOMO-LUMO gaps. [fig_ref] Figure 3: Interaction of D614 and P681 to their NN and NL AAs in... [/fig_ref] shows the calculated total DOS (TDOS) of the current supercell WT SD2-FP containing P681 and D614 with 3654 atoms. The 0.0 eV energy stands for the HOMO state or the top of the occupied valence band. The LUMO is located at approximately 2.5 eV. There exist some gap states within the HOMO-LUMO gap as expected due to some interacting states within this complex biomolecule. There is virtually no difference in the TDOS between the WT model and those that contain the mutated AA. The only difference is a very minute structure in some peaks, where, presumably, the mutated AA has a slightly different energy level. In principle, the PDOS can be resolved into an individual AA or groups of AAs, which will be very useful if a more detailed analysis is necessary, such as making distinctions between mutated and unmutated AAs. The atomic scale interaction must be revealed by a detailed analysis of the calculated electronic structure on relevant AAs, which will be fully revealed in this Section later. [fig_ref] Figure 5: The general workflow of how supervised ML works is shown [/fig_ref] displays the PC of AAs on the solvent-excluded surface of the SD1-FP model [fig_ref] Figure 1: The illustration of SD2-FP model construction [/fig_ref] , with the location of key AAs that undergo mutation marked D614 and P681. Interestingly, D614 is highly negatively charged and P681 is near neutral (0.15 e − ). Other relatively positively charged AAs (colored blue) shown in the [fig_ref] Figure 5: The general workflow of how supervised ML works is shown [/fig_ref] [fig_ref] Table 3: Data obtained by simulating random samples from a Bayesian network [/fig_ref]. There are 141 AAs with a PC range of −0.182 e − to 0.019 e − (grey color) and 63 AAs with a PC range in between 0.020 e − and 0.222 e − (green color). This type of charge distribution is common in such biomolecules. The PC distribution of the HR1-CH WT model is displayed in . The WT D950 has a largely negative PC, which changes to a positive PC when it mutates to N950. The PC for each AA for the HR1-CH model is listed in .
## Interatomic bonding
In contrast to the TDOS discussed above, the actual interatomic interaction in the form of bond order (BO) values (see Computational Methods Section S1 in SM) are fully available for all of the atoms in the supercell used in the DFT calculation with the OLCAO method. These BO values are the basic ingredients for calculating the AABP values, central to this paper. shows the BO vs. BL distribution for every pair in the WT model for BL less than 3.5 Å, including all covalently bonded pairs, as well as the HB. The inset shows the distribution for BL from 2.0 Å to 4.5 Å. This is a very busy figure containing many interesting facts. We succinctly summarize them below:
1.
The first group of covalent bonds are O-H, N-H, and C-H, with BL ranging from less than 1 Å to less than 1.2 Å, and with BO ranging from 0.15 e − to 0.48 e − , depending on the actual structure of the AAs listed in ; it may even be between certain AAs. There are two O-H bonds at 1.33 Å and 1.44 Å. The former bond occurs between two AAs (D808 and K811) and the latter one is from the same AA F592; 2.
The second group of covalent bonds is the usual covalent bond between C, O, and N. We now focus our observations on the inset of for the BL ranging from 2.0 Å to 4.5 Å. It reveals many weaker HBs, with a BO less than 0.03 e − . Even more surprising is the presence of many atomic pairs (H-H, C-H, C-C, N· · · H) that contribute to BO values with weak but nontrivial values of less than 0.02 e − . These bonds are obviously formed between the non-local AAs, which play a critical role in the total AABP values, to be discussed in the next section; 6.
One point we must emphasize is that the use of BO is a relatively new concept advocated by us. The BLs must be interpreted as the distance of separations between a pair of atoms, with the proviso that their interatomic interaction can go beyond the actual atomic pairs labeled as 'BL' due to the quantum effects arising from overlapping orbitals of their nearby atoms. Such subtle issues are usually ignored in biomolecular systems, since they are seldom discussed in the context of quantum mechanical wave functions, but rely on the distances between two atoms quantified by 'BL'. Similar issues have been raised recently in the literature regarding the nature of C-H and C-C bonds [bib_ref] Not Carbon s-p Hybridization, but Coordination Number Determines C−H and C−C Bond..., Vermeeren [/bib_ref].
## Aabp data for mutations in delta variant
The mutations on the Delta variant have been a hot topic that has attracted a lot of attention [bib_ref] SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion, Saito [/bib_ref] [bib_ref] SARS-CoV-2 B. 1.617. 2 Delta variant replication and immune evasion, Mlcochova [/bib_ref] [bib_ref] Effectiveness of COVID-19 vaccines against the B. 1.617. 2 (Delta) variant, Bernal [/bib_ref] [bib_ref] Evolutionary analysis of the Delta and Delta Plus variants of the SARS-CoV-2..., Kannan [/bib_ref] [bib_ref] SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation, Rajah [/bib_ref] [bib_ref] The biological and clinical significance of emerging SARS-CoV-2 variants, Tao [/bib_ref]. Most of these studies focus on the clinical or experimental observations to demonstrate the danger of mutations, especially the P681R near the furin cleavage site in the SD2-FP domain of the S-protein, but, to the best of our knowledge, no theoretical explanation or computational studies have been reported so far. Based on the detailed atomic-scale electronic structure calculation described in the above two sections, we extend the calculation of interactions between AAs involved in the mutations in the form of AABP described in the methods section. The calculated AABP values of mutations are summarized in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref]. Each calculation is considered as a data point labeled by the specifically designed notation that will be instrumental in data mining and machine learning (ML) applications (see Section 5.2). The main observations of [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] are as follows.
To better explain the information present in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] regarding the nature of total AABP values and its components of nearest neighbor (NN) AABP and non-local AABP (NL) values, we show in [fig_ref] Figure 3: Interaction of D614 and P681 to their NN and NL AAs in... [/fig_ref] the sequence of AAs from E592 to S689. [fig_ref] Figure 3: Interaction of D614 and P681 to their NN and NL AAs in... [/fig_ref] in the ribbon form and [fig_ref] Figure 3: Interaction of D614 and P681 to their NN and NL AAs in... [/fig_ref] in the sequential form both show the location of the main mutation sites P681 and D614 (pink circle), the NN AAs to these two mutations are in yellow circles, and the non-local interacting AAs (green circle) connected by lines indicate that they are interacting. The main observations of the data in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] These graphical illustrations demonstrate the complex structural features of mutations and interaction details never revealed before. In particular, the presence or the lack of HBs within the same AA or with other AAs have not been sufficiently elaborated in the current literature in biochemical interactions, except in a few isolated cases. In the same figure, the partial charge on each group is shown in the red boxes. These PC values are obtained by summing PC values of individual AAs in each interacting group. In the WT, both groups involving D614 and P681 have a positive PC. After mutation, G614 and its interacting group have their PC significantly increase, whereas R681 and its interacting group have their positive PC only slightly increase. More surprisingly, in the case of double mutation, G614 with its group have their PC slightly negative and P681 with its group have their positive PC continue to increase. This is another solid instance of evidence for the strong effect of mutation on different AAs that has never been discussed or revealed before. Similarly, the detailed interaction of the 950th site in two HR1-CH models is shown in , with their AABP listed in , their NL bonding listed in , and their results discussed in Section S2 in SM.
# Discussions
## The origins of mutation
Viruses can undergo frequent genetic mutations, including point mutations (the source of genetic variation) and recombination [bib_ref] Recombination in viruses: Mechanisms, methods of study, and evolutionary consequences, Pérez-Losada [/bib_ref]. Mutations are nucleotide changes that result in AA sequence changes implying new phenotype variants, whereas recombination allows for these variants to move across genomes to produce new haplotypes. Recombination occurs when viruses containing variants with different mutations infect the same host cell and exchange the genetic segments [bib_ref] Recombination in viruses: Mechanisms, methods of study, and evolutionary consequences, Pérez-Losada [/bib_ref]. The fate of these genetic changes will be ultimately determined by natural selection and genetic drift, so it is very difficult to forecast when a viral mutation will become globally dominant. Although coronaviruses have an exonuclease enzyme that reduces their replication error rates, they accumulate mutations and generate more diversity via recombination [bib_ref] The biological and clinical significance of emerging SARS-CoV-2 variants, Tao [/bib_ref] [bib_ref] Rates of evolutionary change in viruses: Patterns and determinants, Duffy [/bib_ref] [bib_ref] Recombination, reservoirs, and the modular spike: Mechanisms of coronavirus cross-species transmission, Graham [/bib_ref]. SARS-CoV-2 itself is most likely the result of a recombination between different SARS-related coronaviruses [bib_ref] Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic, Boni [/bib_ref] , with different subsequent mutations affecting their many biological and biomedical properties, such as pathogenicity, infectivity, transmissibility, and/or antigenicity, even though they tend to be either deleterious and quickly purged, or relatively neutral [bib_ref] SARS-CoV-2 variants, spike mutations and immune escape, Harvey [/bib_ref].
One of the most urgent tasks in the virus research is the origin of virus mutations and how to predict new variants even before they occur. We assert that the first task must be related to the interaction between AAs at the atomic level that results in the structural modification in the S-protein due to mutated AAs. It must involve the interaction with nonlocal AAs in addition to the NN AAs. The contribution to AABP from hydrogen bonding is critical, and the role of HB has been recognized by all researchers but seldom explored in detail. In a much broader sense, the origin of mutation is not limited just to SARS-CoV-2 research per se, but is related to broader themes of evolutionary biology, such as the origin of species. This accentuates the importance of a fundamental understanding of biomolecular interactions.
The data in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] reveal that D614G and P681R mutations have lower AABP values than the unmutated WT case. Our results make sense for the following reasons. First, the substitution of D614 with G results in losing the sidechain, leading to the elimination of many intramolecular interactions in the same protomer, as shown in [fig_ref] Figure 4: shows more vividly the non-local AA-AA interactions of mutations in the Delta... [/fig_ref]. More specifically, our result elucidates that this mutation disrupts the non-local network interactions [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref]. This could have large structure consequences in other domains of the S-protein, such as in promoting the up conformation of the S-protein or enhancing the cleavage site, as reported before [bib_ref] SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity, Zhang [/bib_ref]. This enhancement in the cleavage site could be due to an increase in the flexibility of the mutated 614 and 681 sites. In addition to these intra-protomer interactions, it has been structurally demonstrated that the D614G mutation destroys an inter-protomer hydrogen bond between D614 (chain A) and T859 (chain B) [bib_ref] Structural and functional analysis of the D614G SARS-CoV-2 spike protein variant, Yurkovetskiy [/bib_ref]. However, the SD2-FP model alone is insufficient in assessing these significant conformational changes. It is necessary to include all atoms of the S protein trimer, which is currently impossible to perform in a single ab initio calculation. Second, our decomposition of the total AABP into NN and NL AABPs reveals that the R681 increases the NL AABP by forming new HBs and decreases the NN AABP as compared to P681 [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] and [fig_ref] Figure 4: shows more vividly the non-local AA-AA interactions of mutations in the Delta... [/fig_ref]. Importantly, the P681R mutation reduces the local rigidity, as evidenced by the NN AABP values [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref]. Additionally, the proline is well-known as the most rigid AA, and when it is mutated to arginine at position 681, it loses its rigidity. Furthermore, the positive charge associated with R681 appears to alter virus tropism via enhancing S1/S2 cleavage, as previously demonstrated in human airway epithelial cells [bib_ref] SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion, Saito [/bib_ref]. This coincides with our conclusion that mutation decreases AABP but also increases the flexibility of AAs.
Such understanding of the molecular and atomic origins of the individual mutations or their combinations in SARS-CoV-2 can provide deep information to prepare and prevent future outbreaks, such as those reported for AY.4.2. or the just-emerging "Omicron" VOC. It can also play an important role in guiding the development of new drugs. It would also be desirable to have a quantitative scale from 1 (insignificant) to 10 (most dangerous) to quantify the nature of mutations by linking the mutation to specific clinical data or research using other methods, such as experimental or computational.
## Extension to machine learning (ml)
Over the last decade or so, machine learning (ML) has become a very powerful tool that is being applied to many different areas, including image, speech, text and facial recognition, autonomous vehicles, medical image classification, instruction detection, finances, drones, national defense, etc. [bib_ref] Making machine learning trustworthy, Eshete [/bib_ref] [bib_ref] Making machine learning robust against adversarial inputs, Goodfellow [/bib_ref]. In the present work, the word ML is strictly used only for the calculated data of AABP between AAs in the S-protein to predict potential unknown mutations.
One of the major challenges we face in applying ML techniques to our problem is the size of the dataset. This is because each data instance of a Delta variant model is computationally expensive to generate. When dealing with small datasets, deep learning techniques (based on artificial neural networks) will tend to underperform. Hence, conventional ML techniques should be preferred. Our first step is to prepare the data by constructing feature vectors for different Delta variant models. We can represent each model as a five-dimensional feature vector: TAABP (total AABP), NN (NN AABP), NL (non-local AABP), HB (AABP from HB), and NNL (no. of NN AAs). Each feature is a continuous variable. The target vector is denoted by a binary variable MT to represent whether a Delta variant model is unmutated (0) or mutated (1). [fig_ref] Table 2: Data representation for ML [/fig_ref] shows how the data can be represented for the Delta variant models, shown in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] for HR1-CH. Several extensions can be made to the target vector depending on the prediction task. Suppose we wish to predict the site of the Delta variant model (e.g., 614, 681). A categorical variable can be introduced as the target vector. To predict different kinds of mutations (e.g., single, double), another categorical variable can be introduced in the target vector.
Suppose we wish to predict whether a new Delta variant model is mutated or not. Using the feature and target vectors in [fig_ref] Table 2: Data representation for ML [/fig_ref] , a binary classifier can be learned on the data. Classifiers can be built using different techniques, such as (a) a logistic regression classifier, (b) Gaussian naïve Bayes classifier, (c) support vector machine (SVM) classifier [bib_ref] Support-vector networks, Cortes [/bib_ref] , (d) decision tree classifier [bib_ref] Induction of decision trees, Quinlan [/bib_ref] , (e) random forest classifier [bib_ref] Random Forests, Breiman [/bib_ref] , and (f) the extreme gradient boosting (XGBoost) classifier [bib_ref] Xgboost: A scalable tree boosting system, Chen [/bib_ref]. Hyperparameter tuning is necessary for the different classifiers to achieve the best accuracy. Feature importance is another task that can be valuable to researchers. For instance, the XGBoost classifier trained on data in [fig_ref] Table 2: Data representation for ML [/fig_ref] showed that TAABP and HB were the most important features to predict a mutation. XGBoost uses the notion of gain, which is the relative contribution of a feature to the model, to compute feature importance. The above ML techniques assume that the data instances are independently and identically distributed (i.i.d). However, if this assumption is not appropriate, then ML techniques, such as Markov logic networks [bib_ref] Markov logic networks, Richardson [/bib_ref] , should be considered. We can also learn causal relationships among the features using a probabilistic graphical model, such as a Bayesian network. By better understanding the cause-effect relationships among the features, better prediction models can be developed. A Bayesian network can compactly encode the joint distribution of a set of random variables as the product of the conditional probability of the nodes given its parents in the network. Using a Bayesian network, we can also simulate new data that follow the distribution learned by the network. [fig_ref] Table 3: Data obtained by simulating random samples from a Bayesian network [/fig_ref] shows a set of four samples obtained by simulating random samples from a Bayesian network. (This network was learned using the Max-Min Hill Climbing algorithm [bib_ref] The max-min hill-climbing Bayesian network structure learning algorithm, Tsamardinos [/bib_ref] over the data shown in [fig_ref] Table 2: Data representation for ML [/fig_ref] This algorithm first learns the skeleton of a Bayesian network. It then uses a greedy hill-climbing search to orient the edges using a Bayesian scoring function [bib_ref] The max-min hill-climbing Bayesian network structure learning algorithm, Tsamardinos [/bib_ref]. [fig_ref] Figure 5: The general workflow of how supervised ML works is shown [/fig_ref] shows the general steps involved in applying supervised ML to our Delta variant data. In summary, ML can offer unprecedented opportunities to predict mutations in the Delta variant.
## Looking forward
Looking forward, we would like to speculate where our methodology could lead to in the future. Obviously, it can be extended to other mutations in the S-protein, such as RBD and NTD and their interfaces with ACE2, or maybe even those newly emerged mutations, such as AY.4.2and B.1.1.529. The obvious goal is to increase the mutation data points so that a reasonable size of the AABP database can be used in the ML, as discussed in the previous section. It is totally possible to calculate other potential mutations for more data points in different domains based on the insights obtained with the calculations on known mutations. The list is endless, restricted by the large computational resources it demands. Fortunately, the emergence of the next generation of the exa-scale supercomputer is already availableto meet these challenges. However, even more importantly, we posit that our methodology could be extremely valuable for the VOC that appear to be characterized by an unprecedented number of concurrent mutations-as appears to be the case in the latest Omicron VOC-with more than 30 mutations per S-protein. Clearly, in such cases, there must be collective effects tying together several mutations, possibly also enhancing the susceptibility of different AAs to mutations. The mutation quantifier based on AABP, as introduced in this paper, which, by its very nature, already embodies the collective effects of mutations, is clearly a good candidate for such an analysis. In view of this striking development of the SARS-CoV-2 mutational capacity, our research seems to represent a well-placed origin for further elaboration, not only of the effects of single mutations, but, maybe even more importantly, their interaction and synergy.
One of the fundamental questions in biology is to ask if mutations are random or if there are specific reasons for each mutation. We may be able to also shed some light on this issue by accumulating a large database for known mutations and applying the ML protocol to see if any successful prediction can be verified with a global database or clinical data. Thus far, we have six data points, plus another four from RBD, or ten data points altogether.
The current calculation and analysis are restricted to chain A of the S-protein of SARS-CoV-2. An extension to cases with chains A, B, and C in up and down conformations would be highly desirable and important. It is also opportune to start looking at mutations in the RBD of the S-protein, such as those involved in binding to the ACE2 receptor, to provide a deeper understanding of how the virus can mutate and overcome the human defense mechanisms of immune response, intimately related to vaccination. Most neutralizing monoclonal antibodies (mAbs) target either the RBD or the NTD of the S-protein, and mutations in these domains have the greatest impact on neutralization.
The broader implications of the present work would also be in protein-protein interactions, where one could define similar ab initio interaction quantifiers based on single-point calculations as applied to a protein-protein network, protein-protein mapping, application to cancer research, etc. This broader phenomenology also revolves around mutations (replacing AA at a specific site, some AAs are more important than others), but on a much larger scale and with much more detailed interactions. While, so far, such studies were essentially based on experimental or clinical observations, the lack of a firm fundamental theoretical basis is noticeable.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14030465/s1, [fig_ref] Figure 1: The illustration of SD2-FP model construction [/fig_ref] : Detailed schematic of S-protein in SARS-CoV-2 colored by domains: SP, NTD, RBD, SD2, furin cleavage site (S1/S2), FP, HR1, CH, CD, HR2, TM, and CT. The delta variant mutation sites are marked by gray solid line in the bottom. In the present work, our calculations have been mainly carried out on region 3 of SD2 and FP domains; : The 20 amino acids in [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] are divided into 6 functional groups based on the nature of their sidechains: (a,b) for hydrophobic AAs, (c) for neutral polar AAs, (d,e) for charged polar AAs, and (f) for unique AAs; [fig_ref] Figure 3: Interaction of D614 and P681 to their NN and NL AAs in... [/fig_ref] : Calculated total density of stated (TDOS) for WT SD2-FP model; [fig_ref] Table 1: AABP of four SD2-FP models and other information [/fig_ref] : 20 canonical amino acids in alphabetical order. The last column shows their functional group; [fig_ref] Table 2: Data representation for ML [/fig_ref] : Four SD2-FP models (a-d) and two HR1-CH models (e,f) with the number of atoms, total energy and time used in Cori; [fig_ref] Table 3: Data obtained by simulating random samples from a Bayesian network [/fig_ref] : List of PC value for each amino acids with their sequence number for WT SD2-FP; : List of PC value for each amino acids with their sequence number for WT HR1-CH; : NL bonding information for WT SD2-FP shown in [fig_ref] Figure 4: shows more vividly the non-local AA-AA interactions of mutations in the Delta... [/fig_ref] ,b; : NL bonding information for mutated R681 shown in [fig_ref] Figure 4: shows more vividly the non-local AA-AA interactions of mutations in the Delta... [/fig_ref] ,d; : NL bonding information for double mutation R681 & G614 shown in Figure 4e,f; : AABP of two HR1-CH models; : Bonding information for WT and mutated HR1-CH shown in . References [bib_ref] Generalized gradient approximation made simple, Perdew [/bib_ref] [bib_ref] Implication of the solvent effect, metal ions and topology in the electronic..., Poudel [/bib_ref] [bib_ref] Impact of hydrogen bonding in the binding site between capsid protein and..., Poudel [/bib_ref] [bib_ref] Charge distribution and hydrogen bonding of a collagen α2-chain in vacuum, hydrated,..., Eifler [/bib_ref] [bib_ref] Electronic structure and partial charge distribution of doxorubicin in different molecular environments, Poudel [/bib_ref] [bib_ref] Electronic structure, stacking energy, partial charge, and hydrogen bonding in four periodic..., Poudel [/bib_ref] [bib_ref] Structure and electronic properties of a continuous random network model of an..., Adhikari [/bib_ref] [bib_ref] First-principles study in an inter-granular glassy film model of silicon nitride, Ching [/bib_ref] [bib_ref] Thermodynamic Dissection of the Intercalation Binding Process of Doxorubicin to dsDNA with..., Jawad [/bib_ref] [bib_ref] Ab Initio Study of Hydrolysis Effects in Single and Ion-Exchanged Alkali Aluminosilicate..., Baral [/bib_ref] [bib_ref] Electronic population analysis on LCAO-MO molecular wave functions. I, Mulliken [/bib_ref] [bib_ref] Electronic population analysis on LCAO-MO molecular wave functions. II. Overlap populations, bond..., Mulliken [/bib_ref]
[fig] Figure 1: The illustration of SD2-FP model construction. (a) Ribbon of a single protomer in up conformation (chain A) of spike protein SARS-CoV-2 from signal peptide (SP) to central helix (CH) and the associated mutation for Delta variant in different domains, (b) the ribbon structure of SD1-FP model with two marked mutations that is selected for constructing our models, (c) ball and stick of the SD1-FP in (b) and their respective mutations as marked. [/fig]
[fig] Figure 3: Interaction of D614 and P681 to their NN and NL AAs in WT SD2-FP model. (a) Ribbon structure from residue F592 to S689. (b) Sketch of AA sequence from F592 to S689 showing AABP interaction for D614 and P681 with joining lines. Both D614 and P681 are shown in pink color with its NN in yellow and NL interaction in green. [/fig]
[fig] Figure 4: shows more vividly the non-local AA-AA interactions of mutations in the Delta variant. They are divided into six panels in two columns: (a) WT D614 and (b) WT P681; (c) mutated G614 and (d) mutated R681; and (e) double mutation G614-R681 G614 and (f) R681. In each case, the ball and stick sketch of all participating AAs is shown (red, O, grey, C, blue N, white, H). The focused AA is marked light pink. Its two NNs are marked light yellow, and its interacting NL AAs are marked light green. All interactions are marked by solid lines and the dashed lines show HBs. All of these NL interactions with the bonds formed are listed in Tables S5-S7. At the lower part of each figure, the three smaller figures show the same figure rotated for 90 • , 180 • , 270 • from left to right. These figures show some of the most detailed information on the AA-AA interaction at the atomic-scale summarized as follows. [/fig]
[fig] 1: The WT D614 and WT P681 in (a) and (b) at the two different locations in the S-protein have very different features in size, shape, and volume controlled by their interatomic interactions with the two NN and five NL AAs. (2) The mutated AAs G614 and R681 in (c) and (d) are drastically different from the WT case in (a) and (b). (3) The double mutation in (e) and (f) also show significant differences with WT and interacts with the same number of NL AAs as in a single mutation. [/fig]
[fig] Figure 5: The general workflow of how supervised ML works is shown. Here, D and T represent training data and testing data, respectively. [/fig]
[table] Table 2: Data representation for ML. [/table]
[table] Table 3: Data obtained by simulating random samples from a Bayesian network. [/table]
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Mutational processes contributing to the development of multiple myeloma
To gain insight into multiple myeloma (MM) tumorigenesis, we analyzed the mutational signatures in 874 wholeexome and 850 whole-genome data from the CoMMpass Study. We identified that coding and non-coding regions are differentially dominated by distinct single-nucleotide variant (SNV) mutational signatures, as well as five de novo structural rearrangement signatures. Mutational signatures reflective of different principle mutational processesaging, defective DNA repair, and apolipoprotein B editing complex (APOBEC)/activation-induced deaminase activitycharacterize MM. These mutational signatures show evidence of subgroup specificity-APOBEC-attributed signatures associated with MAF translocation t(14;16) and t(14;20) MM; potentially DNA repair deficiency with t(11;14) and t(4;14); and aging with hyperdiploidy. Mutational signatures beyond that associated with APOBEC are independent of established prognostic markers and appear to have relevance to predicting high-risk MM.
# Introduction
Cancers have variable numbers of somatic mutations that have accumulated during the life history of the tumors as a consequence of diverse cellular processes, including defective DNA replication or DNA repair, and exposure to endogenous or exogenous DNA-damaging agents [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] Mechanisms underlying mutational signatures in human cancers, Helleday [/bib_ref]. Each of these processes results in mutational signatures, which serve as proxy for the cellular processes that have gone amiss. Mathematical deconvolution 3 of these mutational signatures in large pan-cancer series has revealed multiple distinct signatures [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] , several of which are associated with known etiologies, but many remain unexplained [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] Mutational signatures associated with tobacco smoking in human cancer, Alexandrov [/bib_ref] [bib_ref] Environmental exposures and mutational patterns of cancer genomes, Pfeifer [/bib_ref]. Hence, studying the mutational signatures of cancers provides a mechanism for gaining insight into the etiological basis of tumor development.
Multiple myeloma (MM) is an incurable malignancy of plasma cells whose pathogenesis is only partially understood [bib_ref] Genomic complexity of multiple myeloma and its clinical implications, Manier [/bib_ref]. Approximately 40% of MM tumors harbor chromosome translocations leading to over-expression of oncogenes (including CCND1, CCND3, MAF, MAFB, WHSC1/MMSET, and FGFR3) through juxtaposition to the immunoglobulin heavy-chain locus [bib_ref] Genomic complexity of multiple myeloma and its clinical implications, Manier [/bib_ref]. Other tumors exhibit hyperdiploidy (HD), which is also considered to be an important initiating event [bib_ref] Genomic complexity of multiple myeloma and its clinical implications, Manier [/bib_ref]. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) studies have so far identified over 40 driver genes that are recurrently altered in MM [bib_ref] Genomic complexity of multiple myeloma and its clinical implications, Manier [/bib_ref] [bib_ref] Whole-genome sequencing of multiple myeloma reveals oncogenic pathways are targeted somatically through..., Hoang [/bib_ref] [bib_ref] Mutational spectrum, copy number changes, and outcome: results of a sequencing study..., Walker [/bib_ref] [bib_ref] Heterogeneity of genomic evolution and mutational profiles in multiple myeloma, Bolli [/bib_ref] [bib_ref] Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy, Lohr [/bib_ref]. However, the molecular mechanisms giving rise to these mutations are yet to be fully elucidated.
Here we report a comprehensive analysis of the mutation signatures of over 800 MM genomes. We identify major mutational signatures in MM reflective of three known principle mutational processes: aging [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] , DNA repair deficiency [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] [bib_ref] Mutational processes molding the genomes of 21 breast cancers, Nik-Zainal [/bib_ref] [bib_ref] Validating the concept of mutational signatures with isogenic cell models, Zou [/bib_ref] [bib_ref] Somatic ERCC2 mutations are associated with a distinct genomic signature in urothelial..., Kim [/bib_ref] [bib_ref] Deficiency of nucleotide excision repair is associated with mutational signature observed in..., Jager [/bib_ref] [bib_ref] Use of CRISPR-modified human stem cell organoids to study the origin of..., Drost [/bib_ref] , and activation-induced deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity (signature 2, 9, and 13) [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] Mutational processes molding the genomes of 21 breast cancers, Nik-Zainal [/bib_ref] [bib_ref] Molecular mechanisms of antibody somatic hypermutation, Di Noia [/bib_ref] [bib_ref] Non-coding recurrent mutations in chronic lymphocytic leukaemia, Puente [/bib_ref]. These mutational signatures show subgroup specificity and are reflective of the molecular mechanisms involved in tumorigenesis. Additionally, we show that information on mutational signatures beyond that associated with APOBEC has relevance to predicting patient prognosis and defining high-risk MM.
# Results
## Genome sequencing of multiple myeloma
To examine the diversity of mutational signatures, we analyzed overlapping WGS and WES data on 850 and 874 MM tumor-normal pairs, respectively, generated by the Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile Study (CoMMpass, IA10 release). The frequency of the MM major subgroups-HD, t , t(4;14), t(14;16), t , and t MYC translocation-is similar to other unselected series of patients who have been reported 6 (Supplementary [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. We used the high-coverage WES data (120-150×, 136,074 single-nucleotide variants to analyze coding regions and the low-coverage WGS data (6-12×, 1,348,881 SNVs and 44,155 structural variants to provide genome-wide insights into clonal mutations associated with early processes underlying tumorigenesis [bib_ref] Whole-genome sequencing of multiple myeloma reveals oncogenic pathways are targeted somatically through..., Hoang [/bib_ref] [bib_ref] Optimizing cancer genome sequencing and analysis, Griffith [/bib_ref].
## Mutational signatures in multiple myeloma
Application of non-negative matrix factorization (NMF) [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] to extract de novo SNV mutational signatures did not identify any novel mutational signatures [fig_ref] Table 2: Summary of characteristics of the seven cluster subgroups SV structural variant, SNV... [/fig_ref] , consistent with a recent analysis on CoMMpass exome dataset [bib_ref] Biological and prognostic impact of APOBEC-induced mutations in the spectrum of plasma..., Maura [/bib_ref]. Overall, a total 9 of the 30 mutational signatures referenced by COSMIC (Catalog of Somatic Mutations in Cancer) were seen at >1% mutational contribution in the WGS data [fig_ref] Table 2: Summary of characteristics of the seven cluster subgroups SV structural variant, SNV... [/fig_ref] ; signature 1 related to aging 1 ; 2 and 13 to activity of the APOBEC family of cytidine deaminases; 9 to polymerase η implicated with the activity of AID during somatic hypermutation [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] Molecular mechanisms of antibody somatic hypermutation, Di Noia [/bib_ref] [bib_ref] Non-coding recurrent mutations in chronic lymphocytic leukaemia, Puente [/bib_ref] ; signature 30 reflective of mismatch repair deficiency 17 ; and signature 16 which has as yet an unknown etiology. We also extracted flat signatures 3, 5, and 8 in tumorsindicative of DNA repair deficiency (homolgous recombination deficiency and nucleotide repair deficiency) [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] [bib_ref] Mutational processes molding the genomes of 21 breast cancers, Nik-Zainal [/bib_ref] [bib_ref] Validating the concept of mutational signatures with isogenic cell models, Zou [/bib_ref] [bib_ref] Somatic ERCC2 mutations are associated with a distinct genomic signature in urothelial..., Kim [/bib_ref] [bib_ref] Deficiency of nucleotide excision repair is associated with mutational signature observed in..., Jager [/bib_ref]. In view of the potential ambiguous assignment of these three signatures 22,23 , we considered them collectively thereafter. We did however identify five novel de novo structural rearrangement signatures (RSs) : RS1 (19% of SVs across samples)-characterized by nonclustered deletions, large-scale tandem duplications and inversions; RS2 (17%)-characterized by clustered translocations; RS3 (13%)-characterized by inversions; RS4 (21%)-characterized by non-clustered small-scale deletions and tandem duplications; RS5 (30%)-characterized by non-clustered translocations. We therefore focused on the nine major SNV and five de novo SV mutational signatures for subsequent analyses.
Following on from this, we examined the contributions of the nine major COSMIC SNV mutational signatures in both WES and WGS datasets. The signature profiles recovered from the analysis of clonal WES and exome-restricted WGS data were highly correlated (r = 1.00, Spearman's correlation, [fig_ref] Figure 4: Mutational signatures associated with driver genes [/fig_ref] the average sensitivity to detect clonal SNVs from the WGS data is 20-35% 7 , these findings indicate that the mutational signatures identified by WGS are valid and representative of early mutational processes in MM. We also observed a high concordance of mutational signature in WES data from CoMMpass and that reported by Walker et al. [bib_ref] Mutational spectrum, copy number changes, and outcome: results of a sequencing study..., Walker [/bib_ref] (r = 0.86, Spearman's correlation, [fig_ref] Figure 5: Integrative clusters based on mutational signatures and patient prognosis [/fig_ref] , Supplementary , reflecting the generalizability of our observations. No significant association between the major COSMIC SNV signatures and those associated with rearrangements was seen (Supplementary .
## Influence of dna replication and transcription on mutational signatures
The impact of DNA replication and transcription on mutational signatures was broadly consistent with observations previously made in the analyses of other cancers [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] [bib_ref] Mutational strand asymmetries in cancer genomes reveal mechanisms of DNA damage and..., Haradhvala [/bib_ref]. Specifically, an overall increased mutation rate in late-replicating regions was shown (P < 1.0 × 10 −4 ) , with the exception of signature 13 having higher mutation rate in earlyreplicating regions (P < 1.0 × 10 −4 , , Supplementary , consistent with generalized replication time-dependent DNA damage mechanisms that operate in other cancers such as those of the breast [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] and liver [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref]. The difference in how replication timing influences mutation rates in signatures 2 and 13, which are both associated with APOBEC activity, suggests an intrinsically different mutational processes linked to DNA replication consistent with the model previously described [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref].
Similarly, as previously documented, strong replicative strand asymmetry (>30% imbalances) [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] was shown with respect to signatures 2 (Q = 4.0 × 10 −16 ) and 13 (Q = Relationship between replication and transcription in mutational processes. a Mutation rates across different DNA replication timing bins for single-nucleotide variants (SNVs). Whole-genome sequencing (WGS) mutation rate (blue) was estimated from low-coverage WGS data (6-12×). Whole-exome sequencing (WES) mutation rate (orange) was estimated from high-coverage WES data (120-150×) with variants called by at least two variant callers. b Proportion of mutations on leading and lagging strands per signature based on WGS data. Asterisks indicate significant asymmetry (Q < 0.05 and strand imbalances >30%). . These findings are consistent with APOBEC activity primarily affecting lagging strands.
Overall, increased mutation rate was associated with increased transcription, suggesting the mutagenic role of the transcriptional process in MM . This contrasts markedly to hepatocellular carcinoma 11 , suggesting that transcription-associated mutagenesis may overwhelm transcription-coupled repair in MM [bib_ref] Genomic evidence for elevated mutation rates in highly expressed genes, Park [/bib_ref]. Moreover, strikingly elevated mutation rates of both SNVs and indels were shown for highly expressed genes . A number of these highly expressed genes (i.e., FPKM (fragments per kilobase of exons per million reads) >100), which are also frequently mutated, including EGR1 26 , XBP1 27 , BTG2 28 , DDX5 29 , and NFKBIA 9 (Supplementary , have wellestablished roles in plasma cell differentiation and MM. The strong replicative, but weak transcriptional mutational asymmetry , Supplementary [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref] seen in MM is consistent with the mutual exclusivity trend of replicative and transcriptional asymmetries shown in many cancers [bib_ref] Mutational strand asymmetries in cancer genomes reveal mechanisms of DNA damage and..., Haradhvala [/bib_ref].
## Mutational signatures in coding and non-coding regions
A significant difference in all mutational signatures within coding and non-coding regions was shown [fig_ref] Figure 3: Contribution of each single-nucleotide variant mutational signature in coding [/fig_ref] , [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref] , implying different genomic regions are subject to specific mutational processes, consistent with earlier observations [bib_ref] Analysis of mutational signatures suggest that aid has an early and driver..., Maura [/bib_ref]. AID-attributed signature 9 predominates in non-coding regions, whereas exonic mutations are dominated by signatures 1, 2, and 13, implicating aging and APOBEC signatures as important.
## Relationship between mutational signatures and kataegis
Local hypermutated regions of tumor genomes, or kataegis, have been observed in MM 9,31 and other B cell malignancies 1 . We examined COSMIC mutational signatures contributing to kataegis (defined on the basis of average inter-mutation distance ≤1 kb; 3,32 [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref] , which were detected in 9% of samples (71/ 874). We did not observe significant and consistent enrichment of COSMIC signatures at kataegis foci compared to other mutations in tumors with and without kataegis detected [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. We identified 70 genes disrupted by kataegis [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref] , including CCND1, CCND3, MAF, and FZD2, which are often affected by chromosomal rearrangements [bib_ref] Genomic complexity of multiple myeloma and its clinical implications, Manier [/bib_ref] [bib_ref] The molecular classification of multiple myeloma, Zhan [/bib_ref]. Globally, 62% of kataegis foci co-localize with 5% of somatic structural arrangement sites (Supplementary , consistent with previous finding that most genomic rearrangements do not feature kataegis in nearby regions 1 .
## Mutational signatures and myeloma subgroups
We observed significant association between specific mutational signatures and MM subgroups [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. Signature 1 was enriched in HD MM (Q = 3.2 × 10 −4 ) consistent with the correlation between age and frequency of HD [bib_ref] Age has a profound effect on the incidence and significance of chromosome..., Ross [/bib_ref] [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref] [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. Although speculative it is possible that the t(4;14) translocation, which leads to up-regulation of histone methyltransferase (MMSET), may affect genomic instability through some as yet undisclosed epigenetic mechanism.
We further explored the links between established prognostic mutational events (1p deletion, 1q gain, 17p deletion, and TP53 mutations) with mutational signatures (Supplementary [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. Associations between chromosome-arm events at 1p and 1q with COSMIC signatures 2, 13, and RS1 (Q < 0.05) and between TP53 mutations tumors with RS1 (Q = 0.033) and RS2 (Q = 7.4 × 10 −3 ) raise the possibility of causal relationships.
## Mutational signatures and driver genes
To identify etiological mutational processes underlying driver mutations in MM, we compared mutational contribution in driver genes to other exonic mutations. Overall, the same diversity of processes in driver mutations was seen as in other coding mutations, but with differences: lower contribution of signatures 2 and 13; and higher contribution of signatures [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] [bib_ref] Heterogeneity of genomic evolution and mutational profiles in multiple myeloma, Bolli [/bib_ref] [bib_ref] Deficiency of nucleotide excision repair is associated with mutational signature observed in..., Jager [/bib_ref] [bib_ref] Analysis of mutational signatures suggest that aid has an early and driver..., Maura [/bib_ref] , and the flat signatures in coding regions of driver genes, compared to other exonic mutations [fig_ref] Figure 4: Mutational signatures associated with driver genes [/fig_ref]. Notably, we observed an over-representation of signatures reflective of aging with CCND1 and DNAH5 mutations, and AID with EGR1 mutations [fig_ref] Figure 4: Mutational signatures associated with driver genes [/fig_ref] , Supplementary [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. In contrast, a relative under-representation of signatures 2 and 13 suggests that APOBEC mutations are ubiquitous mutational processes and they do not specifically affect driver genes. Driver genes were replicated earlier than other coding genes (P < 2.2 × 10 −16 , Wilcoxon's rank-sum test) and we therefore assessed whether this difference could explain enrichment of the signatures. APOBEC signature 2 is enriched in late-replicating regions , Supplementary ; hence, the tendency of driver genes to be replicated early may explain the lower frequency of signature 2 mutations associated with driver genes. [bib_ref] Heterogeneity of genomic evolution and mutational profiles in multiple myeloma, Bolli [/bib_ref] [bib_ref] Deficiency of nucleotide excision repair is associated with mutational signature observed in..., Jager [/bib_ref] [bib_ref] Analysis of mutational signatures suggest that aid has an early and driver..., Maura [/bib_ref] , and the flat signatures were also associated with latereplicating regions , Supplementary , but conversely were more frequently associated with driver gene mutations. To test if the enrichment of mutational processes in driver genes were due to positive selection of certain mutations, we excluded all mutations that occurred at the exact same position in multiple tumors (46% of mutations) and repeated the analysis. Exclusion of recurrent mutations did not change the overall results, inferring that positive selection of specific mutations did not bias the analysis. We did not observe any significant transcriptional strand bias across mutational signatures , suggesting that the differences in mutational contribution between driver genes and other exonic mutations are unlikely to be influenced by transcription.
## Prognostic impact of mutational signatures
We next investigated the prognostic impact of mutational signatures using the prospective data from CoMMpass. The APOBEC signature has previously been reported to be associated with a worse patient outcome [bib_ref] Biological and prognostic impact of APOBEC-induced mutations in the spectrum of plasma..., Maura [/bib_ref] [bib_ref] APOBEC family mutational signatures are associated with poor prognosis translocations in multiple..., Walker [/bib_ref]. In this study after adjusting for age, sex, translocation status, chromosome-arm events, and TP53 status, no statistically significant association was shown, suggesting that APOBEC status does not represent an independent biomarker of patient outcome: progressionfree survival (PFS: hazard ratio [HR] = 2.45, 95% confidence interval [CI] = 0.94-6.37, P = 0.066) and overall survival (OS: HR = 2.81, 95% CI = 0.96-10.10, P = 0.10) (Supplementary [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. We next explored whether incorporating information on major SNVs and SV mutational signatures could further enhance the prediction of patient outcome after taking into account of established prognostic factors. Unsupervised hierarchical clustering provided evidence for seven distinct groups (A-G) associated with both PFS (log-rank P = 3.4 × 10 −4 ) and OS (log-rank P = 0.011) [fig_ref] Figure 4: Mutational signatures associated with driver genes [/fig_ref] , Supplementary , with group C being enriched for HD MM, group G is featuring tumors with 1p deletion, while group D being characterized by APOBEC mutation, enrichment for MAF-translocation subgroups, 1p deletion, and 1q gain [fig_ref] Table 2: Summary of characteristics of the seven cluster subgroups SV structural variant, SNV... [/fig_ref]. Post hoc delineation allowed us to stratify patients in seven groups into low-(A, B, C, and E) and high-risk groups (D, G, and F) (Supplementary [fig_ref] Table 1: Association of major myeloma subgroups and mutational signature [/fig_ref]. Classification of MM based on mutational signatures captured by these seven groups is independent prognosis factors [fig_ref] Table 2: Summary of characteristics of the seven cluster subgroups SV structural variant, SNV... [/fig_ref]. Notably, group F was independently associated with adverse prognosis (PFS: HR = 1.95, 95% CI = 1.35-2.81, P = 3.3 × 10 −4 ; OS: HR = 1.47, 95% CI = 1.02-2.13, P = 0.039) (Supplementary [fig_ref] Table 2: Summary of characteristics of the seven cluster subgroups SV structural variant, SNV... [/fig_ref] , despite not being associated with the high-risk features of APOBEC, t (14;16)/t(14;20), 1p/1q/17p chromosome-arm events or TP53 mutation status, but was typified by non-clustered structural rearrangements [fig_ref] Figure 5: Integrative clusters based on mutational signatures and patient prognosis [/fig_ref] , Supplementary .
# Discussion
Our analysis of over 800 myeloma genomes has afforded a global overview of the mutational processes in MM tumorigenesis. A major finding of this study is that a combination of signatures linked to aging, APOBEC/AID, and indicative DNA repair deficiency-account for around 80% of mutations in MM. Despite the difficulty of assigning flat signatures (3, 5, and 8) [bib_ref] Decon-structSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and..., Rosenthal [/bib_ref] [bib_ref] A practical guide for mutational signature analysis in hematological malignancies, Maura [/bib_ref] , their detection of such profiles in large patient series supports the role of defective DNA repair in MM. By utilizing both WES and WGS data, we were able to extract five novel structural RSs and identify differential prevalent mutational processes in coding (aging and APOBEC) and non-coding regions (AID), consistent with a previous report [bib_ref] Analysis of mutational signatures suggest that aid has an early and driver..., Maura [/bib_ref]. Our work supports previous findings 30 in implying an early role for AID in shaping the MM mutational landscape. We also identified new and validated previously reported subgroup associations with mutational signatures, allowing further categorization of MM beyond simple translocation status and providing additional insight in the etiological processes implicated in tumorigenesis .
Mutations do not occur uniformly over the genome and local mutation rates are modulated by replication, transcription, and chromatin organization 12 . We observed an enrichment of somatic mutations in late-replicating regions, as seen across several cancers 36 , and highly expressed regions. Previous analyses, which have sought to establish the mutational profile of myeloma genomes, have been based on data solely from exome sequencing projects. Here we have sought to provide a more comprehensive analysis; however, we acknowledge that the low coverage of CoMMpass WGS raises the possibility that we may have underestimated the global mutation rate. The strong replicative asymmetry observed is consistent with mutations in MM being predominantly associated with APOBEC family of mutations [bib_ref] Mutational strand asymmetries in cancer genomes reveal mechanisms of DNA damage and..., Haradhvala [/bib_ref]. In addition, we identified that coding drivers are likely to be originated from a number of mutational processes, including aging and DNA repair deficiency. In contrast, while APOBEC enzymes appear to act more ubiquitously within coding regions, they do not specifically affect coding drivers. The different MM translocation subgroups showed striking differences in their mutational signatures, reflective of the cellular processes driving respective clonal expansions . As previously reported, t(14;16) and t (14;20) MM were enriched with APOBEC signatures 2 and 13 [bib_ref] Whole-genome sequencing of multiple myeloma reveals oncogenic pathways are targeted somatically through..., Hoang [/bib_ref] [bib_ref] APOBEC family mutational signatures are associated with poor prognosis translocations in multiple..., Walker [/bib_ref]. This is a consequence of the over-expression of APOBEC genes, specifically APOBEC3A and APOBEC3B, mediated through the over-expression of MAF transcription factors [bib_ref] APOBEC family mutational signatures are associated with poor prognosis translocations in multiple..., Walker [/bib_ref]. The t(4;14) subgroup was also enriched with APOBEC mutational patterns, although only for signature 2 and, to a lesser extent, as compared to MAF-translocation subgroups. Since signatures 2 and 13 are reflective of different mutational processes [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref] , we speculate that the mutational processes associated with t (4;14) are likely to be different from those with MAF-translocation subgroups. In contrast, signatures indicative of DNA repair deficiency were associated with t(11;14) and t(4;14) and aging with HD. DNA breaks unsuccessfully repaired due to defective DNA repair may facilitate the generation of chromosomal translocations [bib_ref] How does DNA break during chromosomal translocations?, Nambiar [/bib_ref]. Because of the flat structure of signatures 3, 5, and 8 robust insight into etiological contribution of DNA repair deficiency to MM tumorigenesis requires assiduous signature fitting and adjustment for confounding covariates [bib_ref] A practical guide for mutational signature analysis in hematological malignancies, Maura [/bib_ref]. The molecular mechanisms responsible for initiating HD in MM are unknown. However, by inference from childhood acute lymphoblastic leukemia [bib_ref] Formation of a hyperdiploid karyotype in childhood acute lymphoblastic leukemia, Onodera [/bib_ref] , it is likely it is a consequence of the simultaneous gain of chromosomes in a single abnormal cell division. Cells failing to execute programmed cell death in response to mitotic failure are likely to divide asymmetrically, resulting in the generation of aneuploidy cells [bib_ref] Cell death by mitotic catastrophe: a molecular definition, Castedo [/bib_ref]. The association between aging with increased cell division errors [bib_ref] Mitotic misregulation and human aging, Ly [/bib_ref] and decreased apoptosis [bib_ref] Programmed cell death in aging, Tower [/bib_ref] further supports a relationship between HD MM and aging. Signatures defined by large-scale structural aberrations were associated to varying degrees with MM subgroups, but clustered translocations and non-clustered deletions, large-scale tandem duplications, and inversions showed a significant association in t(4;14) MM. The APOBEC mutational signatures are inextricably linked to a high mutation load 7,35 and the adverse t(14;16) and t(14;20) MAF-translocation subgroups. We show that molecular classification based solely on APOBEC signatures do not fully differentiate the underlying genomic complexity in MM relevant to predicting patient outcome. Hence, while APOBEC activity is an adverse prognostic factor in MM [bib_ref] Biological and prognostic impact of APOBEC-induced mutations in the spectrum of plasma..., Maura [/bib_ref] [bib_ref] APOBEC family mutational signatures are associated with poor prognosis translocations in multiple..., Walker [/bib_ref] , using it as a sole classifier does not fully capture high-risk MM, which with genetically unstable genome is typified by complex structural variants. Our findings support the need for considering other mutational signatures to refine prediction of patient prognosis.
Our study does, however, suggest that analysis of APOBEC activity together with other molecular features at diagnosis should allow for the identification of high-risk MM patients that may benefit from more intensive treatment. Collectively, these data shed new light on the diversity of cellular processes generating somatic mutations in MM. Moreover, they provide a strong rationale for integration of mutational signatures data in conventional molecular profiling of patient tumors to tailor therapy.
# Materials and methods
## Samples and datasets
All data analyzed were generated as part of the Multiple Myeloma Research Foundation (MMRF) CoMMpass Study (release IA10). WGS data on 850 matched tumornormal baseline newly diagnosed bone marrow samples were downloaded from the database of Genotype and Phenotype (dbGaP). Matched tumor RNAseq processed by HTseq were used for gene expression analysis. WES variants (detected by at least two out of three variant callers-MuTect, Seurat, and Strelka) from 874 samples, RNAseq, copy number variation (CNV), clinical data, and sequencing-based fluorescent in situ hybridization (Seq-FISH) data (MMRF IA10 dataset) were downloaded from MMRF web portal (https://research.themmrf.org/). WES and WGS data were available for 824 samples.
## Somatic mutation calling
Calling of somatic mutations was performed as described previously [bib_ref] Whole-genome sequencing of multiple myeloma reveals oncogenic pathways are targeted somatically through..., Hoang [/bib_ref]. Briefly, raw WGS sequencing data were quality checked using FastQC (v.0.11.4) and aligned using the Burrows-Wheeler Alignment tool 42 (BWA v0.7.12) to the human genome hg19/GRCh37 assembly. SNV Contribution of major mutational processes operative in multiple myeloma. This model represents differential contribution of various identified mutational processes in myeloma. For early mutational processes, activation-induced deaminase (AID) has the overall largest contribution to mutational processes across all subgroups represented by a larger oval. For late mutational processes, major mutational processes with known etiologies associated with aging, apolipoprotein B editing complex (APOBEC), DNA repair deficiency (DRD), and AID are depicted. Larger oval sizes indicate larger relative contribution of the mutational process mutations were called using MuTect 43 (v1.1.7) according to best practices, utilizing data from dbSNP v147 and COSMIC non-coding variants v77 [bib_ref] COSMIC: exploring the world's knowledge of somatic mutations in human cancer, Forbes [/bib_ref]. Mutations were then filtered for oxidation artifacts [bib_ref] Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing..., Costello [/bib_ref] and by quality score as described previously [bib_ref] Whole-genome sequencing of multiple myeloma reveals oncogenic pathways are targeted somatically through..., Hoang [/bib_ref]. Mutations mapping to immune hypermutated regions (429 immunoglobulin and the major histocompatibility complex loci, each region extended by 50 kb, as defined in Ensembl v73) [bib_ref] Genome-wide analysis of noncoding regulatory mutations in cancer, Weinhold [/bib_ref] were excluded to avoid bias from mutation as a consequence of normal B cell development.
## Determination of myeloma karyotype
Translocation status of MM tumors was based on Seq-FISH [bib_ref] A comparison of clinical FISH and sequencing based FISH estimates in multiple..., Miller [/bib_ref]. HD was defined as amplification of 90% of the chromosome in at least two autosomes 7 . Prognostic chromosome-arm events (>1 Mb) were defined as deleted or amplified with abs(log 2 ratio) ≥0.1613 occurring at 1p12, 1p32.3, 1q21.1, 1q23.3, and 17p13 6 .
## Mutational signatures
Characterization of the 30 COSMIC mutational signatures (http://cancer.sanger.ac.uk/cosmic/signatures) and de novo extraction of signatures was performed using Palimpsest [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] [bib_ref] Palimpsest: an R package for studying mutational and structural variant signatures along..., Shinde [/bib_ref] with default parameters. We compared de novo mutational signatures with 30 pre-defined COSMIC signatures by computing their cosine similarities 1 . A de novo mutational signature was assigned to a COSMIC signature if the cosine similarity was >0.75 as previously advocated [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref]. If multiple COSMIC signatures passed this threshold, then the most similar COSMIC signature was assigned to the de novo signature. We compared proportion COSMIC mutational signatures between highcoverage WES clonal mutations (alternate allele ratio >0.9) and low-coverage WGS mutations restricted to exome regions, as well as between CoMMpass exome and Walker et al. [bib_ref] Mutational spectrum, copy number changes, and outcome: results of a sequencing study..., Walker [/bib_ref] exome mutations. Correlations were tested using Spearman's correlation. For those signatures with an apparent flat profile we considered these in concert, by combining the respective contributions of signatures 3, 5, and 8.
We used MANTA to identify somatic SVs from the WGS data adopting default settings [bib_ref] Manta: rapid detection of structural variants and indels for germline and cancer..., Chen [/bib_ref]. We applied the same statistical framework used for signature analysis of SVs implemented in Palimpsest 48 to extract de novo RSs as previously described [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref]. Correlations between SV signatures and major COSMIC pre-defined SNV signatures (>1% mutational contribution in WGS) were tested using Spearman's correlation. No significant correlation was seen after adjusting for multiple testing (i.e., Q > 0.05).
We examined the relationship between mutational signatures and clinico-pathological parameters confining our analysis to the major MM subgroups-HD, t(4;14), t(11;14), t(14;16), t , and t(8;14) MYC. Test of association between each signature and subgroups was based on a two-tailed Fisher's exact test using Benjamini-Hochberg false discovery rate procedure to address multiple testing.
We compared contribution of each mutational signature to coding and non-coding regions using WGS data. To calculate contribution of a mutational signature to a genomic region, we first estimated the probability that each mutation was due to the process underlying each signature and calculated the cumulative probability of all mutations in each region, as per Letouze et al. [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref]. After computing these probabilities, regional differences in trinucleotide composition were accounted for when comparing the contribution of mutational signatures between two genomic regions (regions X and Y). Such normalization was conducted by changing the number of mutations from each mutational category in region X to that expected if the trinucleotide composition of region X was identical to the trinucleotide composition of region Y, assuming a constant rate of mutation at positions of each trinucleotide context. The normalized number of mutations U C;X norm of category C in region X was calculated as:
[formula] U C;X norm ¼ U C;X V C;Y W X V C;X W Y ; [/formula]
where U C,X is the number of mutations of category C observed in region X, V C,X is the number of positions at which a mutation of category C can occur in region X, and W X is the size of region X (in base pairs). As U C;X norm is not necessarily an integer, it is rounded to the closest integer before comparisons are completed. Mutation numbers were normalized within each tumor. Since small numbers of mutations may impact on normalization, in each comparison the larger region was designated as region X, the smaller region designated as region Y.
## Replication timing and replication strand bias
We used replication sequencing (Repli-seq) data generated by the ENCODE consortium for the lymphoblast cell lines GM12878, GM06990, GM12801, GM12812, and GM12813 to define early-and late-replicating regions, as well as leading and lagging DNA strands using Repli-seq signal peaks from GM12801 as previously described [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] [bib_ref] The topography of mutational processes in breast cancer genomes, Morganella [/bib_ref]. Mutation rates across deciles of replication timings were estimated globally using WGS data and for each signature, with each mutation assigned to a single signature by Palimpsest [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] [bib_ref] Palimpsest: an R package for studying mutational and structural variant signatures along..., Shinde [/bib_ref]. The replication timing slope was estimated by linear regression model. To test the null hypothesis that the slope gradients equal zero, the replication timing deciles were permuted 10,000 times.
Empirical P values were calculated as the fraction of permutations with absolute slope values at least as great as the absolute slope value computed using the true replication timing deciles.
Analysis of mutational replication strand bias between leading and lagging strands was performed across all 30 COSMIC signatures as previously described [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] , using WGS data. The Wilcoxon's rank-sum test was used to determine significant difference of mutational contribution from each COSMIC signature between leading and lagging strands. Levels of asymmetry were considered significant if strand imbalances were >30% 12 and Q < 0.05.
## Transcriptional levels and transcriptional strand bias
To correlate mutational processes with gene expression, RNAseq data were normalized to FPKM 11 . For each tumor, genes were partitioned into pentiles based on respective FPKM. Immunoglobulin-related genes and genes known to be highly upregulated in MM as a result of translocations (CCND1, CCND3, FGFR3, MMSET, MAF, MAFB, and MYC) 6 were excluded to mitigate against bias. Mutation rates of genes within each of the five transcriptional level categories were estimated per tumor based on WES called mutations. Average alignability score for highly expressed genes was based on alignability of 75mers defined by the ENCODE/CRG GEM mappability tool [bib_ref] Fast computation and applications of genome mappability, Derrien [/bib_ref]. We examined mutation rates on transcribed and non-transcribed strands globally and for each signature as described previously 11 using Palimpsest [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref] [bib_ref] Palimpsest: an R package for studying mutational and structural variant signatures along..., Shinde [/bib_ref]. Wilcoxon's rank-sum tests, corrected for multiple testing, were used to determine significant difference of mutational contribution from each COSMIC signature between transcribed and non-transcribed strands. Levels of asymmetry were again considered significant if strand imbalances were >30% 12 and Q < 0.05.
## Kataegis
We restricted our kataegis analysis to high-coverage WES data, where we have sufficient coverage to detect local hypermutation. Kataegis foci were defined as having six or more consecutive mutations with an average mutational distance ≤1 kb, as previously described [bib_ref] Deciphering signatures of mutational processes operative in human cancer, Alexandrov [/bib_ref] [bib_ref] Landscape of somatic mutations in 560 breast cancer whole-genome sequences, Nik-Zainal [/bib_ref]. Colocalization of kataegis and structural rearrangements was assessed based on the proportion of SV regions having kataegis foci residing within 10 kb. To examine enrichment of a mutational signature at kataegis regions, we compared mutational contribution of each signature across all mutations at kataegis foci with other mutations in tumors with and without kataegis being detected using Wilcoxon's rank-sum test, corrected for multiple testing and imposed a threshold of Q < 0.05.
## Association of mutational signatures with the mutation of driver genes
For SNV mutational signatures, Wilcoxon's rank-sum tests were used to compare contribution of each mutational signature in coding drivers 7-10 and other exonic mutations, with normalizing for trinucleotide composition as described above. For each somatic mutation, we estimated the probability that it was the result of each mutational process considering the trinucleotide context and the number of mutations attributed to each process in the respective tumor as per Letouze et al. [bib_ref] Mutational signatures reveal the dynamic interplay of risk factors and cellular processes..., Letouze [/bib_ref]. We then compared, for each driver gene and mutational signature, the probability distribution in mutations affecting the driver gene as compared to all other mutations in tumors with and without the driver gene mutated using Wilcoxon's rank-sum tests, imposing Benjamini-Hochberg correction for multiple testing. We evaluated all driver genes identified in previous studies 7-10 with Q < 0.05.
## Association of signatures with clinical features
Multivariate Cox regression was performed to adjust for covariates, including age at diagnosis, sex, translocation status, and APOBEC mutational contribution (COSMIC signatures 2 and 13). We used the ConsensusClusterPlus R package 51 to hierarchically cluster patients based on de novo SV and major COSMIC SNV signatures (>1% contribution) extracted from WGS with default settings 32 . Fisher's exact test was used to test whether clusters were associated with MM subgroups or driver gene mutations, imposing Benjamini-Hochberg correction for multiple testing. The log-rank test was used to assess the differences in PFS and OS between all cluster groups. To delineate clusters into low-and high-risk groups, pairwise comparisons in survival distributions were performed using the pairwise_survdiff function implemented in the survminer R package [bib_ref] The molecular classification of multiple myeloma, Zhan [/bib_ref].
Multivariate Cox regression was performed for each subgroup vs. other subgroups, adjusting for age at diagnosis, sex, translocation status, APOBEC contribution, 1p deletion, 1q gain, 17p deletion, and TP53 nonsynonymous mutations.
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Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41408-019-0221-9).
[fig] Figure 3: Contribution of each single-nucleotide variant mutational signature in coding (blue) and non-coding (orange) regions. Flat signatures include COSMIC signatures 3, 5, and 8 [/fig]
[fig] Figure 4: Mutational signatures associated with driver genes. a Cumulative mutational contribution of mutational signatures across 50 multiple myeloma (MM) driver genes 7-10 (blue, 1679 mutations in total) and other exonic mutations (orange). b Normalized cumulative mutational contribution of signatures with top ten contribution for most frequently mutated MM driver genes (+) vs. other mutations (−) in tumors with the corresponding driver gene being mutated: KRAS (n = 247), NRAS (n = 204), DIS3 (n = 104), TRAF3 (n = 83), CCND1 (n = 78), BRAF (n = 70), FAM46C (n = 70), EGR1 (n = 65), TP53 (n = 52), SP140 (n = 30), PRDM1 (n = 26), and ATM (n = 19); n: number of mutations. Flat signatures include COSMIC signatures 3, 5, and 8 [/fig]
[fig] Figure 5: Integrative clusters based on mutational signatures and patient prognosis. a Heatmap showing proportions of rearrangement signatures and major COSMIC (Catalog of Somatic Mutations in Cancer) signatures in unsupervised hierarchical clusters. Flat signatures include COSMIC signatures 3, 5, and 8. The lower panel shows distribution of translocations, prognostic chromosome-arm events, and TP53 non-synonymous mutations across all samples. b Progression-free survival and c overall survival across different cluster groups. The global P values across all cluster groups were calculated to assess whether there is survival difference between groups [/fig]
[table] Table 1: Association of major myeloma subgroups and mutational signature (Q < 0.05) [/table]
[table] Table 2: Summary of characteristics of the seven cluster subgroups SV structural variant, SNV single-nucleotide variant, APOBEC apolipoprotein B editing complex [/table]
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Background:The probability of undergoing treatment with curative intent according to the hospital of diagnosis varies for esophagogastric cancer in the Netherlands. Little is known about the factors contributing to this variation.This study aimed to improve the understanding of the differences between the
# | introduction
In the Netherlands, almost 4200 patients are annually diagnosed with esophageal and gastric (esophagogastric) cancer, with over 3000 cancer-related deaths per year. 1 Guidelines state that treatment with curative intent should be considered in patients without distant metastasis, depending on the patients' general health status and local extent of disease. The cornerstones of treatment with curative intent for nonmetastatic esophagogastric cancer and early-stage tumors are resection with or without (neo)adjuvant chemo(radiation) therapy and endoscopic resection, respectively. [bib_ref] Oesophageal cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up, Lordick [/bib_ref] [bib_ref] Gastric cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up, Smyth [/bib_ref] [bib_ref] Hospital of diagnosis influences the probability of receiving curative treatment for esophageal..., Landelijke Werkgroep Gastro-Intestinale Tumoren ; Van Putten [/bib_ref] For patients with esophageal cancer, definitive chemoradiation is a reasonable alternative with curative intent for frail patients, patients with unresectable locally advanced cancer, or patients who refrain from surgery. [bib_ref] Oesophageal cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up, Lordick [/bib_ref] Nevertheless, the probability of undergoing treatment with curative intent has been shown to vary considerably depending on the hospital of diagnosis, with a significant effect on survival in 2010-2013 (hazard ratios of hospitals with a high probability of undergoing treatment with curative intent in esophageal cancer 1.15, 95% confidence interval [CI] 1.07-1. [bib_ref] Impoverished diabetic patients whose doctors facilitate their participation in medical decision making..., Golin [/bib_ref] and gastric cancer 1.21, 95% CI 1.04-1.41). [bib_ref] Hospital of diagnosis and probability of having surgical treatment for resectable gastric..., Van Putten [/bib_ref] [bib_ref] Changes in hospital variation in the probability of receiving treatment with curative..., Luijten [/bib_ref] Practice variation can only partially be explained by differences in patient-and tumor-related factors. [bib_ref] Hospital of diagnosis and probability of having surgical treatment for resectable gastric..., Van Putten [/bib_ref] [bib_ref] Changes in hospital variation in the probability of receiving treatment with curative..., Luijten [/bib_ref] Hence, other factors might explain variation in treatment decisions, such as organization of clinical pathways, the emphasis and subsequent advice of multidisciplinary Rob H. A. Verhoeven, Godebaldkwartier 419, 3511 DT Utrecht, The Netherlands. Email: [email protected] multidisciplinary team meeting treatment proposal and the treatment that was actually carried out and to qualitatively investigate the differences in treatment decision-making after the multidisciplinary team meeting treatment proposal between hospitals.
# Methods:
To gain an in-depth understanding of treatment decision-making, quantitative data (i.e., multidisciplinary team meeting proposal and treatment that was carried out) were collected from the Netherlands Cancer Registry.
Changes in the multidisciplinary team meeting proposal and applied treatment comprised changes in the type of treatment option (i.e., curative or palliative, or no change) and were calculated according to the multivariable multilevel probability of undergoing treatment with curative intent (low, middle, and high).
Qualitative data were collected from eight hospitals, including observations of 26 outpatient clinic consultations, 30 in-depth interviews with clinicians, seven focus groups with clinicians, and three focus groups with patients. Clinicians and patients' perspectives were assessed using thematic content analysis.
# Results:
The multidisciplinary team meeting proposal and applied treatment were concordant in 97% of the cases. Clinicians' implementation of treatment decision-making in clinical practice varied, which was mentioned by the clinicians to be due to the clinician's personality and values. Differences between clinicians consisted of discussing all treatment options versus only the best fitting treatment option and the extent of discussing the benefits and harms. Most patients aimed to undergo curative treatment regardless of the consequences, since they believed this could prolong their life.
# Conclusion:
Since changes in the multidisciplinary team meeting-proposed treatment and actual treatment were rarely observed, this study emphasizes the importance of an adequately formulated multidisciplinary team meeting proposal.
## K e y w o r d s
clinicians' perspectives, esophageal and gastric cancer, multidisciplinary team meeting, patients' perspectives, treatment decision-making team meetings, and treatment decision-making during the outpatient clinic visit after the multidisciplinary team meeting, which encompasses a treatment decisionmaking process between physicians and patients. [bib_ref] Impact of a multidisciplinary tumour board meeting for upper-GI malignancies on clinical..., Van Hagen [/bib_ref] [bib_ref] The multidisciplinary team meeting improves staging accuracy and treatment selection for gastro-esophageal..., Davies [/bib_ref] [bib_ref] Effect of multidisciplinary team treatment on outcomes of patients with gastrointestinal malignancy, Du [/bib_ref] [bib_ref] Interventions for improving the adoption of shared decision making by healthcare professionals, Légaré [/bib_ref] Thus, the best available evidence of possible outcomes should be shared with the patient, to guide the patient's considerations on treatment preferences. [bib_ref] Shared decision making: a model for clinical practice, Elwyn [/bib_ref] Hence, one of the aspects that could attribute to practice variation may be a change in the multidisciplinary team meeting proposal caused by this process. Knowledge about these changes could shed light on whether variation in practice between hospitals occurs at the multidisciplinary team meeting level or more during the treatment decision-making process thereafter in the outpatient clinic.
This study aimed to quantitatively investigate the compliance to the multidisciplinary team meeting proposal when compared to the treatment carried out and to gain insight into factors explaining these changes based on qualitative observations during outpatient clinic visits, interviews with clinicians, and focus groups with clinicians and patients.
# | methods
This study is part of a mixed methods multiple case study investigating causes of hospital practice variation in the curative treatment of esophagogastric cancer: the VARIATE-project (Box 1 describes the study design of the VARIATE-project). The current sub-study focuses on identifying compliance with the multidisciplinary team meeting treatment proposal, as a result of the treatment decision-making process during the outpatient clinic visit after the multidisciplinary team meeting combined with quantitative and qualitative data. Quantitative data were used to assess the differences between the multidisciplinary team meeting proposal and actual treatment carried out. Outpatient clinic observations, semi-structured interviews, and focus groups with clinicians were used to gain insight into the clinicians' perspective on treatment decision-making, and patient focus groups were organized to gain insight into the patients' perspectives on treatment decision-making. Clinicians' perspectives included the physicians' convictions and values regarding treatment options, their beliefs and attitudes regarding treatment decision-making, and the input of a patient's proxy. Patients' perspectives included the patients' experiences regarding treatment decision-making during the outpatient clinic visit, reasons for selecting a specific treatment modality, their experiences with the discussions with their doctor, and their treatment experiences.
## | data collection procedures
## | quantitative research
Patients with potentially curable (cT1-4a or cTx, any cN, cM0) esophagogastric cancer diagnosed between 2015 and 2017 were selected from the nationwide population-based Netherlands Cancer Registry (NCR). Patients diagnosed with a gastroesophageal junction tumor were included in the esophageal cancer group. Information on patient characteristics, such as sex, age, modified Charlson Comorbidity Index score, and Eastern Cooperative Oncology Group (ECOG) performance status, and tumor characteristics, such as tumor location, histology, stage, and treatment, were collected from medical records by specially trained data managers of the NCR.
Additional data regarding the final multidisciplinary proposed treatment plan prior to the start of treatment were collected from 38 hospitals for patients with esophageal cancer and 68 hospitals for patients with gastric cancer. As the incidence rate of gastric cancer is lower than that of esophageal cancer in the Netherlands, more hospitals (n = 68) were included as a representative sample.
Differences between the proposed and actual treatments were assessed, and treatments with curative intent were defined as endoscopic resection (endoscopic mucosal resection or endoscopic submucosal dissection), surgical resection of the primary tumor (with or without [neo] adjuvant chemo[radio]therapy), or definitive chemoradiation (for esophageal cancer). Palliative treatment was defined as systemic therapy only, radiotherapy only, and best supportive care (e.g., stent placement).
2.1.2 | Qualitative research: Observations, interviews, and focus groups Eight hospitals were selected based on hospital type (academic resection hospital [n = 3], regional resection hospital [n = 4], and referring non-resecting hospital [n = 1]), probability of offering treatment with curative intent (low [L] n = 2, low/middle [L/M] n = 2, and high probability [H], n = 4), [bib_ref] Changes in hospital variation in the probability of receiving treatment with curative..., Luijten [/bib_ref] and size and geographical location in the Netherlands (see methods regarding hospital selection in Box 1), to achieve a deviant case sampling. 14 A detailed description regarding the calculation of probability of treatment with curative intent classification has been described previously.. [bib_ref] Changes in hospital variation in the probability of receiving treatment with curative..., Luijten [/bib_ref] From January 2019 to November 2020, observations during multidisciplinary team meetings and outpatient clinic visits, interviews with clinicians, and focus groups (with clinicians and patients) were conducted. Data were collected and analyzed iteratively. All data were collected by BOX 1 The VARIATE-study: A mixed methods multiple case study combining qualitative and quantitative research All patients diagnosed with esophageal and gastric cancer in the Netherlands are registered in the Netherlands Cancer Registry (NCR). Previous multivariable multilevel analyses of potentially curable patients diagnosed in the period 2015-2017 have shown that the probability of receiving treatment with curative intent differed according to the hospital of diagnosis. [bib_ref] Changes in hospital variation in the probability of receiving treatment with curative..., Luijten [/bib_ref] Hospitals were divided into three tertiles: low, middle, or high probability of undergoing treatment with curative intent. Patients diagnosed in a hospital with a high probability of receiving treatment with curative intent had a significant better long-term survival. [bib_ref] Changes in hospital variation in the probability of receiving treatment with curative..., Luijten [/bib_ref] In order to obtain in-depth information and knowledge of the underlying mechanisms of hospital practice variation in proposing treatment with curative intent the VARIATE-study (VariAtion in the cuRatIve treatment of esophAgeal and gasTric cancEr) was developed, which was financed by the Dutch Cancer Society. Received treatment with curative intent was defined as endoscopic or surgical resection, initiation of surgery (without resection), definitive chemoradiation (external beam radiotherapy and concurrent chemotherapy; including initiation of chemoradiation). Palliative treatment was defined as: palliative systemic therapy, palliative radiotherapy, and best supportive care.
## Design:
The VARIATE study is a mixed methods multiple case study, which combines qualitative and quantitative research. A purposive sample 43 of eight cases (i.e., hospitals) participated. These hospitals were a representative sample of Dutch hospitals regarding the probability of offering treatment with curative intent (low, low, or middle for gastric or esophageal cancer, and high), hospital type, size, and geographical location. Recruitment: Surgeons or medical oncologists from 11 different hospitals were invited by email. After interest was voiced, JL presented the study during the multidisciplinary team meeting (MDTM) of the eight interested hospital to assess the interest of the multidisciplinary team. All hospitals and team members who saw the presentation wished to participate in this study. This study used an iterative approach for qualitative data collection and analyses, data collection consisted of: 1. Observations of (Upper-GI specific) MDTMs (2-4 MDTMs per hospital) and outpatient clinic visits (minimum of two outpatient clinic visits per hospital) 2. Semi-structured interviews (n = 30) with clinicians involved in the multidisciplinary care for esophageal and gastric cancer (i.e., surgeons (S, n = 8), medical oncologist (MO, n = 6), radiation oncologist (RO, n = 5), gastroenterologists (GE, n = 6), and case managers (CM, n = 5)) 3. Focus groups with clinicians in order to validate and further enrich the results of their own hospital (n = 7). 4. Focus groups with patients diagnosed with potentially curable esophageal-or gastric cancer were organized to explore factors related to their treatment choices (n = 3: low, middle, and high probability hospital). Based on the analysis of the first three hospitals the following decisions regarding the quantitative and qualitative data collection in the further hospitals were made: 1. Depending on the emerging topics from previous interviews the topic list was altered (more focus on: MDTMs, cases of doubt, shared decision-making). 2. Clinicians in the other five hospitals were selected for interviewing through emergent sampling (i.e., gastroenterologist that did not treat early carcinomas were not invited for participation, recent new members in multidisciplinary teams were not invited for participation). 3. Additional quantitative data for potentially curable patients diagnosed in 2015-2017 was gathered in XX hospitals in the Netherlands (i.e., data were gathered by the NCR regarding diagnostics, the MDTM treatment proposal and outpatient clinic visits) in order to gain insight in clinical pathways and alterations in MDTM treatment proposal. The VARIATE-study focusses on the organization of clinical pathways and MDTMs and the outpatient clinic visit. Analyses: 1. Qualitative analyses: Interviews were audio recorded, transcribed per verbatim and summarized (all by JL) and shared with the interviewed clinicians serving as member check. Next, the interviews were reviewed and coded, using open coding as described by Strauss and Corbin.To minimize subjectivity the first 11 transcripts a medical doctor (JL), who was trained to interview, organize focus groups, and analyze the data together with two experienced researchers in the field of qualitative research (LB, MW). Medical oncologists, surgeons, radiation oncologists, gastroenterologists, and case managers (e.g., nurse practitioners, physician assistants) involved in esophagogastric care were all observed and interviewed in the first three hospitals. After the iterative analyses of these three hospitals, emergent themes were discussed within the research team. Thereafter, clinicians in the other five hospitals were selected for interviewing through emergent sampling (see method section Box 1), which implies that sampling decisions were made during data collection as the study progressed. 15
## Observations
Twenty-six outpatient clinic visits in eight hospitals were observed in half-day sessions (approximately 4 h), focusing on how clinicians and patients dealt with the multidisciplinary team meeting proposal, discussed treatment options, discussed benefits and harms, and clinician-patient interaction. Field notes were kept during the observations and summarized at the end of each observation. Observations and informal conversations were helpful in building a relationship of trust (rapport) with the clinicians and were used as inputs for the interviews and focus groups.
## Interviews
Semi-structured interviews with clinicians were conducted using a topic list (Supplementary 1), based on the expertise of the research team and literature, such as the organization of healthcare and multidisciplinary team meetings [bib_ref] The multidisciplinary team meeting improves staging accuracy and treatment selection for gastro-esophageal..., Davies [/bib_ref] [bib_ref] Effect of multidisciplinary team treatment on outcomes of patients with gastrointestinal malignancy, Du [/bib_ref] [bib_ref] Effect of hospital characteristics on outcome of patients with gastric cancer: a..., Siemerink [/bib_ref] and physician attitudes toward treatment options. [bib_ref] Surgeon attitudes and practice patterns in managing small bowel obstruction: a qualitative..., Thornblade [/bib_ref] During all interviews, the opportunity was provided to discuss topics not part of the topic list, exploring new themes that evolved during the interview. Broad topics (Supplementary 1) were discussed during the interviews conducted in the first three hospitals. During the course of this study and through iterative analyses, the topic list evolved, encompassing factors contributing to treatment decision-making during the outpatient clinic visit, such as the physicians' convictions and values regarding treatment options, their beliefs and attitudes regarding shared decision-making, and the input of a patient's proxy. These factors led to more focused interviews in the last five hospitals. Semi-structured interviews were performed by one researcher (JL) with a mean duration of 39 (range 25-54) min. Interviews were audio-recorded and transcribed ad verbatim (JL). All interviews were summarized. Summaries were sent to each interviewed clinician to assess for accuracy, serving as a member check. All clinicians agreed to the summaries' accuracy.
## Clinician focus groups
In seven of the eight hospitals, focus groups were conducted after the observations and interviews with 3-4 clinicians.
In the included referring hospital, only observations and interviews were conducted, and no focus groups were performed, since only two clinicians were involved in the upper gastrointestinal (GI) clinical pathway. Each focus group started with a presentation of the most important results of the observations and interviews, followed by a discussion in which the clinicians were encouraged to further discuss, complement, or contradict the results of the findings in their hospital. Focus groups were physically organized within the hospital (n = 3) or videoconference (n = 4) due to the severe acute respiratory syndrome-coronavirus disease 2019 pandemic and lasted for an average of 1 h and 30 min. The focus groups were observed by one of the two members of the research group (RV, PV) and were all moderated by JL. The focus group moderator and observer discussed all focus groups directly after the focus groups; thereafter, the focus group audio recordings were summarized.
## Patient focus groups
In three resection hospitals, patients who were diagnosed with a curable tumor stage were recruited for the patient focus group. Thus, patients with a curable tumor stage undergoing palliative treatment were excluded in the focus groups. Patients (n = 18) were informed by their treating physician about the VARIATE-project. After consent was provided, the patients were invited to participate in a focus group in the hospital in which they underwent treatment. All but one invited patient agreed to participate.
were independently coded by two researchers (JL, PV) and discussed until consensus was reached. [bib_ref] Series: practical guidance to qualitative research. Part 4: trustworthiness and publishing, Korstjens [/bib_ref] The remaining 19 transcripts were coded by JL. A summary was written for each interview and each hospital. Using thematic content analyses emerging themes were found.Simultaneously, through a constant comparison across and within cases, relations were searched for and themes were identied.The core study group (JL, PV, RV, GN) met weekly to discuss analyses, refine the codebook and identify emerging themes. The coding process was facilitated by Atlas ti 8 software. Quantitative analyses: Quantitative data was analyzed according to the probability of receiving treatment with curative intent using SAS® version 9.4 (SAS Institute). A p-value below 0.05 was considered statistically significant.
One patient did not participate due to logistical reasons (see Supplementary 2 for patient demographics). The focus group guide (Supplementary 3) was used to structure patient's discussion. The focus groups addressed the patients' experiences regarding treatment decisionmaking during the outpatient clinic visit, reasons for selecting a specific treatment modality, their experiences with patient-doctor discussions, and their treatment experiences. Focus groups were organized in a conference room of the hospital and lasted for an average of 1 h and 29 min (range 1 h 15 min-1 h 43 min). Three focus groups were conducted with five to six patients per focus group, moderated by LB, and observed by JL.
## | data analyses
## | quantitative data analyses
The primary quantitative outcome parameter was the proportion of patients in whom the multidisciplinary team meeting proposal differed from the actual treatment carried out. Secondary outcomes were the proportion of patients discussed during a multidisciplinary team meeting, the type of multidisciplinary team meeting proposal, and received treatment according to the hospitals of diagnosis' probability of undergoing treatment with curative intent.
Patients' baseline characteristics and outcome parameters were reported as frequencies with percentages. To evaluate baseline characteristics and outcome parameters, the chi-squared test and Fisher exact test were used, when appropriate.
## | qualitative data analyses
The data used for analyses comprised outpatient clinic field notes, transcripts of the interviews focusing on factors influencing the treatment decision-making during the outpatient clinic visit, and summaries of the clinician focus groups and patient focus groups. A thematic content analysis was used to identify individual and hospital experiences,focusing on the clinicians' perceptions and beliefs regarding treatment decision-making during the outpatient clinic (e.g., aims, beliefs, personal characteristics, discussed treatment options) (see Box 1 for a complete overview of the coding process and the identification of emerging themes and subthemes) and was based on the treatment decision-making model created by Elwyn et al. [bib_ref] Shared decision making: a model for clinical practice, Elwyn [/bib_ref] For each hospital, a similar thematic map summarizing each theme and subtheme per clinician was created. Through constant comparison within and across cases, associations and deviant cases were identified.Preliminary conclusions from the thematic map were thoroughly discussed by the core research team (JL, PV, RV, GN, MW). The thematic map comprised the themes and interrelations between the codes and themes, such as discussed treatment options, influence of patient characteristics, and influence of clinician's personality traits. Thereafter, the themes were discussed with two experts in the field of shared decision-making (LB and LT).
## | ethics
Ethics approval was not required according to the Medical Research Ethics Committees United of the Netherlands (number: W.18.166). All participating hospitals approved this study. All quantitative data collected by the NCR were de-identified and coded. Written informed consent was obtained from all participants prior to the interview or start of the patient focus groups. Privacy and confidentiality were protected through pseudonymization. The transcripts and summaries will be stored pseudonymized for a minimum of 10 years, on the secured network of IKNL, to which only the core research team members have access. This study was funded by the Dutch Cancer Society (project number: 10895). shows patient characteristics according to the hospital's diagnosis probability of undergoing treatment with curative intent. For esophageal cancer, no significant differences were observed between the probability groups; however, for gastric cancer, significant differences were observed in the number of comorbidities, ECOG performance status, and cT tumor stage. Surgery with curative intent was proposed during multidisciplinary team meeting in 63%, 65%, and 72% of patients diagnosed with esophageal cancer in a hospital with a low, middle, or high probability of undergoing treatment with curative intent, respectively. For gastric cancer, the rates were 75%, 82%, and 82%, respectively. Definitive chemoradiation was proposed during the multidisciplinary team meeting in 13%, 12%, and 15% of patients diagnosed with esophageal cancer in a hospital with a low, middle, or high probability of undergoing treatment with curative intent, respectively. In hospitals with a low, middle, or high probability of proposing treatment with curative intent, the proposed treatment plan for patients with esophageal cancer changed by 5%, 4%, and 3% (p = 0.24), respectively, and for gastric cancer, these were 1%, 0%, and 0% (p = 0.18), respectively . Changes in the multidisciplinary team meeting proposal and actual treatment carried out comprised changes in the type of curative treatment option, such as a surgical proposal changing to definitive chemoradiation (n = 42). Nonetheless, no changes from curative intent multidisciplinary team meeting proposals to palliative treatment proposals or vice versa were observed . Of the patients diagnosed with esophageal cancer in which the treatment plan was known (n = 1293), neoadjuvant treatment followed by resection was proposed in 65% of the patients. Of them, 18% did not undergo the proposed treatment plan: 12% underwent primary surgery, 5% underwent definitive chemoradiation, and 1% underwent endoscopic resection. Of the patients diagnosed with gastric cancer in which the treatment plan was known (n = 935), neoadjuvant treatment followed by surgery was proposed in 51% of the patients. Of them, 17% did not receive chemotherapy but directly underwent surgery. Of the patients in which perioperative treatment followed by resection was proposed, 83% underwent neoadjuvant treatment followed by resection of whom 39% did not undergo the adjuvant component. Direct surgery was proposed in 28% of the patients diagnosed with gastric cancer of which 98% underwent surgery .
# | results
## | quantitative results: changes in the multidisciplinary team meeting proposal
## | qualitative results: from treatment
proposal to the actual treatment carried out shows a visualization of the treatment decisionmaking process during the outpatient clinic visit, in which the multidisciplinary team meeting proposal was discussed with the patient. In this process, the patient should become aware of having a choice, understand the treatment options (option communication) with the possible benefits and harms (risk communication), and feel that there is sufficient time and support to consider which treatment option fits most to the patients' preferences (shared decision-support), resulting in decision communication (treatment decisionmaking). A comprehensive description of the factors that play a role in treatment decision-making during the clinicianpatient interaction and deliberation is shown in , such as clinician's aims, personality, and conversation style. These themes and subthemes are described in more detail below and complemented by the patient's perspectives on treatment decision-making, which were expressed during the focus groups displays quotes of the patient focus groups).
## | discussing the multidisciplinary team meeting proposal with the patient
## | context
The setting and participants during the outpatient clinic visit differed between hospitals; for example, some hospitals organized an outpatient clinic visit directly after the multidisciplinary team meeting involving the whole multidisciplinary team, as opposed to other hospitals where patients were seen separately by different clinicians on different occasions after the multidisciplinary team meeting. Additionally, in some hospitals, the referring clinician discussed the multidisciplinary team meeting proposal with the patient, whereas in other hospitals, an upper-GI involved clinician from the expert center discussed the multidisciplinary team meeting proposal with the patient.
## | clinician-patient interaction
## | clinician aims and conversation style
Most frequently, a clinician's intention was to accomplish an informed treatment decision by explaining how the multidisciplinary team meeting decision was substantiated, facilitating the patient to make a balanced decision during treatment decision-making as explained by a surgeon: "If he understands and comprehends what is happening, and is treatable, also in the postoperative phase (i.e., no mental impairment leading to declining nasogastric feeding), then we would perform surgery" (surgeon 3, low/middle probability). Additionally, most clinicians believed that the treatment decision should be made together with the patient and family members, in which they experience respect and trust that the best treatment decision has been made: "I would like the patient and the family to have the feeling that they are heard and that they understand that we are on the same page regarding the treatment aims" (surgeon 1, low probability). Hence, clinicians believed that it is important that the patient feels that sufficient time is available in which the patients is allowed to discuss their arguments. Clinicians believed that the trust between them and the patient was important. Patients mentioned that they trusted to put their lives into the clinician's hands, and since their will to survive prevailed, they agreed with the proposed (curative) treatment proposal.
## F i g u r e 1 treatment decision-making
The observations showed that communication styles varied among clinicians, such as elaborating and discussing treatment and complications in more detail, which was also explained by a case manager during an interview: "They definitely differ; one of them is more short but decisive, and the other elaborates more; however, their aims are similar" (case manager 8, low/middle probability). Empathy or humor was helpful in putting the patient at ease, as explained by other clinicians.
## | deliberation
## | clinician's personality and practice style
The clinician's practice style, communication style, and treatment belief are all part of a clinician's personality, which differed between clinicians and was mentioned to affect discussion. For instance, some clinicians were more inclined to guide the patient toward the multidisciplinary team meeting-proposed treatment plan, whereas others provided more room to let the patient make a treatment decision. According to a gastroenterologist, clinicians should express their vulnerability when discussing uncertainties, as this would benefit the patient's decision-making: "Naturally, at times, patients do not know which choice to make, and at these times perhaps, we should take more responsibility; a lot of doctors are unsure to discuss their uncertainties with patients about choice A or choice B, since they hold the belief that the doctor should provide the patient with a superior choice" (gastroenterologist 3, low/middle probability).
## | option communication
Most clinicians explained that all treatment options, including the multidisciplinary team meeting proposal, should be discussed during treatment option communication, as opposed to others who mentioned that they only explain other treatment options if the patient asked for them: "Only if patients ask for it, then we discuss definitive chemoradiation, but we do tell them that it does not provide the best odds" (case manager 2, high probability). Moreover, in the patient focus groups, patients mentioned that not all treatment options were discussed. Clinicians differed in their discussion of the multidisciplinary team meeting proposal during the outpatient clinic. Based on the observations and interviews, some clinicians only discussed the multidisciplinary team meeting proposal, as opposed to others who discussed their personal treatment beliefs in addition to the multidisciplinary team meeting proposal: "Well, that's the question, ultimately, the multidisciplinary team meeting proposal is only an advice; you can deviate from this advice as treating physician, but you have to motivate your decision […], so if the multidisciplinary team meeting's advice is on the conservative spectrum, and I personally belief a more invasive treatment option is feasible, 'I would like to discuss this with you and then we can make a shared decision'" (surgeon 6, high probability). During treatment decision-making in the outpatient clinic visit, the clinician's personal treatment beliefs and values played a role according to a surgeon: "The person's ethics, the beliefs of the clinician, are important in how that person comes to a decision; perhaps, life experiences play a role as well" (surgeon 2, high probability).
Most clinicians mentioned that providing tailored information is important, such as providing the patient with a realistic perspective: "Occasionally, patients imagine total alopecia due to chemotherapy; that is however no longer the case in patients treated with chemotherapy for esophageal cancer." (case manager 1, low probability). Most clinicians aimed to outline the context, such as the effect of patient age on treatment outcome and the uncertainties associated with certain (experimental) treatment options.
## | risk communication
Most clinicians mentioned that they explain the benefits and harms of the treatment options during risk communication: "In all openness, the benefits and harms are discussed with the patient, weighing what the best treatment option is for this individual patient" (medical oncologist 3, low/middle probability). However, it was observed that the extent of discussing the benefits and harms of the proposed treatment varied among clinicians. Some clinicians only mentioned the probability of postoperative complications, whereas others discussed complications in more detail. Most patients mentioned that they were not concerned about the impact or harms, since they had a strong will to be cured.
## | patient's preferences
During the patient focus groups, most patients mentioned that they did not experience that there was a choice, since their will to be cured prevailed. Some clinicians mentioned exceptions, for instance, patients who felt that they had a complete life or did not want to compromise their quality of life standards by surgery. This was also explained by one of the patients, stressing that preserving quality of life was more important than prolonging life, which made her choose for definitive chemoradiation. Patients' motivation, preferences, home situation, and family opinions were taken into account during treatment decision-making according to most clinicians: "I think that we take the interest of the patient into account, or the patient's opinion, what does the patient want, and I can imagine that there are hospitals that are more guiding" (surgeon 8, low/middle probability). Some clinicians explained that they always stress that the patient always has a choice: "I always try to tell them, "yes, you do have a choice" […] I always discuss that, and that we can also refrain from treatment" [medical oncologist 5, high probability). However, most clinicians explained that refraining from treatment was rarely an option for patients.
## | decision-communication
Most clinicians recognized their ability to influence the patient's treatment decision-making during decisioncommunication: "It is not like I tell the patient to make a choice, as choosing your food at the Chinese restaurant […], yet I realize that we are able to guide the patient's treatment decision-making […] it's not only explaining the options, but you can also influence the patient's decision" (surgeon 1, low probability). Based on the observations, clinicians used different techniques, ranging from describing all treatment options without the treating physician's personal preference to guiding the patient to the definitive treatment plan, which was also mentioned by a surgeon: "I try to get an impression how the patient responds to all the treatment options, how they consider the treatment options, or whether they did their own search on the internet" (surgeon 8, low/middle probability). Some clinicians felt that doctors need a certain maturity to guide the patient properly during treatment decision-making: "It is important that doctors have a certain maturity, a certain confidence, that during uncertainties, they can guide the patient" (gastroenterologist 3, low/middle probability).
## | decision-support
During the focus groups, patients mentioned that they needed time to process the discussed treatment options and their own preferences and were not able to decide during the outpatient clinic visit, which was also mentioned by some clinicians. One patient mentioned that he received a significant amount of information during the visit and needed time to think about what was explained during the consultation. In these situations, the clinician organized a second appointment to support the patient in the treatment decision-making or involved the patient's general practitioner. Most clinicians felt that the patient should discuss the treatment proposal with all involved clinicians prior to a definitive treatment choice is made: "I insist that the patient has also a consultation with the surgeon prior to the start of treatment; this was however not always the case in the beginning" (radiation oncologist 6, high probability). Based on these observations, patients rarely seek contact with peers or provide pamphlets to explain treatment options.
# | discussion
In this mixed method multiple case study, the quantitative results demonstrated that differences between the multidisciplinary team meeting proposal and applied treatment rarely occurred. Despite the qualitative finding that most clinicians held similar opinions regarding the decisional process, the implementation of treatment decision-making in practice varied. Based on the observations, differences comprised discussing all treatment options versus only the best fitting treatment option and the thoroughness of discussing the benefits and harms of treatment options. Most patients aimed to undergo treatment with curative intent and were less concerned about the possible harms.
Based on the principle that multidisciplinary case discussions lead to improved treatment recommendations, multidisciplinary team meetings have been widely implemented in cancer care. The multidisciplinary team meeting structure enhances communication, decisionmaking, and coordination, based on evidence-based and updated knowledge or expert opinions. [bib_ref] Tumor boards: optimizing the structure and improving efficiency of multidisciplinary management of..., El Saghir [/bib_ref] The quantitative results of the present study indicate that changes in the multidisciplinary team meeting proposal and the actual treatment decision during the outpatient clinic visit only rarely occurred. Hence, variation in the probability of undergoing treatment with curative intent between hospitals mainly originates during clinical decision-making during the multidisciplinary team meeting. Furthermore, it can be hypothesized that the multidisciplinary tumor board generally proposes an adequate treatment plan; thus, sufficient information is available during the multidisciplinary team meeting, including patient characteristics, such as the patient's opinion, resulting in a fitting treatment proposal that matches the individual needs of a specific patient. Therefore, based on the results of the current study, adequate organization of a multidisciplinary team meeting and clinical decision-making during a multidisciplinary team meeting is invaluable, since most clinicians mentioned that they value the upper-GI multidisciplinary team meeting treatment proposal and communicate this during the outpatient clinic visit as a treatment advice. Due to the development of multidisciplinary team meetings, the process of individual physician decision-making has shifted to multidisciplinary decision-making, yet the definitive treatment decision is a shared decision between the clinician and patient made during the outpatient clinic visit.
In 1956, three basic models of the physician-patient relationship were described (i.e., activity-passivity, guidance-cooperation, and mutual participation). [bib_ref] A contribution to the philosophy of medicine; the basic models of the..., Szasz [/bib_ref] Recently, treatment decision-making should consist of mutual participation, in which the physician and patient work together as partners toward a mutually agreed treatment plan. First, awareness of equipoise should be created (explaining that there is no best choice), followed by discussing the benefits and harms. Subsequently, the patient's concerns and preferences should be elicited, and the patient should be supported in the process of deliberation. [bib_ref] Shared decision making: really putting patients at the Centre of healthcare, Stiggelbout [/bib_ref] The process of treatment decision-making during the outpatient clinic visit is pivotal, and regardless of the multidisciplinary team meeting proposal, the discussion toward a definitive treatment plan should be personalized to accompany the patient in the treatment decision-making. During the patient focus groups, it became clear that most patients trusted their clinician in the treatment advice and their strong intention to be cured prevailed, which resulted in following the treatment proposal. In line with a previous study, [bib_ref] Challenges in shared decision making in advanced cancer care: a qualitative longitudinal..., Brom [/bib_ref] the current study demonstrated that clinicians expressed that during treatment decisionmaking, a partnership between the clinician and patient in which the patient feels supported and encouraged is important. Furthermore, clinicians explained the importance of the patient having the feeling that they were heard, and that the best treatment decision was made as a joined venture. Importantly, previous studies have demonstrated that greater patient participation during decision-making has a positive effect on patient satisfaction, coping, and treatment adherence. [bib_ref] Impoverished diabetic patients whose doctors facilitate their participation in medical decision making..., Golin [/bib_ref] [bib_ref] How does communication heal? Pathways linking clinician-patient communication to health outcomes, Street [/bib_ref] [bib_ref] Cognitive coping style (monitoring and blunting) and the need for information, information..., Rood [/bib_ref] Hence, although the multidisciplinary team meeting treatment proposal only rarely changed during the outpatient clinic visit, treatment decision-making is an important process in treatment decision-making that enhances patient empowerment and possibly results in increased patient satisfaction and treatment adherence.
During the interviews, it became apparent that most clinicians recognized their ability to influence patients' decision-making during decision-communication. In addition, differences were observed, such as describing all treatment options, including personal preferences, and guiding the patient to the multidisciplinary team meeting proposal or offering treatment options that differed from the multidisciplinary team meeting proposal. In line with previous studies, 27,28 the qualitative results of the present study demonstrate that the clinician's personality, personal beliefs, and values on treatment (subjective considerations) influence treatment decision-making during the process of clinical decision-making during outpatient clinic visits. In 1984, Wennberg reported that subjective physician considerations play a decisive role during clinical treatment decision-making (i.e., the practice style factor). [bib_ref] Dealing with medical practice variations: a proposal for action, Wennberg [/bib_ref] He found that physicians held different opinions regarding the best therapy, and that this was unrelated to scientific controversies. [bib_ref] Dealing with medical practice variations: a proposal for action, Wennberg [/bib_ref] More recent literature has described that the practice style factor reflects a deeply rooted behavioral mechanism regarding convictions of the individual clinician with respect to how to practice medicine, [bib_ref] Practice variation and physician-specific effects, Grytten [/bib_ref] possibly attributed to the observed differences in offering guidance or withholding a personal opinion in the current study, which is in concordance with a previous study. [bib_ref] Communicating shared decision-making: cancer patient perspectives, Thorne [/bib_ref] Furthermore, variation in practice was observed regarding the discussed treatment options and the extent of discussing benefits and harms during the outpatient clinic visit. The frequent absence of addressing alternative options or refraining from treatment and differences in the extensiveness of discussing potential benefits and harms have also been described in previous studies. [bib_ref] Challenges in shared decision making in advanced cancer care: a qualitative longitudinal..., Brom [/bib_ref] [bib_ref] Patient centered decision making in palliative cancer treatment: a world of paradoxes, De Haes [/bib_ref] [bib_ref] Understanding provision of chemotherapy to patients with end stage cancer: qualitative interview..., Buiting [/bib_ref] A potential explanation for this phenomenon may be that clinicians might feel that fostering hope may contribute to the patient's well-being, [bib_ref] Understanding provision of chemotherapy to patients with end stage cancer: qualitative interview..., Buiting [/bib_ref] which might explain the finding that clinicians refrained from discussing alternative treatment options with a lower likelihood of survival. Another suggested explanation is that the therapeutic relationship might be damaged by a clinician sharing information on gaps in scientific knowledge, uncertainties, and controversies. [bib_ref] Partnerships with patients: the pros and cons of shared clinical decision-making, Coulter [/bib_ref] This means that the process of treatment decision-making might be influenced by the individual practice style factor but yet rarely leads to changes in the multidisciplinary team meeting treatment proposal.
## | strengths and limitations
The main strength of this study is the combination of quantitative data and the clinicians' and patients' perspectives, providing a complete understanding of differences between clinicians toward treatment decision-making during outpatient clinic visits. Furthermore, the reliability and validity of the data increased due to data triangulation and member checking. [bib_ref] Qualitative interviews in medical research, Britten [/bib_ref] [bib_ref] A critical appraisal of the role of triangulation in nursing research, Sim [/bib_ref] Nevertheless, this study has some limitations when interpreting the results. For instance, our findings based on the patient focus groups should be interpreted with caution, since only patients who underwent treatment with curative intent were included. It would have been of added value when patients diagnosed with a curable stage, but who refrained from treatment with curative intent, would have been included. However, we were unable to recruit the patient group. Nevertheless, the outcomes of this study provide insight into the perspectives of patients who underwent treatment with curative intent and their reasoning. Additionally, all observations and interviews were performed by one researcher; nonetheless, potential researcher bias was tried to be prevented during data collection and analyses by peer debriefing.Furthermore, the last multidisciplinary team meeting prior to treatment was defined as the multidisciplinary team meeting treatment proposal in the quantitative analyses. Since multiple multidisciplinary team meetings were held in some patients, the patient's feedback on previously discussed treatment options might be used as input during the multidisciplinary team meeting in which the multidisciplinary team meeting proposal is finalized. This means that during multidisciplinary team meeting, information regarding the patient's preference might be known and taken into consideration. In addition, at times, an "if then" treatment decision was made during the multidisciplinary team meeting, and since the data managers were only able to formulate one multidisciplinary team meeting proposal, these cases might have affected the quantitative outcomes of our results.
## | future directions
To overcome the gap in treatment information that is provided to patients, personalized pamphlets (infographics) describing treatment options (i.e., curative and palliative options), and their outcomes (i.e., survival, functional, and quality of life outcomes) could be provided directly after diagnosis, informing patients about all treatment options prior to the outpatient clinic consultation for finally deciding on the treatment. Additionally, decision aids have been helpful in facilitating patient engagement in treatment decision-making and could enhance the patient's question asking behavior. [bib_ref] Decision aids for people facing health treatment or screening decisions, Stacey [/bib_ref] [bib_ref] The characteristics and effectiveness of question prompt list interventions in oncology: a..., Brandes [/bib_ref] [bib_ref] Costutility of an eHealth application 'Oncokompas' that supports cancer survivors in self-management:..., Van Der Hout [/bib_ref] An ongoing trial (the SOURCE trial: NCT04232735) investigates the effects of personalized predictions of treatment outcomes, [bib_ref] SOURCE: a registry-based prediction model for overall survival in patients with metastatic..., Van Den Boorn [/bib_ref] used by caregivers who are trained in using this specific tool on tailored information provision to patients diagnosed with esophagogastric cancer. The outcomes of such studies may improve personalized and tailored information provisions in clinical consultations.
# | conclusion
Since changes in the multidisciplinary team meeting proposal and actual treatment carried out rarely occurred, the multidisciplinary team meeting proposal is pivotal. Differences in the beliefs and personalities of the attending clinicians might have contributed to the variation during the outpatient clinic consultation. Patients who aimed to be cured of their cancer trusted their treating clinician and were less concerned with the harms of a certain treatment.
interpretation of data, acquisition of data, revising manuscript, final approval, accuracy, and integrity; PV: conception, design, analysis, interpretation of data, revising manuscript, final approval, accuracy, and integrity; AW: interpretation of data, revising manuscript, final approval, accuracy, and integrity; FW: interpretation of data, revising manuscript, final approval, accuracy, and integrity; JH: interpretation of data, revising manuscript, final approval, accuracy, and integrity; SM: interpretation of data, revising manuscript, final approval, accuracy, and integrity; JOH: interpretation of data, revising manuscript, final approval, accuracy, and integrity; MvD: interpretation of data, revising manuscript, final approval, accuracy, and integrity; LT: interpretation of data, revising manuscript, final approval, accuracy, and integrity; MH: interpretation of data, revising manuscript, final approval, accuracy, and integrity; HvL: conception, design, interpretation of data, acquisition of finances, revising manuscript, final approval, accuracy, and integrity; CR: conception, design, interpretation of data, acquisition of finances, revising manuscript, final approval, accuracy, and integrity; PS: conception, design, interpretation of data, acquisition of finances, revising manuscript, final approval, accuracy, and integrity; MW: conception, design, analyses, interpretation of data, acquisition of finances, revising manuscript, final approval, accuracy, and integrity; RV: conception, design, analyses, interpretation of data, acquisition of finances, revising manuscript, final approval, accuracy, and integrity; GN: conception, design, analyses, interpretation of data, acquisition of finances, revising manuscript, final approval, accuracy, and integrity.
## Ethics approval
The Medical Research Ethics Committees of the Netherlands confirmed that ethical approval was not required for this study (W.18.166). The participating hospitals approved this study. Written informed consent was obtained from all the participants prior to the interviews and focus groups
# Data availability statement
The data underlying this article cannot be shared publicly due to for the privacy of individuals that participated in the study. Data are however available from the authors upon reasonable request and with permission of Netherlands Comprehensive Cancer Organization (IKNL).
## Orcid
|
Empty follicle syndrome: Successful pregnancy following dual trigger
# Introduction
Empty follicle syndrome (EFS), a much debated enigmatic syndrome, is annoying and highly stressful causing considerable concern for both the clinician and the patient. [bib_ref] Failure to collect oocytes in assisted reproductive technology: A retrospective, Driscoll [/bib_ref] EFS is defined as a condition in which no oocytes are retrieved from mature ovarian follicles following ovulation induction in an in-vitro fertilization (IVF) cycle with apparently normal folliculogenesis and steroidogenesis despite meticulous follicular aspiration and flushing. It cannot be predicted by the pattern of ovarian response to stimulation, either sonographically or hormonally. Consequently, the diagnosis of EFS is retrospective. In view of the uncertainty surrounding the causes of this phenomenon, and the mounting evidence that oocytes can, in some instances, be successfully retrieved, the term EFS is considered inappropriate; yet still in use in current medical literature. [bib_ref] Unsuccessful oocyte retrieval: Technical artefact or genuine 'empty follicle syndrome, Bustillo [/bib_ref] EFS has been classified into two types: "Genuine EFS" (GEFS) and "false" EFS (FEFS). The prevalence of GEFS is 0-1.1%. [bib_ref] Empty follicle syndrome: Successful treatment in a recurrent case and review of..., Beck-Fruchter [/bib_ref] GEFS is defined as failure to retrieve oocytes from apparently normal growing ovarian Empty follicle syndrome: Successful pregnancy following dual trigger was also a case of unexplained infertility, who had undergone three IVF cycles elsewhere with the retrieval of only 1-2 immature oocytes in all the cycles and was advised donor oocytes. Her physical examination was unremarkable with no acne or hirsutism and body mass index of 22 kg/m 2 . Pelvic sonography was normal and her baseline investigations were all within normal limits with follicle stimulating hormone (FSH)-7.5 IU/L, anti-mullerian hormone -2.9 ng/ml, and prolactin-17 ng/ml. Her thyroid stimulating hormone (TSH) was 6.6 µIU/ml for which her thyroxin was upgraded to 75 µg/day with repeat TSH 6 weeks later being 1.9 µIU/ml. Diagnostic hysterolaparoscopy revealed normal findings. Patient's husband was 38 years old with normal semen analysis (total count -69 million/ml, total motility -68%, and normal forms -35%) and normal DNA fragmentation index.
As the patient had failed to conceive in four cycles of Intrauterine insemination, IVF was planned with long luteal agonist protocol. showed evidence of intact follicles in both the ovaries. Following aspiration of the mature follicles from the right ovary, as the follicular fluid in the first three tubes did not show any evidence of granulosa cells or oocytes, we decided to flush the remaining follicles. Despite repeated flushing, neither oocytes nor cumulus ± corona complexes were recovered. Follicular fluid tested for β-hCG was found to be positive. A careful interrogation of the patient and the nurse who had administered the hCG injection revealed that there were no drug-or administration-related problems. We also decided to check the serum levels of hCG and P4 and surprisingly, we found it to be 165 mIU/ml and 14 ng/ml, respectively. As the serum levels were assuring and within the expected range, we proceeded with the aspiration of the other ovary. Unfortunately, no oocytes were retrieved.
A second cycle was planned 4 months later with an antagonist protocol. On day 2 of spontaneous cycle, FSH, LH, E2, and P4 levels were 6.5 IU/L, 4.6 IU/L, 37 pg/ml, and 2.1 ng/ml, respectively, with antral follicle count (AFC) of 6-8 in both the ovaries. Stimulation was started with a combination of R-FSH (Recagon, Organon) 150 IU and 75 IU HMG (Menogon, Ferring Pharmaceuticals Ltd). On stimulation day 5, with lead follicles of 13 mm and 14 mm and E2-456 pg/ml, GnRH antagonist, Ganirelix (Orgalutran, Organon) 0.25 mg s.c. was added which was continued daily till the day of trigger. On day 9 of stimulation with 3 dominant follicles ≥17 mm and peak E2, LH, and P4 levels of 1856 pg/ml, 2.4 IU/L, and 1.01 ng/ml, respectively, R-hCG (Ovitrelle, Serono) 500 mcg was administered s.c. by a qualified nurse and OR was planned at 36 h. TVS at the time of pickup showed intact follicles in both the ovaries and also the LH levels on the day of trigger were reassuring. Even with repeated flushing of the follicles in both the ovaries, neither cumulus complexes nor oocytes were recovered. We were perplexed to find a serum hCG level of 154 mIU/ml and P4 of 19 ng/ml and a repeat problem made us really worry despite taking all the precautions in the second cycle.
After two previous failed IVF attempts, the patient returned only 2 years later for a third cycle. This cycle we choose an antagonist protocol with dual trigger. Her day 2 FSH, LH, E2, P4 levels were 7.9 IU/L, 3.5 IU/L, 43 pg/ml, 1.9 ng/ml, respectively, with an AFC of 5-6 in each ovary. Ovarian stimulation was started with R-FSH, (Gonal F, Serono) 225 IU. On stimulation day 6 with a lead follicle of 13 mm and the rest of the follicles between 9 and 11 mm with E2-301 pg/ml, Recombinant LH, Luveris (Serono) 75 IU along with Gonal F 150 IU and GnRH antagonist, and Ganirelix (Orgalutran, Organon) 0.25 mg s.c., was added which was continued daily till the day of trigger. On day 10 of stimulation with 5 lead follicles 17 mm and E2, LH, and P4: 2415 pg/ml, 2.45 IU/L, and 0.78 ng/ml, respectively. R-hCG (Ovitrelle, Serono) 250 mcg and Triptorelin (Decapeptyl, Ferring) 0.2 mg was administered for final maturation and OR planned at 36 h. Eleven oocytes were retrieved, 10 were mature, 9 fertilized with intracytoplasmic injection, and 9 cleaved further. A total of seven 8-cell good quality embryos were available on day 3 with two 6 celled arrest being discarded. Four 8-cell good quality embryos were cultured to blastocyst and the remaining 3-8 cell good quality embryos were frozen on day 3. Two good quality blastocysts (311, 211) were transferred on day 5. Luteal phase was supported with micronized progesterone 400 mg vaginally twice a day and three doses of hCG 2000 IU every 3 days from the day of embryo transfer. Serum β-hCG 2 weeks later was 426 mIU/l and TVS at 6 + weeks showed a single live intrauterine fetus.
Patient continued well through the first trimester. Thyroid supplementation was altered as per the need for pregnancy and monitored with TSH and free T4 levels. During the second trimester, she developed gestational diabetes and was started on insulin. Elective cesarean section was performed at 38 weeks of gestation delivering a live female baby of 3.1 kg with uneventful intra and postpartum period.
# Discussion
EFS was first reported by [bib_ref] Empty follicle syndrome, Coulam [/bib_ref]. [bib_ref] Empty follicle syndrome, Coulam [/bib_ref] The incidence of EFS has been estimated to be 0.6-7%, [bib_ref] Inappropriate timing of hCG administration: An avoidable cause of empty follicle syndrome..., Khalaf [/bib_ref] [bib_ref] Continuing the debate on empty follicle syndrome: Can it be associated with..., Awonuga [/bib_ref] 2-7%, [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] and 0.5-2% [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] [bib_ref] Empty follicle syndrome due to human errors: Its occurrence in an in-vitro..., Quintans [/bib_ref] of IVF cycles. Unsuccessful OR does not appear to be related to stimulation regimen [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] as the phenomenon is observed in natural as well as stimulated cycles [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] and in almost all IVF cycles because the oocyte yield rarely reaches 100%. However, the terminology is usually used to refer to total failure to retrieve any oocytes. [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] The etiology of EFS is multifaceted, with both procedural factors and ovarian dysfunction contributing as suggested by Bustillo. [bib_ref] Unsuccessful oocyte retrieval: Technical artefact or genuine 'empty follicle syndrome, Bustillo [/bib_ref] FEFS is defined as a condition where no oocytes are retrieved in the presence of low β-hCG due to human errors [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] or pharmaceutical problems [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] or due to reduced bioavailability of hCG. [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] Indeed, EFS has been described as a "pharmaceutical industry syndrome" [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] and accounts to about 67% of cases. [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] GEFS is presumably related to intrinsic ovarian dysfunction [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] and various hypotheses have been suggested which include: (1) Dysfunctional folliculogenesis or premature apoptosis of the oocytes that still continued follicular growth. [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] [bib_ref] Steroid profiles of follicular fluids from a patient with the empty follicle..., Tsuiki [/bib_ref] (2) Defective granulosa cell function. [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] (3) Faulty oocyte development and maturation. [bib_ref] Transcriptional profiling of granulosa cells from a patient with recurrent empty follicle..., Inan [/bib_ref] Abnormal oocytes like immature oocytes that were zona-free or that had zona which was lacking in oocytes could be the cause in some cases. [bib_ref] Transcriptional profiling of granulosa cells from a patient with recurrent empty follicle..., Inan [/bib_ref] (4) Strong attachment of cumulus cell complexes to the follicular wall. (5) Dysfunctional ovulation induction. (6) Rare cases, follicles may need longer exposure to hCG to undergo cumulus expansion and separate from the follicular wall. [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] [bib_ref] Empty follicle syndrome due to human errors: Its occurrence in an in-vitro..., Quintans [/bib_ref] (7) Biological abnormality in the supply of mature oocytes. [bib_ref] Continuing the debate on empty follicle syndrome: Can it be associated with..., Awonuga [/bib_ref] (8) Genetic factors (a) LH/hCG receptor mutations. [bib_ref] Empty follicle syndrome in two sisters with three cycles: Case report, Onalan [/bib_ref] (b) altered expression of genes regulating cumulus expansion. (c) Altered expression of genes involved in cellular processes and apoptosis resulting in increased apoptotic gene expression and reduction in transcripts with lose of oocytes during late folliculogenesis due to apoptosis. (d) Pericentric inversion of chromosome 2. [bib_ref] Empty follicle syndrome in two sisters with three cycles: Case report, Onalan [/bib_ref] (9) Advanced ovarian ageing [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] through altered folliculogenesis which is considered as a risk factor for EFS recurrence. [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] The various reasons for FEFS include: (1) Drug-related causes due to an abnormality in the in-vivo biological activity of some batches of commercially available urinary hCG preparations. [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] (2) Human errors-inappropriate timing, administration, and dosage of hCG. [bib_ref] Continuing the debate on empty follicle syndrome: Can it be associated with..., Awonuga [/bib_ref] [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] [bib_ref] Empty follicle syndrome due to human errors: Its occurrence in an in-vitro..., Quintans [/bib_ref] (3) Low bioavailability resulting from variation in the absorption or clearance of hCG with some batches of urinary hCG. [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] (4) Pharmacological problems. [bib_ref] Continuing the debate on empty follicle syndrome: Can it be associated with..., Awonuga [/bib_ref] (5) Variation in the threshold for follicular response to hCG. (6) Variation in the time needed from hCG exposure to the maturation of oocyte-cumulus complexes. (7) Rapid breakdown of products that contained desialylated hCG by the liver, resulting in a lack of exposure to biologically active hCG. [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] (8) As with any other metabolic process, individuals vary in their rate of clearance of hCG and some may metabolize hCG quite rapidly.
In assisted reproductive technology (ART) for decades, a bolus of hCG is commonly used as a surrogate of the mid-cycle LH surge as its actions are similar to those of endogenous LH. The LH surge initiates a cascade of events resulting in mucification of cumulus cells, facilitating the detachment of the oocyte-cumulus complex from the follicular wall, resumption of meiosis with extrusion of first polar body and subsequent ovulation. [bib_ref] The temporal relationship between the luteinizing hormone surge and human oocyte maturation, Seibel [/bib_ref] The threshold amplitude to define an LH surge is still a matter of debate, but levels more than 10 IU/L are commonly reported although a doubling from basal level could be a more appropriate definition, particularly for patients with high basal LH levels. [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] The LH concentration must be maintained above a threshold for 14-27 h in order to maximize oocyte maturation with metaphase II oocytes obtainable within 28-38 h after the onset of the LH surge. [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] Thus, it is easy to understand those cases of EFS that occur due to faulty techniques. In such cases, a repeat hCG from a different batch or the use of R-hCG with OR scheduled 24 h, [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] 36 h [bib_ref] Empty follicle syndrome due to human errors: Its occurrence in an in-vitro..., Quintans [/bib_ref] later would yield mature oocytes from the intact ovary. [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] Thus, it is important for the clinician to differentiate the two types of EFS and if FEFS has been identified, rescue protocol should be used to salvage the cycle. GEFS patients are unlikely to respond to a rescue protocol. FEFS should not recur, provided caution is taken to minimize its recurrence in the subsequent cycles. [bib_ref] Unsuccessful oocyte retrieval: Technical artefact or genuine 'empty follicle syndrome, Bustillo [/bib_ref] Optimal serum concentrations of hCG to predict EFS and a successful yield of mature oocytes are still not unequivocally defined. Various authors have reported various levels of serum β-hCG as the threshold cut-off value on the day of OR; 106 mIU/ml, [bib_ref] Predicting empty follicle syndrome, Ndukwe [/bib_ref] 40 mIU/ml, [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] 100 mIU/ml, [bib_ref] The empty follicle syndrome: A pharmaceutical industry syndrome, Zegers-Hochschild [/bib_ref] and 98-161 IU/l. [bib_ref] Oocytes in the empty follicle: A controversial syndrome, Aktas [/bib_ref] A serum hCG levels <10 mIU/ml as reported by Ndukwe et al. could predict EFS with a sensitivity and specificity of 100%. [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] Driscoll et al. reported a median serum hCG concentration of 117.1 IU/l (range: 48-249) after R-hCG 250 µg and 83.6 IU/l (range: 32-99) with 5000 IU urinary hCG [bib_ref] A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG..., Driscoll [/bib_ref] due to the immunoassays used to measure serum hCG. Urinary hCG may contain dissociated and oxidized subunits that would be detected by immunoassay but may have no biological activity. R-hCG, due to the absence of contaminant urinary proteins and the exacting standards applied during the production process, may make it possible to predict the risk of unsuccessful OR more accurately. [bib_ref] A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG..., Driscoll [/bib_ref] Various strategies suggested to prevent the occurrence of EFS in a subsequent ART cycle are: (1) Changing the batch of hCG. [bib_ref] Continuing the debate on empty follicle syndrome: Can it be associated with..., Awonuga [/bib_ref] [bib_ref] Empty follicle syndrome due to human errors: Its occurrence in an in-vitro..., Quintans [/bib_ref] [bib_ref] Curing empty follicle syndrome, Ndukwe [/bib_ref] (2) Using R-hCG to trigger an endogenous LH surge. R-hCG with its high purity (≥99%) and consistency between batches, may be a better choice than urinary hCG, which contains miscellaneous urinary proteins and the biological activity of which may be affected by missing peptide bonds and alterations of the glycosylation profile. [bib_ref] A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG..., Driscoll [/bib_ref] (3) shifting from an agonist to antagonist protocol. [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] (4) Use of recombinant LH as trigger. (5) Using GnRHa as trigger in an antagonist cycle. (6) Prolonging the interval between ovulation trigger and OPU. Droesch et al.
reported that retrieval of oocytes at 35-36 h is superior to retrieving at <24 h. [bib_ref] Timing of oocyte retrieval in cycles with a spontaneous luteinizing hormone surge..., Droesch [/bib_ref] In our case report, the serum levels of hCG and progesterone were confirmatory of the correct timing, dose of R-hCG administered in both the IVF cycles. On the day of OR, the serum β-hCG in the first and second IVF cycles was 165 mIU/ml and 154 mIU/ml respectively. Since the threshold values of β-hCG on the day of OR were assuring and in concordance to the levels reported above by various authors, we did not find it necessary on a second occasion to administer GnRHa as trigger.
In both the IVF cycles, folliculometry showed good follicular development and normal steroidogenesis. On the day of trigger, the LH and P4 levels in the first and second cycles were 1.9 IU/l, P4-0.2 ng/ml and LH = 2.4 IU/l, P4 = 1.01 ng/ml, respectively. Also, sonography on the day of oocyte pickup documented intact follicles in both the cycles with assuring peak LH levels precluding premature LH surge and ovulation as a cause of EFS.
Our patient was 32 years old with primary infertility which seems to be a typical profile in these cases as described by Levran et al. [bib_ref] Maturation arrest of human oocytes as a cause of infertility: Case report, Levran [/bib_ref] Her ovarian reserve tests were within normal limits which refutes the hypothesis of ovarian aging as a potential cause of the condition. She was a case of unexplained infertility and in the original report by Coulam et al., all patients were diagnosed as having unexplained infertility where the authors suggested that EFS itself might be a cause of infertility. [bib_ref] Empty follicle syndrome, Coulam [/bib_ref] Though, later studies have shown that EFS could occur in different categories of infertility. [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] Stevenson and Lashen have shown that about 41% of the GEFS couples had male factor infertility, indicating that most of the women who experienced EFS had no potential underlying pathology. [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] In patients with EFS, an altered steroid profile has been suggested to be a sign of dysfunctional ovulation induction. [bib_ref] Steroid profiles of follicular fluids from a patient with the empty follicle..., Tsuiki [/bib_ref] The follicular fluids in these patients showed high levels of estradiol (E2) and androstenedione with low progesterone levels. On the contrary, Zreik et al. reported low levels of estradiol concentrations due to hampered granulosa cell function and/or metabolism. [bib_ref] Empty follicle syndrome: The reality of a controversial syndrome, a systematic review, Stevenson [/bib_ref] In our case, the peak E2 and P4 concentrations in the first and second IVF cycles were: E2-2011 pg/ml; P4-0.2 ng/ml and E2-1856 pg/ml; and P4-1.01 ng/ml, respectively, with no demonstrable alterations as reported by few authors.
We concluded it as case of GEFS and the probable cause was within the cumulus-oocyte complex. Hence, it is possible to assume that a more physiologic induction of final follicular maturation with natural LH and FSH activity directly from the hypophysis and in terms of gonadotropin surge duration may optimize the signaling mechanisms from the surrounding cumulus and the oocyte, resulting in adequate OR and maturation. [bib_ref] Empty follicle syndrome after GnRHa triggering versus hCG triggering in COS, Castillo [/bib_ref] This can be achieved by the use of GnRHa as a trigger in GnRH antagonist cycles, the simultaneous induction of an FSH surge being an added advantage resembling a natural cycle. This FSH surge induces LH receptor formation in luteinizing granulosa cells, promotes oocyte nuclear maturation and cumulus expansion, opens the gap junctions between the oocyte and cumulus cells which are important in signaling pathways, allowing the oocyte-cumulus cell mass to detach from the follicular wall before ovulation. This might explain why some studies have reported retrieval of more mature oocytes after GnRHa trigger compared with hCG trigger. The FSH surge and the direct action of the agonist on the ovarian GnRH receptor might explain the favorable results in our patient.
We may assume that a GnRHa trigger alone in the third cycle may have resulted in adequate oocyte maturation and might have prevented EFS. We decided to give dual trigger so as to mimic the physiological FSH and LH surge and at the same time the addition of hCG would bring LH activity for luteal support to counteract the luteolytic effect seen after GnRHa trigger, "rescuing" the luteal phase insufficiency for a successful outcome. [bib_ref] Luteal phase rescue in high-risk OHSS patients by GnRHa triggering in combination..., Humaidan [/bib_ref] In patients who experience an empty cycle, EFS should be considered as a borderline form of poor response to ovarian stimulation and could be a recurrent event. If EFS has occurred once, the risk of recurrence is 20%. [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] As age advances, the risk of recurrence increases and is about 24% in patients between 35 and 39 years of age whereas it is about 57% for those >40 years of age. [bib_ref] Empty follicle syndrome: Evidence for recurrence, Zreik [/bib_ref] In this subset of population, empty cycle could be a good predictor that a subsequent stimulated cycle will be an unfavorable one and such patients need to be counseled regarding the risk of recurrence and might benefit with oocyte donation.
The relatively small risk of GEFS and the even smaller risk of recurrence indicate that it is a chance occurrence and does not represent a permanent pathophysiological condition [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] as is evident in our case.
It can also not be excluded that the 2 years, which passed between prior treatment failures and the successful treatment, could have influenced the outcome. Thus, EFS may be self-limiting phenomena with most cases occurring only sporadically. [bib_ref] Failure of oocyte retrieval during in vitro fertilization: A sporadic event rather..., Ben-Shlomo [/bib_ref] If EFS has been encountered in the previous cycles, different IVF treatment methods in the subsequent cycles like the use of dual trigger using GnRHa and hCG could modulate the response with successful oocyte recovery, pregnancy and term delivery, as proven in our case.
It is also the first case, whereby a combination of GnRHa and R-hCG was used as trigger for the treatment of GEFS, thereby adding a new management option in the armamentarium to this uncommon, yet distressing, and challenging condition.
## Financial support and sponsorship
Nil.
Journal of Human Reproductive Sciences / Volume 8 / Issue 3 / Jul -Sep 2015
Conflicts of interestThere are no conflicts of interest.
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Fracture of an epicardial left ventricular lead implanted at open‐heart surgery in anticipation of future need for cardiac resynchronization therapy
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.AbstractEpicardial left ventricular leads can be implanted at open-heart surgery for cardiac resynchronization therapy. We report a 2-year-old fractured epicardial left ventricular lead detected at generator implant. It highlights the importance of good surgical implant technique and of rigorous lead evaluation for signs of impending failure at generator implant. K E Y W O R D S cardiac resynchronization therapy, epicardial lead, lead failure, open-heart surgery 384 | LARSEN Et AL. How to cite this article: Larsen JM, Bashir J, Laksman ZW. Fracture of an epicardial left ventricular lead implanted at open-heart surgery in anticipation of future need for cardiac resynchronization therapy. Clin Case
# | introduction
Cardiac resynchronization therapy (CRT) improves survival and reduces morbidity in patients with heart failure with reduced left ventricular ejection fraction (LVEF) and significant interventricular conduction delay. 1,2 Transvenous implantation of a left ventricular (LV) lead via the coronary sinus can be achieved in >90% of patients. [bib_ref] Cardiac-resynchronization therapy for the prevention of heart-failure events, Moss [/bib_ref] Alternatively, a transthoracic epicardial LV lead can be deployed in cases of failed transvenous implantation or de novo at the time of open-heart surgery if a future need for CRT is considered likely. [bib_ref] Clinical outcomes of cardiac resynchronization with epicardial left ventricular lead, Chen [/bib_ref] [bib_ref] Effects of epicardial versus transvenous left ventricular lead placement on left ventricular..., Van Dijk [/bib_ref] However, outcome data on transthoracic epicardial LV leads and modes of failure are sparse.
This case report describes an inactive epicardial LV lead implanted at open-heart surgery in which multiple fractures caused by sternal wires were detected two years later during a CRT device implant. The contemporary literature on clinical outcomes and performance of surgically placed epicardial leads is briefly reviewed, and clinical considerations to detect possible impending epicardial LV lead failure at later CRT device implant are suggested.
## | case report
In 2016, a 77-year-old man underwent open-heart surgery with aortic root replacement using a valve conduit for a stenotic bicuspid aortic valve and associated aortic root dilatation. Preoperatively, the patient had mild nonobstructive coronary artery disease, LVEF 45%, and Left Bundle Branch Block (LBBB) with QRS duration 172 ms. The cardiac surgeon implanted a lateral epicardial bipolar suture-on LV lead (Medtronic 4968-35 Capsure Epi) with the lead body exiting at the suprasternal angle and with the silicone capped header placed in the left pectoral subcutaneous tissue assuming a likely future need for CRT .
Two years later in 2018, the patient developed progressive clinical heart failure New York Heart Association (NYHA) class III, LVEF 35%, and LBBB with QRS duration 176 ms. The patient had a class I indication for CRT. [bib_ref] ACC/AHA/HRS guideline on the evaluation and management of patients with bradycardia and..., Writing Committee Members [/bib_ref] At implant, the header of the capped epicardial LV lead in the subcutaneous tissue was mobilized with electrocautery and with gentle traction, turned out to have multiple overt fractures near the level of the suprasternal notch most likely caused by shredding over the fractured sternal wires . Inadvertent capture of the lead body by the sternal wires at implant may have contributed to the lead damage. Hence, a transvenous LV lead was implanted instead in a posterolateral coronary sinus tributary. All three transvenous leads had excellent pacing parameters and were connected to a CRTpacemaker in the subcutaneous pocket.
# | discussion
The contemporary CRT implanter has a range of possible transvenous coronary sinus delivery sheath systems, and LV lead designs to overcome anatomical challenges and thereby increase the probability of a successful implant. However, in the 2018 AHA/ACC/HRS pacing guidelines, implanting an epicardial LV lead at open-heart surgery is still a class IIb indication if future CRT is considered likely. [bib_ref] ACC/AHA/HRS guideline on the evaluation and management of patients with bradycardia and..., Writing Committee Members [/bib_ref] The present case report illustrates an example of what may be a rare mode of failure of an epicardial lead but highlights several key clinical issues to be considered in case a previously implanted inactive epicardial LV lead is to be used in a contemporary CRT system.
## | how is the clinical response to crt using a surgically placed epicardial lv lead versus a standard transvenous lv lead?
The response to CRT using a transvenous approach with reduced morbidity and mortality is well established. 1,2 Optimal transvenous LV lead placement is limited by the variable coronary venous anatomy, but contemporary transvenous lead delivery systems and use of quadripolar leads yield implant success rates of up to 97% with postoperative possibility of vector optimization and multipoint pacing to optimize CRT response. [bib_ref] Safety and efficacy of multipoint pacing in cardiac resynchronization therapy: the MultiPoint..., Niazi [/bib_ref] The alternate option is to surgically place an epicardial LV lead. This can be performed at concomitant openheart surgery or by less invasive video-assisted thoracoscopy and left-lateral mini-thoracotomy. There are no large randomized trials demonstrating noninferiority of CRT with a surgically placed LV leads compared to a transvenous LV lead. Two randomized trials with only 9 [bib_ref] Comparison of endovascular versus epicardial lead placement for resynchronization therapy, Garikipati [/bib_ref]
## | what are the failure rate and modes of surgically implanted epicardial lv leads?
The reason for lead failure in the current case was most likely mechanical stress by the sternum and shredding of the lead by the fractured sternal wires which inadvertently may have tied down the lead body at implant. An equivalent failure mechanism is subclavian crush of transvenous pacing leads inadvertently caught in the costoclavicular soft tissue. This emphasizes the universal importance of the implanter of pacing leads being to be very diligent to avoid unnecessary mechanical lead stress by exerting good implant technique no matter it being transvenous or epicardial.
Data on electrical performance, failure rates, and modes of failure of surgically implanted epicardial LV leads in patients with an indication for CRT are sparse. The largest report is a single-center studywith 216 leads of the same kind as presented in the current case report (Medtronic 4968 Capsure Epi). They demonstrated excellent pacing thresholds, sensing, and impedance during a mean follow-up of 3 years. Two lead-related implant complications were observed, that is, permanent phrenic nerve damage and coronary artery stenosis with myocardial infarction which underline the importance of meticulous surgical implant technique. The 5-year LV lead failure rate was 1.6% with high pacing thresholds, and the 5-year device infection rate was 9.6%. A small study [bib_ref] Surgical epicardial left ventricular lead versus coronary sinus lead placement in biventricular..., Mair [/bib_ref] with 19 mini-thoracotomy placed epicardial LV leads and 79 transvenous coronary sinus leads has demonstrated lower epicardial pacing thresholds 18 months postimplant with only 1 surgically placed lead needing repositioning. However, another small study 8 with only 9 thoracoscopically placed epicardial leads and 12 transvenous coronary sinus leads demonstrated a contrasting finding with much higher epicardial pacing thresholds 12 months postimplant. This could be explained by challenges with the thoracoscopic implant technique, and myocardial damage at the epicardial implant site may with time result in higher pacing thresholds. A study [bib_ref] Cardiac resynchronization therapy combined with coronary artery bypass grafting in ischaemic heart..., Romanov [/bib_ref] including 91 completely epicardial CRT systems with a mean follow-up of 55 months has demonstrated a low lead failure rate of 1.5% and no infections. Similarly, a small study [bib_ref] Epicardial left ventricular leads via minimally invasive technique: a role of steroid..., Caliskan [/bib_ref] with 32 sutureless screw-in epicardial LV leads placed by thoracoscopy or mini-thoracoscopy has demonstrated excellent sensing, impedance, and thresholds with a mean follow-up of 2.6 years with no clinically significant differences between leads with and without steroid elusion. Overall, limited clinical data suggest that epicardial LV leads have relatively low failure rates, and good implant technique is imperative to keep these at an acceptable level.
## | what should be considered by the crt implanter when planning to utilize a previously implanted epicardial lv lead?
There is an inherent risk of including a failing epicardial LV lead in a new CRT system as an indwelling inactive lead has no electrical track record. The present case report shows complete lead fractures of all conductors and insulation, and lead failure would therefore with certainty have been detected when connected to the device. Impending, but not overt, lead failure can be much more challenging to detect. We suggest the following steps to reduce the risk of introducing a failing epicardial LV lead to a new CRT system: Preprocedure; check available chest X-rays for signs of epicardial LV lead damage to insulation or conductors by using electronic image magnification with special focus on the thoracic exit point to the subcutaneous tissue; Intraprocedure: consider checking lead integrity with high frame rate fluoroscopy with special focus at the thoracic exit point to the subcutaneous tissue, and when testing the electrical integrity, include manual compression of the thoracic exit point of the lead while looking for lead noise and repeat impedance measurements. Furthermore, check that the position of the epicardial lead is basal posterolateral for optimal CRT and that the pacing threshold is acceptably low and without phrenic nerve stimulation. If not, consider implanting a new transvenous LV lead.
# | conclusions
Surgically implanted epicardial LV leads for use in CRT seem to perform well with low failure rates, but focus on good implant technique is imperative to reduce risk of lead failure. There are no large randomized trials directly comparing outcomes in CRT with bipolar surgical epicardial LV leads versus contemporary quadripolar transvenous leads with vector optimization. When possible, transvenous LV leads may, therefore, be preferable. Inactive epicardial LV leads have no electrical track record if implanted at open-heart surgery in anticipation F I G U R E 3 Focused postprocedure chest x-ray after the cardiac resynchronization therapy device implant in 2018 with three transvenous leads in the right atrium, right ventricle, and left ventricle via the coronary sinus, respectively. The abandoned part of the epicardial left ventricular lead was completely fractured at the suprasternal notch (superior arrow) with an isolated short extrathoracic subcutaneous fragment that also was severed from the explanted part of the lead at the level of one of the fractured sternal wires (inferior arrow)
[fig] F: I G U R E 1 Postoperative chest X-ray after open-heart surgery in 2016 with aortic root replacement using a valve conduit and concomitant implant of a bipolar epicardial left ventricular lead with the silicone covered header placed in the subcutaneous tissue in the left pectoral area. No signs of damage to the epicardial lead at implant I G U R E 2 Intraprocedure photo from the cardiac resynchronization therapy device implant in 2018 showing the proximal explanted fragment of the fractured epicardial lead [/fig]
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Renal artery stenosis presenting as preeclampsia
Background: Renal artery stenosis is a notorious cause of secondary hypertension which classically presents as chronic refractory hypertension, recurrent flash pulmonary edema or renal insufficiency after initiation of an angiotensin converting enzyme inhibitor. Rarely, there have been reported cases of pregnant patients presenting with new onset or superimposed preeclampsia secondary to renovascular hypertension. In this subset of patients, renovascular hypertension carries significantly higher risks including obstetric, fetal and medical emergencies and death. Prompt treatment is required. However, the teratogenic risks of radiological investigations and antihypertensive medications limit diagnostic and management options thus posing quite a dilemma. Case presentation: A 38-year-old female, at 33 weeks of gestation, was hospitalized for preeclampsia with severe features. A viable neonate had been expeditiously delivered yet the patient's post-partum blood pressures remained severely elevated despite multi-class anti-hypertensive therapy. Renal artery dopplers revealed greater than 60% stenosis of the proximal left renal artery and at least 60% stenosis of the right renal artery. Renal angiography showed 50% stenosis of the left proximal renal artery for which balloon angioplasty and stenting was performed. The right renal artery demonstrated less than 50% stenosis with an insignificant hemodynamic gradient, thus was not stented. Following revascularization, the patient's blood pressure improved within 48 h, on dual oral antihypertensive therapy. Conclusions: Preeclampsia that is refractory to multi-drug antihypertensive therapy should raise suspicion for renal artery stenosis. Suspected patients can be screened safely with Doppler ultrasonography which can be then followed by angiography. Even if renal artery stenosis does not seem severe, early renal revascularization may be considered in patients with severe preeclampsia who do not respond to antihypertensive management.
# Background
Renal artery stenosis is a notorious cause of secondary hypertension resulting from the activation of the reninangiotensin system in response to reduced renal blood flow. Classic presentations include chronic refractory hypertension, recurrent flash pulmonary edema and renal insufficiency after initiation of an angiotensin converting enzyme inhibitor. Although rare, there have also been reported cases of pregnant patients presenting with new onset or superimposed preeclampsia secondary to renovascular hypertension. In this subset of patients, renovascuar hypertension carries significantly higher risks including obstetric, fetal and medical emergencies and death. Prompt treatment is required. However, the teratogenic risks of radiological investigations and antihypertensive medications such as angiotensin converting enzyme inhibitors or aldosterone antagonists limit management options and poses quite the dilemma. When possible, expedited delivery is beneficial; notwithstanding the fact that there has been success with interventional treatment prior to successful delivery. Furthermore, even after delivery, the mortality risk of pre-eclampsia continues into the post-partum period thus urgent and aggressive treatment strategies should continue to be pursued for these patients including consideration of early revascularization.
## Case presentation
A 38-year-old female, gravida 3 para 2 at 33 weeks of gestation, was hospitalized for preeclampsia with severe features. A viable neonate had been expeditiously delivered yet the patient's post-partum blood pressures remained severely elevated ranging from 230/130 mmHg to 280/ 170 mmHg. She had no antenatal care but reported a history of uncomplicated hypertension during her prior pregnancies and tobacco abuse which was stopped 8 months prior. At the bedside, she complained of mild headaches but denied visual disturbances or upper abdominal pain.
She was alert and well oriented with a pulse of 80 bpm. There was no hyperreflexia, clonus, papilledema, peripheral edema or signs of pulmonary edema. Her examination was otherwise unremarkable including the absence of renal bruits. Apart from an elevated random urine protein to creatinine ratio of 0.7, the laboratory investigations were within normal limits including serum creatinine, electrolytes, platelet count, liver function and coagulation studies. There were no laboratory features of hemolysis. She was treated with multiple anti-hypertensives over the next 72 h including oral nifedipine, labetalol and clonidine as well as intravenous infusions of labetalol, nicardipine, hydralazine. Magnesium was used for eclampsia prophylaxis. Of note, a single dose of intravenous enalapril was given with a subsequent 60% increase in serum creatinine that returned to baseline within 24 h of discontinuation. Renal artery dopplerswere performed which greater than 60% stenosis of the proximal left renal artery and at least 60% stenosis of the distal right renal artery. Computerized tomography angiography showed approximately 50% stenosis of the proximal left renal artery without stenosis of the right renal artery . At this juncture, in the setting of recalcitrant severe preeclampsia and the mortality risk of impending eclampsia, an invasive strategy for better evaluation and possible intervention was deemed net beneficial. Renal angiography showed 50% stenosis of the left proximal renal artery for which balloon angioplasty and stenting was performed. The right renal artery demonstrated less than 50% stenosis with an insignificant hemodynamic gradient, thus was not stented. Following revascularization, the patient's blood pressure improved, ranging from 180/100 mmHg to 160/90 mmHg within 48 h, on dual oral antihypertensive therapy. She was ultimately discharged to titrate further anti-hypertensive therapy as an outpatient.
# Discussion
Renal artery stenosis is a well-established cause of secondary hypertension resulting from the activation of the renin-angiotensin system in response to reduced renal blood flow. Atherosclerosis is the most common etiology and is usually suspected in patients over the age of 45, dyslipidemic patients, or smokers. However, other etiologies such as fibromuscular dysplasia in younger patients or Takayasu's arteritis should be considered. Atherosclerotic stenosis typically affects the proximal main renal artery near the ostium compared to fibromuscular dysplasia which typically affects the distal segments.
Classic presentations include chronic refractory hypertension, recurrent flash pulmonary edema and renal insufficiency-notably after initiating an angiotensin converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB). Although rare, there have also been reported cases of pregnant patients presenting with new onset or superimposed preeclampsia secondary to renovascular hypertension.
Of the different modalities used to investigate renal artery stenosis, doppler ultrasonography is the safest and has a sensitivity of at least 85%, though it frequently overestimates stenoses as in our case. Magnetic resonance or computerized tomography angiography have superior diagnostic accuracy with a sensitivity of 94% but the gold standard remains conventional catheter based angiography. Supplemental studies such as direct renal vein renin, captopril renography or plasma renin activity to aldosterone ratios may be helpful in diagnostic dilemmas, though not currently routinely recommended.
Treatment may involve aggressive medical therapy with statins, antiplatelets and antihypertensives and/or renal artery revascularization. Historically, ACE-I or ARB therapy has been cautioned especially in bilateral renal artery stenosis because of the possibility of reduced post-stenotic renal perfusion pressures and subsequent ischemic nephropathy and renal failure. However, there have been observational studies suggesting a mortality benefit to closely monitored ACE-I or ARB treatment.
In terms of invasive treatment, percutaneous transluminal renal angioplasty with or without stenting has become the standard versus surgical revascularization. Although a recent systematic review showed only marginal benefit to this approach compared to medical therapy alone, there is evidence that select patient do have significant benefits in blood pressure control. Furthermore, studies have shown that usually at least 80% stenosis is required to produce any significant hemodynamic stimulus to the renin-angiotensin system and thus may be a threshold for invasive treatment. However, as in our case, few patients have been shown to benefit from revascularization at stenoses of as Computerized tomography of the right (a) and left (b) proximal renal arteries (arrows). Approximately 50% stenosis of the left renal artery is noted low as 50%. Additionally, these hemodynamic studies were performed in non-pregnant patients. Thus, whilst these data are important to avoid unnecessary procedures, clinical acumen remains necessary for select cases where revascularization of seemingly insignificant stenoses may yet produce a clinical response.
In women with preeclampsia due to renovascular hypertension, there is significant risk for obstetric and medical complications including death especially with severe preeclampsia (blood pressures more than or equal to 160/90 mmHg even without signs of end organ dysfunction or hemolysis). Prompt treatment is required. Yet, the teratogenic risks of radiological investigations and antihypertensive medications such as ACE-I/ARB's limit diagnostic and management options and pose quite a dilemma. When possible, expedited delivery is beneficial. However, there has been some success with interventional treatment prior to delivery. The high mortality risk of eclampsia continues into the the postpartum period and it is uncertain when blood pressures can be expected to normalize in preeclampsia even in the absence of renovascular hypertension. Therefore, an urgent and aggressive management strategy should be pursued for these patients with consideration for early revascularization if a rapid clinical response is not seen with medical management.
# Conclusions
Preeclampsia that is refractory to multi-drug antihypertensive therapy should raise suspicion for renal artery stenosis. Suspected patients can be screened safely with Doppler ultrasonography which can be then followed by angiography. Even if renal artery stenosis does not seem severe, early renal revascularization may be considered in patients with severe preeclampsia who do not respond to antihypertensive management.
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Multifork chromosome replication in slow-growing bacteria
The growth rates of bacteria must be coordinated with major cell cycle events, including chromosome replication. When the doubling time (Td) is shorter than the duration of chromosome replication (C period), a new round of replication begins before the previous round terminates. Thus, newborn cells inherit partially duplicated chromosomes. This phenomenon, which is termed multifork replication, occurs among fast-growing bacteria such as Escherichia coli and Bacillus subtilis. In contrast, it was historically believed that slow-growing bacteria (including mycobacteria) do not reinitiate chromosome replication until the previous round has been completed. Here, we use single-cell time-lapse analyses to reveal that mycobacterial cell populations exhibit heterogeneity in their DNA replication dynamics. In addition to cells with non-overlapping replication rounds, we observed cells in which the next replication round was initiated before completion of the previous replication round. We speculate that this heterogeneity may reflect a relaxation of cell cycle checkpoints, possibly increasing the ability of slow-growing mycobacteria to adapt to environmental conditions. Bacteria must precisely coordinate growth rate with major cell cycle events, including chromosome replication. The bacterial cell cycle can be divided into three periods: the time between cell division and the initiation of replication (B period); chromosome replication (C period); and the time between the termination of replication and the completion of subsequent cell division (D period) 1 . When the doubling time (Td) is shorter than the duration of chromosome replication (C period), a new round of replication begins before the previous round terminates. Thus, newborn cells inherit partially duplicated chromosomes. This phenomenon, termed multifork replication, is well described in fast-growing model organisms 1-11 but has not previously been observed in slow-growing bacteria such as mycobacteria. Mycobacteria exhibit an unusual mode of cell elongation and division[12][13][14][15]. In contrast to Escherichia coli and Bacillus subtilis, they grow by polar extension and often divide asymmetrically to generate daughter cells that differ in size. Recent studies used DnaN-FP (FP stands for fluorescent proteins such as EGFP or mCherry) to mark the sliding clamp and enable visualization of DNA replication forks (replisomes) in mycobacteria16,17. In these studies, the appearance and disappearance of DnaN-FP foci indicate assembly and disassembly, respectively, of the replisome complex and are considered to correspond to the initiation and termination of DNA replication, respectively. These studies found that DNA replication occurs near the midcell and that the replisomes are highly dynamic, frequently splitting over a short distance and then merging back together. These prior reports also revealed that shortly after the initiation of chromosome replication, two copies of a newly replicated chromosomal origin (oriC) are bound by the segregation protein (ParB) and move towards the cell poles. Moreover, it was found that after mycobacteria complete chromosome replication, a new round of replication is initiated prior to cytokinesis of the mother cell (monitored using Wag31-GFP) and is completed in the daughter cells 12 . However, the processes involved in the multifork replication of mycobacteria have not yet been described.Results and DiscussionIn our earlier time-lapse fluorescence microscopic (TLFM) analyses of single-cell replisome dynamics in Mycobacterium smegmatis, we noted that a subfraction of cells had more than two DnaN-FP foci 17 . Moreover, distances between individual DnaN-FP foci were frequently longer than the corresponding distances in cells with two fluorescent spots. The presence of widely separated DnaN foci could reflect that a sliding clamp is associated longer with the template (especially on lagging-strand Okazaki fragments), that DnaN-mediated DNA repair and recombination is underway[18][19][20][21]or that chromosome replication has been re-initiated.
Scientific RepoRts | 7:43836 | DOI: [bib_ref] The midcell replication factory in Bacillus subtilis is highly mobile: implications for..., Migocki [/bib_ref].1038/srep43836
To assess whether the appearance of additional DnaN foci reflects replication reinitiation, we visualized the alpha (catalytic) subunit, which is directly involved in DNA synthesis. We constructed two strains: one encoding the DNA polymerase III alpha subunit fused with EYFP and one encoding the two replisome fluorescent markers, alpha-EYFP and DnaN-mCherry. In both strains, the fusion proteins were expressed from their native loci. A snap-shot analysis revealed that the localization pattern of alpha-EYFP was similar to those observed in the DnaN-FP strains [fig_ref] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to... [/fig_ref]. To test whether fusion of the alpha subunit to EYFP disturbed DNA replication, we used TLFM to compare the cell cycle parameters of the analyzed fluorescent reporter strains. We found that the duration of DNA replication was shorter in the strain expressing the sliding clamp fused with mCherry (120 + /−9 min) than in those expressing the alpha subunit fused to EYFP or both fluorescent replisome markers (145 + /−10 min, . Overall, replication was slightly delayed in strains expressing the catalytic subunit fused to EYFP. However, we did not note any significant difference in the growth rates of the alpha and DnaN strains (see growth curves, [fig_ref] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to... [/fig_ref]. The lack of a difference in growth rate was most likely a consequence of the shortened B and/or D periods of the alpha-labeled strain (see . Next, we used TLFM to simultaneously track the dynamics of both replisome subunits at 2-min intervals. During replisome assembly, both subunits appeared nearly simultaneously [fig_ref] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to... [/fig_ref]. In contrast, the DnaN-mCherry focus disappeared up to 10 min after alpha-EYFP disassembly in most (98%) cells [fig_ref] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to... [/fig_ref]. Thus, our data confirmed that the sliding clamp had a longer association period with the DNA template than the alpha subunit.
Because both the DnaN-mCherry and alpha-EYFP strains have some limitations (longer association with the DNA and delayed DNA synthesis, respectively), parallel studies were performed using fluorescent replisome markers. We tracked the spatiotemporal dynamics of replisomes at different time intervals. The dynamics of alpha-labeled replisomes (Supplementary Movie 1) were similar to those previously described for DnaN-FP-marked replisomes. Our TLFM analysis of both reporter strains revealed that a relatively large M. smegmatis strain Temp.
[°C] . Cell cycle parameters of alpha-EYFP* and DnaN-FP** strains grown in rich medium at various temperatures. *The alpha-EYFP and alpha-EYFP/ParB-mCherry strains were equivalent for the listed parameters; n = 100. **The DnaN-FP (mCherry or EGFP) and ParB-mNeon strains were equivalent for the listed parameters; n 37 = 84, n 30 = 62, n 25 = 61. Generation time was measured as the time between two subsequent initiation events (in mother cell and daughter cells). This corresponds to the total of C + B + D duration. fraction of cells (more than 10% of each) contained multiple alpha-or DnaN-FP foci and that these were often widely separated [fig_ref] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to... [/fig_ref]. A careful examination of cells with widely separated foci revealed that the appearance of new alpha-or DnaN-FP-marked replisomes preceded the disappearance of earlier foci, suggesting the onset of another round of replication (see [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref] -c and Supplementary Movies 2 and 3). For DnaN-FP, we only considered cells in which an additional replication round began a minimum of 10 min before the previous replisome spot(s) disappeared (i.e., longer than the interval between the disappearances of alpha-EYFP and DnaN-mCherry). The initiation of a new round of replication should be followed by the subsequent separation of daughter oriCs, which can be monitored by visualizing ParB complexes. As ParB-DNA complexes do not disassemble at any stage of the cell cycle, ParB-FP represents a reliable marker of oriC [bib_ref] Characterization of the mycobacterial chromosome segregation protein ParB and identification of its..., Jakimowicz [/bib_ref]. To directly monitor the duplication of oriC regions, we replaced the parB gene with a parB-mneon or parB-mcherry fusion gene in the DnaN-mCherry or alpha-EYFP strains, respectively. In canonically dividing cells, the ParB-FP focus will duplicate once per cell cycle, soon after the appearance of DnaN-mCherry (or alpha-EYFP). Thereafter, both nascent foci move towards the cell poles [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref]. In multifork cells (Supplementary Movies 4 and 5) during the first round of replication, we observed that the oriC is duplicated (similar to cells with non-overlapping rounds of replication) almost immediately after replisome appearance (phase I in [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref] ; the sister oriCs segregate to positions near the cell poles. In these cells, however, we observed that an additional replisome focus appeared at one of the segregated oriCs (phase II); there was a subsequent additional oriC duplication event (phase III). In a substantial fraction of these reinitiating cells (90%), only one of the two previously segregated oriCs underwent a second duplication event, which occurred several frames (taken at 5-or 10-min intervals) before the first round of replication terminated (i.e., the first replisome disappeared). As a consequence of this asynchronous reinitiation, three oriC regions (i.e., ParB foci) were observed per single cell: two near the cell poles and a third close to the midcell (see [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref]. In the remaining 10% of reinitiating cells, both sister oriCs were reduplicated. We observed a larger fraction of multifork cells in the alpha-EYFP/ParB-mCherry strain (18%, n = 205) than in the DnaN-mCherry/ParB-mNeon strain (11%, n = 417). Presumably, this difference indicates that the C-period is longer and the B and/or D periods are shorter in the alpha-EYFP strain compared to the DnaN-mCherry strain. Together, our findings clearly demonstrate that in a subset of M. smegmatis cells, a new round of replication begins before the previous one has terminated. Interestingly, in mycobacteria only one of the two sister oriC regions is typically reduplicated, while in fast-growing bacteria such as E. coli and B. subtilis, reinitiation is triggered at both oriCs.
To exclude the possibility that the fusion of a fluorescent protein with a replisome subunit could affect replication dynamics and trigger the observed reinitiation, we examined this phenomenon using a strain that expressed only the ParB-mNeon fusion. Staining with the membrane-specific dye FM5-95 revealed that even though most dividing cells (89%) had two ParB-mNeon foci, there were a number of cells (11%) in which additional duplication(s) of ParB foci preceded septum formation, usually resulting in three distinct ParB-mNeon foci per cell [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref]. The division of mother cells with three ParB foci usually gave rise to two daughter cells of unequal size: one with a single ParB complex and one with two ParB complexes [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref]. Thus, one cell inherited a complete chromosome, while the other contained two partially replicated chromosomes (phase IV in [fig_ref] Figure 2: Multifork DNA replication in mycobacteria [/fig_ref].
The replication reinitiation observed in a fraction of M. smegmatis cells raises an interesting question: Do these cells exhibit different growth and/or division patterns? A closer look revealed that cells exhibiting multifork replication were longer at birth than their non-reinitiating counterparts (5.9 μ m vs. 4.9 μ m, respectively, p = 1.72e-06 by t-test; [fig_ref] Figure 3: Comparison of various features between canonically replicating and over-replicating cells [/fig_ref]. In addition, septum placement was more often asymmetric (p = 6.84e-08; [fig_ref] Figure 3: Comparison of various features between canonically replicating and over-replicating cells [/fig_ref] , the elongation velocity was greater (2.0 μ m h −1 vs. 1.7 μ m h −1 , respectively, p = 1.818e-05 by t-test; [fig_ref] Figure 3: Comparison of various features between canonically replicating and over-replicating cells [/fig_ref] , and the doubling time was shorter (136 vs. 158 min, respectively, p = 0.0030; [fig_ref] Figure 3: Comparison of various features between canonically replicating and over-replicating cells [/fig_ref] in multifork-replicating cells than in cells with non-overlapping C-cycles. When we compared the duration of the C-period in cells that undergo multifork replication with the corresponding period in the mother cell (i.e., from the previous generation), we failed to detect a statistically significant difference between the two groups. Thus, reinitiation was not triggered in daughter cells as a means to compensate for a longer replication time in the mother cell. As it was difficult to observe the dynamics of multiple foci within a single cell, we could not exclude the possibility that reinitiation may compensate for elongated replication in the same generation in which a multifork event occurs. Analyses of the growth parameters of reinitiating cells suggested that this reinitiation might be advantageous: cells with overlapping C-cycles grow faster and divide more frequently than normal cells.
In E. coli and B. subtilis, multifork replication occurs under optimal temperature and nutrient conditions 3,23,24 . To test whether these parameters are also crucial for the occurrence of over-replication in M. smegmatis, we reduced the temperature in the TLFM analyses. As expected, the time required for chromosome replication was found to be longer at lower temperatures when we compared the C-period obtained at 37 °C (120 + /−9 min) with those obtained at 30 °C (170 + /−16 min) and 25 °C (226 + /−24 min) . However, we still observed multifork replication at lower temperatures. Surprisingly, the highest fraction of cells with overlapping C-periods was observed at the lowest tested temperature (25% of cells at 25 °C vs. 11% at 37 °C). At both non-optimal temperatures, cells with overlapping C-cycles were significantly longer and had a significantly higher elongation velocity compared to their normally growing counterparts (p < 0.005 for all comparisons; [fig_ref] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to... [/fig_ref]. We also analyzed the replication dynamics of cells grown in minimal medium (M9 broth supplemented with 0.05% Tyloxapol and 0.1% glycerol) at 37 °C. Even under such conditions, we still observed multifork replication (data not shown), although the fraction of reinitiating cells was lower (6%) than the corresponding fraction of cells growing in rich medium (11%). Thus, unlike E. coli, mycobacteria do not appear to employ replication reinitiation exclusively under optimal growth conditions. Our observation that there is cell-to-cell variation in the replication dynamics of M. smegmatis raises the following question: Is this heterogeneity generated by stochastic or deterministic mechanisms? This is particularly interesting considering the peculiar mode of growth and division of mycobacteria, which results in daughter cells that differ in both size and elongation rate. A recent study indicated that there is a deterministic component to this cell-to-cell heterogeneity in size and elongation rate [bib_ref] Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic..., Aldridge [/bib_ref]. However, we observed only minor differences in the multifork replication frequencies between cells that inherited new and old poles (57% vs. 43%, respectively). Thus, our data suggest that chromosome replication reinitiation may be a random process in M. smegmatis. Moreover, we speculate that asynchronous reinitiation may be a consequence of competition between sister cell origins for limiting replication factor(s), such as proteins responsible for replication initiation and/or pools of precursors that are required for DNA synthesis in slow-growing bacteria. Asynchronous reinitiation may also be a consequence of the absence of a sequestration mechanism. We cannot exclude the possibility that heterogeneity in replication dynamics may be a consequence of relaxed cell cycle checkpoints. It may be tempting to speculate that such relaxation may be associated with a mycobacterial ability to enter a dormant state and/or return to active cell division.
In summary, we unexpectedly discovered multifork replication in a slow-growing bacterium, namely M. smegmatis. This phenomenon is even more intriguing when we consider that under optimal growth conditions, the doubling time is longer than the C-period. Moreover, our studies demonstrate for the first time that a clonal population of M. smegmatis exhibits cell-to-cell heterogeneity in replication dynamics: some cells exhibit multifork replication, while others do not. Surprisingly, we observed a larger fraction of over-replicating cells under unfavorable growth conditions compared to optimal conditions (24% vs. 11% when grown at 25 °C and 37 °C, respectively). Over-replicating cells extended more quickly and had shorter doubling times than canonically dividing cells. We speculate that the cell-to-cell heterogeneity in chromosome replication dynamics may result from relaxed cell cycle checkpoints and that this helps M. smegmatis adapt to different environmental conditions (e.g., nutrient limitations or lower temperatures). Our results can be extrapolated to other species of Mycobacterium, including M. tuberculosis, because most of the cell cycle proteins, including those involved in chromosome replication, are nearly identical. For example, DnaN protein from M. smegmatis shares high homology with the corresponding protein from M. tuberculosis (99% identity).
# Methods
Bacterial strains and plasmids. All plasmids used for mycobacterial transformation were propagated in the E. coli DH5α strain. Cells were incubated in LB broth or on LB agar plates (Difco) supplemented with proper antibiotic(s) (ampicillin, kanamycin) and/or other compounds (X-Gal, isopropyl-β -D-1-thiogalactopyranoside [IPTG]) according to standard procedures. Mycobacterial strains were cultured either in 7H9 broth or on 7H10 agar (Difco) supplemented with OADC (BD) and 0.05% Tween80 and/or proper antibiotics. Strains, plasmids and primers are listed in .
The allelic replacement of genes encoding the alpha (Msmeg_3178) or beta (dnaN, Msmeg_0001) subunits of polymerase DNA III with either alpha-eyfp or dnaN-mcherry fusion genes, as well as the replacement of the parB gene with a parB-mneon green/parB-mcherry fusion gene, was performed as previously described [bib_ref] Use of a flexible cassette method to generate a double unmarked Mycobacterium..., Parish [/bib_ref]. The eyfp gene with a short DNA fragment that encoded a 10-amino acid sequence at the 5ʹ -terminus was PCR-amplified using the primers L_EYFP_Eco_Fw and L_EYFP_STOP_Nhe_Rv. Flanking sequences containing the Msmeg_3178 gene were amplified using mc 2 155 (WT) chromosomal DNA as a template and the primer pairs alpha1_Bam_Fw and alpha1_Bsr_Nhe_Rv and alpha2_Bsr_Nhe_Fw and alpha2_Pac_Rv (Supplementary . The latter two products were cloned into a p2NIL (kan R ) vector, and an EYFP-encoding fragment was inserted at the EcoRV and NheI restriction sites. The obtained plasmids were verified by sequencing. Finally, the pGoal17 cassette was cloned into the PacI site of the p2NIL derivative. The p2NILdnaN-mCherry-pGoal plasmid was constructed as previously described. To prepare the p2NilparB-mneon vector, the mneon green gene preceded by a 19-aa encoding linker was amplified from pVV04 (kindly provided by Dr. R. Reyes-Lamothe) using the primers KK_Neon_ Fw and KK_Neon_Rv. Next, the vector p2NILparB-mcherry 27 was amplified with the primers KK_ParBNeon_Fw and KK_ParBNeon_Rv. Gibson Assembly (NEB) was used to assemble the two PCR products, and the pGoal17 cassette was cloned into the PacI site of the p2NIL derivative. M. smegmatis cells were transformed with 100 ng of NaOH/EDTA-treated plasmid DNA, and the unmarked mutants were selected as previously described [bib_ref] Enhanced gene replacement in mycobacteria, Hinds [/bib_ref].
The alpha-EYFP and ParB-mNeon strains were generated by transformation with p2NIL alpha-EYFP-pGoal and p2NIL-parB-mNeon-pGoal, respectively. The alpha-EYFP/DnaN-mCherry and ParB-mNeon/ DnaN-mCherry strains were obtained by transforming the alpha-EYFP and ParB-mNeon strains with p2NIL DnaN-mCherry-pGoal. Alpha-EYFP/ParB-mCherry was obtained by transforming alpha-EYFP with p2NILparB-mCherry-pGoal [bib_ref] ParA of Mycobacterium smegmatis co-ordinates chromosome segregation with the cell cycle and..., Ginda [/bib_ref]. The correct allelic replacement and incorporation of integration vectors were confirmed via PCR, Southern blotting and Western blotting. The fusion of the functional fluorescent protein was confirmed using semi-native SDS-PAGE followed by analysis on a Bio Rad PharosFX-Plus Molecular Imager. Western blotting was performed with polyclonal anti-mCherry and/or monoclonal anti-GFP antibodies (Santa Cruz Biotechnology) using standard procedures Microscopy. For live-cell snap-shot imaging, M. smegmatis was grown to mid-log phase (OD 600 = 0.5) in liquid medium, centrifuged (8000 rpm, 5 min), resuspended in PBS, and smeared onto microscope slides. The samples were dried, mounted on coverslips using 5 μ l of PBS-glycerol (1:1) solution, and visualized using a Delta Vision Elite imager (GE Healthcare) equipped with softWoRx software (provided with GE Healthcare equipment). Analyses were performed using the "R"and FIJI software platforms (Image J; https://imagej. nih.gov). For all measurements, two-sided, parametric Student's t-test was applied. To avoid generation of false assumptions in the case of non-normal distributions, the statistical significance of differences in measured values was confirmed with the non-parametric two-sided Wilcoxon test with minimum 0.995 confidence intervals. For time-lapse observations of the ParB-mNeon strain stained with FM5-95 (membrane dye), cells in mid-log phase were spread on 7H10 agar (supplemented with 10% OADC, 0.5% glycerol, and 0.25 μ g/ml FM5-95) and placed in an ibidi 35-mm dish (ibidi GmbH, Germany). Images were acquired at 10-min intervals at 37 °C.
Microfluidic experiments. TLFM was performed as previously describedusing the CellASIC Onix platform (Merck Millipore). For all experiments, 1.5 psi pressure was applied to the incubation plate. The data were collected using a Delta Vision Elite imager equipped with SoftWorx software and analyzed with the "R"and FIJI software platforms.
[fig] Figure 1: Kinetics of the appearance and disappearance of DnaN-mCherry foci with respect to alpha-EYFP foci. (top panel) Initiation of DNA replication; t = 0 indicates the simultaneous appearance of both replisome subunits. (bottom panel) Termination of DNA replication; t = 0 indicates the simultaneous disappearance of both replisome subunits (n = 53). Scientific RepoRts | 7:43836 | DOI: 10.1038/srep43836 [/fig]
[fig] Figure 2: Multifork DNA replication in mycobacteria. (a) Replisome patterns of canonically replicating and multifork cells. In most (90%) non-canonically replicating cells, only one of the two previously segregated origins (oriC) undergoes a second duplication. Chromosomes within cells (gray line), oriC/ParB (magenta dot) and replisomes (green dot) are shown schematically. Examples of cells from different cell cycle phases (I-IV) are shown below (b and c). (b and c) Replisome dynamics in cells with overlapping rounds of DNA replication. TLFM of cells expressing alpha-EYFP (green) (b) or DnaN-mCherry (magenta) (c). The oriC region was visualized using ParB protein fused to mCherry (b) or mNeon (c). Scale bar, 5 μ m. d, Origin localization in canonically replicating and multifork cells. TLFM of cells expressing ParB-mNeon (green focus). Cell walls were stained with FM 5-95 (styryl dye, magenta color). Scale bar, 5 μ m. [/fig]
[fig] Figure 3: Comparison of various features between canonically replicating and over-replicating cells. (a) Cell length. (b) Shifting of the division site with respect to the midcell. (c) Elongation velocity. (d) Doubling time (n = 50). Doubling time was measured as time between two subsequent "snapping" events. [/fig]
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Objective Prediction of Hearing Aid Benefit Across Listener Groups Using Machine Learning: Speech Recognition Performance With Binaural Noise-Reduction Algorithms
The simulation framework for auditory discrimination experiments (FADE) was adopted and validated to predict the individual speech-in-noise recognition performance of listeners with normal and impaired hearing with and without a given hearing-aid algorithm. FADE uses a simple automatic speech recognizer (ASR) to estimate the lowest achievable speech reception thresholds (SRTs) from simulated speech recognition experiments in an objective way, independent from any empirical reference data. Empirical data from the literature were used to evaluate the model in terms of predicted SRTs and benefits in SRT with the German matrix sentence recognition test when using eight single-and multichannel binaural noisereduction algorithms. To allow individual predictions of SRTs in binaural conditions, the model was extended with a simple better ear approach and individualized by taking audiograms into account. In a realistic binaural cafeteria condition, FADE explained about 90% of the variance of the empirical SRTs for a group of normal-hearing listeners and predicted the corresponding benefits with a root-mean-square prediction error of 0.6 dB. This highlights the potential of the approach for the objective assessment of benefits in SRT without prior knowledge about the empirical data. The predictions for the group of listeners with impaired hearing explained 75% of the empirical variance, while the individual predictions explained less than 25%. Possibly, additional individual factors should be considered for more accurate predictions with impaired hearing. A competing talker condition clearly showed one limitation of current ASR technology, as the empirical performance with SRTs lower than À20 dB could not be predicted.
# Introduction
With worldwide increasing life expectancy and (agerelated) hearing disorders, a steadily increasing demand for high-performance hearing aids (HA) with effective algorithms that improve the everyday communication will occur. To make the development of HA algorithms more efficient and to improve the prescription of a certain HA processing scheme to the individual hearing-impaired (HI) user, valid and reliable objective methods for the prediction of the individual benefit are highly desirable. The current article therefore proposes and evaluates a new method for pursuing this task by extending the recently proposed framework for auditory discrimination experiment prediction applied to the individual aided speech-in-noise recognition performance with and without a given HA algorithm in realistic binaural listening conditions. This extension includes the simulation of the respective HA processing for the individual user, an approximate simulation of the effect of the individual hearing loss , and an approximation of the binaural hearing performance by considering the better ear listening effect.
Many signal processing schemes have been proposed with the aim of improving speech recognition in noisy environments, with the most promising ones using multiple microphone input signals (as opposed to singlechannel, monaural processing schemes, see . Because all HA algorithms operate best under certain assumptions about the desired target signal and undesired interfering sound components, the real-life performance of these algorithms with arbitrary acoustic situations is hard to predict, especially for real users with impaired hearing. To assess the utility of a hearing device, comprehensive performance tests have to be carried out in a variety of acoustical situations and a variety of individual users with a hearing impairment both in aided and unaided conditions. This makes the determination of the optimum HA processing and, even more so, the development of new and more effective hearing devices, very time and effort consuming. Consequently, suitable and accurate models of the binaural speech recognition for listeners with impaired hearing that are applicable to unaided and aided realistic conditions are desired to make the development-evaluation cycle of HA algorithms more efficient.
As outlined in more detail in [fig_ref] Table 1: Classification of Models That Can Be Used to Predict the Speech Recognition... [/fig_ref] , numerous models that can be used to predict the performance of human listeners in speech recognition tests in noise consider either the effect of hearing impairment or (nonlinear) signal preprocessing (e.g., by HAs). Fewer approaches were proposed which consider a combination of, and hence, the interplay between both. Many models were not designed to be used with fluctuating noise maskers, which are prevalent in many realistic communication environments. Finally, only few models can work natively on binaural signals. In addition to possible use cases, models can be characterized by their assumptions and the underlying a priori information entering the prediction of speech reception thresholds (SRTs) that are quite diverse and can limit their applicability. Two distinct categories of assumptions can be identified that have far-reaching implications:
. Superhuman a priori knowledge of various degrees, such as access to separated speech or noise signals in any other way than it would be required to perform the speech test, that is, to generate mixed signals with a defined signal-to-noise ratios (SNRs). Such a priori knowledge could include, for example, frozen signal parts, ideal binary masks, or calculation of time/frequency-dependent SNRs. We will refer to this category as models with intrusive classification. . Outcome calibration using an empirical reference to predict the outcome of a speech recognition test, which generally means that empirical speech recognition data are used to set parameters of the model. This is accompanied by the assumption that the model correctly reflects desired properties of the real world in the reference condition because the prediction in the reference condition is by definition ''correct.'' We will refer to this category as (empirical) reference-based models.
Here, we define single-ended classification as nonintrusive and double-ended classification as intrusive, in close analogy to the case of empirical measurements of SRTs. Classification in this context refers to the decision for the recognized items, for example, words, or any mapping to a scale in the evaluation of the recognized items, for example, word recognition rates. By this definition, measurements of empirical SRTs are nonintrusive, even if the listeners heard the clean sentences before performing the speech recognition test. [fig_ref] Figure 1: Characterization and examples for intrusive and nonintrusive classification [/fig_ref] illustrates the definition with some examples. Hence, the best example for nonintrusive classification is the measurement of SRTs with human listeners. To perform the speech test, separate speech and noise signals are required to mix them at a desired SNR and optionally process them with some arbitrary algorithm before presenting the mixed and processed signal to the listener. During measurements of SRTs, listeners have access only to the mixed and processed signals. Prior to the measurements with the matrix sentence test, a few training lists usually allow the listeners to adapt to the speech material; and even clean speech material might be presented. The measurement itself is intrusive because the separate signals are required to perform it, but the classification (here the human listener) is not because she or he does not rely on signals prior to mixing and processing to solve the speech recognition task, for example, by listening to the clean speech or noise signal on one ear. An example for a possibly nonintrusive measurement of an SRT with human listeners would be to ask them to estimate the SRT.
Models with intrusive classification solve a different task than the human listeners, as they have access to more information about the solution. Please note, however, that access to the separate signals does not reveal the empirical SRT. In other words, knowing the exact testing conditions is different information than knowing the empirical outcome of the corresponding test when it is performed with human listeners.
The category of models with intrusive classification can be subdivided into those models that require (parts of) the separate signals (1) after or (2) before a potential nonlinear signal processing (e.g., compression, clipping, or frequency shifts by HAs). The first subdivision allows for a combination with arbitrary signal processing algorithms, for example, to predict the benefit of HAs, while the second subdivision does not directly provide this Here, this property describes the dependency of the classifier, that is, detector, on signals that are typically not available to human listeners during a speech recognition test. From this perspective, nonintrusive means that the classification is single-ended, that is, the classification is performed on mixed and processed data, while intrusive means that the classification is double-ended, that is, it requires additional signals. By this definition, the measurement of SRTs with human subjects is nonintrusive. Intrusive classification can require additional assumptions about the signal processing (Intrusive (1)), for example, by estimating the speech and noise portions of the signal [bib_ref] A method to measure the effect of noise reduction algorithms using simultaneous..., Hagerman [/bib_ref] , or just use the signal processing but take the clean speech or noise signal as further inputs (Intrusive (2)). SRMR ¼ speech-to-reverberation modulation energy ratio; STOI ¼ Short-Time Objective Intelligibility; HASPI ¼ Hearing-Aid Speech Perception Index. option because adding signal and noise and the nonlinear processing is generally not permutative. A couple of modeling approaches that can be used to predict the speech recognition performance in noise of human listeners are presented in [fig_ref] Table 1: Classification of Models That Can Be Used to Predict the Speech Recognition... [/fig_ref] together with the corresponding classification whether . they were designed to take hearing-impairment into account (HI), . they can be used with nonlinear hearing-aid algorithms (HA), . they were designed to be used with fluctuating noise maskers (F), . they can be used in binaural conditions (B), . they use intrusive classification (I), . or reference-based (R).
Please note that [fig_ref] Table 1: Classification of Models That Can Be Used to Predict the Speech Recognition... [/fig_ref] attempts a coarse technical classification of existing modeling approaches, reflecting the necessary requirements for aided predictions in realistic binaural listening conditions and contains by no means statements about the extensibility nor prediction accuracy for any particular model or task.
The overview in [fig_ref] Table 1: Classification of Models That Can Be Used to Predict the Speech Recognition... [/fig_ref] reveals that many models are designed for rather specific use cases, and no approach fulfilled all the technical requirements for aided predictions in realistic binaural listening conditions. SNRbased models, such as the Speech Transmission Index [bib_ref] A physical method for measuring speech transmission quality, Steeneken [/bib_ref] [bib_ref] Basics of the STI-measuring method, Steeneken [/bib_ref] , the speech intelligibility-weighted signal-to-noise ratio (iSNR; [bib_ref] Intelligibilityweighted measures of speech-to-interference ratio and speech system performance, Greenberg [/bib_ref] , the Speech Intelligibility Index (SII; ANSI, 1997), and similar or derived methods, such as the extended SII [bib_ref] Modelling the speech reception threshold in non-stationary noise in hearing-impaired listeners as..., Rhebergen [/bib_ref] , the revised Binaural Speech Intelligibility Model (BSIM; [bib_ref] Revision, extension, and evaluation of a binaural speech intelligibility model, Beutelmann [/bib_ref] , the multiresolution speech-based Envelope Power Spectrum Model [bib_ref] A multiresolution envelope-power based model for speech intelligibility, Jørgensen [/bib_ref] , or the multiresolution Generalized Power Spectrum Model [bib_ref] The role of short-time intensity and envelope power for speech intelligibility and..., Biberger [/bib_ref] , derive an estimate of the SNR or an internal SNR in different frequency or modulation bands. These approaches aim to represent signal properties that are highly correlated with speech intelligibility and hence with speech recognition performance. To calculate these properties, they require separate speech and noise signal parts after possible signal processing. This is why they cannot be used with nonlinear signal processing algorithms without significant modifications, such as using the separation method from [bib_ref] A method to measure the effect of noise reduction algorithms using simultaneous..., Hagerman [/bib_ref] cf. [fig_ref] Figure 1: Characterization and examples for intrusive and nonintrusive classification [/fig_ref]. Hence, their mapping, that is, classification, is Intrusive (1). Some of these models specifically make assumptions about the speech material, such as the SII with the band importance functions that were determined on empirical speech recognition results.
The Glimpsing Model from [bib_ref] A glimpsing model of speech perception in noise, Cooke [/bib_ref] calculates (binary) SNR masks from time-frequency bins of a spectrotemporal representation of the clean speech and noise signals, and the Hidden Markov Model (HMM)-based approach from [bib_ref] An information theoretic approach to predict speech intelligibility for listeners with normal..., Stadler [/bib_ref] builds descriptive models from the separate clean speech and noise signals. The way they require the separate signals in their presented configuration does not allow using them without modifications with nonlinear signal processing algorithms, similar to the first group of models. Hence, these approaches also use Intrusive (1) classification. In contrast to the first group, these approaches train automatic speech recognizer (ASR) models and (could) perform a speech recognition task. They have the potential to make predictions of SRTs without relying on empirical reference data, which, however, was not shown.
Correlation or coherence-based models, such as the coherence SII [bib_ref] Coherence and the speech intelligibility index, Kates [/bib_ref] , the Short-Time Objective Intelligibility (STOI) measure [bib_ref] An evaluation of objective measures for intelligibility prediction of time-frequency weighted noisy..., Taal [/bib_ref] , the extended STOI [bib_ref] An algorithm for predicting the intelligibility of speech masked by modulated noise..., Jensen [/bib_ref] , or the Hearing-Aid Speech Perception Index [bib_ref] The hearing-aid speech perception index (HASPI), Kates [/bib_ref] quantify the presence of properties (often certain modulation patterns) of the clean input speech signal in the noisy processed output signal. Similar to the first group, they aim to represent signal properties that are highly correlated with speech intelligibility. In contrast to the first group, they do not require the separate signal parts after but before signal processing. Hence, they can be used in combination with nonlinear signal processing algorithms without further modification, which was also one of their design goals. Because their classification still requires additional signals apart from the noisy processed speech signal, it is Intrusive (2). Further, the signals before and after processing need to be carefully aligned in time, which excludes all signal processing algorithms that do not allow a temporal alignment, such as accelerated speech. STOI and extended STOI do not take individual impaired hearing into account. Further, coherence SII and Hearing-Aid Speech Perception Index were designed to consider individual impaired hearing but were not shown predict the effect of fluctuating noise maskers.
The only measure that does not use intrusive classification is the speech-to-reverberation modulation energy ratio [bib_ref] Non-intrusive objective speech quality and intelligibility prediction for hearing instruments in complex..., Falk [/bib_ref] , which is calculated solely from the noisy processed signal. It is even able to predict an SRT without any knowledge about the clean speech or noise signal, but still not able to predict speech recognition scores or SRTs without empirical reference data. Furthermore, this approach was not designed for modulated maskers and binaural conditions. All these models do not predict the outcome of a speech recognition test directly-although some could-but a (possibly highly) correlated objective measure. For a direct comparison with empirical data, these objective measures need to be mapped to the outcome of a speech-in-noise recognition experiment, which is either a recognition rate depending on the SNR or directly an SRT. This mapping is generally calibrated with or fitted to empirical data from speech recognition experiments and hence, reference-based.
There are fundamental disadvantages of models with reference-based mappings or intrusive classification. The disadvantage of exploiting information that is not accessible to the human listener, who must perform the task with noisy processed stimuli, is a potential uncontrolled bias of the prediction towards unrealistically high performance levels. For human listeners, the limited information results in a desired natural lower boundary for their performance in speech recognition tests, which is commonly quantified with the SRT. Models with intrusive classification intentionally circumvent the difficulties associated with solving the task, possibly at the cost of universality. The disadvantage of using empirical reference data is that a part of the model is undetermined by design and needs to be filled in with empirical data that compensates for any unexplained detail. Apart from requiring empirical measurements prior to predictions, the selection of a specific empirical reference makes implicit assumptions about the generalization ability towards unknown conditions and the model construction. These assumptions might eventually be violated if the predicted conditions differ from the reference condition (e.g., different noise type, different SNR, different vocabulary, different speaker, or different nonlinear channel characteristics, etc.). If the prediction accuracy depends on the selection of the reference (which often is the case), the universality of the model is clearly limited. Further, the models are not strictly objective when empirical reference data from speech recognition experiments influence the outcome of the predictions. An unpleasant ''synergistic'' effect in models that use reference-based mapping and intrusive classification is that the former conceals any indication of wrong assumptions of the latter. In other words, a bias due to superhuman knowledge can neither be detected nor quantified.
A first step to address these drawbacks was already taken by [bib_ref] Speech intelligibility prediction in hearing-impaired listeners based on a psychoacoustically motivated perception..., Holube [/bib_ref] , who used a dynamic-time-warp (DTW)-based speech recognizer with a modified version of the perception model (PEMO) from [bib_ref] A quantitative model of the ''effective'' signal processing in the auditory system...., Dau [/bib_ref] as a front-end to estimate word recognition rated depending on the SNR, hence measuring the psychometric function of what can be considered a very simple ASR system. In its initially presented form, it uses nonintrusive classification because it performs the speech recognition task (on a word level) comparing noisy test words with noisy response alternatives. Therefore, it could be even used with nonlinear signal processing. Further, it is the only discussed approach that does not need empirical reference data to predict the outcome (recognition probability depending on the SNR). The approach in the current study shares many properties and goals with the modeling approach from [bib_ref] Speech intelligibility prediction in hearing-impaired listeners based on a psychoacoustically motivated perception..., Holube [/bib_ref]. However, to achieve an acceptable prediction accuracy on a logatome recognition task, [bib_ref] Microscopic prediction of speech recognition for listeners with normal hearing in noise..., Ju¨rgens [/bib_ref] found that the approach required knowledge about the clean speech samples when performing the speech recognition task, that is, using intrusive classification. Its use with fluctuating noise maskers did not provide satisfactory prediction accuracies, and there are reasonable doubts that the approach in its presented form would generalize to realistic fluctuating noise conditions. In other words, the system was not sufficiently robust against fluctuating noise maskers.
In accordance with this observation, [bib_ref] Microscopic multilingual matrix test predictions using an ASR-based speech recognition model, Scha¨dler [/bib_ref] recently found that independent frequency bands (as assumed, e.g., with the DTW þ PEMO approach) for speech recognition in fluctuating noise masker seem to be an untenable assumption. In the domain of ASR, and much in contrast to many models of auditory signal processing, integration across frequency bands is regarded an essential feature to solve the task of speech recognition in noise, and particularly, in fluctuating noise conditions. In other words, the currently used front-ends in ASR and the vast majority of speech intelligibility models do not agree on which information of an audio signal is most relevant for robust speech recognition. To improve the DTW-based approach from [bib_ref] Speech intelligibility prediction in hearing-impaired listeners based on a psychoacoustically motivated perception..., Holube [/bib_ref] , it seems appropriate to use a robust ASR system to improve the recognition performance and achieve better predictions of human speech recognition performance at the same time.
It is important to differentiate the aims of auditorymotivated feature extraction (like in computational auditory scene analysis [CASA]), robust automatic speech recognition, and the prediction of human speech recognition performance. The aim of CASA is to extract highlevel information from an audio signal, where high-level information here means anything that makes sense to us humans. The solutions can be technical and don't-need to-adhere to what is commonly understood as auditory principles, which is why it is also referred to as computational acoustic scene analysis. However, many of these high-level information extraction tasks can be performed extremely well by human listeners, which is why they serve as a ''model'' as long as they perform better than their technical counterparts. Hence, the interest there is limited to understand the principles of-or even just mimic-the model solution to better solve the task of information extraction. The same is true for any form of bionics. From this perspective, robust ASR is a special problem in CASA, which addresses the transcription of recordings of spoken speech, an extremely challenging and useful high-level information extraction task. Still, the criterion for a good solution is not adherence to any auditory signal processing principles but the raw recognition performance in relevant conditions. Coincidentally, some parts of robust ASR systems implement basic principles of auditory signal processing, such as a frequency analysis on a Mel or Bark scale, the compression of amplitude values with a logarithm and the extraction of spectral (e.g., Mel-frequency cepstral coefficients) and-less prominent-temporal (e.g., temporal derivatives) modulation patterns. Again, this is the current state because it is the best technical solution in terms of recognition performance; still no generally superior solution has been found. Despite recent advances in the field of ASR (primarily due to the use of deep neural networks, efficient training algorithms, hardware acceleration, and large databases), robust speech recognition of spontaneous speech in realistic multitalker environments is still a stronghold of human listeners, turning humans into models for robust speech recognition. Because of the many nonlinearly interacting components of ASR systems, it is still unknown why human listeners perform better, that is, which components of an ASR system must be improved to put it on a par with human listeners. The prediction of human speech recognition performance by means of simulation with ASR will inherit many of these limitations from the ASR systems. The aim of predicting the speech recognition performance of human listeners is an end in itself, and the criteria for good predictions are the prediction error and-increasingly valued-the assumptions that are required to achieve them.
It is worth noting that full knowledge of the test conditions, that is, the speech/noise signals, is not critical for modeling human speech recognition, while knowledge about the empirical reference SRT data in that conditions is. In contrast, for robust ASR tasks, full knowledge of the test conditions, which is also called matched training, is critical, while the human performance on the same task is irrelevant for the recognition performance of the ASR system. The opportunities of using robust ASR systems as models of human speech recognition have hardly been studied. The opportunities that lie in combining both schools of though were outlined by, who found that the missing bit to close the gap between human and machine speech recognition performance in important speech recognition tests was matched training.
There, as a starting point for a convergence and to overcome the fundamental limitations of referencebased models, FADE [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref] was proposed, which simulates (speech) recognition experiments with a simple matched-trained ASR system. Because it uses nonintrusive classification, it can be tested with nonlinearly processed signals just like human listeners. To maximize the chances of generalization, FADE makes only very basic assumptions, such as how the audio signals are represented (e.g., spectrotemporal representation) and which tokens (e.g., words) need to be discriminated using this representation. FADE performs the same task as humans in the listening experiments, that is, the recognition of words/sentences presented in the presence of noise at different SNRs. The desired outcome, for example, an SRT at 50% correct word recognition rate, can be calculated directly from the evaluation of the transcripts of the ASR system, without using any empirical data. Because the SRT predictions do not depend on empirical reference data, they are objective. The nonintrusive classification of noisy signals with FADE puts a natural lower bound on the recognition performance that strongly depends on the signal representation and was found to be in good agreement with empirical measurements of listeners with normal hearing . The approach was shown to predict the outcome of the matrix sentence test in different languages in stationary and fluctuating noise conditions [bib_ref] Microscopic multilingual matrix test predictions using an ASR-based speech recognition model, Scha¨dler [/bib_ref]. The auditory signal representation, which was originally designed for robust automatic speech recognition, was extended by common signal processing deficiencies to model impaired hearing [bib_ref] Individual speech recognition in noise, the audiogram and more: Using automatic speech..., Kollmeier [/bib_ref]. The impairment can be individualized using the individual absolute hearing threshold.
In the current work, FADE is evaluated for the first time as an objective method for SRT predictions in aided conditions for normal-hearing and HI listeners in binaural realistic listening conditions. The extent to which this model can explain the human behavior is assessed with the empirical data from [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] who measured SRTs of 10 listeners with normal hearing and 12 listeners with aided impaired hearing with eight different noise-reduction algorithms in three binaural acoustic conditions. There, the empirical data were modeled with the SII-based BSIM from [bib_ref] Revision, extension, and evaluation of a binaural speech intelligibility model, Beutelmann [/bib_ref]. BSIM only predicts SRTs relative to a reference condition, which is why its predictions are always biased to some extent by the empirical reference data. Because this model requires separate speech and noise signals, which are not available anymore after the nonlinear processing of the algorithms, [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] did not perform individual aided predictions. This limitation could be circumvented by using a technique called shadow filtering, which attempts to separate the signal parts by applying the nonlinear processing to the speech and noise signals separately. However, shadow filtering would have introduced additional assumptions about the linearity of the signal processing.
To enable objective predictions of SRTs in the cafeteria condition for listeners with normal hearing, the FADE approach is extended towards binaural conditions by implementing a simple better ear listening strategy. Further, to enable individual predictions of SRTs, the model is individualized by taking into account the absolute hearing thresholds (left and right) given by the audiograms and the configuration of the hearing devices (left and right). The individual and group predictions for unaided and aided conditions for listeners with normal hearing and impaired hearing are compared with the empirical data in terms of SRTs and benefit in SRT, that is, the improvement in SRT due to activation of noise suppression scheme compared with the reference condition (with deactivated noise suppression).
# Methods experiment
The stimuli and the empirical SRTs were taken from [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref]. There, SRTs of 10 listeners with normal hearing and 12 listeners with aided impaired hearing were adaptively measured with the German matrix sentence test in a binaural cafeteria environment that was simulated with head-related impulse responses from [bib_ref] Database of multichannel in-ear and behind-the-ear head-related and binaural room impulse responses, Kayser [/bib_ref] with eight different binaural preprocessing strategies. Three different noises were employed to determine SRTs in a condition where the target speaker was located in front of the listener: (a) A 20-talker babble noise (20T), (b) the ambient sound in the cafeteria in which the head-related impulse responses were recorded (cafeteria ambient noise [CAN]), and (c) a single interfering talker (SIT). In this study, the focus was on the natural, recorded ambient sound the cafeteria, which was assumed to represent the most realistic condition. The preprocessing algorithms included adaptive differential microphones (ADM), a coherence filter, single-channel noise reduction (SCNR), and binaural beamformers and are listed in Section Binaural Preprocessing Algorithms. In addition, a condition with no preprocessing (NoPre) served as a reference. To provide listeners with impaired hearing with audible signals in all conditions, the multiband dynamic compressor of the Master Hearing Aid (MHA) was used to compensate their HL with the nonlinear fitting procedure by the National Acoustic Laboratories [bib_ref] NAL-NL1 procedure for fitting nonlinear hearing aids: Characteristics and comparisons with other..., Byrne [/bib_ref] , even in the reference condition with no binaural preprocessing. For the group of listeners with normal hearing, no amplification was provided. The group of listeners with impaired hearing consisted of eight male and four female listeners with ages ranging from 21 to 66 years. Their hearing thresholds were 43.9 dB higher on average over normal-hearing listeners between frequencies of 500 Hz and 4 kHz and ranged from 20 dB to 55 dB HL. For more details on the audiometric data and the compensation of HL, please refer to [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref].
## Prediction of srts with fade
The outcome of the German matrix test was predicted with FADE [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref]. The condition here is defined by the task (matrix sentence recognition), the speech signals, the noise signal, the individual signal processing, and the individual audibility. Therefore, the ASR system is trained and tested for each condition separately, that is, there is no interdependence regarding the conditions. It is particularly noteworthy that no prior information about any empirical SRT is required to obtain a predicted SRT. In other words, no reference to another condition is required, and an SRT can be predicted for an isolated condition.
The procedure to obtain the predicted outcome in one condition with FADE is as follows:
Gaussian Mixture Model (GMM) and HMM-based ASR systems are trained on all available matrix sentences; one for each considered SNR (À24 dB to 6 dB in 3-dB steps). Therefore, random portions of the noise signal were chosen to generate the training data. The mixed recordings were then processed with the (individual) HA using the MHA [bib_ref] The master hearing aid: A PC-based platform for algorithm development and evaluation, Grimm [/bib_ref] with the algorithm that corresponds to the condition. From the processed signals, features were extracted that incorporate the individual hearing thresholds as explained in Section Model of Impaired Hearing. The feature extraction is based on separable Gabor filter bank (SGBFB) features with mean-and-variance normalization. The SGBFB feature set was shown to robustly extract information of speech signals from audio recordings and to be suitable for modeling tone detection and speech recognition experiments with normal-hearing listeners [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref]. SGBFB features encode a range of spectrotemporal modulation patterns, where mean-and-variance normalization removes information about the absolute level and the overall dynamic range of the recording. For the acoustic model, word models with six states were used. One GMM/HMM word model was trained for each word and SNR. For each SNR, the 50 word models were combined with a language model that describes the (language-independent) syntax of the matrix test; that is, 5 words with 10 options each.
The ASR systems are then used to recognize all available matrix sentences at all considered SNRs (À24 dB to 6 dB in 3-dB steps). Like for the training data, random portions of the noise signal were chosen to generate the testing data. The mixed recordings were, again like for the training data, processed with the (individual) HA using MHA with the algorithm that corresponds to the condition. From the processed signals, features that incorporate the individual hearing thresholds were extracted, like for the training data. This training/testing scheme is also known as matched training, an ideal training condition. It is employed to get an estimate of the lowest achievable SRT in the given condition. Recognition performance of the ASR system is subject to the same limitations that trained human listeners encounter due to the task, the speech material, the noise signal, the individual signal processing, and the individual audibility. From the transcripts, the word recognition rates in percent are determined for each combination of training and testing SNR and compiled into a square matrix, with training SNR in one dimension and testing SNR in the other dimension. This matrix is referred to as the recognition result map (cf. [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref]. Usually the testing SRT depends on the training SNR. The lowest testing SNR at which 50% of the words were correctly recognized was interpolated between the 3 dB-spaced samples and taken as the predicted SRT. For further details about the simulations, please refer to the original publications [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref]. The concept of how the predictions with FADE were individualized is illustrated in .
The feature extraction and HAs were individualized based on the audiogram, which is explained in Sections ''Model of Impaired Hearing'' and ''Model of Binaural Hearing.'' FADE is free software and available online. 1
## Model of impaired hearing
The feature extraction of the original publication from [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref] was modified to implement an absolute hearing threshold. The implementation of the hearing threshold is identical to the one from , where the absolute hearing threshold and a suprathreshold parameter were successfully used to predict individual outcomes of the German matrix sentence test in noise for listeners with impaired hearing. In this work, only the hearing thresholds were considered because no suitable empirical data existed to determine individual estimates of the suprathreshold component. To account for the loss of sensitivity, which is usually assessed by the audiogram, the individual hearing threshold was applied to the spectrotemporal representation, the log Mel-spectrogram, by setting any input spectrogram level to the audiogram level if it was below this value. The effect of this implementation of the hearing threshold on the log Melspectrogram of a noisy speech sample is illustrated in [fig_ref] Figure 3: Illustration of the combined effect of an additive noise and a typical... [/fig_ref]. Applying an individual threshold has the effect that any patterns below have no effect on the feature vector anymore. Hence, part of the speech information is concealed from the recognizer, which naturally results in individually lower recognition performance and increased SRTs.
## Model of binaural hearing
The measurements in [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] were dichotic, which to model required a stage that integrated the two acoustic signals from both ears. Different models of binaural hearing and signal processing were proposed in the literature (e.g., [bib_ref] Revision, extension, and evaluation of a binaural speech intelligibility model, Beutelmann [/bib_ref] ; however, due to using intrusive classification, none of them works blindly. In other words, these models require information that is not accessible in the current setup, where only noisy, processed signals are available, and hence could not be used. Here, to limit the complexity of the approach, independent feature vectors were calculated for each ear, which were then concatenated to form one binaural feature vector. This very simple binaural model assumes a late, that is, cortical, integration of binaural information in speech processing. Because the recognizer can learn a better ear during the training stage, we denominate it as automatic better ear listening. An implementation of the feature extraction is available online. 2 . Illustration of the individualization of predictions with FADE. The gray box represents the common logic to measure psychometric functions that is used for human listeners as well as for FADE-based predictions. To humans, only the mixed (noisy) output is available to perform the speech recognition task. For the recognition, the speech recognizer does not need access to additional data from within the gray box and uses nonintrusive classification. The feature extraction transforms the time series into a spectrotemporal representation and thereby limits the information that is available to the recognizer for decision-making. It can be individualized based on the audiogram to remove the parts that are not audible. Further, hearing aids (blue boxes) with-potentially several-individualized, nonlinear algorithms can be inserted into the signal path, prior to feature extraction. In this way, interactions between hearing aid and hearing impairment can be modeled naturally, that is, without further modifications. FADE ¼ framework for auditory discrimination experiments; SNR ¼ signal-to-noise ratio.
## Binaural preprocessing algorithms
The eight binaural signal preprocessing strategies were taken from [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] and included (1) No preprocessing (NoPre), (2) ADM [bib_ref] A simple adaptive firstorder differential microphone, Elko [/bib_ref] , (3) ADM with coherence-based noise reduction (ADM þ coh; [bib_ref] Increase and subjective evaluation of feedback stability in hearing aids by a..., Grimm [/bib_ref] , (4) SCNR (e.g., [bib_ref] Improved a posteriori speech presence probability estimation based on a likelihood ratio..., Gerkmann [/bib_ref] , (5) fixed binaural minimum variance distortionless response (MVDR) beamformer (e.g., [bib_ref] Beamforming: A versatile approach to spatial filtering, Van Veen [/bib_ref] , (6) adaptive binaural MVDR beamformer (e.g., [bib_ref] An alternative approach to linearly constrained adaptive beamforming, Griffiths [/bib_ref] , (7) common postfilter (com PF) based on fixed MVDR (e.g., [bib_ref] Post-filtering techniques, Simmer [/bib_ref] , (8) com PF based on adaptive MVDR (e.g., [bib_ref] Post-filtering techniques, Simmer [/bib_ref] , and (9) individual postfilter (ind PF) based on adaptive MVDR (e.g., [bib_ref] Post-filtering techniques, Simmer [/bib_ref]. The algorithms were described and instrumentally evaluated byin the same experimental conditions. ADM and SCNR are monaural algorithms and do not require a binaural link, that is, they work independently on both ears. The remaining six algorithms are binaural preprocessing strategies, where the signal input on one side can influence the output on the other side. All algorithms aim to improve speech intelligibility; however, they achieve that goal only to a varying degree . The goal of the current study is to predict this benefit, which can be measured in improvement in SRT. For details on the individual algorithms please refer to [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] ,, or the corresponding original publications. It is assumed that this selection provides a good representation of modern binaural speech and audio signal processing algorithms, and hence also reflects typical effects, nonlinearities, and artifacts involved in such processing. In the current study, the signal processing was performed by using the MHA, which was also used by [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] to carry out the measurement of the empirical data. For the individual predictions, the same individual configuration that was used for the empirical measurements was used to process the noisy training and testing data (cf. Section ''Prediction of Speech Reception Thresholds With FADE'') before individual feature vectors that considered the individual hearing thresholds were extracted (cf. Section ''Model of Impaired Hearing'').
# Results
## Predicted srts
The predicted SRTs are plotted against the empirical SRTs in [fig_ref] Figure 4: Predicted versus empirical SRTs for listeners with normal and impaired hearing with... [/fig_ref] for the 20T condition and the CAN condition, and median SRTs for the groups of listeners with normal hearing and aided impaired hearing in all conditions are presented in numerical form in [fig_ref] Table 2: Median Empirical and Predicted SRTs for the Group of Listeners With Normal... [/fig_ref]. It should be noted that the FADE approach in its current form is not suitable to predict the recognition thresholds for the conditions with an SIT. The model seems to be unable to differentiate between the two competing speakers, and no a priori information was provided to the algorithm which speaker is the target and which is the interferer. Without a cue to differentiate the speakers, the FADE model can only perform the recognition task at much higher SNRs than achieved by human listeners. This is reflected by the finding that the predicted SRTs for listeners with normal hearing in the SIT condition were about 10 dB higher than the empirical ones. Reasonable predictions for the generally very homogeneous group of listeners with normal hearing can be considered a prerequisite to performing reasonable predictions for the generally less homogeneous group of listeners with impaired hearing, which is why no predictions were performed for listeners with impaired hearing in this condition. Hence, in the following, the extreme (SRTs below À20 dB) SIT condition is not considered, and the analysis focuses on the artificial 20T condition and, primarily, on the realistic CAN condition. However, for completeness, the available data for the SIT condition are provided in numerical form. The middle panel shows the same sentence but with the cafeteria noise added at 0 dB SNR, which represents the information that would be available to listeners with normal hearing according to the model. The lower panel shows the same signal and noise but with the typical audiogram N4 from [bib_ref] Standard audiograms for the IEC 60118-15 measurement procedure, Bisgaard [/bib_ref] , masking portions of the speech and noise signal. ASR ¼ automatic speech recognizer; SNR ¼ signal-to-noise ratio; SPL ¼ sound pressure level.
For the 20T and CAN conditions, the median empirical SRTs of both groups (normal and impaired hearing) show an effect size of about 5 dB due to the different binaural preprocessing algorithms. The median predicted SRTs show a very similar effect size. The median empirical SRTs for listeners with normal hearing are lower than the ones for listeners with impaired hearing, which is also correctly predicted by the model. The median predictions for both groups (black symbols) were mostly underestimated, that is, the predicted SRTs were higher than the empirical ones. This deviation is more pronounced in the CAN than in the 20T condition. However, the predicted values seem to be scattered around a parallel to the diagonal, which indicates an Note. Individual predictions for listeners with impaired hearing in the SIT condition were not performed. The numbers in the header identify the algorithms:
(1) no preprocessing (NoPre), (2) adaptive differential microphones (ADM), (3) ADM with coherence-based noise reduction (coh), (4) single-channel noise reduction (SCNR), (5) fixed binaural minimum variance distortionless response (MVDR) beamformer, (6) adaptive binaural MVDR beamformer, (7) overall offset (bias) but correct relations of the predictions. For the median prediction of the group of listeners with impaired hearing, the deviation from the diagonal is more pronounced in the 20T than in the CAN condition. Individual predictions (cf. colored symbols in [fig_ref] Figure 4: Predicted versus empirical SRTs for listeners with normal and impaired hearing with... [/fig_ref] were overestimated as well as underestimated, where the predictions for a specific listener, indicated by the same color, seem to tend to either one. A statistical analysis of the median and individual SRTs is provided in [fig_ref] Table 3: Statistical Analysis of the Predicted SRTs for the Normal-Hearing Group [/fig_ref]. The high bias of 13 dB in the SIT condition quantifies the extensive gap between the empirical and predicted group performance with a pure speech masker, and the p value > .05 shows that the predicted and the empirical data were not significantly correlated. In contrast, in the 20T and CAN condition, all predicted outcomes (individual and group) were found to be significantly correlated with the empirical data. However, only the group predictions explained more than 50% of the variance of the data (R 2 , assuming normally distributed data) due to the use of the different preprocessing algorithms. For the group data of listeners with normal hearing, the predictions explained 87% of the variance in the 20T condition and 92% of the variance in the CAN condition. For the-usually less homogeneous-group of listeners with impaired hearing, the group predictions still explained 63% and 83% in the 20T and the CAN condition, respectively. The individual predictions, although significantly correlated, explained less than 20% of the variance in the individual data. The bias of the predictions, which also determines the lower limit of the root-mean-square (RMS) prediction error, mainly depended on the noise condition and was found to be about 1.5 dB in the 20T condition and less than 3 dB in the CAN condition.
## Predicted benefits in srt
The benefit in SRT was calculated as the inverted difference between the SRT in a condition and the SRT in the reference (NoPre) condition and reflects the improvement due to using an algorithm (higher values are better). Note that this calculation was performed independently for the predicted and the simulated data, and each individual. Hence, the predictions of benefits did not depend on empirical data (apart from the individual audiograms), and the median values are calculated from differences of SRTs, that is, they are not differences of median SRTs. While the analysis in this section focuses on the realistic CAN condition, the results for the 20T and SIT condition are reported in numerical form. In [fig_ref] Figure 5: Predicted versus empirical benefits in SRT [/fig_ref] , the predicted individual and group benefits are plotted against the empirical individual and group benefits in the CAN condition for listeners with normal and aided impaired hearing. For listeners with normal hearing (left panel), the data points were closely aligned to the diagonal, which indicates highly accurate predictions. For the group of listeners with impaired hearing (right panel), the deviations from the diagonal were more pronounced than for listeners with normal hearing. This was an expected result due to less homogeneous nature of this group. Nonetheless, the group's median values were found to be well aligned to the diagonal, which indicates still accurate predictions of the benefit for the group of listeners with impaired hearing in the CAN condition. The predicted group benefits with the different algorithms are directly compared with the empirically determined ones in for the group of listeners with normal hearing and in for the group of listeners with aided impaired hearing. For listeners with normal hearing, the predicted benefits were found to be in very good agreement with the median of the empirical data. The largest deviations were observed for the algorithms with the adaptive MVDR beamformer, which is the most complex algorithm and also resulted in a relatively large empirical interquartile range of about 2 dB. It is notable that the model correctly predicted that the SCNR did not improve speech intelligibility. Note. Pearson's correlation coefficients (R 2 ) are reported including their 95% confidence intervals according toalong with the root-mean-square (RMS) prediction error and the bias (B) for predicted SRTs in dB. SRT ¼ speech reception threshold.
For listeners with impaired hearing, the median of the individually predicted benefits is in agreement with the empirical data, however, not to the same degree than it was found for listeners with normal hearing.
Interestingly, the interquartile ranges of the predictions were found to be consistently larger than those of the empirical data. This is probably due to the interaction of the hearing threshold, which is implemented as a hard . Median predicted and empirical benefits in SRT (difference to reference condition) for the group of listeners with normal hearing in a cafeteria ambient noise condition with eight different binaural preprocessing algorithms: ADM) adaptive differential microphones, þ coh) with coherence-based noise reduction, SCNR) single-channel noise reduction, MVDR) binaural minimum variance distortionless response beamformer, com PF) common postfilter based, and ind PF) individual postfilter based. The error bars of the empirical data indicate the first and third quartile of the corresponding individual data, and their length the interquartile range. The error bars of the predicted data indicate an assumed symmetric interquartile range of 1 dB. SRT ¼ speech reception threshold.
## Predicted benefit in srt / db
Empirical benefit in SRT / dB threshold as opposed to the probably smoother transition area from inaudible to audible in human listeners, with the compressive amplification scheme. Small deviations of the hard threshold will have stronger interactions with compressed signal levels than human listeners with smooth transition zones. This effect is reinforced by the low accuracy (AE5dB) of threshold measurements in audiograms, which adds an additional noise source to the predictions not present in the empirical data. The benefit of the best and worst performing algorithms, fixed MVDR and SCNR, respectively, was overestimated by about 1 dB, while the deviations in the other conditions were smaller. Again, the predicted results did not suggest a benefit from using the SCNR algorithm. The group benefits in SRT for all conditions are presented in numerical form in [fig_ref] Table 4: Median Empirical and Predicted Benefits in SRT [/fig_ref] , and a statistical analysis of the individual and group benefits in SRT is provided in [fig_ref] Table 5: Statistical Analysis of the Predicted Benefits [/fig_ref]. The proportion of explained variance of the empirical group data was similar in the 20T and the CAN condition. It was higher (about 90%) for the group of listeners with normal hearing than for the group of listeners with impaired hearing (about 75%). The bias and RMS error were particularly low in the CAN condition, with less than 0.5 dB and less than 1.0 dB, respectively. These were found to be slightly higher in the babble noise condition, with about 1 dB and less than 2 dB, respectively. In the SIT condition, the median predicted benefits were not significantly correlated with the empirical data. The predicted benefits for the individual listeners with aided impaired hearing were found to be significantly correlated with the empirical data. However, the proportion of explained variance was, with less than 25%, low, and the RMS prediction error, with more than 2.4 dB, comparatively high.
## Correlation of prediction errors across test conditions
To test if the pronounced individual prediction errors (difference between the predicted and the corresponding empirical SRT) for listeners with aided impaired hearing in the 20T and the CAN condition (cf. left panel in [fig_ref] Figure 5: Predicted versus empirical benefits in SRT [/fig_ref] are systematic, that is, due to a common cause that the model did not consider, the individual prediction errors are plotted against each other in [fig_ref] Figure 8: Individual prediction errors [/fig_ref]. The prediction errors were found to be significantly correlated (R 2 ¼ .69, p < .01), clearly indicating a systematic error. In other words, when the model over/ underestimated an individual SRT in one condition, it also over/underestimated it in the other conditions.
# Discussion
The main finding of this article is that the average benefit from several binaural noise-reduction strategies can be surprisingly well predicted using the comparatively simple, ASR-based FADE approach. FADE provides an objective estimate of the SRT that can be individualized to suit an individual listener with hearing impairment. . Median predicted and empirical benefits in SRT (difference to reference condition) for the group of listeners with aided impaired hearing in a cafeteria ambient noise condition with eight different binaural preprocessing strategies (cf. for an explanation of the abbreviations). The error bars the indicate the first and third quartile of the corresponding individual data, and their length the interquartile range. SRT ¼ speech reception threshold; ADM ¼ adaptive differential microphone; SCNR ¼ single-channel noise reduction; MVDR ¼ minimum variance distortionless response; PF ¼ postfilter. [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] modeled the same empirical data with the BSIM [bib_ref] Revision, extension, and evaluation of a binaural speech intelligibility model, Beutelmann [/bib_ref] and found that the difficult-but required for BSIM predictionspost hoc separation of the processed signal into a speech and noise part was a major problem for those conditions where nonlinear signal processing algorithms were used.
## Intrusive classification and objectivity
The problems of Intrusive (1) classification concerns many speech intelligibility models (cf. [fig_ref] Table 1: Classification of Models That Can Be Used to Predict the Speech Recognition... [/fig_ref]. To overcome this problem in modeling, [bib_ref] Comparing binaural pre-processing strategies III: Speech intelligibility of normal-hearing and hearing-impaired listeners, Vo¨lker [/bib_ref] proposed to use FADE, which was successfully employed in this study. Another-often neglected-problem with many other prediction methods for human speech recognition performance is the lack of objectivity due to being based on empirical reference data, which also makes the Note. Individual predictions for listeners with impaired hearing in the SIT condition were not performed. The numbers in the header identify the algorithms:
(1) adaptive differential microphones (ADM), (2) ADM with coherence-based noise reduction (coh), (3) single-channel noise reduction (SCNR), (4) fixed binaural minimum variance distortionless response (MVDR) beamformer, (5) adaptive binaural MVDR beamformer, (6) common postfilter (com PF) based on fixed MVDR, (7) com PF based on adaptive MVDR, and (8) individual postfilter (ind PF) based on adaptive MVDR. Please note that the group predictions of the benefits in SRT do not have to coincide with the corresponding differences in the group predictions of SRTs provided in [fig_ref] Table 2: Median Empirical and Predicted SRTs for the Group of Listeners With Normal... [/fig_ref]. SRT ¼ speech reception threshold. Note. Pearson's correlation coefficients (R 2 ) are reported including their 95% confidence intervals according toalong with the root-meansquare (RMS) prediction error and the bias (B) in dB for the predicted SRTs. SRT ¼ speech reception threshold; RMS ¼ root-mean-square; iSNR ¼ intelligibility-weighted signal-to-noise ratio; FADE ¼ framework for auditory discrimination experiment. interpretation of prediction errors difficult. To the best of our knowledge, no other model with nonintrusive classification that allows the objective, that is, empirical reference-free, prediction of SRTs in aided conditions with fluctuating noise maskers exists at this time of writing.
Nonintrusive classification. FADE requires only the same information as needed to perform the matrix sentence test with human listeners. This includes the ability to generate mixtures at defined SNRs that normally requires the separate availability of the clean matrix sentences and the noise signal. However, it does not give the recognizer (here, the ASR system) access to the separate signals. This closely resembles experiments with human listeners, where only noisy signals are presented to determine the outcome. Hence, FADE is a modeling approach with nonintrusive classification. In contrast to many other models (cf. [fig_ref] Table 1: Classification of Models That Can Be Used to Predict the Speech Recognition... [/fig_ref] , FADE works naturally on mixed signals, which allows to apply arbitrary signal processing to the mixed signals prior to presenting them to the recognition system (just like in the empirical measurements). The employed algorithm can be treated as a black box to which no access-apart from the signal input and the signal output-is needed.
Objective. FADE directly predicts the SRT as the primary outcome of the matrix sentence test employed. Indexbased models predict an index value between 0 and 1 for each SNR. Assuming a strictly monotonic relationship between SNR and index, which is not always true, each index value can be mapped to an SNR. Very often, a reference value for the index that corresponds to the SRT, that is, the SNR at 50% correct, is determined by using the empirical data from one of the conditions that the model aims to explain. One implication of that procedure is that no single condition can be predicted. Another-more far-reaching-implication is that the predictions then depend on the reference condition and hence on (a) the complexity of the speech material, (b) the language, (c) the talker, (d) the noise, (e) the reverberation of the reference condition, and probably many more parameters whose influence is not predicted by the model. As a consequence, these SRT predictions are not strictly objective because they strongly depend on empirical measurements. In contrast, FADE can perform SRT predictions for single conditions solely based on fairly basic principles. For the predicted SRT to match the empirically determined one, it must correctly predict the joint effect of all relevant factors, for example, the complexity of the speech material, the language, the talker, the noise, possible binaural interactions, and of the signal processing. In doing so without using any empirical reference SRT data, it provides a truly objective measure.
## Predicted outcomes
Predicted outcomes of the German matrix sentence test were compared with empirical outcomes, whose differences are the prediction errors. An interpretation of the prediction error is a prerequisite for well-founded speculations on their causes, and hence on the causes of potential shortcomings of the model. Objective SRT predictions in combination with nonintrusive classification are presumably helpful for deriving a meaningful interpretation of the prediction errors. For example, if FADE cannot explain some aspect, the recognition performance gets worse and the predicted SRTs increase, hence underestimating the empirical recognition performance. This performance limitation is similar to most ASR systems that perform worse than listeners with normal hearing when confronted with situations for which they were not designed. Hence, a significant overestimation of the empirical performance (not observed here) indicates that the model was (a) provided with more information or (b) made better use of the information that is required to perform the corresponding recognition task. A significant underestimation of the empirical performance of listeners with normal hearing, on the other hand, indicates a suboptimal-or missing-implementation of at least one of the required principles to perform the corresponding recognition task. A small underestimation of the median empirical performance was observed in Section ''Predicted SRTs'' for the 20T and, slightly more pronounced, in the CAN condition. This could be due to the simplistic binaural better-ear strategy adopted by the current approach that might not be able to separate the target speaker from the noises as efficiently as listener with normal binaural hearing. The interaction of the spatial scene with binaural signal processing and binaural (impaired) hearing could also be the cause of the variability in the deviation of predicted median SRTs for listeners with impaired hearing in the 20T condition. A more sophisticated, true binaural processing stage with a direct across-ear processing of the input signal (e.g., [bib_ref] Combining binaural and cortical features for robust speech recognition, Spille [/bib_ref] might improve the model behavior here.
A larger systematic underestimation of the recognition performance was observed in Section ''Predicted SRTs'' for the SIT condition, which resulted in large positive biases in [fig_ref] Table 3: Statistical Analysis of the Predicted SRTs for the Normal-Hearing Group [/fig_ref] for the performance of listeners with normal hearing in all conditions. The considerable bias of 13 dB indicates that the model had a severe problem with voiced speech maskers. The system was neither able to learn nor provided with information about which speech portion is considered as target and which has to be considered as nonattended interferer. Human listeners achieve very low SRTs ð5À 20 dB) primarily by being able to listen in the dips of the masking speech. Probably, human listeners are better able to segregate the talkers based on their pitch and spatial cues than the model, an ability that is often referred to as the cocktail-party effect. The necessary signal processing to perform such a task by machines has been studied under the term computational acoustic scene analysis (CASA) and is largely missing in current ASR systems as well as in FADE. Emerging technical solutions to this problem could potentially improve ASR systems and lead to improved predictions in the SIT condition.
Because the predicted SRTs in the SIT condition were not found to be in agreement with the empirical data for normal-hearing listeners, no individualization, that is, prediction for listeners with impaired hearing was performed. In the 20T and CAN condition, the predictions for the group of listeners with impaired hearing showed the same bias (AE0.2 dB) and hence higher SRTs than for the group of listeners with normal hearing (cf. [fig_ref] Table 3: Statistical Analysis of the Predicted SRTs for the Normal-Hearing Group [/fig_ref] in agreement with the empirical data. According to the empirical data, some listeners with impaired hearing outperformed the average listener with normal hearing, by up to % 2 dB. However, the individual predicted SRTs were not found to be more than 0.5 dB lower than the corresponding predictions without individualization, that is, for listeners with normal hearing. This can be partly observed in [fig_ref] Figure 4: Predicted versus empirical SRTs for listeners with normal and impaired hearing with... [/fig_ref] comparing the lowest individual predictions for listeners with impaired hearing (colored stars) to the lowest prediction for listeners with normal hearing (black squares) and also holds for the other algorithms (not shown). An explanation might be the inevitable uncertainty or statistical measurement error in the individual empirical data that could sum up to % 2 dB depending in the noise condition. This expected error in the empirical data is due to small training effects, limited and varying attention of the listeners, and the small number of presented sentences (at most, 20) on which the SRT calculation is based. It should be noted that this statistical error is not-or to a much lesser extent-found in the predictions because SRTs are calculated based on results from all 120 sentences.
Apart from the already discussed bias, which also dominated the RMS prediction errors, the predicted group SRTs correlated significantly with the empirical data, explaining between 63% and 92% of the variance across algorithms in the 20T and CAN condition, respectively. The individual predictions for listeners with impaired hearing did not explain more that 16% of the variance across individuals and algorithms. These results already hint at good group predictions in general, but unsatisfactory individual predictions for listeners with impaired hearing. However, individual offsets originating from nonacoustical causes which are not modeled here (e.g., central neurosensory component of the HL or varying cognitive abilities) may affect all algorithms alike. They may cause a spread of the individual empirical SRTs and hence result in a lower correlation between the predicted and empirical data. To test this hypothesis and remove an individual bias, the benefits in SRT with respect to the reference condition (NoPre) were calculated individually for all listeners (to be discussed later).
## Objective instrumental evaluation of algorithms
The high prediction accuracy (RMS prediction error 41 dB) and the low absolute biases (< 1 dB) of the benefits in SRT for the group of listeners with normal hearing (cf. [fig_ref] Figure 5: Predicted versus empirical benefits in SRT [/fig_ref] , and [fig_ref] Table 5: Statistical Analysis of the Predicted Benefits [/fig_ref] , respectively) indicate a solid basis for individualized predictions. Further, the predicted benefits for listeners with normal hearing do not depend on any empirical data, which is why FADE provided a near-optimal objective evaluation in this regard. Notably, in the realistic CAN condition, the model correctly predicted the fixed MVDR to result in the highest benefit and the SCNR in the lowest, where the benefit of the latter was correctly predicted to be negative, that is, it increased the SRT. While the ADM was predicted to perform worse than the fixed MVDR in the artificial 20T condition, which was not observed empirically, the negative benefit of the SCNR was still correctly predicted. The differentiation between improvement and deterioration is very valuable to algorithm developers and users alike.
In the instrumental evaluation of the algorithms from, the iSNR, STOI, and perceptual evaluation of speech quality (PESQ; , which can be considered representative for the methods that are used to objectively assess the performance of (speech) signal processing algorithms, were used to predict the benefit. None of them predicted the negative effect of the SCNR algorithm in the artificial 20T condition and the realistic CAN condition for listeners with normal hearing. There, benefits in SNR were only predicted with the iSNR because the output of the other measures were indexes that could not be mapped to SNRs or benefits in SNR due to the lack of empirical reference data in that study. This reflects a common, fundamental problem of reference-based models. For objective predictions of the benefit with the iSNR, a working point, that is, an input SNR at which the benefit was predicted, had to be chosen independently from the empirical data, which was 0 dB. If the empirical working point is unknown, which usually is the case when objective measures are used, a guess of 0 dB seems still reasonable, particularly if the predictions should also represent the behavior of listeners with impaired hearing or cochlear implants. These limitations, that is, the requirement of guessing or knowing the outcome prior to any prediction, clearly shows one advantage of FADE that can predict an SRT without any empirical reference. The statistical analysis of the predicted outcomes with the iSNR fromfor the group of normal-hearing listeners in the 20T and CAN condition is shown in [fig_ref] Table 5: Statistical Analysis of the Predicted Benefits [/fig_ref]. For the artificial 20T condition, the iSNR predictions explained about 60% of the variance in empirical data and the RMS prediction error exceeded 3 dB. For the realistic CAN condition, the iSNR predictions were not significantly correlated with the empirical data and the RMS prediction error also exceeded 3 dB. Compared with these results, the FADE predictions of benefits in SRT for normal-hearing listeners provided a much more reliable objective instrumental measure with RMS prediction errors of 1 dB and less in the respective 20T and CAN conditions. The mediocre performance of the iSNR, STOI, and PESQ could be partly due to the method (phase inversion according to [bib_ref] A method to measure the effect of noise reduction algorithms using simultaneous..., Hagerman [/bib_ref] that was used to separate the speech from the noise signal after processing and that might not achieve a perfect separation. This seems probable because no true separate processed signals exist whereby the goodness of the estimation cannot be assessed. The phase inversion could be possibly replaced by a more sophisticated separation method, such as shadow filtering. However, even if this would lead to better predictions, it would not overcome the fundamental limitation that this kind of separation of the mixed signals can lead to uncontrolled biases, which are typically concealed by a calibration to empirical reference data. Because the classification with FADE is nonintrusive and works on mixed signals, predictions can be performed with virtually any signal preprocessing without additional modifications to the model. It seems worthwhile to further validate the suitability of FADE for the objective evaluation of algorithms on a broader basis of applications.
## Individual aided performance predictions
Individualization was performed using the audiograms, that is, the individual hearing thresholds of both ears, where listeners with normal hearing were assumed to have no HL. This is also why there were individual measurements from listeners with normal hearing but no individual predictions for them. The individualization for listeners with impaired hearing was reflected in the personalized HA setting: Here, it had consequences for the fitting of the multiband compressor of the MHA, where the HL was (partially) compensated (cf. Section ''Binaural Preprocessing Algorithms'') and the feature extraction of FADE, where the individual hearing threshold was simulated (cf. Section ''Model of Impaired Hearing''). Therefore, assuming an appropriate compensation for the main component of the moderate HLs encountered in our rather homogeneous group of listeners with impaired hearing, no strong individual deviation from the compensated main component of the HL should be expected.
However, despite similar audiograms and individually compensated HLs, the empirical results show (a) that the variance of the individual benefits in SRT were in the same order of magnitude as the effect size of the algorithms (cf. [fig_ref] Figure 5: Predicted versus empirical benefits in SRT [/fig_ref] and (b) that the median SRTs for the group of listeners with impaired hearing were about 1 to 2 dB higher than for the group of listeners with normal hearing. The individual variance of the listeners with impaired hearing could not be explained by the model considering individual absolute hearing thresholds, where at most 23% of the variance could be explained with a considerable RMS prediction error of 2.4 dB (cf. ''HI indiv.'' predictions in the CAN condition in [fig_ref] Table 5: Statistical Analysis of the Predicted Benefits [/fig_ref]. However, the general trend, that the SRTs for the group of listeners with impaired hearing were higher than those for listeners with normal hearing, was predicted in accordance with the empirical data (cf. [fig_ref] Table 2: Median Empirical and Predicted SRTs for the Group of Listeners With Normal... [/fig_ref]. Also, the predictions of benefits in SRT for the group of listeners with impaired hearing (median of the corresponding benefits in SNR) explained about 75% of the variance in the empirical data with RMS prediction error of 1.5 dB and 0.9 dB in the 20T and CAN condition, respectively.
A hypothesis is that the listeners with impaired hearing are not sufficiently characterized by the audiogram, as already suggested by [bib_ref] Individual speech recognition in noise, the audiogram and more: Using automatic speech..., Kollmeier [/bib_ref]. According to [bib_ref] Individual speech recognition in noise, the audiogram and more: Using automatic speech..., Kollmeier [/bib_ref] , a (monaural) suprathreshold distortion component was important to accurately model the individual effect of HL on speech recognition tasks, and according to [bib_ref] Auditory handicap of hearing impairment and the limited benefit of hearing aids, Plomp [/bib_ref] , such a distortion component would affect the individual SRTs similarly in different conditions. This behavior could be observed in [fig_ref] Figure 8: Individual prediction errors [/fig_ref] , where the prediction errors in the 20T and CAN condition were plotted against each other and showed a significant and strong correlation (R 2 ¼ .69, p < .01). Any effect that was not explained by the model is accumulated in the prediction error, that is, deviations due do not considering a suprathreshold distortion component, missing binaural interaction, or neglected cognitive effects, as well as any measuring inaccuracies (apart from the audiograms). Errors in the measurement of the audiograms would affect the predictions in all conditions similarly, while errors in the measurement of SRTs can be assumed to be independent and hence would not lead to systematic errors. One possible explanation for the strong correlation of prediction error across conditions could be systematic biases due to errors in the measurement of the audiograms. Another possibility is that there could be another individual factor-apart from the audiogram-missing to accurately predict individual benefits in SRT with FADE. Such an uncontrolled individual distortion component could be responsible for up to 4 dB (cf. point spread in [fig_ref] Figure 8: Individual prediction errors [/fig_ref] , which would translate to an average group effect of up to approximately 2 dB. This is in agreement with the average differences in SRT between the group of listeners with impaired hearing and the group of listeners with normal hearing, which were empirically found to be 1.8 dB in the 20T and 1.3 dB in the CAN condition, and predicted to be 1.7 dB and 1.2 dB, respectively.
In its current form, FADE was able to accurately predict the benefit of algorithms for the groups of listeners with normal hearing and impaired hearing. Individual (aided) performance, however, could not be predicted based on an individualization with the audiogram alone. In future work, a possible individual distortion component, for example, as suggested by [bib_ref] Individual speech recognition in noise, the audiogram and more: Using automatic speech..., Kollmeier [/bib_ref] in the form of a level uncertainty, should be considered to possibly enable accurate individual predictions of the aided performance of listeners with impaired hearing.
Another (related) explanation for the low correlation of the individual predictions could be an insufficient consideration of the (possibly even individual impaired) binaural interaction. The current approach considers better ear listening that does not include true binaural interactions across the signals presented to both ears. An individual component regarding the efficiency of the binaural processing would have the same effect as the (monaural) suprathreshold distortion component discussed before. In the end, a combination of both seems likely.
## Limitations and opportunities of the approach
The FADE modeling approach has been extensively evaluated with the matrix sentence test-mainly because of its very good test-retest reliability-but still not with other speech tests. Because ASR systems are trained for each prediction on the corresponding speech material, there are no fundamental reasons why the same approach should not work with other speech material or vocabulary. In fact, it was already shown that the very same model predicts the outcome of the matrix sentence test in other languages [bib_ref] Microscopic multilingual matrix test predictions using an ASR-based speech recognition model, Scha¨dler [/bib_ref] in different noise conditions without additional assumptions. However, larger vocabularies and more complex grammatical structures will probably increase the already high computational demands; currently, about 5 min per prediction can be achieved on high-end 3 desktop PCs. It should be investigated in the future if the approach generalizes in this regard, that is, if it is able to predict the effect of the complexity of a speech test.
Also, these properties allow the interpretation of the prediction error. This might help to better understand which cues are missing in the feature vector for different kinds of tasks in comparison with full-blown complex ASR experiments where single factors cannot be isolated as easily, let alone the required resources to perform these experiments. A natural evolution of FADE could be the stepwise integration of methods/models from research on ASR, CASA, psychoacoustics, and even human physiology. Such extensions might be valid as long as the predicted SRTs for listeners with normal hearing do not increase. This appears to be a simple criterion that does not depend on empirical data and preserves the objectivity of FADE while being compatible with research on robust ASR.
If objectivity and interpretability of prediction errors are of no concern, FADE can be turned into a referencebased model by calibrating the predictions with empirical reference data. For example, if SRTs are empirically found to be 4 dB higher for a more complex speech test than for the matrix sentence test in a reference condition, the reference-free predictions for the matrix sentence test can be increased by 4 dB to predict the outcome of the more complex speech test. This approach is very similar to the calibration of reference-based models. It would allow performing reference-based predictions with FADE for data sets which otherwise could only be predicted with other reference-based models. Evaluations of predictions with FADE for other speech tests should consider testing the validity of a constant-offset assumption.
For individual predictions, the SRTs are allowed to increase depending on (dys-)functional parameters such as the audiogram or putative suprathreshold processing deficiencies. It is open for further research, which modification of the standard signal preprocessing and feature extraction best reflects the properties of listeners with hearing impairment. However, traditional auditory models are not expected to provide a sufficiently robust signal representation to achieve human performance levels with FADE in a fluctuating interferer [bib_ref] A simulation framework for auditory discrimination experiments: Revealing the importance of across-frequency..., Scha¨dler [/bib_ref] , a prerequisite for impaired predictions. This was attributed to the assumption of independent frequency bands and the resulting lack of across-frequency integration.
# Conclusions
The most important findings of this work can be summarized as follows:
. FADE explained about 90% of the variance of the empirical speech recognition threshold (SRT) data resulting from eight different binaural preprocessing strategies in a realistic cafeteria environment for listeners with normal hearing. It predicted the corresponding benefits in SRT with an RMS error of only 0.6 dB without requiring any a priori knowledge of empirical data to perform those predictions. This result makes FADE a promising candidate for the objective evaluation of complex (speech) signal processing algorithms. . A voiced speech masker (competing talker) at very low SRTs (<À20 dB in SNR) posed a problem to the model in its current form as it probably would to any ASR system. This led to pronounced underestimation of the empirically determined recognition performance. Further improvements in robust ASR features and the combination with CASA might help to overcome this problem in modeling. . Median predictions across the group of listeners with impaired hearing-where individual factors average out-explained about 75% of the variance in the empirical data. Basing individual SRT benefit predictions only on audiograms for listeners with impaired hearing explained only an unsatisfactory small fraction (< 25%) of the variance in the empirical data. The high correlation of prediction errors between different noise conditions across listeners indicate that at least one further individual factor, such as a suprathreshold processing deficiency, should be accounted for to improve the accuracy of individual predictions with impaired hearing. . The predictions with FADE showed a general trend to underestimate the empirical recognition performance. This bias indicates that the feature extraction stage and the simple binaural better ear listening strategy as well as the ASR model employed may not be optimal. This contrasts with the inherent assumption of FADE to estimate the lowest achievable SRT and shows a limitation of the current approach. However, the existence of the bias is due to two desired properties of FADE: (a) Its classification is nonintrusive, which prevents trivial manipulations of the bias towards lower SRTs using superhuman knowledge, and (b) its objectivity allows direct predictions of the outcomes and avoids the bias-removing necessity of a calibration to a reference condition.
[fig] Figure 1: Characterization and examples for intrusive and nonintrusive classification. [/fig]
[fig] Figure 3: Illustration of the combined effect of an additive noise and a typical hearing threshold on the individual spectrotemporal representation of the speech signal, from which the features for the ASR system are calculated. The upper panel shows the log Melspectrogram of the German matrix sentence ''Wolfgang malt drei alte Messer,'' presented at about 75 dB SPL. [/fig]
[fig] Figure 4: Predicted versus empirical SRTs for listeners with normal and impaired hearing with eight different binaural preprocessing algorithms (plus the no processing reference condition) in a 20-talker babble noise condition (left panel) and a cafeteria ambient noise condition (right panel). The median SRTs are plotted with black squares and circles for listeners with normal (NH) and impaired hearing (HI), respectively. The filled symbols indicate the no preprocessing (NoPre) condition. The individual predictions for listeners with impaired hearing are plotted with colored stars, where the color indicates the listener. The dash-dotted lines indicate perfect prediction. SNR ¼ signal-to-noise ratio; SRT ¼ speech reception threshold. [/fig]
[fig] Figure 5: Predicted versus empirical benefits in SRT (difference to reference condition, higher values are better) for listeners with normal (NH, left panel) and aided impaired (HI, right panel) hearing in a cafeteria ambient noise condition with eight different binaural preprocessing algorithms. The individual predictions for listeners with impaired hearing are plotted with colored stars, where the color indicates the listener. The dash-dotted lines indicate perfect prediction. The error bars of the median predictions indicate the first and third quartile of the corresponding individual data, where for the predictions for normal hearing listeners, these were assumed to be the median AE 0.5 dB. SRT ¼ speech reception threshold. [/fig]
[fig] a: The dt of the iSNR-bsed predictions were tken fromBumgärtel et l. (2015). [/fig]
[fig] Figure 8: Individual prediction errors (difference between the predicted and the empirical SRT) for listeners with aided impaired hearing in a cafeteria ambient noise condition plotted against a 20talker babble noise condition. SRT ¼ speech reception threshold; CAN ¼ Cafeteria Ambient Noise; HI ¼ hearing impaired. [/fig]
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Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing
Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self.
# Introduction
The most prevalent type of RNA editing in metazoans is the adenosine to inosine (A-to-I) modification, mediated by a highly conserved protein family known as adenosine deaminases acting on RNA (ADAR; [bib_ref] The ADAR protein family, Savva [/bib_ref]. Acting on RNAs with substantial degrees of doublestrandedness (dsRNAs), ADAR enzymes have the capacity to bind and modify specific adenosines to inosines in short and imperfect dsRNA substrates [bib_ref] Functions and regulation of RNA editing by ADAR deaminases, Nishikura [/bib_ref]. Conversely, in long and perfectly base-paired dsRNA molecules, ADARs exhibit a promiscuous catalytic activity that modifies up to 40-50% of adenosines, a phenomenon referred to as hyper-editing [bib_ref] RNA editing by adenosine deaminases that act on RNA, Bass [/bib_ref]. Inosine nucleosides in RNA are interpreted as guanosines by the cellular machineries involved in RNA metabolism, including polymerases, the spliceosome and ribosome [bib_ref] Synthetic polynucleotides and the amino acid code, Basilio [/bib_ref]. Thus, ADAR-mediated modifications in RNA molecules inherently alter RNA metabolism on multiple levels, generating an increased variety of transcriptional outputs that enhance eukaryotic molecular complexity and serve as a source of variation for the generation of evolutionary novelty. Characterization of RNAediting landscapes from a broad range of phyla, using next-generation sequencing technologies, suggests that ADAR modifications are more widespread than previously thought and are distributed throughout genomes in highly species-specific patterns. Despite variations between RNA-editing landscapes across model organisms, editing sites are observed in both coding and non-coding regions of the genome, with the latter being the most prevalent. Similarly, RNA-editing sites in humans are over-represented in non-coding Alu repeats [bib_ref] Accurate identification of human Alu and non-Alu RNA editing sites, Ramaswami [/bib_ref] [bib_ref] A-to-I RNA editing occurs at over a hundred million genomic sites, located..., Bazak [/bib_ref] , the most abundant transposable element existing in the genome, corresponding to approximately 10% of its content [bib_ref] Initial sequencing and analysis of the human genome, Lander [/bib_ref].
RNA-editing enzymes are enriched in the nuclear compartment and expressed predominantly within the nervous system [bib_ref] The ADAR protein family, Savva [/bib_ref]. This specific localization pattern suggests a pivotal role of these enzymes for proper nervous system function. Indeed, the primary function of ADARs is thought to be in preserving nervous system integrity, as exemplified by neurological phenotypes of RNA-editingdeficient genetic models. Specifically, invertebrates lacking all ADAR activity exhibit severe neurological defects and behavioral abnormalities. For example, loss of function of the single Adar in Drosophila leads to frequent seizures, chronic uncoordination, and age-dependent neurodegeneration [bib_ref] A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system..., Palladino [/bib_ref]. Furthermore, Caenorhabditis elegans (C. elegans) lacking RNA-editing activity through the deletion of both encoded adar genes exhibit defects in chemotaxis [bib_ref] RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans, Tonkin [/bib_ref]. In contrast to ADAR deficiencies in invertebrates, mice lacking either ADAR1 or ADAR2 editing enzymes result in lethal phenotypes. Deletion of ADAR1 leads to embryonic lethality accompanied by elevated cellular apoptosis [bib_ref] Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene, Wang [/bib_ref] , while mice lacking ADAR2 exhibit severe seizure episodes characteristic of juvenile-onset epilepsy that result in lethality early in life [bib_ref] Point mutation in an AMPA receptor gene rescues lethality in mice deficient..., Higuchi [/bib_ref]. Two observations suggest that RNA-editing systems are functionally pleiotropic in regulating distinct physiologically essential RNA pathways. First, the chemotaxis defect exhibited by adar null C. elegans is rescued by mutants impaired in RNA interference (RNAi) response [bib_ref] Mutations in RNAi rescue aberrant chemotaxis of ADAR mutants, Tonkin [/bib_ref]. This observation suggests that without RNA editing, improper dsRNAs enter the RNAi pathway and trigger spurious silencing responses. Such a result is confirmed by the analysis of small RNAs generated in these adar null animals. In this study, the absence of adar caused the appearance of new classes of small interfering RNAs (siRNAs) from what the authors named, adar-dependent loci (ADLs; [bib_ref] Competition between ADAR and RNAi pathways for an extensive class of RNA..., Wu [/bib_ref]. Second, the lethality observed in ADAR2 null mice can be rescued by the edited version of the glutamate receptor GluR2 [bib_ref] Point mutation in an AMPA receptor gene rescues lethality in mice deficient..., Higuchi [/bib_ref]. Therefore, depending upon species context, some of the numerous fates of edited RNA molecules are more physiologically relevant compared to others operating within the same transcriptome.
## General fates of edited rnas
## Genomic recoding
Since ribosomes interpret inosines as guanosines, the capacity to generate protein products that are not literally encoded by genomic DNA is enabled by RNA editing in coding regions. Also known as genomic recoding, this phenomenon extends the genetic information potential through diversification of the protein repertoire, analogous to alternative splicing [bib_ref] Expansion of the eukaryotic proteome by alternative splicing, Nilsen [/bib_ref]. In the nucleus and during the synthesis of nascent transcripts, ADAR enzymes bind to dsRNA structures generally formed by highly conserved intronic sequences, which are complementary with the exon to be edited. Occurring co-transcriptionally [bib_ref] Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila, Rodriguez [/bib_ref] , the short and imperfectly base-paired nature of the dsRNA substrates allows ADARs to choose specific adenosines for modification within exonic sequences [bib_ref] RNA editing by adenosine deaminases that act on RNA, Bass [/bib_ref] , directed by loops and bulges in the dsRNA structure. Edited RNA templates are subsequently exported to the cytoplasm and translated by the ribosomal machinery. For instance, a specific RNA-editing event within a glutamic acid codon (CAG(Q) −→ CIG) is interpreted by the ribosome as CGG(R) [fig_ref] FIGURE 1 |: General fates of edited RNAs [/fig_ref] , and results in the insertion of the charged residue arginine in the polypeptide chain rather than the encoded polar residue glutamine. Genomic recoding is extensively utilized in Drosophila as a means of neuronal proteome diversification , while in vertebrates this kind of RNA editing seems to be limited [bib_ref] Limited RNA editing in exons of mouse liver and adipose, Lagarrigue [/bib_ref]. Intriguingly, proteins involved in neurotransmission are the major targets of specific editing [bib_ref] Nervous system targets of RNA editing identified by comparative genomics, Hoopengardner [/bib_ref]. However, recent studies suggest that RNA editing can additionally target transcripts encoding proteins involved in a variety of cellular functions including transcription, RNA splicing, protein metabolism, and DNA replication [bib_ref] The developmental transcriptome of Drosophila melanogaster, Graveley [/bib_ref] [bib_ref] Genome-wide analysis of A-to-I RNA editing by singlemolecule sequencing in Drosophila, St Laurent [/bib_ref]. Generally, genomic recoding events can influence protein function, and in some cases this fine-tuning effect can have broad cellular consequences. For example, a specific RNA-editing event that leads to a quite conservative missense amino acid substitution dramatically alters the rate of inactivation in human potassium (K + ) channels [bib_ref] Control of human potassium channel inactivation by editing of a small mRNA..., Bhalla [/bib_ref] , while a single RNA-editing site within the Adar transcript in Drosophila reshapes the global landscape of editing events in a manner that impacts complex adult behaviors [bib_ref] Auto-regulatory RNA editing fine-tunes mRNA re-coding and complex behaviour in Drosophila, Savva [/bib_ref] and has even more dramatic consequences for heterochromatic gene silencing .
## Rna splicing
Since RNA editing occurs cotranscriptionally, specific ADAR modifications can influence downstream RNA processing events. Given that the majority of editing sites are found within intronic sequences, editing has the capacity to influence RNA splicing. More specifically, due to the canonical nature of 5 splice donor sites (GU) and 3 splice acceptor sites (AG), specific RNA editing in introns can generate novel splicing signals (e.g., AU-to-IU −→ GU (donor) or AA-to-AI −→ AG(acceptor); Nishikura, 2010). In mammals, the ADAR2 RNA-editing enzyme modifies its own transcript to generate a novel splicing signal that results in the inclusion of 47 nucleotides in the mature RNA. (C) Hyper-editing of dsRNA molecules leads to inefficient Dicer processing. Hyper-editing antagonizes RNA-mediated silencing responses through the generation of fewer siRNAs. (D) Specific RNA editing near the base of miRNA precursors leads in the inhibition of Drosha cleavage, which prevents the processing of mature miRNAs. In addition, specific RNA editing within the "seed" region of the mature miRNA may result in redirection to a new target.
For example, RNA editing in an intronic Alu element within the human nuclear prelamin A recognition factor generates a novel 3 splicing acceptor site regulating its exonization in a tissuespecific pattern [bib_ref] RNA-editing-mediated exon evolution, Lev-Maor [/bib_ref]. Similarly, the mammalian ADAR2-editing enzyme generates a new acceptor site in its own transcript that results in a 47 nucleotide inclusion [fig_ref] FIGURE 1 |: General fates of edited RNAs [/fig_ref] and subsequent generation of a hypomorphic allele [bib_ref] Regulation of alternative splicing by RNA editing, Rueter [/bib_ref] , which leads to reduction in RNA-editing levels at multiple adenosine targets [bib_ref] Altered RNA editing in mice lacking ADAR2 autoregulation, Feng [/bib_ref]. Correspondingly, RNA editing in a 3 acceptor splice site (AG-to-IG −→ GG) can prevent its recognition by the spliceosome machinery. The observation that ADAR1 knockdown leads to aberrant exonization of an Alu element in the seryl-tRNA synthetase transcript, and suggests that RNA editing also plays a destructive role in eliminating unwarranted acceptor sites to ensure proper splicing [bib_ref] Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome, Sakurai [/bib_ref].
## Rna silencing
Cellular defense against endogenous and exogenous parasitic nucleic acids (such as transposons and viruses, respectively) is achieved by dsRNA molecules, which trigger a highly conserved biological response known as RNAi [bib_ref] RNA interference, Hannon [/bib_ref]. In addition to its safeguarding roles, RNAi also regulates gene expression through target transcript cleavage or by translation repression. Central to RNAi-mediated gene regulation are two kinds of small RNA species, siRNAs and microRNAs (miRNAs; [bib_ref] Small silencing RNAs: an expanding universe, Ghildiyal [/bib_ref]. These small RNA species are generated via Dicer processing of dsRNA triggers [bib_ref] Role for a bidentate ribonuclease in the initiation step of RNA interference, Bernstein [/bib_ref] , whose formation is mediated by endogenous genomic sequences with near-perfect base complentarity. For the biogenesis of siRNAs, Dicer enzymes process long and perfectly base-paired dsRNA molecules usually formed by transposable element sequences [bib_ref] Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells, Kawamura [/bib_ref]. Such dsRNA sources can also serve as ADAR substrates. Similarly, RNA-editing enzymes are capable of binding the same substrates as Dicer, hyper-editing the dsRNA molecules and destroying their near-perfect duplex nature and leading to inefficient Dicer processing [fig_ref] FIGURE 1 |: General fates of edited RNAs [/fig_ref] ; [bib_ref] Editor meets silencer: crosstalk between RNA editing and RNA interference, Nishikura [/bib_ref]. Thus, hyper-editing has the capacity to antagonize cellular RNAi responses through interfering with siRNA biogenesis [bib_ref] RNAi is antagonized by A->I hyperediting, Scadden [/bib_ref]. This assertion is borne out by the observation that a significant fraction of siRNA effector molecules found in AGO2 RISC complexes contain a single A-to-G mismatch, implying the action of ADAR on siRNA precursors does not completely prevent their appearance in silencing complexes [bib_ref] Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells, Kawamura [/bib_ref]. Intriguingly, ADAR activity can regulate heterochromatin formation by associating with dsRNA sources produced from transposable elements, altering gene expression of neighboring genomic regions . Contrary to the siRNA pathway, the dsRNA triggers for miRNA biogenesis are shorter and usually contain bulges and loops, resulting in specific editing [bib_ref] RNA editing of a miRNA precursor, Luciano [/bib_ref] [bib_ref] RNA editing of human microRNAs, Blow [/bib_ref]. Moreover, specific editing regulates the miRNA pathway at numerous levels [fig_ref] FIGURE 1 |: General fates of edited RNAs [/fig_ref]. First, as miRNA precursors are edited, pre-miRNA cleavage and other processing steps of the pathway can be inhibited [bib_ref] Modulation of microRNA processing and expression through RNA editing by ADAR deaminases, Yang [/bib_ref]. Second, the targeting step, which requires complementarity between a miRNA and its target sequence, may potentially reroute the miRNA target site recognition to new mRNAs due to specific editing within the miRNA "seed" region. Indeed, a single RNA-editing event is sufficient to redirect a miRNA to a new complementary target [bib_ref] Redirection of silencing targets by adenosine-to-inosine editing of miRNAs, Kawahara [/bib_ref]. Finally, the observation that ADAR modifications are abundant at miRNA target sites suggests that RNA editing can regulate gene expression by destruction of miRNA/target complementarity [bib_ref] Canonical A-to-I and C-to-U RNA editing is enriched at 3 UTRs and..., Gu [/bib_ref].
## Rna editing and circular rnas
Transcriptional profiling studies in metazoans have revealed mysterious new RNA species comprising unbroken circles [bib_ref] Circular RNAs are a large class of animal RNAs with regulatory potency, Memczak [/bib_ref] [bib_ref] Detecting and characterizing circular RNAs, Jeck [/bib_ref] [bib_ref] Circular RNAs: diversity of form and function, Lasda [/bib_ref]. Termed circular RNAs (circRNAs), these RNA species are generated through a non-linear splicing mechanism in which a canonical 5 donor site of an exon is spliced to an upstream 3 acceptor splice site [bib_ref] circRNA biogenesis competes with pre-mRNA splicing, Ashwal-Fluss [/bib_ref] [bib_ref] Exon circularization requires canonical splice signals, Starke [/bib_ref]. While in humans circRNAs can be detected in diverse cell types [bib_ref] Circular RNAs are the predominant transcript isoform from hundreds of human genes..., Salzman [/bib_ref] , recent evidence suggests that they are highly enriched in the nervous system, specifically at synapses [bib_ref] Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties..., Westholm [/bib_ref] [bib_ref] Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically..., Rybak-Wolf [/bib_ref]. A hallmark for circRNA biogenesis in mammals is the presence of complementarity between inverted intronic sequences that flank the exon destined for circularization. Mechanistically, these sequences can base pair extensively forming stem-loop dsRNA molecules, wherein the exon(s) to be circularized are sequestered within the loop sequences. Alu repetitive sequences are highly associated with exon circularization [bib_ref] Circular RNAs are abundant, conserved, and associated with ALU repeats, Jeck [/bib_ref] [bib_ref] Complementary sequence-mediated exon circularization, Zhang [/bib_ref]. Strikingly, A-to-I substitutions in introns flanking splice sites of circularization are highly enriched [bib_ref] Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals, Ivanov [/bib_ref]. Moreover, ADAR knockdown leads to accumulation of circRNAs in human cells [bib_ref] Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals, Ivanov [/bib_ref]. Similarly, several mouse circRNAs are upregulated upon decreasing ADAR expression [bib_ref] Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically..., Rybak-Wolf [/bib_ref]. This antagonistic effect between RNA-editing enzymes and circRNA biogenesis is a conserved feature in invertebrates. First, intronic sequences flanking circRNAs are enriched for hyper-editing events in C. elegans [bib_ref] Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals, Ivanov [/bib_ref]. Second, Drosophila raised at 29 - C exhibit elevated levels of circRNAs when compared to flies maintained at 18 - C [fig_ref] FIGURE 1 |: General fates of edited RNAs [/fig_ref]. The accumulation of circRNAs at higher temperatures is thought to occur due to a decrease in ADAR expression at these temperatures, as a recent study shows elevated temperature dynamically downregulates levels of the ADAR protein [bib_ref] Dynamic response of RNA editing to temperature in Drosophila, Rieder [/bib_ref]. Clearly, circRNA biogenesis is highly regulated through ADAR activity; however how this is achieved mechanistically is not yet clear. While the biological roles of circRNAs are currently unknown, evidence suggests that circRNAs can act as sponges for the assembly of miRNAs and accumulate during the aging of the nervous system [bib_ref] Natural RNA circles function as efficient microRNA sponges, Hansen [/bib_ref] [bib_ref] Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties..., Westholm [/bib_ref]. Nevertheless, whatever the functions of these circRNAs might be, RNA editing has the capacity to finetune downstream biological phenomena by antagonizing the biogenesis of circRNAs .
## Rna editing and innate immune responses to dsrnas
Cellular infection by viruses activates a protective mechanism that involves an inflammatory response [bib_ref] Origin and physiological roles of inflammation, Medzhitov [/bib_ref]. The physiological role of this inflamatory response is to provide necessary stimuli required by the host to establish a potent defense mechanism. Referred to as the innate immune response, it is triggered by bacteria and viruses without the need for adaptive immunity. In particular, the recognition of foreign dsRNAs generated during the initial cycles of viral replication is a potent inducer of innate immune pathways [bib_ref] Pathogen recognition and innate immunity, Akira [/bib_ref]. In vertebrates, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) operate as sensors for the recognition of foreign dsRNA molecules [bib_ref] Pattern recognition receptors and inflammation, Takeuchi [/bib_ref]. In the cytoplasm, RIG-I recognizes dsRNAs that are up to 1 kb in legth, while MAD5 senses longer RNA duplexes. Despite this discriminatory recognition, both sensors trigger the actions of a mitochondrial antiviralsignaling protein, MAVS, which signals for the activation of a cascade of events. This cascade involves several factors that orchestrate the expression of cytokines and type I interferons (IFNs) genes required for multiple defense responses [bib_ref] Pattern recognition receptors and inflammation, Takeuchi [/bib_ref]. It has been recently realized that genomes are transcribed in their entireties through pervasive transcription [bib_ref] The transcriptional landscape of the mammalian genome, Carninci [/bib_ref] [bib_ref] Landscape of transcription in human cells, Djebali [/bib_ref] , generating a myriad of RNA species that participate in diverse cellular functions. Moreover, the identification of endogenous RNA-based silencing pathways suggests that dsRNA molecules are generated by a variety of genomic sources [bib_ref] Endo-siRNAs: yet another layer of complexity in RNA silencing, Nilsen [/bib_ref]. Due to their nature, endogenously generated dsRNA duplexes are highly structurally similar to those generated through viral replication Frontiers in Genetics | www.frontiersin.org FIGURE 2 | Novel roles for RNA editing. (A) The biogenesis of circular RNAs involves the formation of dsRNA duplexes mediated by intronic sequences flanking the exon(s) destined for circularization. These dsRNA molecules can serve as ADAR substrates. It is currently thought that hyper-editing destroys the dsRNA nature of such substrates, which promotes linear splicing and restricts exon circularization. Examples of how RNA editing antagonizes the biogenesis of circRNAs formed by single exon and two exons are depicted. (B) In the cytoplasm, RIG-I and MDA5 act as sensors for dsRNA molecules. Upon recognition, these sensors activate MAVS, which triggers the innate immune response. MAVS-mediated signals result in the translocation of interferon-regulatory factors (IRFs) and NF-κB into the nucleus, which initiates the transcription of ISG and cytokine genes. Hyper-editing of immunoreactive dsRNAs prevents detection by RIG-I and MDA5, inhibiting aberrant innate immune responses toward endogenous dsRNA sources.
after infection. This raises the question of how cells are able to discriminate endogenous dsRNA molecules from exogenous and therefore avoid aberrant immune responces. Previous observations suggest that endogenous RNAs marked with specific nucleoside modifications avoid detection by sensor proteins of the immune response [bib_ref] Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification..., Kariko [/bib_ref]. Intriguingly, synthetic dsRNAs containing multiple IU pairs, which mimic the hyperediting activity of RNA-editing enzymes, fail to induce innate immune responses [bib_ref] Double-stranded RNAs containing multiple IU pairs are sufficient to suppress interferon induction..., Vitali [/bib_ref]. Thus, it was proposed that the presence of IU pairs in dsRNA duplexes interfere with the detection process mediated by the RIG-I and MAD5 sensors, suggesting that RNA editing in principal can regulate innate immune responses. Indeed, two recent studies have uncovered that RNA editing regulates the cascade of events that lead to the activation of innate immune responses. Moreover, this pathway acts upstream to hyper-edit naturally occuring (self) dsRNA duplexes in order to avoid detection as foreign dsRNA (non-self) that otherwise would elicit abberant immune responses.
In these studies, it was reasoned that the elevated cellular apoptosis exhibited by ADAR1 null mice is due to induction of immune/inflammatory responses triggered by non-edited, endogenous dsRNA sources. In agreement with this notion, [bib_ref] The RNA-editing enzyme ADAR1 controls innate immune responses to RNA, Mannion [/bib_ref] examined the transcriptional profiling in ADAR1 null embryos and observed that transcripts of interferon-stimulated genes (ISGs) were elevated significantly. Moreover, the Adar null (Adar1 (−/−) ) mouse embryonic lethal phenotype was substantially rescued to pup birth in Adar1 (−/−) /Mavs (−/−) double-mutant mice. Finally, transfection of dsRNA species containing multiple IU pairs was unable to elicit an immune response in fibroblast cells of ADAR1-deficient mice, which indicates that editing activity is necessary for blocking the response. In a similar study, [bib_ref] RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself, Liddicoat [/bib_ref] generated an ADAR1 editing-activity-deficient mouse allele (Adar1 E861A/E861A ). Through transcriptional profiling of homozygous mutant mice, they observed an atypical upregulation of ISGs, highly similar to the one observed in the ADAR1 protein null mice [bib_ref] RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself, Liddicoat [/bib_ref]. Furthermore, through analysis of the ADAR1 editing landscape they revealed that the major substrates of this RNA-editing ezyme are dsRNA duplexes formed by 3 UTR sequences. They reasoned that the absence of IU pairs from these dsRNAs can elicit aberrant immune responces through MDA5 sensing. Indeed, generating Adar1 E861A/E861A /MDA5 (−/−) double-mutant mice also rescued the embryonic lethality phenotype suggesting that MDA5 is the principal sensor of non-edited endogenous dsRNAs. Clearly, these observations suggest a pivotal role for RNA editing in the regulation of innate immune responses toward self-generated dsRNA molecules. More importantly, ADAR1 acts hierarchically upstream in the immune cascade and through RNA editing it fingerprints dsRNAs as endogenous, thus preventing undesired cellular immune responses and allowing robust measures to be taken against invading non-self dsRNA signaling events .
# Conclusion
A universal property of RNA, and trait of transcriptomes in general, is the generation of RNA molecules with varying degrees of double-strandedness that orchestrate diverse cellular functions. Despite variation in length, many endogenous molecules with dsRNA character intersect with the A-to-I RNAediting system, since no sequence specificity is required for ADAR binding and editing.
Rather, ADAR modification conceptually generates two radically opposed forces determined by substrate structure. When structures are imperfect dsRNA, only specific editing events occur. Because, in general, these imperfect structures involve conserved secondary and tertiary RNA interactions of exquisite detail, specific editing must be seen as a creative force in the evolutionary tool-kit -a novelty generator. Conversely, less specific hyper-editing of near-perfect duplexes serves a preventative or intervening role, competing with other enzymes that have different designs on the fate of long perfect dsRNA. Deconvolution of these opposing roles of editing in organismal phenotype will remain a challenge into the near future.
RNA-editing enzymes have the inherent capacity to regulate many dinstinct pathways that are involved in RNA metabolism and serve to fine-tune and optimize transcriptional outputs [bib_ref] Genomic analysis of ADAR1 binding and its involvement in multiple RNA processing..., Bahn [/bib_ref]. Although, anomalies in RNA editing are associated with multiple neurological disorders such as epilepsy, schizophrenia, and amyotrophic lateral sclerosis (ALS; [bib_ref] Adenosine-to-inosine RNA editing and human disease, Slotkin [/bib_ref] , the molecular determinants of ADAR-mediated pathogenesis still remain elusive. In principle, mutations that affect editing activity [bib_ref] Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B,..., Crow [/bib_ref] or lead to abnormal ADAR sequestration [bib_ref] RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention, Donnelly [/bib_ref] can both lead to disease phenotypes due to detrimental impacts imposed on the known general fates of edited RNAs. In addition, as these recent studies suggest, a severe reduction in RNA editing may lead to the abnormal accumulation of circular RNAs and elicit aberrant innate immune responses. Future research should aim to understand how these novel RNA-editing roles may contribute to the etiology of neurological disorders.
Previously, multiple missense mutations in the Adar1 gene were identified in individuals diagnosed with AicardiGoutières syndrome (AGS), an autoimmune disorder [bib_ref] Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon..., Rice [/bib_ref]. In addition, individuals with AGS-associated Adar1 mutations exhibited high levels of expression for a number of IFNstimulated genes, a phenotype observed previously in ADAR1deficient mice [bib_ref] ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon..., Hartner [/bib_ref]. Therefore, it was proposed that ADAR1 theoretically could suppress immunoreactive dsRNA molecules through hyper-editing and control aberrant immune reponses. Certainly, the new role for the A-to-I RNAediting system provided by [bib_ref] The RNA-editing enzyme ADAR1 controls innate immune responses to RNA, Mannion [/bib_ref] and [bib_ref] RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself, Liddicoat [/bib_ref] contributed significantly to a better understanding of how Adar1 mutations can result in autoimmune diseases. Thus, it is becoming increasingly clear that ADAR-mediated modifications occupy a central role in cellular physiology. Therefore, a more comprehensive understanding on how RNAediting systems regulate global cellular RNA metabolism can shed light in the development of therapeutic strategies of ADAR-mediated human diseases by providing insights into their pathogenesis.
# Author contributions
All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.
[fig] FIGURE 1 |: General fates of edited RNAs. (A) Specific RNA editing in coding region of a pre-mRNA. ADAR-mediated hydrolytic deamination of the adenine base within the glutamic acid codon (Q) is interpreted by the ribosomal machinery as an arginine codon (R), leading to an amino acid substitution. (B) Specific RNA editing in non-coding regions can generate novel 3 splice acceptor sites (AA −→ AG). [/fig]
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An overlapping module identification method in protein-protein interaction networks
Background: Previous studies have shown modular structures in PPI (protein-protein interaction) networks. More recently, many genome and metagenome investigations have focused on identifying modules in PPI networks. However, most of the existing methods are insufficient when applied to networks with overlapping modular structures. In our study, we describe a novel overlapping module identification method (OMIM) to address this problem. Results: Our method is an agglomerative clustering method merging modules according to their contributions to modularity. Nodes that have positive effects on more than two modules are defined as overlapping parts. As well, we designed de-noising steps based on a clustering coefficient and hub finding steps based on nodal weight. Conclusions: The low computational complexity and few control parameters prove that our method is suitable for large scale PPI network analysis. First, we verified OMIM on a small artificial word association network which was able to provide us with a comprehensive evaluation. Then experiments on real PPI networks from the MIPS Saccharomyces Cerevisiae dataset were carried out. The results show that OMIM outperforms several other popular methods in identifying high quality modular structures.
# Background
In general, a good understanding of protein families provides us with further views on biological processes. Previous studies have shown that modular structures are densely connected internally but sparsely interacting with others in PPI networks [bib_ref] A network of interacting proteins in yeast, Schwikowski [/bib_ref] [bib_ref] Protein complexes and functional modules in molecular networks, Spirin [/bib_ref]. Modules can be understood as independent sub-networks and proteins in the same module always interact more frequently and show stronger functional dependencies. These days, more and more people are likely to address biological problems with graphic models, where proteins or genes are viewed as nodes and their pair wise interactions as edges in a network [bib_ref] MINE: module identification in networks, Rhrissorrakrai [/bib_ref] [bib_ref] An algorithm for finding functional modules and protein complexes in protein-protein interaction..., Cui [/bib_ref].
Several methods have been proposed for module identification in the last decade. In 2003, Bader and Hogue proposed a molecular complex detection method (MCODE), which can separate densely connected regions by assigning a weight to each protein [bib_ref] An automated method for finding molecular complexes in large protein interaction networks, Bader [/bib_ref]. A Markov clustering method (MCL) which is based on flow simulation and high-flow areas corresponding to protein complexes was applied to detect protein families in 2002 [bib_ref] An efficient algorithm for largescale detection of protein families, Enright [/bib_ref]. A network module mining method (NeMo) proposed by Yan et al. identifies frequent dense sub-graphs in input networks using coherent edge frequencies, which can lose statistical power in sparse networks with few edges [bib_ref] A graph-based approach to systematically reconstruct human transcriptional regulatory modules, Yan [/bib_ref]. However, most of the existing methods cannot identify overlapping modules in PPI networks. As far as we know, some proteins may be included in multiple complexes and component parts of a complex could be activated at a specific time or location [bib_ref] What do we learn from high-throughput protein interaction data?, Titz [/bib_ref] [bib_ref] Exploring hierarchical and overlapping modular structure in the yeast protein interaction network, Liu [/bib_ref].
In 2006, a clique percolation method (CPM) was used for the first time to identify overlapping modules in PPI networks by finding fully connected sub-graphs of different minimum clique sizes [bib_ref] CFinder: locating cliques and overlapping modules in biological networks, Adamcsek [/bib_ref]. But its high computational complexity (O(exp(n))where n represents the number of nodes in the network) hindered its application to large scale networks.
Based on these considerations, we propose the OMIM, which is able to partition large scale PPI networks with overlapping modular structures. OMIM first clusters all nodes using a Newman algorithm [bib_ref] Fast algorithm for detecting community structure in networks, Newman [/bib_ref] and then defines nodes that have comparatively positive effects on the modularity of more than two modules as overlapping ones. Moreover, we designed de-noising steps through assigning a weight to each edge. Hubs can also be found according to their nodal weight. OMIM is a method that is able to identify highly interconnected modules and has few control parameters, allowing it to be applied to many types of networks. We evaluate OMIM as applied to an artificial network and a PPI network. The results showed that it outperforms several other current methodologies.
# Methods
## Overview
As we know, a PPI network can be described as an undirected and unweighted graph, G=(V,E), where V and E represent nodes (proteins) and edges (interactions) in the network. In our method, we first assign weights to all edges according to their importance to the network and remove those with lower weights as noise. Then the steps for identifying overlapping modules are performed. The main idea of identifying overlapping parts in OMIM is to find nodes that have comparatively positive effects on different modules. In addition, hubs were also found according to connections with their neighbors [bib_ref] Hubs of knowledge: using the functional link structure in Biozon to mine..., Shafer [/bib_ref].
## De-noising
In general, data in PPI networks are obtained from highthroughput protein-protein interaction experiments. So far, the most frequently used protein-protein interaction detection methods are yeast-2-hybrid, tandem affinity purification, mass spectrometry technology and protein chip technology. Although these high-throughput detection methods make for easy experimentation, they bring about noise and incompleteness [bib_ref] Geometric de-noising of protein-protein interaction networks, Kuchaiev [/bib_ref] [bib_ref] Sequence-based prediction of protein-protein interactions by means of rotation forest and autocorrelation..., Xia [/bib_ref] [bib_ref] Predicting protein-protein interactions from sequence using correlation coefficient and high-quality interaction dataset, Shi [/bib_ref].
The main idea in our de-noising step is to assign a weight to each edge of a PPI network to reflect the reliability of the corresponding interactions. In our study, we use a popular metric from graph theory, i.e., clustering coefficient. A clustering coefficient is a measure that represents the interconnectivity in the neighborhood of a node [bib_ref] An ensemble framework for clustering protein-protein interaction networks, Asur [/bib_ref]. The clustering coefficient of node i with degree k i can be described as
[formula] CC i = 2n i k i (k i − 1)(1) [/formula]
where n i denotes the number of triangles that go through node i.
The weight between nodes i and j can be assigned according to the following equation: [bib_ref] Protein complexes and functional modules in molecular networks, Spirin [/bib_ref] where CC' represents the clustering coefficient after the edge between i and j is removed. According to the viewpoint of Asur et al. [bib_ref] An ensemble framework for clustering protein-protein interaction networks, Asur [/bib_ref] , if two nodes are not actually connected in the original network, then the SCC(i,j) value should be small or equal to zero. Here, we define a threshold a, and remove edges that are smaller than a as noise.
[formula] SCC(i, j) = CC i + CC j − CC i − CC jSCC(i, j) ≤ α(3) [/formula]
Overlapping module identification method Newman algorithm
Because OMIM is a variant of the Newman algorithm, we first introduce the Newman algorithm briefly. This is a hierarchical agglomerative method based on the idea of modularity [bib_ref] Fast algorithm for detecting community structure in networks, Newman [/bib_ref]. We know that modularity is a measure of the quality of a particular division of a network and a large value of modularity always corresponds to good network division [bib_ref] Finding and evaluating community structure in networks, Newman [/bib_ref]. If we let e rk be the fraction of edges in the network, connecting nodes in group r to those in group k and let a r = k e rk , then
[formula] Q = r e rr − a 2 r(4) [/formula]
where Q is a quality function representing modularity. The physical meaning of Eq. (4) is that modularity is equal to the fraction of edges that fall within modules, minus the expected value of the same quantity if edges fall at random without regard to its modular structure [bib_ref] Fast algorithm for detecting community structure in networks, Newman [/bib_ref]. The Newman algorithm is a method for optimizing Q in order to discover the best modular structure.
The steps of the Newman algorithm can be summarized as follows.
Step 1. Initialize each node in the input data to be a module, define a matrix e and a vector a according to Eqs. [bib_ref] An automated method for finding molecular complexes in large protein interaction networks, Bader [/bib_ref] and [bib_ref] An efficient algorithm for largescale detection of protein families, Enright [/bib_ref].
[formula] a i = k i /2m(5) [/formula]
where m represent the total number of edges in the network.
Step 2. Calculate the change of modularity ΔQ according to:
[formula] Q = 2(e ij − a i a j )(7) [/formula]
Merge module pairs with the maximum value of ΔQ. Update matrix e by adding the rows and columns of the corresponding merged modules.
Step 3. Repeat Step 2, until the entire network has become one big module.
From this description, the progress of the Newman algorithm can be represented as a dendrogram. If we choose to cut at different levels, different modular structures can be obtained. Actually, Newman chooses to cut at the maximum value of Q to obtain the best modular structure.
## Identifying overlapping parts
It should be noted that complexes in PPI networks are not static and proteins can be included in different modules. Therefore, identifying overlapping parts between different modules is necessary. We first perform the Newman algorithm to the input data. Then we try to identify overlapping nodes according to their contribution to modularity. The detailed steps are as follows.
Step 1. Perform Newman algorithm. All nodes are clustered without overlapping parts.
Step 2. Define nodes, whose neighbors belong to more than two modules, to be candidate nodes.
Step 3. Randomly select node i from the set of candidate nodes. Assume that i is in module A and one of its neighbors,j, in module B. Copy i to B and a new module B' is obtained. If Eq. (8) is satisfied, then i is an overlapping node.
[formula] Q B > Q B(8) [/formula]
where Q B and Q B' is the modularity of B and B'.
Step 4. Repeat Steps 2~3 until all overlapping parts are identified.
## Discovering hubs
Jordan et al. first found hubs when they studied the evolution of protein and referred to the proteins with large number of partners as hubs [bib_ref] No simple dependence between protein evolution rate and the number of protein-protein..., Jordan [/bib_ref]. Han et al. divided hubs into two classes: party hubs and date hubs [bib_ref] Evidence for dynamically organized modularity in the yeast protein-protein interaction network, Han [/bib_ref]. Party hubs are hubs that interact with their partners at the same time, whereas date hubs either bind their different partners at different times or at different locations. According to their study in a network with a modular structure, date hubs always organize the proteome, while party hubs function inside modules. We propose a computational method to detect the hubs far easier.
First, we defined party hubs as those proteins that have maximal nodal weight (w i ) in a module, i.e.,
[formula] w i = j SCC(i, j), j ∈ {neighbor of i} (9) party hub r = arg max w i i ∈ r,(10) [/formula]
where partly hub r means a party hub of module r. Date hubs are defined as proteins that bind at least three modules. We set a variable ACC i to denote the number of modules to which i is bound. The computational method of ACC i is
[formula] ACC i = n r r=1 f (i) (11) [/formula]
where n r is the total number of modules in the network and f(i) is defined as follows:
[formula] f (i) = 1, i connect to at least one node in r 0, else(12)Algorithm 1. de-noising input: G=(V,E); a for all nodes i(i V) in G compute the clustering coefficient CC i end for all edges (i,j)((i,j) E) in G compute the weight SCC(i,j) if SCC(i,j)<a remove edge (i,a i = k i /2m [/formula]
# Results and discussion
## Data sources
In our experiments, we validated our method on two datasets, i.e., a small-scale artificial dataset and a largescale PPI dataset. The artificial dataset is derived from the South Florida Word Association database [bib_ref] The University of South Florida word association, rhyme, and word fragment norms, Nelson [/bib_ref] , with 151 nodes and 155 edges in the network . The eight core nodes playing important roles are month, sunshine, camp, sleep, work, enjoy, long and sunny respectively, which are connected by the key word day. The yeast (Saccharomyces Cerevisiae) PPI networks used in our study are from the MIPS Comprehensive Yeast Genome Database (CYGD) (PPI_18052006) [bib_ref] CYGD: the Comprehensive Yeast Genome Database, Güldener [/bib_ref]. The dataset contains 4989 proteins and 13583 interactions after removing isolated nodes and self-cycled edges. The on-line annotation tool, GO term finder (version 0.83), is from the SGD database (Saccharomyces Genome Database), which contains 7292 genes as a background set.
Methods used for comparisons in our experiments are Newman, MCL and CPM. There are two main reasons for this selection. In first instance, these are three classical clustering algorithms that have been widely used in many fields. Their use makes for clearer comparisons. Secondly, these algorithms represent the most appropriate methods in different aspects for comparison with OMIM. According to Brohée et al. [bib_ref] Evaluation of clustering algorithms for proteinprotein interaction networks, Brohee [/bib_ref] , MCL outperforms many other algorithms, especially in partitioning PPI networks. CPM is a widely known classical method for identifying overlapping modules and the Newman algorithm is the ancestor of OMIM. Artificial word association dataset. The artificial word association dataset is a small scale network used to validate OMIM. It can be seen as a double layer network. 9 words constitute the first layer, in which the word 'day' works as a hub. The second layer consists of 8 subnetworks that center on other 8 words in the first layer, i.e., month, sunshine, camp, sleep, work, enjoy, long and sunny.
Among these three methods, MCL was executed as an embedded program of BioLayout Express 3Dand the CPM algorithm was performed by using of CFiner, a tool created for clustering based on CPM [bib_ref] Uncovering the overlapping community structure of complex networks in nature and society, Palla [/bib_ref].
## Performance on an artificial dataset
Three evaluation indices, i.e., accuracy (AC), overlapping rate (OL) and average degree (AVD) were used.
[formula] AC = n i=1 m i j=1 x i (j) n(13) [/formula]
where node j is a neighbor of node i, m i represents the total number of neighbor nodes of i, num_V(r) and num_E(r) represent the number of nodes and edges in module r respectively. x i (j) is a function defined as follows: if j is classified correctly, x i (j)=1; else, x i (j)=0. [fig_ref] Table 1: Results of the comparison on the word association dataset [/fig_ref] shows that the OMIM performed better than the other methods on accuracy. Although CPM is an algorithm which is able to find overlapping modular structures, it performed worst on the artificial dataset. The reason for this is that, the CPM filtered too much useful nodes during its execution. MCL discovered one more module than OMIM. The discrepancy is primarily due to the fact that MCL cannot deal with hierarchical networks and regards the first layer as another module. Note that the OL value of Newman is 1, which is a result of its inability to identify overlapping module structures.
Eight party hubs were found by OMIM, i.e., month, sunshine, camp, sleep, work, enjoy, long and sunny. The date hub is day. Besides, we also discovered four overlapping nodes: moon, outside, delight and walk. Compared with the original network shown in , our results can correctly cluster all nodes, verifying the effectiveness of our method.
## Performance on ppi networks p-value
According to the SGD database, the P-value is an index to determine the statistical significance of the association of a particular GO term with a group of genes. It has been widely used in bioinformatics in recent years [bib_ref] An algorithm for finding functional modules and protein complexes in protein-protein interaction..., Cui [/bib_ref] [bib_ref] Finding motif pairs in the interactions between heterogeneous proteins via bootstrapping and..., Kim [/bib_ref]. In general, its values are between 0 and 1. The closer the P-value is to zero, the more significant the particular GO term associated with the group of genes, i.e.:
[formula] P − value = n ol n − n 2 n 1 − ol n n 1(16) [/formula]
where n represents the size of the entire network, n 1 is a cluster obtained from the experiment, n 2 the number of proteins annotated with a specific GO term and ol the number of proteins in n 1 that can be annotated with the specific GO term.
In our experiments, P-values that higher than 0.01 were eliminated. We used the negative natural logarithms (-log P-value) to substitute for P-value.
## Cluster frequency
Cluster frequency is another index used in the SGD database which indicates the number of proteins in the experimental group annotated in a specific GO term. Although it is not as meaningful as P-value to represent the significance of a cluster to a specific GO term, its statistical value reflects the proportion of proteins that can reasonably be annotated, i.e.:
cluster frequency = ol n 2 (17)
## Discard rate
The discard rate represents the proportion of proteins not assigned to any module. In general, this rate reflects the filtering ability of the algorithm.
discard rate = 1 − number of output data number of input data [bib_ref] No simple dependence between protein evolution rate and the number of protein-protein..., Jordan [/bib_ref] Size distribution of PPI modules obtained by OMIM After setting the minimum module size to 4, we obtained 115 modules (Additional file 1) with a maximum value of Q=0.3616. is the size distribution of modules obtained by OMIM. shows that most modules are small, with very few modules that are extremely large. This coincides with the scale-free property of PPI networks, where most proteins interact with few partners, while a few proteins interact with many partners. The degree distribution of the PPI dataset in is able to explain the property. From we can see that, like most scale-free networks, the degree of the distribution of the PPI dataset follows the power law relationship P (K)~K -r with r≈2.5.
# Enrichment analysis
Enrichment analysis is an important index for protein function annotation. We used the GO term finder to assign a main function that corresponding to the best Pvalue to each module. 10 modules were selected randomly to demonstrate the results of the enrichment analysis (Additional file 2). [fig_ref] Table 2: Enrichment analysis of 10 randomly selected modules Main functions [/fig_ref] shows that most modules are able to be annotated to reliable functions on three Gene Ontology categories, i.e., molecular functions, biological process and cellular component. According to the P-values in [fig_ref] Table 2: Enrichment analysis of 10 randomly selected modules Main functions [/fig_ref] , the most significant module is # 21, with -log Pvalues of 75.04, 44.07 and 83.63 respectively. However, there are also some modules which do not clearly belong to any GO term functions, such as module # 98. In addition, we can infer proteins with unknown functions according to their membership in a module. In module # 12, RRP4/RRP42/RRP43/SKI6 are with unknown molecular function. However, their neighbor, DIS3, has the following molecular functions: 3'-5'-exoribonuclease activity, tRNA binding and endoribonuclease activity. Consequently, we can infer that RRP4/RRP42/ Size distribution of PPI modules obtained by OMIM. In , the abscissa indicates the size of the modules, i.e, the number of proteins in each module. The ordinate shows the number of modules with the size corresponding to abscissa. Degree distribution of PPI dataset. In , K represents the degree of protein and the ordinate P(K) the fraction of proteins in the network with degree K. RRP43/SKI6 may be associated with one or more molecular functions of DIS3.
# Cluster frequency analysis
Cluster frequency analysis is another evaluation criterion for protein module construction, indicating the proportion of proteins in an experimental group annotated in a specific GO term (Additional file 2). [fig_ref] Figure 4: Cluster frequency of 115 modules on category BP, MF and CC [/fig_ref] is the cluster frequency of 115 modules obtained by OMIM. [fig_ref] Figure 4: Cluster frequency of 115 modules on category BP, MF and CC [/fig_ref] shows that most modules have a very high cluster frequency. In fact, 26 modules have a cluster frequency of 100% in the category of biological process. The result shows that most proteins in these modules have a common reliable function in OMIM.
## Comparison of omim with other algorithms on ppi dataset
In order to validate the OMIM on the PPI dataset, we compared it with the Newman, MCL and CPM algorithms. The results for the Saccharomyces cerevisiae PPI dataset are summarized in [fig_ref] Table 3: Comparison OMIM with other competing algorithms on PPI dataset [/fig_ref]. The performance was largely measured by the discard rate and the enrichment analysis of Gene Ontology (molecular functions, biological process and cellular component). [fig_ref] Table 3: Comparison OMIM with other competing algorithms on PPI dataset [/fig_ref] shows that OMIM and Newman discard the least number of proteins (44.26%) for constructing modules compared with the other two methods. Moreover, OMIM is superior to Newman and MCL according to the enrichment analysis of Gene Ontology categories (BP, MF and CC). Although it has higher -log P-values on BP and CC than OMIM, CPM filtered too many proteins (about 85.51%) which may result in losing much useful information.
# Conclusions
The studies on an artificial and a PPI dataset verify the effectiveness of our method. In the experiment on the artificial dataset, the OMIM can find all modules correctly with an accuracy of 1.0000. All hubs that play key roles in the artificial networks are found precisely. In the experiment on the PPI dataset, we evaluated the performance of OMIM by enrichment analysis, cluster frequency analysis and in comparisons with other competing algorithms. All of the evaluation measures resulted in good performances. In addition, 30% of the hub proteins found by OMIM could directly be verified by the study of Han et al. [bib_ref] Evidence for dynamically organized modularity in the yeast protein-protein interaction network, Han [/bib_ref]. However, since the degree distribution of the PPI dataset follows a power law, the discrepancy on modular sizes was quite large, which is not rational. In our future work, we will try to settle the problem of unbalanced clustering.
# Additional material
Additional file 1: A list of 115 potential functional modules.pdf. This file contains all potential functional modules obtained by OMIM. For module #111 and 113, we did not list their members. The reason is that, their extremely large module sizes, 695 and 392, make them unreliable.
Additional file 2: Enrichment and cluster frequency analysis of 115 modules.pdf. The best P-values and its corresponding cluster frequencies of 115 modules obtained by SGD Go term finder. The empty cells in this table denote 'No significant ontology term can be found for this module'.
[fig] Figure 4: Cluster frequency of 115 modules on category BP, MF and CC. The abscissa indicates the module number and the ordinate the cluster frequency (%) in . Cluster frequency on three main functions BP (biological process), MF (molecular functions) and CC (cellular component) were marked by different colors. [/fig]
[table] Table 1: Results of the comparison on the word association dataset [/table]
[table] Table 2: Enrichment analysis of 10 randomly selected modules Main functions: the GO term that obtained according to -log P-values of all modules for biological process (BP), molecular functions (MF) and cellular component (CC). [/table]
[table] Table 3: Comparison OMIM with other competing algorithms on PPI dataset [/table]
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Engaging culture and context in mhGAP implementation: fostering reflexive deliberation in practice
# Abstract
In 2002, WHO launched the Mental Health Gap Action Programme (mhGAP) as a strategy to help member states scale up services to address the growing burden of mental, neurological and substance use disorders globally, especially in countries with limited resources. Since then, the mhGAP program has been widely implemented but also criticised for insufficient attention to cultural and social context and ethical issues. To address this issue and help overcome related barriers to scale-up, we outline a framework of questions exploring key cultural and ethical dimensions of mhGAP planning, adaptation, training, and implementation. This framework is meant to guide mhGAP activity taking place around the world. Our approach is informed by recent research on cultural formulation and adaptation, and aligned with key components of the WHO implementation research guide (Peters, D. H., Tran, N. T., & Adam, T. (2013). Implementation research in health: a practical guide. Implementation research in health: a practical guide.). The framework covers three broad domains: (1) Concepts of wellness and illness-how to examine cultural norms, knowledge, values and attitudes in relation to the "culture of the mhGAP"; (2) Systems of care-identifying formal and informal systems of care in the cultural context of practice.; and (3) Ethical space: examining issues related to power dynamics, communication, and decision-making. Systematic consideration of these issues can guide integration of cultural knowledge, structural competence, and ethics in implementation efforts.
# Background
In 2002, WHO launched the Mental Health Gap Action Programme (mhGAP) to address the growing burden of mental, neurological and substance use disorders. Globally, there is a significant gap between individuals in need of mental healthcare and those receiving it.The expressed aim of mhGAP is to provide health planners, policy-makers and donors with programmes and tools to support implementation and scale-up of mental health services and care, especially in low-income and middle-income countries (LMICs).The most recent iteration of this programme is presented in the WHO's Comprehensive Mental Health Action Plan 2013-2020 with the objectives of: (1) strengthening effective leadership and governance for mental health;
Summary box ► There is continued debate about efforts to provide standardized mental health care across cultures and contexts with concerns about: (1) inadequate identification and integration of local modes of expression of distress and healing practices; (2) risks of medicalizing everyday forms of distress; and (3) broader ethical questions about the effects of imposing of biomedical frameworks. ► The need for better understanding of culture in global mental health care has long been recognized. However, there is a need to develop approaches to guide the process of integration of culture and context in local adaptation and implementation of mental health protocols and interventions like mhGAP. ► This framework outlines points of entry to engage with culture and context by presenting a series of questions that can guide critical reflexive deliberation at all stages of mhGAP implementation as well as other efforts to address culture and context in global health work. ► The questions are organized around three key domains: concepts of wellness, systems of care, and ethical space. They focus on participants' knowledge, attitudes and assumptions about mental health, as well as local social and cultural context of mental health care, specific cultural and contextual issues relevant to the mhGAP intervention guide, and key ethical issues related to the power dynamics of health service implementation and delivery. ► Engagement with these questions creates opportunities for diverse voices to be heard, to promote knowledge exchange and allow key ethical questions to be addressed, whether for implementation, policy or practice. This framework can guide efforts to promote power-sharing and co-construction of knowledge in ways that are beneficial to diverse populations and communities and has the potential to improve implementation, uptake, and sustainability of global mental health programs.
## Bmj global health
(2) providing comprehensive, integrated and responsive mental health and social care services in communitybased settings; (3) implementing strategies for promotion and prevention in mental health and (4) strengthening information systems, evidence and research for mental health. A key component of mhGAP involves training non-specialised health professionals, and other service providers to address mental health needs in their practice. The mhGAP Intervention Guide (mhGAP-IG) was launched in 2010 to support this training initiative and has been used in more than 90 countries.The guide aims to equip service providers with clinical algorithms to diagnose and treat common mental and neurological disorders.
The mhGAP initiative and the broader global mental health (GMH) movement have been engaged in a debate about the appropriateness of globalising standardised mental healthcare across cultures and contexts.Critiques of GMH include concerns about: (1) inadequate identification and integration of local modes of expression of distress and related healing practices; (2) risks of medicalisation or psychiatrisation of everyday forms of distress and (3) broader ethical concerns about the imposition of biomedical frameworks.Despite efforts to acknowledge culture and context in recent mhGAP materials, critics have argued that in practice mhGAP implementation tends to prioritise a biomedical approach to the relative exclusion of alternative, locally grounded, approaches to care. These and other critiques have spurred discussion of alternative approaches to GMH emphasising greater engagement with social and cultural context. Notwithstanding these conceptual critiques and practical challenges, the mhGAP programme continues to be implemented in varied settings. The programme has been used to train a range of groups including primary healthcare staff, physicians, schoolteachers and others, and the diagnostic algorithms have been adopted by healthcare workers and traditional healers in countries in Africa, Asia and South America.The need to address culture and context stems from assumptions that are embedded in mhGAP tools and approaches about what constitutes a mental health problem and what counts as relevant knowledge for evidence-based practice.The urgent task of better understanding and addressing culture, care and mental health has long been recognised.Prior to the development of the cultural formulation framework, 21 constructs like 'cultural competence','cultural responsiveness','cultural safety'and 'cultural humility' 25 had been advanced as a way of drawing attention to the importance of differences in interpreting the causes of distress and illness and approaches to care. Recent Lancet commissions have argued that engaging with the unique features of social contexts is necessary to adequately prevent, diagnose or treat disease, attain high-quality health systems by improving user experience and trust, foster collaboration across sectors, facilitate access and increase use of care to reduce preventable disease, and overcome barriers and errors encountered in programme scale-up.Like many approaches aimed at standardising and systematising practice across contexts, there is a risk that conceptual constructs oversimplify or neglect crucial factors in healthcare including such as local belief systems, patterns of care and support, and subjective experiences. Simplistic approaches to 'culture' as stereotyped individual traits are common. Implicit assumptions about the meaning of symptoms and appropriate treatments often result in barriers to implementation when they come into conflict with local values or ways of knowing and doing. Our premise is that when these assumptions are made explicit, they can inform the process of local adaptation of interventions through dialogue, planning and action research, enhancing the ability to integrate local idioms of distress, ways of coping, and approaches to care. Our aim in this paper is to outline and operationalise meaningful engagement with culture and context through a series of critically reflexive questions organised around three key domains: concepts of wellness, systems of care and ethical space.
Our framework introduces a series of questions meant to guide reflexive deliberation at all stages of implementation. Reflexivity is a practice rooted in the critical theory tradition that 'goes beyond pragmatic reflection to embrace a critical dimension and to carefully interrogate the very conditions under which knowledge claims are accepted and constructed'.Reflexivity is a justiceoriented practice that attends to the ways that power reproduces modes of thinking and doing and, importantly, points to the ways that this reproduction can neglect alternatives, both deliberately and inadvertently.While some questions in our framework can yield crucial information for specific stages of mhGAP implementation, the questions are meant to drive enquiry, exploration, discussion, reflection and introspection throughout the implementation process by attending to local ways of thinking and doing. Knowledge of local systems and of one's role in the process of implementation are basic to training. Implementers and trainers function within multicultural spaces and contexts, each with their own unique personal and national culture. An explicit understanding of how one's own implicit ways of knowing and presumptions can affect mhGAP implementation will serve to enrich all aspects of mhGAP programming.
## Questions to guide ethical and culturally sensitive deliberation
The framework and questions presented here are based on recent work in cultural psychiatry,including the Diagnostic and Statistical Manual of Mental Disorders-5 Outline for Cultural Formulation and the Cultural Formulation Interview, 21 work on structural competenceand cultural competence,as well as insights from work on cultural safety in Indigenous healthcare.The questions are organised around three key domains that focus BMJ Global Health on participants' knowledge, attitudes and assumptions about mental health, as well as local social and cultural context of mental healthcare, specific cultural and contextual issues regarding the mhGAP-IG, and key ethical issues related to the power dynamics of health service implementation and delivery box 1. Domain 1 (Concepts of Wellness and Illness) considers how cultural knowledge, practice and values influence expectations about the nature, causes, and course of wellness, illness and recovery.Domain 2 (Systems of care) invites participants to identify formal (Formal health systems are comprised of practices, institutions and professionals regulated by laws) and informal systems of care (Informal systems of care are those operating outside health systems regulations including in some cases traditional healers, but also supports provided by social and spiritual networks, neighbours, friends and family members) in the cultural context of their practice, including specific spiritual, family, kinship-based and gendered dynamics of access to care. Having highlighted importance of culture and context and suggested ways to start considering these in a more systematic manner, Domain 3 (Ethical Space) provides a guidance to examine potential tensions and challenges in implementation related to gaps between the cultural assumptions of mhGAP and those of local cultures and systems of care.These questions should be viewed as a starting point for dialogue and reflection-to be adapted, tested and refined in local contexts. Ultimately, the focus here is on the process of engagement with these questions rather than on finding specific answers or acquiring specific skills.
## Concepts of wellness and illness
While the mhGAP-IG V.2.0 47 48 addresses adaptation in more detail than previous versions and includes attention to local terms to improve communication with users and service providers as well the recommendation to include all stakeholders in the process (although it makes no mention of traditional healers), it does not consider many of the assumptions of western psychiatric nosology that are built into mhGAP and, in consequence, may foreground certain symptoms or problems while failing to recognise others.Reflection on the taken-for-granted aspects of culture is particularly important in the area of mental health because concepts of mind, self and personhood vary across cultures with consequences for the experience and expression of illness as well as for definitions and thresholds of normality and pathology. The epistemic critique of the GMH movement, namely that interpretations of distress and responses to this distress are varied across culture emphasises that the evidence base that supports biomedical modes of care is limited and of uncertain generalisability. In box 2, we illustrate this point by using examples from work on the idiom 'thinking too much'.These examples illustrate the often socially rooted ways that distress is interpreted, quite distinct from strictly biomedical categories of pathology.
The examples presented in box 2 point to the importance of understanding local idioms of distress to ensure cultural and contextual fit in diagnostic assessment and intervention.In this case, a narrowly biological or psychological assessment of pathology may fail to identity the social origins of suffering or distress. There are numerous examples of the ways that culture shapes experience and interpretative frames for individual and collective suffering. For example, Pedersen et almapped the multiple forms and expressions of distress of an indigenous community in the Peruvian highlands in relation to political violence experienced in the 1980s and concluded that, although the diagnostic category of Post-traumatic Stress Disorder (PTSD) had some utility, 'no intervention or rehabilitation programme can neglect the reconstruction of the social fabric as its primary concern' (p. 214). Critical examination of the relationship between mhGAP and local concepts of wellness and illness is essential to develop appropriate and effective systems of care.
## Systems of care
Most healthcare interventions implemented in LMIC, including the mhGAP-IG,were developed largely in European and North American contexts.The literature on mental health services in LMIC suggests that interventions developed and evaluated in one context may not yield the same results in another setting. However, despite more than 50 years of research on cultural variations in mental health and illness, there have been few practical tools to integrate culture in a systematic way in the routine implementation of evidence-based interventions in GMH. As we noted above, failing to consider the role of culture and context when training local non-professionals as part of task-shifting approach can lead to a loss of opportunity to incorporate local explanatory models and idioms into regular clinical practice and may risk imposing inappropriate, ineffective and insufficient models of care. Local or indigenous healthcare systems have their own resources and modes of intervention. One of the major risks of neglecting locally meaningful cultural idioms and social systems that frame the experience of distress and wellness, and expectations for care, is that effective local processes of healing, coping and recovery may be missed or discounted.Delivering interventions in context involves engaging the formal, traditional and informal healthcare systems, which may have their own pathways to care and diagnostic and treatment practices, including culturally grounded interventions as well as culturally adopted and adapted interventions.Inadequate attention to culture can create situations in which individuals in need of support are unable or reluctant to access services. If they do access services, these services may fail to recognise the core issues or to address appropriately for example by medicalising social suffering or offering alienating medical solutions.Studies on palliative care, for example, have found that
# Introduction
The initial questions ask participants to locate themselves in the mhGAP process. i. What is your role in the mhGAP (or other) programme being implemented? ii. How might your knowledge, professional training, experience, positionality (Positionality is the social, cultural and political context that shapes your identity in terms of ethnicity, gender, sexuality, socioeconomic and ability status vis-à-vis another person. It also describes how your identity influences and biases your perspectives, understanding and experience of the world) and values influence the ways that you approach the implementation?
## Concepts of wellness and illness
This section focuses on local concepts of wellness and illness, beginning with a general invitation to consider how cultural knowledge and values influence expectations about the nature, causes, and course of wellness, illness and recovery. Practitioners are encouraged to examine their own knowledge, values and attitudes in relation to the taken-for-granted assumptions and knowledge structures behind mental health models and interventions, including mhGAP-what can be termed 'the implicit culture' of the mhGAP-as well as potentially relevant culturally specific knowledge, values, and norms in the context of their practice. The mhGAP diagnostic approach acknowledges the concepts of wellness and illness are culturally determined and may be distinct among populations. This domain encourages the reader/implementer to explore, discuss and consider whether their conceptualisations of wellness and illness differs from those of local colleagues and stakeholders. It also invites the reader/implementer to seek out answers explicitly and to review the content of the mhGAP and their own views accordingly. As a result, the guide adaptation and the diagnostic approach in mhGAP can be guided by culturally-informed adaptations that result in locally meaningful health service provision.
1a. Recognise your own knowledge, values and attitudes in context i. What knowledge, values and experiences (including your personal and professional background) influence the ways you think about mental health and illness? ii. In your view, how do specific factors (eg, biological, psychological, social, cultural, spiritual, etc) contribute to mental illness and recovery? iii. Where might your views (or those expressed in the mhGAP materials) align or misalign with the social and cultural context where you plan to use mhGAP?
1b. Identify local knowledge, values and attitudes i. What are local cultural models of how to be a healthy person? (eg, maintaining family, kinship or other social norms and expectations, religious or spiritual practices, individual goals and aspirations, etc). ii. What cultural and contextual factors influence local concepts of illness, including the causes and course of illness and the process of healing and recovery (eg, biological, psychological, social, moral, spiritual, etc)? iii. What are common local ways of expressing distress that may be related to mental health problems? How do these modes of experiencing, expressing and explaining distress influence coping and help seeking?
## Systems of care
This section invites participants to identify formal (formal health systems are composed of practices, institutions and professionals regulated by laws) and informal systems of care (Informal systems of care are those operating outside health systems regulations including in some cases traditional healers, but also supports provided by social and spiritual networks, neighbours, friends and family members) for self and others in the cultural context of their practice, including specific spiritual, family, kinship-based and gendered dynamics of access to care. i. What are the local cultures and systems of care and how are they accessed by different groups of people?
## Continued
## Bmj global health
hospital policies that prevent families from gathering with a dying family member, or conducting ceremony with the family member lead to a decrease utilisation of such services in Indigenous communities.In many Western contexts those experiencing mental illness often feel isolated from the wider society, leading to a renewed emphasis on fostering social inclusion through recoverybased models.Integrating local systems of care in mhGAP implementation can begin by ensuring those involved have opportunities to explore and clarify: (1) the local systems of healthcare and their cultural practices, as well as how they are accessed by different groups of people;where or to whom people tend to go locally when experiencing distress specifically related to mental health problems; (3) the role of families, communities and institutions including indigenous healers, religious or spiritual groups; (4) how local cultural knowledge, values, practices and institutions influence help seeking, access to and provision of healthcare.
# Ethical space
Critical reflexivity can begin by identifying and interrogating the social, institutional and administrative structures that shape participation in mhGAP implementation. This involves an examination of who is participating, for what reason and, crucially, who is being excluded and why. Asking the group to 'take note of who is invited to the training and why', helps consider local power dynamics and hierarchies within the healthcare system. It may also help clarify what role the training process plays in the larger strategy of implementation and to what extent the wider context of existing models of care have been considered and respected. The responses to this question may also provide insight into the decision-making process at various steps of implementation. The process of participant selection may reflect planners' and administrators' views of mental health and can also influence participants' response to the trainer and the programme. For example, in Kenya, Musyimi, Mutiso, Ndetei and their team of colleagues have engaged faith healers and traditional healers in the mhGAP training and monitoring the impact of this training on service delivery.By bringing together different groups of providers and creating a space for dialogue across approaches, they were able to encourage mutual recognition and greater willingness to work together for service delivery.Although challenging, this reflection is important because the knowledge produced in mental health settings is shaped by the power dynamics of the clinical encounter and the healthcare system, as well as the larger institutional agendas of Non-Governmental Organizations (NGOs), governments, and international agencies. This kind of reflection requires what Indigenous scholars have called 'ethical space'and 'cultural safety', in which past structures of silencing and oppression associated with colonial regimes and other institutions, are recognised and deliberate efforts are made to ensure that diverse perspectives can be articulated and considered. This begins with determining that key stakeholders and community representatives are included and that the working group has a shared understanding of history, culture and context. Research with Indigenous persons in Western healthcare settings continues to undercover ways that colonialism and systemic racism shape healthcare experiences, underscoring the need to create space to share, listen and respond to these forms of structural violence.The notion of ethical space starts with the explicit aim of understanding 'what the other is thinking', by acknowledging the different histories, experiences, cultures and subjectivities of the particular groups and individuals involved.This orientation then extends to a collective process of identifying and interrogating the structural barriers to recognising and integrating local knowledge and experience. For example, Brunger et al 69
## Box 1 continued
ii. Where is care locally provided for mental health problems? In addition to the formal healthcare system, what is the role of families, communities, and institutions including indigenous healers, religious or spiritual groups? iii. What local cultural knowledge, values, practices and institutions influence help seeking, access to and provision of healthcare?
# Ethical space
This section focuses on the processes involved in integrating culture into mhGAP training and other programme or policy development. These issues should be considered at each stage of care, illness experience and healing systems. i. Who identified the need for implementing the mhGAP and what are the explicitly stated objectives? What are the mechanisms to identify the needs on the ground? To what extent are the objectives aligned with local needs and is there a mechanism for reconfiguring the objectives if needed? ii. At what stage in the process of mhGAP implementation was the local community invited to participate? How was the engagement negotiated? What individuals, institutions, interests or other factors may be influencing or constraining this engagement? iii. Which local stakeholders were invited to participate, and which ones may have been excluded? Was there adequate representation of local, regional and/or ethnocultural and socioeconomic groups, genders and sexualities in participants. How were differences in power and perspectives between these groups taken into account? iv. How have local cultural knowledge, values and assumptions underlying the process of wellness, illness experience and healing been explored and integrated into the training? v. What are the potential synergies or tensions with other locally available pathways of care? What are the mechanisms to address these tensions? vi. What power relations may be changed by the implementation? What are the health and social implications of these changes? BMJ Global Health drew from the idea of ethical space to bring together representatives from Indigenous communities in Eastern Canada and stakeholders from government and other relevant institutions to 'critically interrogate' how research in these indigenous communities is governed. The workshop involved presentations, case-based discussion and dialogue on key concepts and logistics. Importantly, the workshop created opportunities to recognise and discuss different viewpoints, resulting in immediate changes in the governance of research work.
# Conclusion
We have outlined a framework to support cultural and contextual adaptation in mhGAP implementation. This framework can serve as a starting point for fostering critical reflexivity and dialogue among stakeholders on key ethical, cultural and pragmatic challenges relevant to the local adaptation and implementation of the WHO mhGAP in LMICs.
The questions we have introduced can guide mhGAP programme planning and implementation, including team development and functioning; situation analysis and needs assessment; implementation planning; training and supervision; and monitoring and evaluations. In each of these different aspects of implementation, a different approach to using these questions can be taken. For example, during the phase of situation analysis these questions may simply be used as a planning guide to help orient the team to questions of inclusion and participation. Another example may be for an individual in charge of say developing an implementation plan to go through the questions by themselves to considering their relevance and impact for the activity planned. Alternatively, as part of the training and supervision process dyads or small teams could be asked to engage with the questions to guide their practice. Finally, during the initial stages of an mhGAP adaptation process, these questions could be used in a more in-depth manner as part of an adaptation workshop. These questions can also be applied beyond mhGAP to other interventions. For example, in the adaptation process of other mental health intervention programmes such as Problem Management Plus or even as part development of mental health awareness and prevention strategies within public health initiatives.
We believe the resultant process of reflexive discussion and knowledge exchange has the potential to improve implementation, uptake, and sustainability of the programme and to advance the goals of GMH equity. This reflexive approach and framework can also contribute to broader global health initiatives. This framework is being presented at a time when the assumptions underpinning global health initiatives are being questioned and the direction of knowledge generation and implementation is being critiqued,where scholars are increasingly drawing on postcolonial theory to reconfigure how we think about global health. As noted, one common critique of GMH programmes has been the lack of sufficient attention to local knowledge, values and practices. In part, this stems from emphasising an evidence-based approach that may discount local knowledge. A more inclusive and sustainable approach to mental health system development and service delivery begins by recognising the diverse knowledges of stakeholders and the hierarchies of power that may privilege some voices while silencing others. Facilitating knowledge exchange requires cultural safety and 'ethical space' to establish a framework in which difference and diversity are respected. The questions proposed here can guide implementers to consider available alternatives. The aim is to move from unidirectional knowledge translation or mobilisation, to knowledge exchange or coproduction and dialogic decision making. The process of dialogue that we advocate encourages interrogation of structural barriers to recognising and integrating local knowledge
## Box 2
Kaiser et al 50 conducted a systematic review to establish the crosscultural applicability and variability of the idiom 'thinking too much'. In a qualitative synthesis of 138 publications (from 1979 to 2014), they assessed descriptive epidemiology, phenomenology, aetiology and course of the idiom and contrasted them to psychiatric constructs. They found that 'thinking too much' did not map onto any one single psychiatric construct and warn against reducing these idioms to a single psychiatric diagnosis. Instead, they consider the idioms of distress as 'heterogeneous lay categories' with a complexity within and across contexts that should be recognised and preserved. Across contexts 'Thinking too much' idioms were more saliently used to communicate distress with reference to locally meaningful ethnopsychological constructs, value systems and social structures. Instead of displacing these idioms with psychiatric constructs, which is often the case, they propose that these idioms may have a role for stigma reduction, clinical communication and therapeutic intervention. They propose this as a starting point to incorporate lessons learnt in other cultural contexts into European/North American psychiatry.
Below are two concrete examples from work reviewed by Kaiser et al 50 that indicate the ethics of insufficiently or inappropriately attending to culture and context, and concretely reinforce the need to systematically avoid the blurring of social suffering and psychiatric care, even though deeply related:
In Nicaragua, Yarris' 73 analysis of 'pensando mucho' (thinking too much) suggests that the idiom 'communicates a certain moral ambivalence in the context of transformed social lives and its embodiment as 'dolor de cerebro' (brainache) reflects failure to achieve moral ideals of unity and solidarity within the family' .Sakti's 52 ethnographic work in East Timor on the idiom 'hanoin barak' ('thinking too much') exposes how this idiom-interpreted by biomedical practitioners as a psychological reaction to traumatic experiences-is better understood as a reaction to disruptions in typical communication and reconciliation practices with ongoing implications for the everyday social fabric of communities.
The consequences of these findings for diagnosis and interventions go beyond the dilemmas of inaccurate diagnosis and potentially inappropriate treatment. The failure to comprehend local modes of expressing distress may lead care providers to miss essential aspects of the problem.
## Bmj global health
and experience. Systematic approaches like the one we propose can support research on cultural and contextual adaptation of interventions and the process of reflexivity essential to advance efforts to develop culturally appropriate mental health interventions globally.
In most settings, mental healthcare involves multiple institutions, actors and practices that extend well beyond biomedical and psychological treatment modalities fostered by the mhGAP programme. These practices include care outside formal health systems, including extended family, community support or healing rituals and ceremonial religious or spiritual practices. Given the diversity of contexts in which mental health services are needed, implementing a generic or standardised training programme must include place and time for safe and inclusive dialogue with local communities. This dialogue aims to create opportunities for diverse voices to be heard, to promote knowledge exchange, and allow key ethical questions to be addressed. In this way, the implementation process itself can begin a process of power-sharing and co-construction of knowledge beneficial to diverse populations and communities.
AcknowledgementsThe authors wish to acknowledge Dr Nicole D'Souza for her
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The Molecular ‘Myc-anisms’ Behind Myc-Driven Tumorigenesis and the Relevant Myc-Directed Therapeutics
MYC, a well-studied proto-oncogene that is overexpressed in >20% of tumors across all cancers, is classically known as "undruggable" due to its crucial roles in cell processes and its lack of a drug binding pocket. Four decades of research and creativity led to the discovery of a myriad of indirect (and now some direct!) therapeutic strategies targeting Myc. This review explores the various mechanisms in which Myc promotes cancer and highlights five key therapeutic approaches to disrupt Myc, including transcription, Myc-Max dimerization, protein stability, cell cycle regulation, and metabolism, in order to develop more specific Myc-directed therapies.Int. J. Mol. Sci. 2020, 21, 9486 2 of 28 transcription factors[12][13][14]. Furthermore, Myc can amplify transcriptional signals by accumulating at the promoters of active genes, even in those with low-affinity E-box-like sequences[15,16]. There is still debate of whether Myc drives global amplification of transcription[15,16]or if global amplification is an indirect consequence of Myc's selective regulation of gene targets[17][18][19].In addition to the E box binding motif, the basic helix-loop-helix leucine zipper (bHLHZip) domain is crucial for Myc's activity. To take on its role as a transcription factor, Myc must heterodimerize with Myc-associated factor X (Max); Myc is incapable of homodimerizing and is inactive as a monomer. Max binds to Myc at the bHLHZip domain[20,21], and this heterodimerization is required to bind to the E box consensus sequence and activate transcription[22,23]. However, overexpression of Max leads to transcriptional repression as the Max homodimers antagonize Myc/Max heterodimers[22,24]. Mad, a transcriptional repressor, can also reduce Myc-driven transcription by dimerizing with Max [5].Dysregulation of Myc Leads to CancerNormally, Myc expression is tightly controlled at each molecular level (transcriptionally, post-transcriptionally, translationally, and post-translationally via protein stability, and via protein interactions), and has a short half-life of 20-30 min[25][26][27][28][29]. Given that there are many levels of regulation, as a consequence, there are many opportunities for which control of MYC can go awry. For instance, point mutations, chromosomal translocations, and gene amplification, or other factors that activate transcription or stabilize Myc, have been found in a wide range of cancers, which are further described by Meyer and Penn and Kalkat et al. [30,31]. This oncogenic activation, which leads to sustained levels of Myc, contributes to tumorigenesis and evasion of tumor-suppressive checkpoints leading to uncontrolled cell growth. MYC expressing tumors thus become addicted to and depend on the oncogene, as shown in cancer models with conditional activation of MYC [32]. On the contrary, inactivation of MYC leads to tumor regression in transgenic mouse models, displaying Myc's vital role in tumor initiation and maintenance[33][34][35].MYC amplification is found in 21% of patients across 33 different cancers [36], particularly breast cancer, lung squamous cell carcinoma, uterine carcinoma, esophageal carcinoma, and ovarian cancer [25](Figure 1). The highest rates of amplification are seen in high-grade serous ovarian cancer wherein greater than 50% of tumors harbor this genomic alteration. MYC translocation affects several hematological malignancies, including multiple myeloma, Burkitt's lymphoma, diffuse large cell lymphoma, and T-cell acute leukemia[37]. Alternatively, some tumors that do not display MYC amplification show extreme phosphorylation levels which aid in Myc stability[38][39][40][41].
## Myc's role as a transcription factor
Think about any key cellular process, and the Myc family most likely has a role in it: proliferation, metabolism, differentiation, and apoptosis. The Myc transcription factor family consists of c-Myc, N-Myc, and L-Myc. Discovery of c-Myc led to finding N-Myc, primarily expressed during development or in neuroblastoma, and L-Myc, expressed in lung tissue and small-cell lung cancer. Although the family shares stretches of homologous regions and some related targets, N-Myc and L-Myc are less characterized. c-Myc (hereon referred to as Myc), is the most well-studied family member as it is an influential protooncogenic transcription factor that binds to about 15% of genes. In order to regulate gene expression, Myc recruits or interacts with many different cofactors, including histone acetyl transferases (CBP, p300, GCN5/TRAPP), P-TEFb and polymerases, and chromatin remodelers (BRD4, the SWI/SNF complex, SIRT1). It is important to note that Myc can also repress gene expression by binding to the promoter region and interacting with MIZ1 and SP1 to displace co-activators or by recruiting DNA methyltransferases. Furthermore, there are two structural components of the MYC gene that are essential to drive its role as a transcription factor: the E box and the basic helix-loop-helix leucine zipper domain.
The canonical Myc E Box DNA binding motif (5 -CACGTG-3 ) is one of the most frequent regulatory motifs in the human genome. Although Myc is not the only transcription factor that can occupy this motif, elevated levels of Myc will replace the other bound transcription factors, demonstrating how Myc can influence the transcription of many genes and diverse processes in proliferating cells. A Myc core signature of 50 common target genes across four human cancer cell types and human embryonic stem cells revealed Myc's influence in RNA processing and ribosome biogenesis. Other diverse functions of Myc target genes include cell cycle regulation, metabolism, cell adhesion, and signal transduction.
However, Myc does not exclusively bind to the E-box to modulate transcription. In repression of gene transcription, cofactors recruit Myc to the promoters lacking the E-box and interfere with active accumulating at the promoters of active genes, even in those with low-affinity E-box-like sequences. There is still debate of whether Myc drives global amplification of transcriptionor if global amplification is an indirect consequence of Myc's selective regulation of gene targets.
In addition to the E box binding motif, the basic helix-loop-helix leucine zipper (bHLHZip) domain is crucial for Myc's activity. To take on its role as a transcription factor, Myc must heterodimerize with Myc-associated factor X (Max); Myc is incapable of homodimerizing and is inactive as a monomer. Max binds to Myc at the bHLHZip domain, and this heterodimerization is required to bind to the E box consensus sequence and activate transcription. However, overexpression of Max leads to transcriptional repression as the Max homodimers antagonize Myc/Max heterodimers. Mad, a transcriptional repressor, can also reduce Mycdriven transcription by dimerizing with Max.
## Dysregulation of myc leads to cancer
Normally, Myc expression is tightly controlled at each molecular level (transcriptionally, posttranscriptionally, translationally, and post-translationally via protein stability, and via protein interactions), and has a short half-life of 20-30 min. Given that there are many levels of regulation, as a consequence, there are many opportunities for which control of MYC can go awry. For instance, point mutations, chromosomal translocations, and gene amplification, or other factors that activate transcription or stabilize Myc, have been found in a wide range of cancers, which are further described by Meyer and Penn and Kalkat et al.. This oncogenic activation, which leads to sustained levels of Myc, contributes to tumorigenesis and evasion of tumor-suppressive checkpoints leading to uncontrolled cell growth. MYC expressing tumors thus become addicted to and depend on the oncogene, as shown in cancer models with conditional activation of MYC. On the contrary, inactivation of MYC leads to tumor regression in transgenic mouse models, displaying Myc's vital role in tumor initiation and maintenance.
MYC amplification is found in 21% of patients across 33 different cancers, particularly breast cancer, lung squamous cell carcinoma, uterine carcinoma, esophageal carcinoma, and ovarian cancer. The highest rates of amplification are seen in high-grade serous ovarian cancer wherein greater than 50% of tumors harbor this genomic alteration. MYC translocation affects several hematological malignancies, including multiple myeloma, Burkitt's lymphoma, diffuse large cell lymphoma, and T-cell acute leukemia. Alternatively, some tumors that do not display MYC amplification show extreme phosphorylation levels which aid in Myc stability. With Myc's prominent role across many cancers, the idea of Myc as a clinical target is too good to be true. Although targeted inhibition of MYC via siRNA reduces tumor burden in mice with very few toxicities despite Myc's influence on global transcription, global MYC knockout is embryonic lethal in mice. Thus, cautious measures in observing side effects of disrupting Myc need to be addressed. The less expected problem is that direct inhibition of Myc is not possible with current therapeutic approaches-Myc lacks both enzymatic activity and an active site for a small molecule to disrupt protein-protein interactions. Myc's primary nuclear localization further escalates the problem. Nonetheless, scientific discoveries led to creative ways to downregulate Myc. This review focuses on how Myc's oncogenic activation leads to tumorigenesis through initiating transcription, increasing stability, and influencing cell cycle and metabolism, coupled with descriptions of the indirect inhibitors of Myc that target each mechanism. The molecular changes in which MYC becomes an oncogene (mutations, translocations, and amplification) is beyond the scope of this review. With Myc's prominent role across many cancers, the idea of Myc as a clinical target is too good to be true. Although targeted inhibition of MYC via siRNA reduces tumor burden in mice with very few toxicities despite Myc's influence on global transcription, global MYC knockout is embryonic lethal in mice. Thus, cautious measures in observing side effects of disrupting Myc need to be addressed. The less expected problem is that direct inhibition of Myc is not possible with current therapeutic approaches-Myc lacks both enzymatic activity and an active site for a small molecule to disrupt protein-protein interactions. Myc's primary nuclear localization further escalates the problem. Nonetheless, scientific discoveries led to creative ways to downregulate Myc. This review focuses on how Myc's oncogenic activation leads to tumorigenesis through initiating transcription, increasing stability, and influencing cell cycle and metabolism, coupled with descriptions of the indirect inhibitors of Myc that target each mechanism. The molecular changes in which MYC becomes an oncogene (mutations, translocations, and amplification) is beyond the scope of this review. Schematic presenting the various cellular processes to target through inhibition or reactivation in the nucleus (left) or cytoplasm (right) upon Myc-induced tumorigenesis. All will be described in detail in this review.
## Disrupting myc stability to inhibit its actions as a transcription factor
In cancer, Myc's aberrant function as a transcription factor leads to increased cell proliferation, cell differentiation, cell adhesion, and angiogenesis. Here we will focus on inhibiting. Schematic presenting the various cellular processes to target through inhibition or reactivation in the nucleus (left) or cytoplasm (right) upon Myc-induced tumorigenesis. All will be described in detail in this review.
## Disrupting myc stability to inhibit its actions as a transcription factor
In cancer, Myc's aberrant function as a transcription factor leads to increased cell proliferation, cell differentiation, cell adhesion, and angiogenesis. Here we will focus on inhibiting transcription, disrupting Myc/Max dimerization, and enhancing protein degradation as strategies to disrupt Myc gene and protein stability and therefore Myc-driven tumorigenesis.
## Myc drives aberrant transcription
As discussed, MYC amplification is common among many cancer types. This amplification of MYC results in increased binding of Myc to promoters and enhancers of active genes, which magnifies the transcriptional signaland as a consequence, increases global transcription. During transcription, Myc recruits the transcriptional pause-release complex P-TEFb (a heterodimer of cyclin-dependent kinase 9 (CDK9) and cyclin T1, T2, or K). P-TEFb leads to activation of transcriptional elongation by phosphorylating RNA polymerase II (Pol II) via CDK9, stimulating pause release. Furthermore, BRD4, part of the bromodomain and extra-terminal motif (BET) protein family, also recruits P-TEFb to promoters to initiate transcription elongation. The overlapping roles of BET proteins and Myc in recruiting P-TEFb suggests BET proteins or CDK9 as therapeutic targets. First, BET proteins are known to regulate MYC transcription. A recent study demonstrated in normal cells that BRD4 has even more control over Myc by binding and phosphorylating Threonine 58 on Myc, leading to degradation. However, Myc is also capable of regulating BRD4 s histone acetyl transferase activity. Additional studies are needed to better understand how this circular balance may be affected in cancer. CDK9 is a potential target as it is part of P-TEFb, is necessary for proliferation and maintenance of MYC-overexpressing hepatocellular carcinoma, and is required for maintenance of gene silencing in several cancer cell lines. Another tumorigenic feature of Myc is looping to tumor-specific super-enhancers (sites defined by multiple enhancers abnormally bound by a plethora of transcription factors, such as BRD4 and CDK9). Therefore, inhibiting MYC transcription indirectly via BET inhibitors or affecting transcription of Myc target genes by inhibiting CDK9 are promising strategies that have shown efficacy in Myc-driven cancers. transcription, disrupting Myc/Max dimerization, and enhancing protein degradation as strategies to disrupt Myc gene and protein stability and therefore Myc-driven tumorigenesis.
## Myc drives aberrant transcription
As discussed, MYC amplification is common among many cancer types. This amplification of MYC results in increased binding of Myc to promoters and enhancers of active genes, which magnifies the transcriptional signaland as a consequence, increases global transcription. During transcription, Myc recruits the transcriptional pause-release complex P-TEFb (a heterodimer of cyclin-dependent kinase 9 (CDK9) and cyclin T1, T2, or K). P-TEFb leads to activation of transcriptional elongation by phosphorylating RNA polymerase II (Pol II) via CDK9, stimulating pause release. Furthermore, BRD4, part of the bromodomain and extra-terminal motif (BET) protein family, also recruits P-TEFb to promoters to initiate transcription elongation. The overlapping roles of BET proteins and Myc in recruiting P-TEFb suggests BET proteins or CDK9 as therapeutic targets. First, BET proteins are known to regulate MYC transcription. A recent study demonstrated in normal cells that BRD4 has even more control over Myc by binding and phosphorylating Threonine 58 on Myc, leading to degradation. However, Myc is also capable of regulating BRD4′s histone acetyl transferase activity. Additional studies are needed to better understand how this circular balance may be affected in cancer. CDK9 is a potential target as it is part of P-TEFb, is necessary for proliferation and maintenance of MYC-overexpressing hepatocellular carcinoma, and is required for maintenance of gene silencing in several cancer cell lines. Another tumorigenic feature of Myc is looping to tumor-specific super-enhancers (sites defined by multiple enhancers abnormally bound by a plethora of transcription factors, such as BRD4 and CDK9). Therefore, inhibiting MYC transcription indirectly via BET inhibitors or affecting transcription of Myc target genes by inhibiting CDK9 are promising strategies that have shown efficacy in Myc-driven cancers. Targeting MYC Transcription-BET Inhibitors, BRD4 Degraders, CDK9 Inhibitors
The BET proteins, BRD2, BRD3, BRD4, and testis-specific BRDT, are epigenetic readers and histone acetyl transferases that activate transcription via binding to specific acetylated lysine residues on histone tails. The bound BET proteins regulate chromatin remodeling via H3K122 acetylation and act as scaffolds to form transcription complexes by recruiting transcriptional activators such as P-TEFb. Furthermore, BRD4 influences mitotic progression by binding selectively to The BET proteins, BRD2, BRD3, BRD4, and testis-specific BRDT, are epigenetic readers and histone acetyl transferases that activate transcription via binding to specific acetylated lysine residues on histone tails. The bound BET proteins regulate chromatin remodeling via H3K122 acetylation and act as scaffolds to form transcription complexes by recruiting transcriptional activators such as P-TEFb. Furthermore, BRD4 influences mitotic progression by binding selectively to transcriptional start sites of M/G1 genes. Oncogenes, such as MYC, have a transcriptional dependency on BRD4 and recent findings suggest additional non-transcriptional functions of BRD4 in cancer. Bromodomains have a mostly hydrophobic pocket with aromatic rings and is an ideal size for protein-protein interactions, making bromodomains attractive and obtainable therapeutic targets, unlike Myc. BET protein inhibitors compete for access to the bromodomain and upon binding, disrupt chromatin remodeling and prohibit expression of target genes, including MYC. Filippakopoulos et al. and Nicodeme et al. independently designed some of the first bromodomain inhibitors, known as JQ1 and iBET respectively, that are highly specific towards the BET protein family. Initial studies showed efficacy of JQ1 downregulating both MYC expression and Myc's transcriptome genome-wide in Myc-addicted hematological malignancies, and solid cancers. iBET's proof-of-concept in preventing BET proteins from binding to acetylated histones was demonstrated in an inflammation context, although a follow-up study exhibited that iBET was capable of downregulating MYC expression, but to a lesser extent than JQ1. It is important to note that both JQ1 and iBET lack specificity for a particular BET protein family member, which limits their therapeutic availability. Therefore, these BET inhibitors serve best as tools to improve our understanding of targeting bromodomains and the effects on MYC. The discovery of JQ1 and iBET inspired the development of additional BET inhibitors, with 10 inhibitors being assessed in clinical trials, including MK-8628/OTX015. Phase Ib trials included six solid tumors such as NUT midline carcinoma (NMC), which harbors an oncogenic form of BRD4, known as BRD4-NUT. The trial (NCT02259114) completed with a recommended dose for Phase II studies, although the NMC patients that initially responded, relapsed several months after treatment. BET inhibitors as a whole currently appear to have limited therapeutic response and dose-limiting toxicities. More preclinical research will increase the biological knowledge on mechanisms of action and resistance of BET inhibitors.
In addition to BET inhibitors, there are also BET degraders that utilize a concept designed in 2000: PROteolysis TArgeting Chimeric (PROTAC). PROTAC protein degraders link the protein of interest to an E3 ligase in order to ubiquitinate the protein of interest for degradation. This approach has been adapted to a variety of targets, including the androgen receptor, estrogen receptor, BCL2, CDK9, and BET proteins to name a few. PROTAC technology has entered clinical trials, including Arvinas's ARV-110 for patients with metastatic castration-resistant prostate cancer (NCT03888612) which has shown efficacy and a promising safety profile in Phase I. The first PROTAC BET degraders, including MZ1, a BRD4-specific degrader, were designed in 2015 and demonstrated increased apoptotic response compared to nonspecific BET inhibitors, but a modest decrease in MYC expression.
It appears the antitumor efficacy of both BET inhibitors and BET degraders is most likely due to global transcription downregulation, rather than downregulation in MYC transcripts specifically. Devaiah et al. recently discovered crucial molecular differences in Myc stability between BET inhibitors and BET degraders. Since endogenous BRD4 destabilizes Myc, treatment with a BRD4 degrader, such as MZ1, enhances Myc stability, but treatment with a BET inhibitor, such as JQ1, does not affect BRD4 s phosphorylation of Myc and therefore Myc's half-life is unaffected while MYC transcription is downregulated. Several PROTAC BRD4 degraders demonstrate robust decreases of MYC expression throughout 3-24 h, though there are no current clinical trials on BET/BRD4 degraders. Perhaps later timepoints and investigation of phophorylated-S62-c-Myc expression will aid in understanding long-term effects of BET degraders on Myc stability. For further reading, detailed reviews on bromodomains and their inhibitors are cited.
CDK9 is another potential therapeutic target, given its kinase activity in the P-TEFb complex which releases paused RNA Pol II to initiate transcription. CDK9 inhibitors demonstrate efficacy in downregulating MYC transcripts and Myc stability across hepatocellular carcinoma, mixed-lineage leukemia, diffuse large B-cell lymphoma, acute myeloid leukemia, and pancreatic cancer. Although preclinical studies have shown efficacy in targeting CDK9, the sequence similarity to other cyclin-dependent kinases made specificity difficult. However, several groups succeeded in creating CDK9-specific inhibitors and PROTAC degraders. A recent study demonstrated that CDK9-specific inhibitor, MC180295, downregulates MYC and leads to reactivation of epigenetically silenced tumor suppressor genes. Thus, downregulation of MYC is not due to off-target effects of nonspecific CDK inhibition. Initial nonselective CDK inhibitors did not succeed in clinical trials, most likely due to toxicities from off-target effects. These trials included patients of many cancer types and were not selective to MYC-amplified patients. However, CDK9-specific inhibitors, such as BAY 1143572 (NCT01938638), are beginning to enter clinical trials in patients with advanced cancer and will evaluate MYC expression as a biomarker.
Additionally, combining CDK9 and BET inhibitors synergistically improves anti-proliferative activity in several cancers, with no hematological toxicity or weight loss shown in vivo. Of the same note, BET inhibitors were also efficacious when paired with additional inhibitors, such as PI3K, ERK, or BCL2 inhibitors. Readers are referred to reviews further discussing targetable Myc cofactors that aid in tumorigenesis, such as G quadraplex stabilizers. In all, BET and CDK9 inhibitors vastly affect transcription and as a result, downregulate MYC expression indirectly; improving their specificity is expected to increase their therapeutic benefit.
## Myc/max dimerization
Another way of affecting Myc transcriptional stability is by preventing Myc from interacting with DNA. Myc must dimerize with Max in order to drive gene expression, though a recent structural study demonstrates that against previous belief, Myc is stabile in the absence of binding DNA. Although, Max heterodimerization with Myc is required for Myc's oncogenic activity. Therefore, inhibiting Myc and Max dimerization prevents Myc from initiating gene transcription. There are two immediate challenges: (a) targeting the bHLHZip domain is nonspecific to Myc/Max and therefore could present off-target effects and (b) there are no apparent pockets for which a small molecule can bind. Despite this, there has been success in disrupting the Myc/Max interaction with several mini-proteins or molecules, including Omomyc, 10058-FA, 10074-G5, KJ-Pyr-9, MYCMI-6, and KI-MS2-008. study demonstrated that CDK9-specific inhibitor, MC180295, downregulates MYC and leads to reactivation of epigenetically silenced tumor suppressor genes. Thus, downregulation of MYC is not due to off-target effects of nonspecific CDK inhibition. Initial nonselective CDK inhibitors did not succeed in clinical trials, most likely due to toxicities from off-target effects. These trials included patients of many cancer types and were not selective to MYC-amplified patients. However, CDK9-specific inhibitors, such as BAY 1,143,572 (NCT01938638), are beginning to enter clinical trials in patients with advanced cancer and will evaluate MYC expression as a biomarker.
Additionally, combining CDK9 and BET inhibitors synergistically improves anti-proliferative activity in several cancers, with no hematological toxicity or weight loss shown in vivo. Of the same note, BET inhibitors were also efficacious when paired with additional inhibitors, such as PI3K, ERK, or BCL2 inhibitors. Readers are referred to reviews further discussing targetable Myc cofactors that aid in tumorigenesis, such as G quadraplex stabilizers. In all, BET and CDK9 inhibitors vastly affect transcription and as a result, downregulate MYC expression indirectly; improving their specificity is expected to increase their therapeutic benefit.
## Myc/max dimerization
Another way of affecting Myc transcriptional stability is by preventing Myc from interacting with DNA. Myc must dimerize with Max in order to drive gene expression, though a recent structural study demonstrates that against previous belief, Myc is stabile in the absence of binding DNA. Although, Max heterodimerization with Myc is required for Myc's oncogenic activity. Therefore, inhibiting Myc and Max dimerization prevents Myc from initiating gene transcription. There are two immediate challenges: (a) targeting the bHLHZip domain is nonspecific to Myc/Max and therefore could present off-target effects and (b) there are no apparent pockets for which a small molecule can bind. Despite this, there has been success in disrupting the Myc/Max interaction with several mini-proteins or molecules, including Omomyc, 10058-FA, 10074-G5, KJ-Pyr-9, MYCMI-6, and KI-MS2-008.
## Disrupting myc/max dimerization
The most well-known, and perhaps the first, Myc/Max dimerization inhibitor is Omomyc, a dominant negative mutant of Myc's bHLHZip domain with 4 amino acid mutations in the leucine zipper that prevents Myc/Max heterodimerization. Omomyc was a laboratory tool developed to bind and inhibit Myc. Over the past two decades, research produced a better understanding of
## Disrupting myc/max dimerization
The most well-known, and perhaps the first, Myc/Max dimerization inhibitor is Omomyc, a dominant negative mutant of Myc's bHLHZip domain with 4 amino acid mutations in the leucine zipper that prevents Myc/Max heterodimerization. Omomyc was a laboratory tool developed to bind and inhibit Myc. Over the past two decades, research produced a better understanding of how the molecular tool functions: Omomyc reduces the amount of Myc that can bind to promoters by either heterodimerizing with Myc in the cytoplasm, heterodimerizing with Max, or homodimerizing. Recent data show Omomyc preferentially binds to Max or homodimerizes. The Omomyc homodimers or Max/Omomyc heterodimers are transcriptionally inactive complexes that bind specifically to E-box sequences and displace Myc/Max heterodimers resulting in decreased Myc-driven transcription. Importantly, Omomyc is specific towards Myc's function and does not suppress gene expression of other E-box-binding transcription factors.
Omomyc has shown efficacy in several tumor studies when it is conditionally or transiently expressed in the cell or linked with a cell penetrating Phylomer. However, Omomyc is indeed capable of penetrating cells, including non-small cell lung cancer, neuroblastoma, glioblastoma, and melanoma cell lines, due to its basic region. Until recently, in vivo proof-of-concept was lacking. Beaulieu et al. show Omomyc downregulates Myc target gene expression and prevents tumor progression in lung adenocarcinoma in vivo models via intranasal administration (2.37 mg/kg) over four weeks. Similarly, in a lung adenocarcinoma xenograft model, paclitaxel combined with Omomyc administered intravenously diminished tumor growth over 30 days. Both models showed no significant changes nor toxicities in blood counts or pathology reports of all major organs. In non-tumor-bearing mice, Demma et al. show Omomyc injected intravenously (5.22 mg/kg) primarily distributes to the liver and kidneys and has a short half-life in plasma. Although this study used a higher dosage of Omomyc than the cancer study, toxicities of Omomyc in normal cells must be considered in future preclinical studies. Dr. Soucek, who created and studied Omomyc over the past 20 years, created the company Peptomyc to develop and sponsor Omomyc-derived clinical candidates; the first clinical trial is anticipated to start in 2021.
While. However, in vivo studies show both 10058-FA and 10074-G5 are rapidly metabolized and lack anti-tumor activity. Therefore, these compounds best serve as molecular tools and a starting point for new compound development. More recent Myc/Max inhibitors include KJ-Pyr-9 and MYCMI-6. KJ-Pyr-9 has sufficient pharmacokinetic properties to penetrate tissue and prevent tumor growth, but cannot reduce existing tumors; its inability to decrease tumor size may be due to residual Myc activity as an effect of incomplete Myc inhibition. MYCMI-6 was identified in 2018 as a promising Myc/Max-specific inhibitor that halts Myc-driven transcription, induces apoptosis, and reduced tumor proliferation in vivo. Interestingly, these different Myc/Max inhibitors all initiate different biological effects.
Recently, Han et al. identified novel Myc-binding inhibitors, MYCi361 and MYCi975, that appear to act through disrupting Myc/Max dimers and increasing Threonine (T)58 phosphorylation of Myc, which leads to Myc degradation. MYCi-induced degradation could be a result of changes in Myc confirmation as it interacts with MYCi; it is important to note that not all Myc/Max inhibitors lead to Myc degradation. Treating cells with proteasome inhibitor MG132 or exposing non-phosphorylatable Myc (T58A mutant) cells to MYCi361 rescues or prevents the MYCi361-induced Myc degradation. In vivo studies utilized a Myc-driven prostate cancer mouse model, MycCaP, in which tumors were significantly decreased upon MYCi361 treatment. Additional studies are required to determine its efficacy in other cancers. Given that MYCi treatment modified the tumor microenvironment through increased expression of PD-L1, Han et al. demonstrated synergistic effects with MYCi361 and anti-PD1 in the MycCaP model, despite the model's documented resistance to anti-PD1 therapy. MYCi975 performs similarly to MYCi361 but has a higher therapeutic index and better tolerability in vivo of up to ten time the anti-tumor efficacious dose. There is promise for future studies on MYCi975 due to its inhibition of cancer cell growth and reduction of Myc target gene expression in vitro and decreased tumor growth in vivo with high tolerability. These compounds represent a new class of directly targeting Myc and inhibiting Myc/Max dimers, which led to Myc degradation.
Agents that inhibit Myc/Max from binding DNA have also been pursued, although they lack in vivo data and specificity towards Myc/Max. One approach to prevent Myc binding to DNA is by targeting one of the many cofactors that recruits Myc to its target genes. WDR5 is an adapter protein that interacts with histone methyltransferase and serves as a scaffold for chromatin; it recruits Myc to chromatin and the Myc-WDR5 interaction is required for Myc-driven tumorigenesis. recently discovered that WDR5 stabilizes the Myc/Max interaction with DNA and a mutant Myc that cannot bind to WDR5 leads to tumor regression in a Burkitt lymphoma in vivo model. However, the mutant Myc was capable of binding to chromatin, suggesting that targeting WDR5 does not affect Myc's ability to interact with DNA. Given the antitumor effect and the druggable pockets within WDR5, it is a viable anti-Myc contender to pursue; additional recent advances with WDR5 are described in the Metabolism section of this review.
Alternative approaches that are not widely explored include stabilizing Max. In 2019, Struntz et al. discovered KI-MS2-008, which stabilizes Max homodimers while decreasing both Myc binding at promoters and Myc protein levels. KI-MS2-008 proved efficacious in T cell acute lymphoblastic leukemia and hepatocellular carcinoma in vivo models with a reduction in tumor burden and no toxicities in liver or kidney. Further studies are needed to determine the mechanism of action to optimize for in vivo use, but for now, KI-MS2-008 serves as an instrument to investigate the importance of Max dimerization in cancer.
Exploring the Myc/Max interaction has been a popular avenue for disrupting Myc-driven transcription. Sammak et al.'s high resolution crystal structure of the Myc and Max heterodimer in the absence of DNA will aid in development of future Myc-targeting therapeutics. Pursing additional compound libraries, such as Carabet and colleagues' computational screen to discover inhibitors of Myc-max in silico, can further broaden our understanding of inhibiting Myc/Max dimers. Future Myc/Max dimerization inhibitors must overcome challenges faced by current therapeutics such as fast metabolism, poor penetrability, and nonspecific targets.
## Myc protein stability
The short half-life of Myc is evidence for Myc's highly controlled turnover. Myc's stability is regulated by phosphorylation on serine 62 (S62) and threonine 58 (T58) by several proteins through the Raf-MEK-ERK kinase and phosphatidylinositol-3 kinase (PI3K)-Akt pathways. First, extracellular signal-regulated kinase (ERK), CDK1, or growth signals stabilize Myc by phosphorylating S62. Glycogen synthase kinase 3 (GSK3) is recruited to phosphorylate T58, which is required for Myc degradation. In brief, Pin1 isomerizes proline 63 on Myc, in which protein phosphatase 2A (PP2A), a serine/threonine phosphatase, can now dephosphorylate Myc at S62. The unstable Myc, with only T58 phosphorylation remaining, becomes ubiquitinated by Fbw7 and is sent for degradation. Again, these many levels of regulation provide multiple opportunities for cancer hijacking. In cancers that lack MYC amplification, there are increases in the stabilizing pS62-Myc and decreases in the degrading pT58-Myc, therefore promoting Myc's stability and activity. Studies show that mutating T58 to alanine, a non-phosphorylatable residue, results in stable Myc expression and tumorigenic properties, suggesting Myc stability has a role in transformation.
In cancer, we see faulty regulation of these proteins that modify the phosphorylation on Myc and promote stabilization. We will describe three scenarios-activation of PI3K/AKT signaling (which inhibits GSK3B), overexpression of Pin1, and suppression of PP2A activity-that stabilize Myc. PI3K, PTEN, and upstream components of the PI3K/AKT pathway are commonly mutated in cancer to promote pathway activation. Activated AKT phosphorylates (and therefore inhibits) GSK3, which in turn enhances Myc stabilityas GSK3 cannot phosphorylate T58-Myc. This is just one example of how an upstream signaling pathway (MAPK, Wnt, Notch) can quickly trickle down to promoting cancer through Myc.
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), an isomerase that specifically recognizes the serine/threonine-proline motif, is overexpressed in several cancers including pancreatic, breast, and prostate and its expression correlates with poor clinical outcomes. Furthermore, Pin1 promotes several hallmarks of cancer through inactivating 26 tumor suppressors and activating 56 oncogenes. By catalyzing the cis/trans conformational change of the target protein, such as Myc, isomerases like Pin1 gain control of the target protein's stability, activity, and localization. As mentioned, Myc's stability is regulated through phosphorylation on S62 and T58 and these sites are recognized by trans-specific phosphatases; therefore, Pin1 can stabilize Myc in the cis-confirmation and prevent degradation. On the contrary, Pin1 can revert Myc back to the trans-confirmation after phosphorylation of T58, which allows PP2A to remove phosphorylation from S62 to promote Myc degradation. However, another consideration is that PP2A is commonly inactivated in cancers (described below), and so even if Pin1 reverts Myc back to the trans-confirmation, Myc would unlikely get degraded in the absence of PP2A activity. More research is needed to better understand how T58 phosphorylation affects Pin1 activity and S62 dephosphorylation. Additionally, Pin1 can promote self-ubiquitination of Fbw7, the E3 ubiquitin ligase that ultimately degrades Myc. Pin1's influence on Myc's transcriptional activity and stability potentiates tumorigenesis and is a potential therapeutic target for MYC-overexpressing cells.
Lastly, PP2A is a ubiquitously expressed tumor suppressor that accounts for a majority of the phosphatase activity in cells and dephosphorylates a range of substrates such as Akt, p53, β-catenin, and Myc. The holoenzyme can contain a variety of different scaffold (A) and regulatory (B) subunits with a common catalytic (C) subunit, with multiple isoforms for each subunit. Inactivation of PP2A through PP2A inhibitor okadaic acid results in tumorigenesis and cellular transformation. PP2A is commonly inactivated in cancer, including lung, colon, breast, skin, cervix, and ovarian. This PP2A inactivation occurs through phosphorylation, somatic mutation, or increased expression of endogenous inhibitors such as SET and CIP2A. In the case of Myc, PP2A inactivation prevents dephosphorylation of S62, therefore stabilizing Myc and promoting transformation. In sum, inhibition of GSK3 through PI3K, overexpression of Pin1, and inactivation of PP2A promote stability of Myc. Although there are many opportunities to increase Myc stability, many of these proteins are also potential therapeutic targets to promote Myc's degradation. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), an isomerase that specifically recognizes the serine/threonine-proline motif, is overexpressed in several cancers including pancreatic, breast, and prostate and its expression correlates with poor clinical outcomes. Furthermore, Pin1 promotes several hallmarks of cancer through inactivating 26 tumor suppressors and activating 56 oncogenes. By catalyzing the cis/trans conformational change of the target protein, such as Myc, isomerases like Pin1 gain control of the target protein's stability, activity, and localization. As mentioned, Myc's stability is regulated through phosphorylation on S62 and T58 and these sites are recognized by trans-specific phosphatases; therefore, Pin1 can stabilize Myc in the cis-confirmation and prevent degradation. On the contrary, Pin1 can revert Myc back to the trans-confirmation after phosphorylation of T58, which allows PP2A to remove phosphorylation from S62 to promote Myc degradation. However, another consideration is that PP2A is commonly inactivated in cancers (described below), and so even if Pin1 reverts Myc back to the transconfirmation, Myc would unlikely get degraded in the absence of PP2A activity. More research is needed to better understand how T58 phosphorylation affects Pin1 activity and S62 dephosphorylation. Additionally, Pin1 can promote self-ubiquitination of Fbw7, the E3 ubiquitin ligase that ultimately degrades Myc. Pin1′s influence on Myc's transcriptional activity and stability potentiates tumorigenesis and is a potential therapeutic target for MYC-overexpressing cells.
Lastly, PP2A is a ubiquitously expressed tumor suppressor that accounts for a majority of the phosphatase activity in cells and dephosphorylates a range of substrates such as Akt, p53, β-catenin, and Myc. The holoenzyme can contain a variety of different scaffold (A) and regulatory (B) subunits with a common catalytic (C) subunit, with multiple isoforms for each subunit. Inactivation of PP2A through PP2A inhibitor okadaic acid results in tumorigenesis and cellular transformation. PP2A is commonly inactivated in cancer, including lung, colon, breast, skin, cervix, and ovarian. This PP2A inactivation occurs through phosphorylation, somatic mutation, or increased expression of endogenous inhibitors such as SET and CIP2A. In the case of Myc, PP2A inactivation prevents dephosphorylation of S62, therefore stabilizing Myc and promoting transformation. In sum, inhibition of GSK3 through PI3K, overexpression of Pin1, and inactivation of PP2A promote stability of Myc. Although there are many opportunities to increase Myc stability, many of these proteins are also potential therapeutic targets to promote Myc's degradation. trans-specific enzyme from binding to Myc. Furthermore, PP2A is inactivated in several cancers, and therefore S62 remains phosphorylated. All of this leads to high Myc stability. (B) Inhibition of PI3K allows for GSK3 to phosphorylate T58 on Myc, which is required for degradation. Pin1 inhibitors and PP2A activators allow for PP2A to recognize and remove the phosphorylation of S62, leading to low stability and Myc's degradation.
## Enhancing degradation of myc
Given the various levels regulating Myc degradation, numerous compounds have been developed to enhance Myc degradation through inhibition of PI3K or Pin1 and re-activation of PP2A. Becker and collogues demonstrated efficacy in combining a PI3K inhibitor with a microtubule destabilizer in high-Myc expressing cells. First, they eloquently demonstrated that unphosphorylated S62-Myc binds to mitotic tubules and is protected from degradation. Given this interaction, treatment with a microtubule destabilizer, vincristine, drastically reduced Myc protein and P493-6 B-cell lymphoma cells with ectopic Myc expression were more sensitive to colony forming unit inhibition than Myc low-expressing cell lines. Since PI3K/AKT inhibits GSK3B activity and therefore stabilizes Myc, Becker and collogues investigated the addition of PI3K inhibitor idelalisib following the G2-M arrest induced by vincristine. Treating first with vincristine followed by idelalisib led to higher cell death and decreased clonogenic growth than either compound alone across 16 Burkitt lymphoma and DLBCL cell lines. Furthermore, this combination lead to reduction of Myc and tumor viability in two lymphoma in vivo models, in which the compounds as single agents were not effective. These results suggest a novel avenue of disrupting Myc stability via microtubule destabilizers followed by PI3K inhibition to further decrease Myc protein levels. Another targetable signaling pathway that influences Myc degradation is the MEK/ERK pathway. As mentioned, ERK maintains S62 phosphorylation of Myc, which promotes Myc's stability. Therefore, inhibition of the MEK/ERK pathway through MEK inhibitor U0126 reduced Myc expression and growth in rhabdomyosarcoma cell lines. Furthermore, inhibition of the MEK/ERK pathway or the consequent decrease in Myc expression, a known driver of radioresistance, sensitizes cancer cells to radiation therapy.
Aside from Pin1 s influence over Myc's stability, there are several other mechanisms in which Pin1 can promote tumorigenesis such as sustaining proliferative signaling and downregulating tumor suppressors. More than ten Pin1 inhibitors have been developed that demonstrate anticancer activity, including sensitizing various cancer cells to chemotherapy. We will discuss two Pin1 inhibitors-All-trans retinoic acid (ATRA) and KPT-6566, that have more favorable specificity and safety profiles than other Pin1 inhibitors. ATRA is clinically used for acute promyelocytic leukemia (APL), although its drug target was unknown. Through a mechanism-based high throughput screen, Wei and collogues discovered ATRA directly binds and degrades Pin1. ATRA was capable of decreasing Pin1 and tumor growth in APL mouse models and APL human patients' bone marrow, along with in vivo models of triple negative breast cancerand acute myeloid leukemia; both cancers overexpress Pin1. However, ATRA has a short half-life of 45 minutes and moderate anti-cancer activity. Yang and collogues developed an improved, controlled-release formulation of ATRA (ATRA-PLLA microparticles) that demonstrated selectivity for Pin1 inhibition and improved anti-cancer efficacy in xenografts of hepatocellular carcinoma, a cancer that is enhanced by Pin1. Several other liposomal ATRA delivery methods have been developed and performed well in clinical trials for APL patients, although it appears trials for solid tumors utilizing the improved ATRA formulation are lacking. Additionally, these studies did not specifically investigate the effects of ATRA and Myc. Several older studies across small cell lung cancer, breast cancer, and colon cancer demonstrated treatment with ATRA decreased Myc expression at the gene or protein level. Selective Pin1 inhibitor KPT-6566, which was also identified through a mechanism-based screen, sets Pin1 for degradation. When KPT-6566 binds to the catalytic site of Pin1, reactive oxygen species are produced and DNA damage occurs, leading to cell death particularly in Pin1-overexpressing cancer cells. There are no data on KPT-6566 decreasing tumor volume in vivo, but in mice injected with MDA-MB-231 cells, KPT-6566 daily treatment reduced metastatic spread and showed no toxicities in vital organs. Again, these studies did not investigate the effects of Pin1 inhibition on Myc. More development is necessary to improve efficacy and drug-likeness of Pin1 inhibitors, especially in the context of Myc-driven cancers.
Compounds that target PP2A, which is the main phosphatase the regulates Myc stability, have shown promise in promoting Myc degradation and cell death. There are several methods published on indirectly activating PP2A as an anti-cancer treatment, such as antagonizing the endogenous PP2A inhibitors SET (via OP449or FTY720, and CIP2A (via bortezomib, erlotinib, or celastrol) or disrupting PP2A post translational modifications. SET-inhibitor OP449 increased PP2A activity dose-dependently and OP449-treated leukemia xenografts had a two-fold reduction of tumor burden. In breast cancer, OP449 decreased both phosphorylation levels of S62-Myc and Myc transcriptional activity across several cell lines in vitro. OP449 additionally induced apoptosis while reducing tumor volume and increasing PP2A activity in vivo. In terms of disrupting CIP2A, the described inhibitors were primarily discovered as a proteasome inhibitor (bortezomib), EGFR kinase inhibitor (erlotinib), or anti-cancer (celastrol), but indirectly or independently reduce CIP2A expression or activity. Small molecule activators of PP2A (SMAPs) have also emerged as a new class of validated compounds that re-activate PP2A through binding to the A scaffolding subunit of PP2A. As PP2A reactivates, S62-Myc becomes dephosphorylated and Myc is sent for degradation. Recently, SMAPs demonstrated efficacy through binding to PP2A in in vivo models of Burkitt's lymphoma, non-small cell lung cancer, and triple-negative breast cancer-all Myc-driven cancers, representing Myc amplification, post-translational stabilization, and overexpression. SMAPs also display efficacy in prostate and pancreatic cancer models. Dr. Narla, one of the developer of SMAPs, serves as Chief Scientific Officer for Rappta Therapeutics to further develop these anti-cancer molecules that reactive PP2A. In all, targeting Myc's protein stability may help reduce toxicity that is expected with a complete loss of Myc.
## Taking advantage of myc overexpression to initiate synthetic dosage lethality in the context of cell cycle
Since transcription factors pose as difficult drug targets, leveraging synthetic lethality offers an alternative approach of antitumoral therapy. Synthetic lethality occurs when a mutation or inhibition of two specific genes leads to cell death, but a mutation or inhibition of just one gene does not affect viability. Synthetic dosage lethality is when manipulation of expression levels leads to cell death; for example, overexpression of gene A and presence of gene B is viable, but the combination of gene A overexpression and loss or lower expression of gene B results in cell death. Therefore, synthetic lethality, or more specifically synthetic dosage lethality, can be advantageous in cancer as the tumors already have mutations or oncogenic addiction, such as overexpression of MYC. A synthetic lethal approach affects the mutated tumor cells and spares the normal cells.
Synthetic lethal targets are identified in an unbiased, high-throughput fashion through RNA interference (RNAi) or CRISPR screens on isogenic cells-cells that differ by a mutation in a single gene. Although the idea sounds swift, identifying clinically relevant synthetic lethal interactions have proven difficult due to validation of lethal mutants by recovery, condition-dependent interactions, and rarity. However, PARP inhibitors successfully demonstrated this concept clinically when given to cancer patients with BRCA mutations, such as in breast and ovarian cancer.
Understanding the biological results of MYC overexpression will help identify second-site targets that lead to synthetic lethality. Reports show that MYC-overexpressing cancer cells have increased sensitivity to apoptosis in response to cytotoxic drugs or radiation. However, the opposite appears to be true in melanoma, in which lower MYC expression improves susceptibility to chemotherapy and radiation due to reactive oxygen species production and mismatch repair protein inhibition. As Myc is a master regulator of cell proliferation and metabolism, genes affiliated with these processes offer a promising avenue to identify synthetic lethal targets.
Cells overexpressing MYC have more mitotic abnormalities, such as altered spindle morphology and mitotic timing. During mitotic stress, Myc worsens mitotic dysfunction and enhances apoptosis, which explains the many cell cycle proteins as targets for synthetic lethality. In normal conditions, advancing through the cell cycle phases of G1, S, G2, and M requires four heterodimers of cyclin-dependent serine/threonine kinases (CDK) and cyclins: CDK1, 2, 4, 6, and cyclins A, B, E, D, all of which are Myc target genes. Cyclin B1 binds to CDK1 at the G2-M transition, activating the complex to promote mitosis. CDK1 is the only essential CDK required for cell cycle progression and it is rarely dysregulated in cancer. Inhibiting CDK1 typically results in a G2 arrest, but in MYC-overexpressing cells, CDK1 inhibition leads to apoptosis. Cyclins and CDKs just scratch the surface of proteins involved in the cell cycle.
Myc also induces expression of Aurora A kinase, which reciprocally stabilizes Myc in addition to its role in cell cycle. Aurora kinases A and B direct cell cycle progression through G2-M. Aurora A aids in centrosome function, spindle assembly, and mitotic entry while Aurora kinase B is the catalytic component within the chromosomal passenger protein complex (CPPC) to control chromosomal condensation and cytokinesis. Aurora A and B kinases are overexpressed in breast and colon cancers, along with sarcoma, esophageal, and stomach cancers. Myc is known to upregulate Aurora kinase A and B expression in B-cell lymphomas, which is necessary to maintain the lymphoma. Similarly, overexpressing MYC in medulloblastoma cell lines with low MYC expression led to an associated increase in Aurora B expression. Overall, cell cycle proteins such as Aurora kinases or CDK1 in MYC-overexpressing cells are potential therapeutic targets as inhibition leads to synthetic dosage lethality. Cells overexpressing MYC have more mitotic abnormalities, such as altered spindle morphology and mitotic timing. During mitotic stress, Myc worsens mitotic dysfunction and enhances apoptosis, which explains the many cell cycle proteins as targets for synthetic lethality. In normal conditions, advancing through the cell cycle phases of G1, S, G2, and M requires four heterodimers of cyclin-dependent serine/threonine kinases (CDK) and cyclins: CDK1, 2, 4, 6, and cyclins A, B, E, D, all of which are Myc target genes. Cyclin B1 binds to CDK1 at the G2-M transition, activating the complex to promote mitosis. CDK1 is the only essential CDK required for cell cycle progression and it is rarely dysregulated in cancer. Inhibiting CDK1 typically results in a G2 arrest, but in MYC-overexpressing cells, CDK1 inhibition leads to apoptosis. Cyclins and CDKs just scratch the surface of proteins involved in the cell cycle.
Myc also induces expression of Aurora A kinase, which reciprocally stabilizes Myc in addition to its role in cell cycle. Aurora kinases A and B direct cell cycle progression through G2-M. Aurora A aids in centrosome function, spindle assembly, and mitotic entry while Aurora kinase B is the catalytic component within the chromosomal passenger protein complex (CPPC) to control chromosomal condensation and cytokinesis. Aurora A and B kinases are overexpressed in breast and colon cancers, along with sarcoma, esophageal, and stomach cancers. Myc is known to upregulate Aurora kinase A and B expression in B-cell lymphomas, which is necessary to maintain the lymphoma. Similarly, overexpressing MYC in medulloblastoma cell lines with low MYC expression led to an associated increase in Aurora B expression. Overall, cell cycle proteins such as Aurora kinases or CDK1 in MYC-overexpressing cells are potential therapeutic targets as inhibition leads to synthetic dosage lethality. Targeting Cell Cycle Proteins CDK1: Purvalanol is a potent CDK1 inhibitor that has selectivity for CDK1 over CDK2 at a low concentration of 4 nM. Goga et al. discovered inhibiting CDK1 pharmacologically with purvalanol or genetically using a cell line with temperature-sensitive Cdk1 allele results in apoptosis in MYC-overexpressing cells. This synthetic lethal interaction led to decreased tumor growth in MYC-expressing lymphoma and hepatoblastoma in vivo models. However, purvalanol is not suitable clinically as it is poorly soluble; new variations are needed to pursue CDK1 inhibitors as a clinical candidate. Additionally, Goga et al. also demonstrated promise for targeting survivin, an endogenous inhibitor of apoptosis and known CDK1 target. CDK1 inhibition via purvalnol degraded survivin, and depleting survivin independently resulted in similar results to CDK1 inhibition in that MYC-overexpressing cells were more sensitive to survivin degradation via peptide inhibitors. Targeting Cell Cycle Proteins CDK1: Purvalanol is a potent CDK1 inhibitor that has selectivity for CDK1 over CDK2 at a low concentration of 4 nM. Goga et al. discovered inhibiting CDK1 pharmacologically with purvalanol or genetically using a cell line with temperature-sensitive Cdk1 allele results in apoptosis in MYC-overexpressing cells. This synthetic lethal interaction led to decreased tumor growth in MYC-expressing lymphoma and hepatoblastoma in vivo models. However, purvalanol is not suitable clinically as it is poorly soluble; new variations are needed to pursue CDK1 inhibitors as a clinical candidate. Additionally, Goga et al. also demonstrated promise for targeting survivin, an endogenous inhibitor of apoptosis and known CDK1 target. CDK1 inhibition via purvalnol degraded survivin, and depleting survivin independently resulted in similar results to CDK1 inhibition in that MYC-overexpressing cells were more sensitive to survivin degradation via peptide inhibitors. Given that loss of p53 influences decreased apoptosis in MYC-overexpressing cells, the authors explored effects of p53 status on the efficacy of the CDK1 inhibitor. Through the use of wildtype and p53 −/− MYC overexpressing mouse embryonic fibroblasts, Goga et al. determined p53 status is independent of purvalanol-induced apoptosis. This is advantageous as many cancers have p53 deficiencies.
A second study from the same group reported that triple-negative breast cancer (TNBC) with elevated MYC expression displayed efficacy with CDK1 inhibition (purvalanol, dinaciclib, or siRNA), compared to lines with low MYC expression. Although third-generation CDK inhibitor dinaciclib inhibits CDK1, CDK2, CDK5, and CDK9, efficacy in TNBC cell lines was shown to be specific to CDK1 inhibition by knocking down Cdk1 via siRNA. Furthermore, dinaciclib, which has improved pharmacokinetic and pharmacodynamic properties than previous CDK inhibitors, decreased tumor volume by about 50% in TNBC xenograft mice. Several clinical trials testing dinaciclib in mainly hematologic malignancies recently completed and are pending results. In 2015 studies, 11% of relapsed multiple myeloma patients partially responded and 54% of lymphocytic leukemia patients partially responded. In a randomized phase II in TNBC patients, dinaciclib failed to outperform capecitabine, the standard of care. These studies did not consider MYC expression but offer promising results to continue dinaciclib in clinical research. An active Phase I clinical trial, NCT01676753, is assessing MYC overexpression with dinaciclib + pembrolizumab efficacy in advanced breast cancer .
Aurora Kinases: Given the similarities and genetic overlap between Aurora kinases A and B, many aurora kinase inhibitors are nonselective, other than Alisertib (MLN8237), which is specific to Aurora A and Barasertib (AZD1152), which is specific to Aurora B. VX-680 and AZD1152 have been studied specifically in MYC-overexpressing cancers preclinically. Yang et al. demonstrated proof of concept of synthetic lethality with non-specific aurora kinase inhibitor VX-680. Pulse treatment of VX-680 in Myc-driven models of lymphoma resulted in a 3-fold increase in survival. Yang and collogues propose synthetic lethality is a result of failed spindle checkpoint due to inhibition of Aurora B (resulting in a compromised CPPC) and MYC overexpression leads to polyploidy; the combination of the two proceeds to apoptosis. Yang and Goga both acknowledged defects in CPPC, through either survivin or Aurora B inhibition, which led to synthetic lethality in MYC-overexpressing cells. This suggests inhibition of other CPPC components as additional avenues to explore. Furthermore, similar to Goga et al.'s findings, Yang et al. reported that the synthetic lethal interaction between Aurora kinase inhibitor and Myc is also independent of p53.
Lastly, another p53-deficient cancer, small cell lung cancer (SCLC), is also susceptible to Aurora kinase-Myc synthetic lethality. Helfirch et al. demonstrated MYC amplification is a good biomarker for predicting in vitro and in vivo growth inhibition of SCLC upon AZD1152 treatment. Similarly, MYC-overexpressing medulloblastoma cells treated with AZD1152 were more sensitive to apoptosis than the low-expressing parent cell line, as was true for medulloblastoma cells with endogenous MYC overexpression. Medulloblastoma xenograft models treated with AZD1152 had decreased tumor growth and prolonged survival.
Several Aurora kinase inhibitors have been tested clinically, including MK-0457 (or VX-680), AZD1152, PHA-739358, and MLN8237. Clinical trials that included MYC expression as a biomarker appear to be limited to Aurora A inhibitor Alisertib . Recent in vivo studies of Alisterib in MYC-overexpressing lymphoma xenografts demonstrated synthetic lethality by caspase-independent cell death and complete tumor regression when paired with chemotherapy cyclophosphamide.
In vivo reports of CDK1 and Aurora kinase inhibitors support the inclusion of MYC overexpression as a recruitment factor in clinical trials or at least warrant further investigation of MYC a biomarker for these inhibitors. This stratification may result in an improved clinical outcome. Additional potential synthetic lethal interactions in Myc-driven cancers are described by Cermelli et al..
## Myc drives metabolism through its target genes
Cellular proliferation is closely related to metabolism. In cancer, metabolic reprogramming, such as prompt ATP synthesis, increased anabolism of macromolecules, and redox homeostasis, support the rapidly proliferating cancer cells. Understandably, Myc, the master regulator of growth, also aids in metabolic reprogramming. Several Myc target genes are involved in metabolic pathways, including glucose transporter GLUT1 (SLC2A1), glutaminase (GLS), hexokinase 2 (HK2), phosphofructokinase (PFKM), enolase 1 (ENO1), peroxisome proliferator-activated receptor gamma coactivator 1-beta (PGC-1β), nuclear respiratory factor 1 (NRF1), and inosine monophosphate dehydrogenase (IMPDH1/2). Through Myc's many target genes, it can regulate aerobic glycolysis (the Warburg effect), mitochondria, and ribosome biogenesis, and metabolism of nucleotides, amino acids, and lipids, which contributes to its oncogenic function.
Cancer cells have increased levels of guanosine triphosphate (GTP), an energy source and signaling molecule. Both GTP and the rate-limiting enzyme for GTP synthesis, inosine monophosphate dehydrogenase (IMPDH), are Myc target genes. MYC and IMPDH expression significantly correlated and IMPDH overexpression has been observed in several cancers, including glioblastoma, leukemia, colorectal cancer, and small cell lung cancer. Manipulating IMPDH expression in glioblastoma cells results in the same change in MYC expression. Furthermore, Myc activates GTP synthesis and it has been shown in small cell lung cancer (SCLC), that IMPDH is depended upon by naïve and chemoresistant high-MYC SCLC cells. In addition, IMPDH links Myc's role in nucleotide biosynthesis and ribosome biogenesis as IMPDH-dependent GTP synthesis is needed for Pol I synthesis of pre-ribosomal RNA.
Glutaminase, another target gene of Myc, converts the abundant glutamine into glutamate. Myc-driven cancers depend on glutamine metabolism rather than glucose, especially when deprived of oxygen. Furthermore, glutamine-depletion-induced apoptosis is dependent on Myc activity, and on the other hand, Myc-induced renal adenocarcinoma depends on glutaminase. Shen et al. explored the concept of glutaminase dependency in the context of ovarian cancer, in which >45% of patients' tumors overexpress MYC. Elevated MYC expression correlated with glutaminase in immortalized cell lines and primary cultures, and overexpression of MYC and GLS were associated with chemoresistance and worse disease outcome.
We will discuss inhibiting the proteins of metabolic Myc target genes, IMPDH and glutaminase, along with epigenetic cofactor WDR5 that recruits Myc to drive ribosome biogenesis. Details of Myc's role in cancer metabolism are beyond the scope of this review and are summarized in several articles. Additional potential Myc-driven metabolic targets are described by Dong et al..
## Myc drives metabolism through its target genes
Cellular proliferation is closely related to metabolism. In cancer, metabolic reprogramming, such as prompt ATP synthesis, increased anabolism of macromolecules, and redox homeostasis, support the rapidly proliferating cancer cells. Understandably, Myc, the master regulator of growth, also aids in metabolic reprogramming. Several Myc target genes are involved in metabolic pathways, including glucose transporter GLUT1 (SLC2A1), glutaminase (GLS), hexokinase 2 (HK2), phosphofructokinase (PFKM), enolase 1 (ENO1), peroxisome proliferator-activated receptor gamma coactivator 1-beta (PGC-1β), nuclear respiratory factor 1 (NRF1), and inosine monophosphate dehydrogenase (IMPDH1/2). Through Myc's many target genes, it can regulate aerobic glycolysis (the Warburg effect), mitochondria, and ribosome biogenesis, and metabolism of nucleotides, amino acids, and lipids, which contributes to its oncogenic function.
Cancer cells have increased levels of guanosine triphosphate (GTP), an energy source and signaling molecule. Both GTP and the rate-limiting enzyme for GTP synthesis, inosine monophosphate dehydrogenase (IMPDH), are Myc target genes. MYC and IMPDH expression significantly correlated and IMPDH overexpression has been observed in several cancers, including glioblastoma, leukemia, colorectal cancer, and small cell lung cancer. Manipulating IMPDH expression in glioblastoma cells results in the same change in MYC expression. Furthermore, Myc activates GTP synthesis and it has been shown in small cell lung cancer (SCLC), that IMPDH is depended upon by naïve and chemoresistant high-MYC SCLC cells. In addition, IMPDH links Myc's role in nucleotide biosynthesis and ribosome biogenesis as IMPDH-dependent GTP synthesis is needed for Pol I synthesis of pre-ribosomal RNA.
Glutaminase, another target gene of Myc, converts the abundant glutamine into glutamate. Mycdriven cancers depend on glutamine metabolism rather than glucose, especially when deprived of oxygen. Furthermore, glutamine-depletion-induced apoptosis is dependent on Myc activity, and on the other hand, Myc-induced renal adenocarcinoma depends on glutaminase. Shen et al. explored the concept of glutaminase dependency in the context of ovarian cancer, in which >45% of patients' tumors overexpress MYC. Elevated MYC expression correlated with glutaminase in immortalized cell lines and primary cultures, and overexpression of MYC and GLS were associated with chemoresistance and worse disease outcome.
We will discuss inhibiting the proteins of metabolic Myc target genes, IMPDH and glutaminase, along with epigenetic cofactor WDR5 that recruits Myc to drive ribosome biogenesis. Details of Myc's role in cancer metabolism are beyond the scope of this review and are summarized in several articles. Additional potential Myc-driven metabolic targets are described by Dong et al.. . IMPDH catalyzes GDP to GTP and glutaminase converts glutamine (Gln) to glutamate (Glu), a major energy source in cancer. Lastly, epigenetic co-factor WDR5 recruits Myc to chromatin to express genes involved in biomass accumulation. (B) Although Myc still has control over target genes IMPDH, GTP, and GLN, the function of these proteins can be inhibited. Cancer's energy supply can be depleted by inhibiting IMPDH, which prevents GTP production, or by inhibiting glutaminase, which will limit the pool of glutamate. Finally, inhibiting WDR5 will prevent Myc's target gene expression of biomass related genes.
## Targeting metabolism through myc target genes and cofactors
Myc's effects on metabolism are highlighted by the negative impact of various metabolic inhibitors in Myc-driven cancer models. First, there are two clinically available IMPDH inhibitors: mycophenolic acid (MPA) and mizoribine. Both are clinically used as an immunosuppressants to prevent organ transplant rejection, but additional research is necessary to explore their anticancer properties. MPA appears to primarily serve as an anticancer tool; it has been preclinically tested in several cancers, but has dose-limiting toxicity due to gastro-intestinal side effects. However, mizoribine has higher tolerability. Studies connected the anticancer efficacy of IMPDH inhibitors to Myc; it appears that Myc is needed for the antiangiogenic properties of MPA. Myc-driven SCLC and hepatoblastoma demonstrate sensitivity to mizoribine and MPA, confirming a dependence on IMPDH. Importantly, mizoribine was capable of decreasing tumor growth in immunocompetent mice, despite its immunosuppressive properties. This study warrants further testing and development of these clinically-available IMPDH inhibitors in Myc-driven cancer models.
A second approach is targeting glutaminase in MYC-overexpressing cells. The concept of pharmacologically inhibiting glutaminase has been discussed since 1975, but there were concerns of targeting a major metabolic component. Allosteric glutaminase inhibitor Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) has been studied extensively. Treatment with BPTES increases reactive oxygen species production and hinders cell bioenergetics, leading to cell death. In an in vivo renal adenocarcinoma model, BPTES reduced tumor growth by 32%. However, modifications must be made to BPTES to improve its therapeutic potential as it has a moderate potency and poor solubility. The newest glutaminase inhibitor, CB-839, is a BPTES derivative and is currently in Phase II clinical trials for several cancers including colorectal cancer, acute myeloid leukemia, and triple negative breast cancer. Shen et al. demonstrated preclinically that ovarian tumor xenografts treated with CB-839 resulted in increased sensitivity to PARP inhibitor olaparib as glutaminase inhibition led to replicative stress. A Phase Ib/II study investigating CB-839 in combination with PARP inhibitor talazoparib for solid tumors is currently recruiting patients (NCT03875313) and a Phase I study combining CB-839 with PARP inhibitor niraparib in platinum resistance BRCA-wildtype ovarian cancer has posted (NCT03944902).
As mentioned, Myc's role in metabolism primarily stems from expression of its target genes that are involved in metabolic pathways. The previously described Myc metabolic therapeutics inhibit Myc's target genes, but not Myc's activity. Thomas et al. discovered that epigenetic cofactor WDR5 recruits Myc to chromatin to promote expression of genes involved in biomass accumulation. An inducible exon swap system in a Burkitt lymphoma cell line was created to study the interaction between Myc and WDR5 by implementing a mutant Myc that could not interact with WDR5. Inhibition of WDR5 prevented Myc's function as a transcription factor by disrupting gene binding, which decreased transcription of translational machinery, including ribosome protein subunits and nucleolar RNAs. When the exon swap system was assessed in vivo, switching to the WDR5-interaction-defective Myc resulted in apoptosis, decreased tumor volume, and improved survival. Thomas and collogues additionally reported that targeting the "WIN" site in WDR5 may also be a valuable target to displace Myc from chromatin. WDR5 inhibitors are currently being synthesized to further study the anticancer effects of disrupting the Myc-WDR5 interaction. Targeting an epigenetic cofactor that aids in Myc's activity will prevent Myc's target gene from being transcribed; this route may be more beneficial than inhibiting already-transcribed genes under Myc's control. This study opens an avenue outside the context of metabolism to explore other targetable Myc-interacting cofactors to prevent Myc binding to chromatin.
# Conclusions
This review summarizes the main mechanisms by which c-Myc promotes tumorigenesis and the different therapeutic approaches that directly/indirectly target Myc. Broadly, we described inhibitors that prevent Myc's actions as a transcription factor through altering Myc stability (transcription, dimerization, degradation), inducing synthetic lethality via cell cycle targets, and inhibiting Myc target genes involved in metabolism. There are other methods of disrupting Myc's activity not listed here, including inhibiting Myc-recruited cofactors or epigenetic mechanisms. Additionally, Myc's robust control on microRNA expression is another area of interest, given Myc's ability to activate oncogenic miRNAs and repress tumor suppressive miRNAs. Modulating expression of the miRNAs to promote anticancer effects as a therapeutic option is being explored. When clinically testing these therapeutics in Myc-driven cancers, it is important to consider patients' MYC expression in the case of stratifying patients and identifying clinically relevant subgroup results. Furthermore, although this review was limited to well-studied c-Myc, the Myc family of c-Myc, N-Myc and L-Myc, can be functionally redundant, and therefore inhibition of the Myc family rather than one specific Myc may be required. All in all, new discoveries improved our understanding of the "myc-anisms" behind Myc-driven cancers and enhanced the potential of targeting the "undruggable" Myc.
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High-Density Epicardial Activation Mapping to Optimize the Site for Video-Thoracoscopic Left Ventricular Lead Implant
Optimization of Left Ventricular Lead Position. Background:The left ventricular (LV) lead local electrogram (EGM) delay from the beginning of the QRS complex (QLV) is considered a strong predictor of response to cardiac resynchronization therapy. We have developed a method for fast epicardial QLV mapping during video-thoracoscopic surgery to guide LV lead placement.Methods: A three-port, video-thoracoscopic approach was used for LV free wall epicardial mapping and lead implantation. A decapolar electrophysiological catheter was introduced through one port and systematically attached to multiple accessible LV sites. The pacing lead was targeted to the site with maximum QLV. The LV free wall activation pattern was analyzed in 16 pre-specified anatomical segments.Results: We implanted LV leads in 13 patients with LBBB or IVCD. The procedural and mapping times were 142 ± 39 minutes and 20 ± 9 minutes, respectively. A total of 15.0 ± 2.2 LV segments were mappable with variable spatial distribution of QLV-optimum. The QLV ratio (QLV / QRSd) at the optimum segment was significantly higher (by 0.17 ± 0.08, p < 0.00001) as compared to an empirical midventricular lateral segment. The LV lead was implanted at the optimum segment in 11 patients (at an adjacent segment in 2 patients) achieving a QLV ratio of 0.82 ± 0.09 (range 0.63-0.93) and 99.5 ± 0.6% match with intraprocedural mapping.Conclusion: Video-thoracoscopic LV lead implantation can be effectively and safely guided by epicardial QLV mapping. This strategy was highly successful in targeting the selected LV segment and resulted in significantly higher QLV ratios compared to an empirical midventricular lateral segment. (J Cardiovasc Electrophysiol, Vol. 25, pp. 882-888, August 2014) cardiac resynchronization therapy, left ventricular lead, epicardial mapping, video, thoracoscopic implantation, heart failure, implantable cardioverter defibrillator R. Polasek reports receiving lecture fees from St. Jude Medical and Medtronic. J. Kautzner reports receiving payment for board membership and lecture fees from Biosense Webster,
# Introduction
Cardiac resynchronization (CRT) is the established therapy of chronic systolic heart failure in patients with intraventricular conduction delay-wide QRS complex. [bib_ref] Cardiac resynchronization in chronic heart failure, Abraham [/bib_ref] [bib_ref] Feldman AM: Cardiac-resynchronization therapy with or without an implantable defibrillator in advanced..., Bristow [/bib_ref] Approximately 30% of patients, however, do not respond to this therapy clinically; and in 50% of patients, CRT is not associated with left ventricular (LV) reverse remodeling. [bib_ref] The problem of non-response to cardiac resynchronization therapy, Birnie [/bib_ref] Left ventricular pacing lead position is closely associated with the response to CRT. Several methods have been advocated for optimization of its position. However, only two of them have been studied more extensively. One comprises echocardiographic local mechanical delay, [bib_ref] Targeted left ventricular lead placement to guide cardiac resynchronization therapy: The TARGET..., Khan [/bib_ref] [bib_ref] Optimal left ventricular lead position predicts reverse remodeling and survival after cardiac..., Ypenburg [/bib_ref] [bib_ref] Impact of left ventricular lead position in cardiac resynchronization therapy on left..., Becker [/bib_ref] [bib_ref] Relative merits of left ventricular dyssynchrony, left ventricular lead position, and myocardial..., Delgado [/bib_ref] [bib_ref] The impact of left ventricular lead position on left ventricular reverse remodelling..., Kristiansen [/bib_ref] while the other consists of time interval between the onset of QRS complex and local LV lead electrogram (EGM) during spontaneous ventricular activation (QLV). [bib_ref] Characterization of left ventricular activation in patients with heart failure and left..., Auricchio [/bib_ref] [bib_ref] The use of epicardial electrogram as a simple guide to select the..., Fatemi [/bib_ref] [bib_ref] The relationship between ventricular electrical delay and left ventricular remodelling with cardiac..., Gold [/bib_ref] [bib_ref] Left ventricular lead electrical delay predicts response to cardiac resynchronization therapy, Singh [/bib_ref] [bib_ref] Role of intraoperative electrical parameters in predicting reverse remodelling after cardiac resynchronization..., Zucchelli [/bib_ref] [bib_ref] Local electrogram delay recorded from left ventricular lead at implant predicts response..., Polasek [/bib_ref] The evidence from observational studies is mounting that more optimal LV lead position (at the site of more delayed contraction and longer QLV) predicts better clinical response and reverses LV remodeling. Inappropriate LV lead position with QLV shorter than one-half of the QRS duration was associated with higher mortality in a small retrospective study. [bib_ref] Left ventricular lead electrical delay predicts response to cardiac resynchronization therapy, Singh [/bib_ref] Reduced mortality and reduced heart failure hospitalization rate (combined endpoint) were observed in patients randomized to echocardiographically optimized LV lead position in the TARGET trial. [bib_ref] Targeted left ventricular lead placement to guide cardiac resynchronization therapy: The TARGET..., Khan [/bib_ref] Unlike transvenous LV lead implantation, which is limited by the anatomy of the coronary sinus and its tributaries, minimally invasive surgical video-thoracoscopic approach has fewer constraints. In such situations, empirical selection of the LV pacing site, which is usually a central lateral segment of the LV according to previous hemodynamic studies, [bib_ref] Effect of resynchronization therapy stimulation site on the systolic function of heart..., Butter [/bib_ref] [bib_ref] Surgical epicardial left ventricular lead versus coronary sinus lead placement in biventricular..., Mair [/bib_ref] and endocardial activation mapping in patients with left bundle branch block (LBBB), [bib_ref] The spectrum of inter-and intraventricular conduction abnormalities in patients eligible for cardiac..., Peichl [/bib_ref] may not be optimal. Therefore, we proposed a new method for fast epicardial mapping of QLV during video-thoracoscopic surgery to optimize the LV lead position. This study was primarily aimed at assessing the feasibility and safety of this approach. In addition, we hypothesized that the benefit of this technique could be indirectly demonstrated.
# Methods
## Patient population
All patients who were indicated for video-thoracoscopic LV lead implantation were eligible for the mapping study if they had preserved atrio-ventricular (AV) conduction with LBBB or intraventricular conduction delay (IVCD). They were recruited from among those in whom transvenous CRT device implantation was unsuccessful or in whom an LV lead was implanted but malfunctioning because of technical issues. In addition, nonresponders to CRT after a 12-month post-implant with QLV ratio <0.7 at the time of CRT implantation were screened for study eligibility. Nonresponders were defined by both New York Heart Association (NYHA) class improvement <1 and absence of LV reverse remodeling (<15% reduction in LV end-systolic volume or <10% reduction in LV end-systolic diameter). Generally, severe LV dilatation/dysfunction, atrial fibrillation and a history of open-heart surgery were not considered exclusion criteria. Eligibility for the video-thoracoscopic procedure was assessed by managing physician and implanting surgeon. Some fragile patients having excessive surgical risk were not considered like those with advanced age, multiple comorbidities or progressive end-stage heart failure.
This study was approved by the local ethics committee and was performed in accordance with the guidelines proposed in the Declaration of Helsinki. All patients gave written informed consent.
## Mapping and implantation technique
A standard, 3-port thoracoscopic approach was used for LV free wall mapping, and subsequent LV lead implantation. After deflation of the left lung, CO 2 insufflation at a pressure of 8-10 mmHg was combined with single lung ventilation. Port positions were chosen according to size and anatomy of the heart. In most cases, the third and fifth intercostal space in the anterior axillary line were used for tools (5-and 10-mm ports), and the fourth intercostal space, between the middle and posterior axillary line, was employed for the scope (10-mm port). The pericardium was opened posterior and anterior to the phrenic nerve, and the vessels on the heart surface were identified. These pericardial incisions were wider (6-8 cm) compared to those used in an empiric implant procedure without mapping. A decapolar electrophysiological catheter was introduced through one port and systematically attached to multiple accessible LV sites in a step-by-step fashion to access all 16 predefined segments of the LV free wall [fig_ref] Figure 1: A 16-segment model of the LV free wall [/fig_ref]. The recordings started at the LV base with the catheter tip directed anteriorly. Then the catheter was shifted to a middle and apical position, with the tip still directed anteriorly. From the apical position, the catheter was rotated counter-clockwise across the apex posteriorly and further shifted back to middle and basal position, with the catheter tip now directed posteriorly. Bipolar LV EGMs during spontaneous ventricular activation were recorded and analyzed at a sweep speed of 200 mm/sec with band-pass filtering of 30-500 Hz (Cardiolab System, Prucka Engineering, GE Healthcare, Little Chalfont, UK) [fig_ref] Figure 2: Epicardial LV activation mapping assessed by decapolar catheter [/fig_ref]. At each position of the catheter, a 30-second recording was performed and 5 bipolar signals were analyzed simultaneously during the same spontaneous ventricular depolarization. QRS mor- phology was inspected to exclude variation of the activation pattern. Attention was paid to select a heart cycle within a period of stable sinus rhythm and with good quality of all 5 local bipolar electrograms. Corresponding QLV intervals were assigned to appropriate LV segments. A bipolar, sutureless epicardial pacing lead (Myopore C , Greatbatch Medical, NY, USA) was implanted at the site with maximum QLV. The procedure was considered successful when the LV free wall epicardial map was nearly completed (at least 12/16 segments) or when the maximal mappable QLV ratio was ࣙ0.90, and when the LV lead was implanted to the optimum or an adjacent LV segment. At the end of the procedure, QLV was measured at the implantation site directly from the newly implanted LV lead. Procedural descriptive data were collected and patients were followed for complications until discharge from the hospital.
## Data processing and statistical analysis
Epicardial EGMs were assessed both in real time for the guidance of LV lead implantation, and off-line for the purpose The values are mean ± standard deviation (range) or number (proportion). of detailed analysis of LV free wall activation wavefront. The analysis was done per prespecified segments in which QLV readings were averaged and standardized to QRS complex duration (QLV ratio = QLV/QRSd). QLV ratios in individual segments (and groups of segments) were compared using a 2-tailed t-test for dependent samples, as well as by ANOVA for repeated measures, with a Newman-Keuls test for post hoc comparisons. In particular, we assessed the difference in QLV ratio between a hypothetical empirical implantation site (central lateral segment of LV, M3), and the individual optimum LV segment (with the longest QLV). The accuracy of optimum site targeting was quantified by QLV match value (QLV of implanted LV lead divided by QLV in the segment with longest average QLV). A P-value <0.05 was considered significant.
# Results
Thirteen patients were included in the study over a 14month period. The rationale for surgical intervention was unsuccessful transvenous LV lead implantation in 5 patients, and LV lead malfunction (phrenic nerve capture or high pacing threshold) in 3 patients. All patients who were offered the surgical mapping procedure agreed to participate in the study. In addition, 10 CRT nonresponders with suboptimal LV lead position, who would benefit from the surgical LV electrode re-implantation according to general clinical judgment, were asked to enter the study. Five of them consented and thus were included in the study.
The baseline characteristics of the study population are shown in the [fig_ref] TABLE 1: Baseline Characteristics [/fig_ref]. Epicardial mapping and LV lead implantation were successfully completed in all patients. Conversion to minithoracotomy or sternotomy was not necessary in any patient. In 2 cases, an additional thoracoscopic port had to be introduced to implant the LV lead in the selected region. The procedural and mapping times were 142 ± 39 minutes and 20 ± 9 minutes, respectively, with an average hospital stay of 6.6 ± 3.0 days.
## Epicardial mapping
A total of 15.0 ± 2.2 (range: 8-16; median: 16; interquartile range [IQR]: 15-16) LV segments were mappable through video-thoracoscopic access. In 1 patient, multiple segments (n = 8) were not accessible because of pericardial adhesions due to previous coronary artery bypass graft surgery (CABG). The average number of mapping points per patient reached 53 ± 16 (range: 14-73; median: 54; IQR: 49-58). The average number of mapping points per mappable segment was 3.5 ± 1.9 (range: 1-10; median: 3; IQR: 2-4).
We have found large interindividual variability of the LV free wall spontaneous activation pattern with widely distributed QLV-optimum segments. Only in 1 patient the best site was determined to be in the 2 anterior-most rows of segments (#1 and #2). In another subject, the best site was determined to be at the LV apex region. The QLV ratio was significantly higher at the optimum segment in comparison with an empirical M3 segment by 0.17 ± 0.08 (P = 0.000002). Despite the variability of LV activation pattern, an averaged QLV ratio map derived from pooled data revealed a clear gradient of improvement (i.e., increase of QLV ratio) in the direction from anterior to posterior segments, [fig_ref] TABLE 2: QLV Ratio in Individual LV Free Wall Segments [/fig_ref]. On the contrary, when the true apex region with incomplete data and usually low QLV ratio was excluded, no apparent gradient in QLV ratio was documented along the LV long-axis. Significance of this observation is supported by ANOVA statistics . On average, the highest QLV ratio was found at the M5 segment (posterolateral or posterior). The QLV ratio in this segment was significantly higher than that in an empirical M3 segment by 0.10 ± 0.01 (P = 0.003). The best segment in individual subjects was still better than the best average segment M5 by 0.08 ± 0.07 (P = 0.003).
## Lv lead placement
An LV lead was successfully implanted at the segment with maximum QLV in 11 patients; it was implanted in an adjacent segment in 2 patients. In one case, the LV apex, which had the maximum QLV, was avoided because of significant scarring in this region and an adjacent apical segment was chosen instead. In the other case, the optimum segment was found to be at the very posterior part of a dilated LV, which was not accessible by thoracoscopic approach. The average QLV match was 99.5 ± 0.6% (P = 0.77 from 100%). The LV lead QLV ratio was 0.82 ± 0.09 (range 0.63-0.93) and was significantly higher (by 0.17 ± 0.08) than the average QLV ratio at an empirical M3 segment (P = 0.000005). In 5 CRT nonresponders with a previously implanted transvenous LV lead and QLV ratio of 0.55 ± 0.04, epicardial re-implantation improved the QLV ratio to 0.77 ± 0.11 (P = 0.02).
## Safety
Two major procedural complications were observed in a single patient: A pneumothorax that did not require suction and an episode of ventricular fibrillation successfully terminated by DC shock, with hospital stay prolongation by 10 days. The performance of LV leads at a 6-month follow-up visit was correct in all patients.
## Figure 3. four examples of variable left ventricular activation pattern. color-coded depictions of activation sequences are shown (green for early and red for late activation). the color scale for the qlv ratio is individualized for each case. note the activation map in a patient with ivcd (panel b) with the most delayed activation outside of the mappable region, and the absence of bracketing of the most delayed activation in another patient (panel c).
# Discussion
This study showed that epicardial LV lead implantation can be optimized by simple epicardial QLV mapping during the standard video-thoracoscopic procedure. This approach is safe and allows successful targeting the optimum LV segment in the majority of cases with acceptable mapping time and total procedural time.
In this study, the use of a decapolar catheter, and electrophysiological recording system enabled us to perform high density mapping of the LV free wall and obtain higher amount of activation sites compared to previously published mapping techniques. The former studies utilized temporary LV lead placement in several locations (total number was not specified) during surgical implantation in order to maximize the right ventricle (RV) pace-LV delay 18 or atrial sense-LV delay. [bib_ref] Minimally invasive left ventricular epicardial lead placement: Surgical techniques for heart failure..., Navia [/bib_ref] Both of them, as well as atrial pace-LV delay, can be measured by device programmers independently of surface ECG acquisition. The atrial pace/sense-LV delay, which is closely related to QLV, is less precise because of tiny variations in AV nodal conduction (or change in AV delay). It cannot also be used in patients with atrial fibrillation. The RV pace-LV interval seems theoretically more relevant than QLV for LV pacing site optimization, especially when intrinsic infrahisian conduction is not about to be utilized for CRT. However, we preferred QLV because larger amount of data is available for predictive value of QLV compared to RV pace-LV interval.
Both parameters QLV [bib_ref] The relationship between ventricular electrical delay and left ventricular remodelling with cardiac..., Gold [/bib_ref] [bib_ref] Local electrogram delay recorded from left ventricular lead at implant predicts response..., Polasek [/bib_ref] and QLV ratio [bib_ref] The use of epicardial electrogram as a simple guide to select the..., Fatemi [/bib_ref] [bib_ref] Left ventricular lead electrical delay predicts response to cardiac resynchronization therapy, Singh [/bib_ref] were used in previous studies and both were significantly associated with clinical outcome. QLV interval reflecting simultaneously the position of LV lead and QRS duration, which is independent predictor of CRT response per se, may be the valid choice in observational studies. Mapping results in individual patients are clearly invariant to the use of QLV or QLV ratio. QLV ratio only (as a standardized measure of LV lead position) is an optimum choice when mapping results are averaged across patients with dissimilar QRS duration like in our study.
Mapping time was not excessively long and achieved total procedural time was even shorter (142 minutes) than reported for video-thoracoscopic procedures in a larger cohort published by Navia et [fig_ref] Figure 2: Epicardial LV activation mapping assessed by decapolar catheter [/fig_ref]. [bib_ref] Minimally invasive left ventricular epicardial lead placement: Surgical techniques for heart failure..., Navia [/bib_ref] The manipulation with mapping catheter was not difficult for operator skilled in thoracoscopic procedure. This is in line with 20-minute mapping time for registering of, on average, 11 catheter positions with signal recording time of at least 30 seconds. Only in 1 patient after open-heart surgery, pericardial adhesion prevented to create a complete epicardial map. Fortunately, a segment with very high QLV ratio >0.90 was accessible even in this patient.
The average hospital stay of 6.6 days appears to be longer than expected. By excluding the patient with procedural pneumothorax, it would be 5.8 ± 0.9 days. In addition, the hospital stay was also exploited for other cardiac examination and adjustment of medical therapy in these patients with advanced heart failure. Majority of patients were discharged [fig_ref] TABLE 2: QLV Ratio in Individual LV Free Wall Segments [/fig_ref].
## . summary of results for epicardial mapping. top paneldistribution of qlv optimum segments (asterisks for individual patients). bottom panel-averaged qlv ratio map from all 13 cases. the color scale corresponds to the qlv ratio differences between individual segments and central lateral m3 segment. absolute numerical results are provided in
on third or fourth postoperative day, which is standard of care after video-thoracoscopic procedure in our institution.
We believe that before this method can be safely applied in clinical practice, further research should prove its validity. The ability to develop the necessary skill set is, however, a reasonable expectation of a tertiary cardiovascular center.
The TARGET trial 4 was the first randomized interventional trial that in 200 patients demonstrated the superiority of tailored LV lead placement as compared with empirical pacing site selection, both in terms of CRT response as well as combined death and heart failure hospitalization endpoint. Despite the fact that our experience with video-thoracoscopic left ventricular lead implantation was obtained as part of a preliminary feasibility study that was not designed to investigate the clinical outcome of patients, some technical aspects of QLV-guided implantation can be compared with the echocardiographically-guided technique of LV lead positioning that was investigated in the TARGET trial. In our study, the LV free wall was mapped very precisely with more than 50 mapping points in 16 predefined segments, while 12segment classification (echocardiography and fluoroscopybased) was used for the assessment of local mechanical delay and implantation site in the TARGET trial. Selection of the optimum site using echocardiography (ECHO) was done offline prior to the implantation procedure and LV lead placement was likely dependent on adequate correspondence between echocardiography and fluoroscopy imaging. [bib_ref] Chest radiography is a poor predictor of left ventricular lead position in..., Rickard [/bib_ref] In our study, epicardial activation mapping was performed during the implantation procedure by a single operator and was lim- The numbers are QLV ratios (mean ± SD) for all patients. #1 = anteriormost row of segments in clockwise short-axis segmentation; #5 = posteriormost row of segments in clockwise short-axis segmentation. Apex-Apical-Middle-Basal denotes segmentation in the long axis.
## Table 3
QLV Differences Between Segments Grouped Along LV Long Axis "1" "2" "3" "4" "5"
"1" 0.002 0.00002 0.000008 0.00002 "2" 0.00007 0.00002 0.000008 "3" 0.19 0.04 "4" 0.26 "5"
The numbers are P-values for mutual differences in QLV ratio between individual groups of segments (ANOVA, Newman-Keuls post hoc test). The true apex was excluded from the analysis. "1" = group of the anterior-most segments; "5" = group of the posterior-most segments.
ited to the accessible LV free wall. The lack of "bracketing" of QLV intervals in the resulting QLV map in some of our patients might indicate that the posterior-most segments of LV (perhaps even more electrically delayed) may have been missed in our study. We demonstrated considerably high QLV match of the final LV lead position and segment with optimum QLV ratio (99%). On the contrary, transvenous implantation in the TARGET trial was associated with a noticeably lower success rate of implantation into the optimum segment (63%).
Both the distribution of maximal mechanical delay in the TARGET trial and the maximal electrical delay in our study revealed high variability of the spatial distribution of the optimum LV segment. This is in line with original observation of a variable endocardial activation pattern in LBBB, resulting from differently located lines of functional conduction block. [bib_ref] Characterization of left ventricular activation in patients with heart failure and left..., Auricchio [/bib_ref] There are limited data on correlation of epicardial and endocardial LV activation in such patients, and comparison of these would be an interesting topic of further research, especially taking into account new investigational endocardial LV lead implantation techniques. A study by Spragg et al. [bib_ref] Optimal left ventricular endocardial pacing sites for cardiac resynchronization therapy in patients..., Spragg [/bib_ref] showed high interindividual variability of optimum LV endocardial pacing sites assessed by acute hemodynamic response to temporary CRT in 11 patients. Overall, these findings could explain why the predictive value of anatomical location of LV lead (except the LV apex) for CRT response was generally low, if any, in previous studies, [bib_ref] Fluoroscopic left ventricular lead position and the long-term clinical outcome of cardiac..., Foley [/bib_ref] [bib_ref] Long-term clinical outcome and left ventricular lead position in cardiac resynchronization therapy, Kronborg [/bib_ref] [bib_ref] Influence of left ventricular lead location on outcomes in the COMPANION study, Saxon [/bib_ref] [bib_ref] Left ventricular lead position and clinical outcome in the multicenter automatic defibrillator..., Singh [/bib_ref] [bib_ref] Sites of left and right ventricular lead implantation and response to cardiac..., Thebault [/bib_ref] [bib_ref] Relationship between left ventricular lead position using a simple radiographic classification scheme..., Wilton [/bib_ref] which has resulted in underestimation of the importance of LV lead position. Another technique of LV lead pacing site optimization was published by Dekker using a special conductance catheter and evaluating the pressure-volume relationship. [bib_ref] Epicardial left ventricular lead placement for cardiac resynchronization therapy: Optimal pace site..., Dekker [/bib_ref] Four to 6 LV free wall positions of temporary LV leads were compared according to pressure-volume loops registered in LV during biventricular pacing. Significant differences were found between various LV lead locations in terms of acute hemodynamic response.
However, such a technically demanding concept does not seem suitable for routine use.
Adjacent segment relationships were not analyzed in our study because of small population size, which was not powered to detect the ANOVA-differences in activation time between individual segments. That is why we pooled the segments "by rows" from anterior-most to posterior-most in order to statistically confirm the presence of overall activation gradient in this direction, which was also depicted in(lower panel). Overall, conduction pattern was homogeneous and physiological meaningful in individual patients, and not completely random in investigated population with aggregation of optimum sites at more posterior segments of LV free wall. A large interindividual variability of activation pattern was caused mainly by inclusion of 2 patients with IVCD (one of them shown in . But even in LBBB patients the site of the latest LV activation was rather variable, upper panel) supporting the value of activation mapping for LV lead placement.
Our data clearly suggest that the posterior part of the LV surface is more delayed than the central lateral segment that is typically chosen during stand-alone video-thoracoscopic LV lead implantation also because of implicitly easy access. However, outcome studies are needed in order to demonstrate the clinical value of this strategy.
# Study limitations
This was a small, single-center study that aimed at establishing the feasibility of a new mapping technique for optimization LV lead position. The acute impact of implantation site optimization was not validated by the LV dP/dt measurement. The study was also too small and not designed to evaluate the clinical benefit of this LV lead placement strategy. Despite the fact that QLV has been identified in several studies as a good predictor of clinical outcome, its correlation with measured changes in dP/dt seems to be modest. [bib_ref] Baseline left ventricular dP/dtmax rather than the acute improvement in dP/dt max..., Bogaard [/bib_ref] However, in that acute study the measured hemodynamic response was not associated with long-term clinical outcome. [bib_ref] Baseline left ventricular dP/dtmax rather than the acute improvement in dP/dt max..., Bogaard [/bib_ref] Therefore, the randomized study of empirical versus electrophysiologically-based LV lead implantation with clinical endpoints is warranted as the next step.
From a technical point of view, it is important to emphasize that the operator has rather limited visual control of the tip of the mapping catheter that is introduced through the pericardial incision. In some instances it was not clear whether the tip could reach the borders of LV free wall. This was especially true for very posterior parts of large left ventricles. In this case the map of the LV free wall may be partially incomplete. Only 1 patient after open-heart surgery was enrolled; more experience is needed in order to identify whether this approach is also feasible for such patients.
# Conclusions
This study showed that video-thoracoscopic LV lead implantation could be effectively and safely guided by simplified epicardial QLV mapping, which can be implemented in heart centers with advanced experience with thoracoscopic techniques and expertise in clinical cardiac electrophysiology. We demonstrated variable activation patterns of the LV free wall in individual patients with varying spatial distribution of optimal pacing sites. This strategy was highly suc-cessful in targeting the selected LV segment. Compared to empirically chosen pacing site in central lateral position, epicardial QLV mapping allowed to achieve significantly longer QLV ratio for the implanted epicardial lead.
[fig] Figure 1: A 16-segment model of the LV free wall. A lateral view of the LV divided into 4 sectors and a short-axis cross-section of the LV with clockwise segmenation into 5 sectors on the free wall. [/fig]
[fig] Figure 2: Epicardial LV activation mapping assessed by decapolar catheter. Both surface ECG and epicardial EGMs recording at a sweep speed of 200 mm/second are depicted. I, II, III, aVR, aVL, aVF = ECG leads; LV 1.2 -LV 9.10-5 bipolar local EGMs registered from a decapolar catheter. Note the maximum QLV at the distal bipole (LV 1.2). QLV and QRSd are measured by an electronic caliper. QLVr = QLV/QRSd. [/fig]
[fig] Figure 4: Summary of results for epicardial mapping. Top paneldistribution of QLV optimum segments (asterisks for individual patients). Bottom panel-averaged QLV ratio map from all 13 cases. The color scale corresponds to the QLV ratio differences between individual segments and central lateral M3 segment. Absolute numerical results are provided in Table 2. [/fig]
[table] TABLE 1: Baseline Characteristics (n = 13) [/table]
[table] TABLE 2: QLV Ratio in Individual LV Free Wall Segments [/table]
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Topographic analysis of K-ras mutations in histologically normal lung tissues and tumours of lung cancer patients
[bib_ref] Chemical and physical carcinogenesis: advances and perspectives, Harris [/bib_ref] [bib_ref] Genetic evidence for an independent origin of multiple preneoplastic and neoplastic lung..., Sozzi [/bib_ref] [bib_ref] Molecular aspects of chemical carcinogenesis: the roles of oncogenes and tumor suppressor..., Stanley [/bib_ref] [bib_ref] Molecular themes in oncogenes, Bishop [/bib_ref] [bib_ref] mutations in human cancers, Hollstein [/bib_ref] [bib_ref] The molecular basis of human lung cancer, Gazdar [/bib_ref] [bib_ref] Molecular aspects of chemical carcinogenesis: the roles of oncogenes and tumor suppressor..., Stanley [/bib_ref] [bib_ref] Molecular themes in oncogenes, Bishop [/bib_ref] [bib_ref] The ras signal transduction pathway, Khosravi-Far R [/bib_ref] [bib_ref] Detection of ras gene mutations in human lung cancer by single-stranded conformational..., Suzuki [/bib_ref] [bib_ref] ras gene mutation as a prognostic marker in adenocarcinoma of the human..., Sugio [/bib_ref] [bib_ref] Clinical significance of ras oncogene activation in human lung cancer, Rodenhuis [/bib_ref] [bib_ref] ) K-ras mutations in human adenocarcinoma of the lung: association with smoking..., Husgafvel-Pursiainen [/bib_ref] [bib_ref] Molecular staging of non-small cell lung cancer according to K-ras genotypes, Rosell [/bib_ref] [bib_ref] Detection of K-ras mutations in lung carcinomas: Relationship to prognosis, Keohavong [/bib_ref] [bib_ref] Aberrant crypts: putative preneoplastic foci in human colonic mucosa, Minna [/bib_ref] [bib_ref] Detection of ras gene mutations in human lung cancer by single-stranded conformational..., Suzuki [/bib_ref] [bib_ref] ras gene mutation as a prognostic marker in adenocarcinoma of the human..., Sugio [/bib_ref] [bib_ref] Clinical significance of ras oncogene activation in human lung cancer, Rodenhuis [/bib_ref] [bib_ref] ) K-ras mutations in human adenocarcinoma of the lung: association with smoking..., Husgafvel-Pursiainen [/bib_ref] [bib_ref] Molecular staging of non-small cell lung cancer according to K-ras genotypes, Rosell [/bib_ref] [bib_ref] Detection of K-ras mutations in lung carcinomas: Relationship to prognosis, Keohavong [/bib_ref] [bib_ref] Detection of low-fraction K-ras mutations in primary lung tumors using sensitive methods, Keohavong [/bib_ref] [bib_ref] ras gene mutation as a prognostic marker in adenocarcinoma of the human..., Sugio [/bib_ref] [bib_ref] ) K-ras oncogene activation in lung adenocarcinomas from former smokers, Westra [/bib_ref] [bib_ref] Analysis of K-ras gene mutations in malignant and nonmalignant endobronchial tissue obtained..., Clements [/bib_ref] [bib_ref] High frequency of K-ras mutations in normal appearing lung tissues and sputum..., Yakubovskaya [/bib_ref] [bib_ref] Codon 12 Ki-ras mutation in non-small-cell lung cancer: comparative evaluation in tumoural..., Urban [/bib_ref] [bib_ref] Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent..., Keohavong [/bib_ref] [bib_ref] Detection of codon 12 K-ras mutations in non-neoplastic mucosa from bronchial carina..., Urban [/bib_ref] [bib_ref] K-ras mutations are relatively late event in the pathogenesis of lung carcinomas, Sugio [/bib_ref] [bib_ref] K-ras oncogene activation in atypical alveolar hyperplasia of the human lung, Westra [/bib_ref] [bib_ref] K-ras mutations are relatively late event in the pathogenesis of lung carcinomas, Sugio [/bib_ref] [bib_ref] High frequency of K-ras mutations in normal appearing lung tissues and sputum..., Yakubovskaya [/bib_ref] [bib_ref] Codon 12 Ki-ras mutation in non-small-cell lung cancer: comparative evaluation in tumoural..., Urban [/bib_ref] [bib_ref] ) c-K-ras and p53 mutations occur very early in adenocarcinoma of the..., Li [/bib_ref] [bib_ref] Analysis of K-ras gene mutations in malignant and nonmalignant endobronchial tissue obtained..., Clements [/bib_ref] [bib_ref] Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent..., Keohavong [/bib_ref] [bib_ref] Detection of codon 12 K-ras mutations in non-neoplastic mucosa from bronchial carina..., Urban [/bib_ref] [bib_ref] K-ras mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients, Zhu [/bib_ref] [bib_ref] Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent..., Keohavong [/bib_ref]
# Materials and methods
## Patients and tissue specimens
Pulmonary lobectomy specimens were obtained from 48 patients with lung cancer, including 38 adenocarcinoma and 10 squamous cell carcinoma. The demographic and clinical information is shown in [fig_ref] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of... [/fig_ref]. These specimens were formalin-fixed and paraffin-embedded and stored at the University of Pittsburgh Medical Center between 1990 and 1999. The diagnosis of lung adenocarcinoma and squamous cell carcinoma was confirmed, using established morphologic criteria. For DNA analysis, 5 µm histologic sections were prepared from multiple paraffin blocks representative of tumour and those representative of matched histologically normal lung tissues (located between 2.0 cm and 5.0 cm from the tumour edge). These normal tissues corresponded to either bronchial or bronchiolar pseudostratified columnar epithelial tissues that matched tumours (adenocarcinoma or squamous cell carcinoma) of a bronchial origin, or alveolar walls that matched tumors (adenocarcinoma or bronchoalveolar type) of an alveolar origin. All tissue sections were individually deparaffinized in xylene and then in ethanol, rinsed with water, and airdried. All slides were stained with hematoxylin and eosin and reviewed to confirm the presence of histologically normal tissue and/or tumour. The microdissection was performed by a trained pathologist using a laser capture microdissection microscope (PixCell II LCM System, Arcturus Engineering, Mountain View, CA). With the aid of this new technique, individual cells within areas of interest of a tissue section were isolated [bib_ref] Laser capture microdissection, Emmert-Buck [/bib_ref]. Areas of tumour tissue and histologically normal tissue were prepared and microdissected separately to avoid any possible cross-contamination between tissues. Samples of 100-200 histologically normal cells or malignant cells were 'lasercaptured' on separate 'Caps' built to fit a 0.5 ml microcentrifuge tube (Acturus Engineering, Mountain View, CA). Each cell sample was considered to be 100% 'homogeneously' normal-appearing or malignant, as determined by a careful microscopic visualization of the cells captured on each Cap.
## Dna extraction and k-ras codon 12 mutation analysis
The captured cells were lysed by adding directly on each Cap 20 µl of lysis solution (40 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% Tween-20, and 0.5 µg µl -1 proteinase K) [bib_ref] K-ras mutations are relatively late event in the pathogenesis of lung carcinomas, Sugio [/bib_ref]. The Cap was enclosed into a 0.5 ml microcentrifuge tube (in an upsidedown position) and incubated at room temperature for 48 hours with occasional gentle shaking. The resulting cell lysate was recovered in the tube by a quick and gentle spinning in a microcentrifuge then heated at 95°C for 5 minutes to inactivate the proteinase K. Half (10 µl) of each cell lysate was used as template for a first round of PCR to amplify a 75-bp genomic fragment containing the 5′ half of exon 1 of the K-ras gene and adjacent flanking intron sequence. PCR was carried out in a final 50 µl reaction mixture containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 50 mM KCl, 100 µM each dNTP, 0.5 µM each primer and 2.5 units of Gold AmpliTaq DNA polymerase (Perkin Elmer, CT). The mixture was heated at 95°C for 9 minutes then subjected to 15 cycles (94°C/1 minute, 53°C/1 minute, 72°C/1 minute), using the primers KI1-1 (sense): 5′-TATTATAAGGCCTGCTGAAA-3′ and PKB (antisense): 5′-AGGCACTCTTGCCTACGGCA-3′. For the second round of PCR, 2 µl of the PCR products from the first round were diluted into a final 25 µl reaction mixture containing the same buffer composition as above except that 0.25 µl of [α-32 P]-dATP (3000 Ci/mmol, New England Nuclear, Boston, MA) was added and primer KI1-1 was replaced by primer PKGC: 5′-GCCGCCTGCAGCCCGCGCCCCCCGTGCCCCCGCCCCGC-CGCCGGCCCGGCGCCTATAAGGCCTGCTGAAAATG-3′. The mixture was heated at 95°C for 9 minutes then subjected to 35 PCR cycles (94°C/1 minute, 60°C/1 minute, 72°C/1 minute). The resulting PCR products were separated by gel electrophoresis and autoradiographed. Band containing the expected-size 126-bp fragment was excised from the gel. DNA was eluted from the gel slice and analysed by DGGE (Bio-Rad, CA) under the conditions described previously [bib_ref] Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent..., Keohavong [/bib_ref]. Mutant alleles were isolated from the denaturing gradient gel and further characterized by sequencing. This method allows us to clearly detect a Kras mutant allele present at a mutant fraction of 5% (1 mutant allele among 20 wild-type alleles) [bib_ref] Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent..., Keohavong [/bib_ref]. This level of sensitivity should be sufficient to identify K-ras mutations in DNA samples from an equivalent 50-100 original cells. In the cases where mutant alleles appeared ambiguous by using the above PCR+DGGE method, a more sensitive version of this method which has a detection limit of 10 -3 -10 -4 (one mutant allele in 10 3 -10 4 wild-type allele) was used. This method requires an additional step for mutant allele enrichment involving a prior treatment of the 126-bp amplified fragment with Ban I restriction enzyme which cleaves fragments with a non-mutated K-ras codon 12. The digestion-resistant fragment was gel-purified and analysed by DGGE as described previously [bib_ref] Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent..., Keohavong [/bib_ref]. [fig_ref] Figure 1: An example of tissue site sampling from a formalin-fixed and paraffin-embedded tissue... [/fig_ref] shows a representative example of a target-site sampling from a case of lung adenocarcinoma. The use of a laser capture microscopy allowed an accurate morphologic analysis and precise sampling of cells from areas of interest in paraffin-embedded tissue sections. Histologically normal cells were individually resected by laser capture from the tissue area located between 2 cm and 5 cm from the edge of the tumour area. Between 100 and 200 cells were captured on each Cap after a careful histologic examination and determination of their benign morphology. All cells appeared homogeneously normal-looking and there were no tumour cells present. Paraffin-embedded tissue blocks representative of tumour tissue were topographically sampled in a similar manner. The cells captured from the tumour tissue also all appeared malignant histologically. After microdissection, each captured cell sample was compared to the matched tissue section prior to and after microdissection to confirm the accuracy of target selection (see [fig_ref] Figure 1: An example of tissue site sampling from a formalin-fixed and paraffin-embedded tissue... [/fig_ref]. Using this approach, we prepared 38 cell samples from histologically normal tissue and 38 cell samples from matched malignant tissue from paraffin-embedded lung tissue sections selected from 38 patients with lung adenocarcinoma. We also prepared cell samples from histologically normal tissue, matched metaplasia and tumour tissue from paraffinembedded tissue sections obtained from 10 patients with lung squamous cell carcinoma.
# Results
## Lung tissue sampling and microdissection
# Mutation analysis
Molecular analysis of the above cell samples using the PCR+DGGE method yielded 10 lung cancer cases showing K-ras mutant alleles in tumour cells and/or paired histologically normal cells, all of which corresponded to lung adenocarcinoma (26.3% or 10 of 38 cases). shows an example of DGGE separation of K-ras mutant alleles from the wild-type allele for 6 of the 10 K-ras mutant-positive cases. The mutant alleles were isolated from the gel and the nature of the mutations was determined by [fig_ref] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of... [/fig_ref] by using a laser capture microdissection microscope (magnification × 100). The figure shows a tissue section obtained from a block representative of a histologically normal lung tissue pre-(A) and post-(B) topographic microdissection of normal-appearing cells from an area of interest of the tissue section. The cells were 'captured' on a Cap (C) and reviewed further for cell population homogeneity and then treated with proteinase K. (D) shows the same cell sample after treatment with proteinase K, with the cellular content being totally lysed. Under the lysis conditions shown in Materials and Methods, between 95% and 100% of the captured cells were completely lysed sequencing. No mutants were identified in any of the cell samples corresponding to tumour tissue or paired histologically normal tissue obtained from the 10 cases of squamous cell carcinoma (data not shown). These results are in line with previous studies showing that K-ras mutations were frequently identified in lung adenocarcinoma but were rarely so in lung squamous cell carcinoma [bib_ref] Clinical significance of ras oncogene activation in human lung cancer, Rodenhuis [/bib_ref] [bib_ref] ) K-ras mutations in human adenocarcinoma of the lung: association with smoking..., Husgafvel-Pursiainen [/bib_ref] [bib_ref] Detection of K-ras mutations in lung carcinomas: Relationship to prognosis, Keohavong [/bib_ref]. As shown in , the 2 control samples of known normal tissues (C1 and C2) revealed only the K-ras codon 12 wild-type homoduplex (wt). Compared with the controls, patients 3 and 5 showed each a mutant pattern in the tumour tissue but not in the paired histologically normal tissue. These patients harboured each a GGT to TGT mutation in their lung tumour tissue. In addition, in patient 5, the wild-type allele appeared only as a small fraction of the mutant allele in this tumour sample (T). This observation may indicate that some tumour cell in this sample may have lost 1 allele of K-ras gene while containing a mutation in the remaining allele. This loss of the wild-type allele appeared to occur at a specific area of the tumour tissue because further analysis of tumour cells taken from 2 separate sites of tumour tissue from this patient showed not only the TGT mutation but also the wild-type allele at a fraction higher than the TGT mutant allele in both samples (data not shown). Nevertheless, the presence of a loss of 1 allele and a mutation in the other allele of the K-ras gene has not been reported previously. It may be that the deletion of the wild-type allele occurred as a secondary event that accumulated during the progression of cells with already a K-ras mutated gene in one allele into tumour. Patients 2, 4 and 6 each harboured a genotypically identical mutation in both the tumour tissue and the paired histologically normal tissue, corresponding to a GGT to TTT, a GGT to GTT, and GGT to TGT, respectively. Patient 1 showed 4 mutant/wild-type heteroduplexes in the histologically normal tissue (N), which were found to correspond to 2 different mutations, a GTT to ATT in codon 9, and a GCT to ACT in codon 11, of the K-ras gene. This patient also showed two mutant/wild-type heteroduplexes in the matched tumour tissue (T), which corresponded to a GGT to GAT mutation in codon 12 of the K-ras gene.
## Multiple site analysis of k-ras mutations
In order to investigate in more detail the topographic distribution of mutations in each lung tumour tissue and histologically normal tissue, all 10 K-ras mutation-positive adenocarcinomas as well as 3 K-ras mutation-negative adenocarcinomas were re-examined further for K-ras mutations. Toward this end, between 3 and 5 additional successive tissue sections were prepared from original tissue blocks representing tumour-surrounding histologically normal tissue as well as those representing tumour tissue for each cancer case. [fig_ref] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of... [/fig_ref] summarizes the final results of K-ras mutations identified at multiple sites in both the histologically normal tissue and the matched tumour tissue for the 10 cases of K-ras mutation-positive lung adenocarcinomas. The cell samples obtained from the tumour tissue and histologically normal tissue for the 3 selected K-ras mutation-negative patients were all found to be negative for K-ras mutations (data not shown), confirming the results of the first experiment. 4 of the 10 K-ras mutationpositive cases (Patients 3, 5, 7 and 8) contained each a mutation (a GGT to TGT) in only the tumour tissue. This mutation was present in all malignant cell samples taken from 3 different areas of the tumour tissue from each of these patients. Patients 2 and 6 each contained a genotypically identical mutation (a GGT to TTT and GGT to TGT, respectively) in both the tumour tissue and matched histologically normal tissue. These mutations were present in all cell samples taken from 3 different tissue blocks representative of the normal-appearing tissue and those representative of the matched tumour tissue obtained from these patients, in agreement with the results of the first experiment shown in for these patients. Likewise, multiple site analysis helped confirm that Patient 4 contained a genotypically identical mutation, a GGT to GTT, in both the tumour tissue and histologically normal tissue. In addition, as shown in , a second mutation, a GGT to AGT, was detected in the histologically normal tissue. However, while the GGT to GTT mutation was found in all tissue blocks representative of the histologically tissue (see lanes N1, N2 and N3 and n1 n2, and n3), as well as those representative of the tumour tissue (see T1, T2 and T3), the GGT to AGT mutation was found in only cell samples taken from one specific area of the histologically normal tissue (see lanes N1 and n1). This tissue area was defined at the margin of resection located 5 cm from the edge of the tumour. In the remaining 3 patients (Patients 1, 9 and 10; [fig_ref] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of... [/fig_ref] , the mutations were identified at a low fraction as shown by the fact they were identified only in 1 or 2 of the total 6 cell samples taken from the histologically normal tissue and/or the tumour tissue and by only using a more sensitive version of the Figure 2 DGGE analysis of K-ras mutations, K-ras gene exon 1 is amplified from cellular DNA isolated from each cell sample microdissected from histologically normal (N) and tumour (T) areas of a selected lung tissue section as shown in [fig_ref] Figure 1: An example of tissue site sampling from a formalin-fixed and paraffin-embedded tissue... [/fig_ref]. The amplified DNA is analysed by DGGE to separate any K-ras mutant allele that may be present in each cell sample from the wild type allele. The figure shows sequence patterns of K-ras mutant alleles for 6 of 10 lung adenocarcinoma that were found to be positive for Kras mutation in the tumour tissue and/or the histologically normal tissue. The wild-type allele was present in excess over mutant allele(s) in each DNA sample, except for the tumour DNA sample in patient 5, and was detected as a homoduplex fragment (wt). The presence of a mutant allele was detected as 3 additional fragments, including a mutant homoduplex and the 2 respective mutant/wild-type heteroduplexes that separated from the wild-type fragment in lower denaturant concentrations from the bottom to the top of the gel, respectively. In patient 1, the histologically normal DNA sample showed 4 mutant/wild-type heteroduplexes that corresponded to the presence of 2 different mutant alleles (see patient 1 in [fig_ref] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of... [/fig_ref]. Lanes C show the sequence patterns of negative control DNA samples corresponding to a known K-ras mutation-negative benign lung tissue (C1) and infant normal intestine tissue (C2). The histologically normal tissue sample (N) in patient 6 contained several faint heteroduplex bands. Further experiments showed that only the 2 heteroduplexes identical to those appearing in the matched tumour sample (T) were repeatedly detected. Therefore, the presence of the other heteroduplexes in the histologically normal tissue sample (N) was presumably due to experimental variation PCR + DGGE method (see Materials and Methods). Patient 1 contained 2 mutations in codon 12 (a GGT to GAT and GGT to AGT) in tumour tissue and 2 mutations, 1 in codon 9 (a GTT to ATT) and 1 in codon 11 (a GCT to ACT), in histologically normal tissue. These mutations were identified each in only 1 or 2 of the 6 samples taken from either the tumour tissue or the histologically normal tissue. In Patient 9, 2 mutations (a GGT to GAT and GGT to AGT) were found in histologically normal tissue and 1 mutation (a GGT to AGT) was found in tumour tissue. In Patient 10, 1 mutation (a GGT to AGT) was found in 2 of 6 samples taken from the histologically normal tissue and in none of 6 malignant cell samples.
# Discussion
Although K-ras mutations have been frequently found in lung tumours and tumour cell lines and implicated in the development of lung cancer, the timing of these mutations in lung carcinogenesis remains poorly understood. There have been several but conflicting reports on whether or not K-ras mutations are present in non-neoplastic or normal-appearing lung tissues, which, in the affirmative, would indicate that they occur early during lung cancer development and can provide an early lung cancer detection marker. We applied a laser capture microdissection microscope to morphologically review and sample cells at multiple sites from both lung tumour tissue and histologically normal tissue adjacent to as well as those distant from the tumour. Our results, based on independent experiments using successive lung tissue sections from multiple tissue blocks, demonstrated that K-ras mutations were present in histologically normal tissue in 6 of 10 (60%) K-ras mutation-positive lung adenocarcinomas, or 6 of all 38 (15.8%) lung adenocarcinomas examined. Therefore, K-ras mutations are relatively frequent in histologically normal lung tissue surrounding malignant tissue in lung adenocarcinomas. These mutations can be grouped into high fraction mutations and low fraction mutations in tumour and/or histologically normal tissue. Low fraction K-ras mutations have been previously reported in lung tumour DNA by us and others [bib_ref] Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma, Mills [/bib_ref] [bib_ref] Detection of low-fraction K-ras mutations in primary lung tumors using sensitive methods, Keohavong [/bib_ref]. They were frequently identified in tumour tissue as well as in histologically normal tissue and metastasis [bib_ref] High frequency of K-ras mutations in normal appearing lung tissues and sputum..., Yakubovskaya [/bib_ref]. Our present study showed that mutations in patients 1, 9 and 10 were detected in only some cell samples taken from malignant tissue and/or matched histologically normal tissue, suggesting that they were not initiating or early events in lung carcinogenesis but rather secondary events accumulating in Except for the 2 mutations found in histologically normal tissues in patient 1, all mutations shown involved codon 12 of the K-ras gene.
wt Patient 4 C1 C2 T1 T2 T3 N1 N2 N3 n1 n2 n3 c1 c2 An example of DGGE analysis of K-ras mutations at multiple sites of lung tissue sections for patient 4 shown in [fig_ref] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of... [/fig_ref]. The figure shows the GGT to GTT mutant sequence pattern in cells sampled at 3 regions of the tumour tissue (T1, T2, T3) and 3 regions of the histologically normal tissue (N1, N2, N3) corresponding to patient 4. The histologically normal cells were sampled at 5 cm (N1 and N2) and 2 cm (N3) from the edge of the tumour. Lanes C1 and C2 contained DNA with non-mutated K-ras gene corresponding to benign lung tissue and infant intestinal tissue, respectively. Shown in lanes n1, n2, n3, and c1 and c2, are the same DNA from N1, N2, N3, and C1 and C2, that had been first subjected to BanI restriction enzyme digestion to eliminate any non-K-ras codon 12 mutant allele, including the wild-type allele (wt), and thereby to enrich for K-ras codon 12 mutant alleles, then analysed by DGGE [bib_ref] K-ras mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients, Zhu [/bib_ref]. The positions of the mutant homoduplexes (second fragment from wt) and the 2 respective mutant/wildtype heteroduplexes (third and fourth fragments from wt) were indicated by arrows for the GGT to GTT mutation in lanes T1-T3, and by arrowheads for the GGT to AGT mutation in lanes N1-N3 and n1. After mutant alleleenrichment by BanI digestion, the GGT to AGT mutant pattern in lane N1 appeared more clearly visible in lane n1. As expected, control DNA shown in lanes c1 and c2 did not contain any K-ras codon 12 mutant allele subclones during tumour progression or as a result of continued exposure to tobacco smoke [bib_ref] Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma, Mills [/bib_ref] [bib_ref] High frequency of K-ras mutations in normal appearing lung tissues and sputum..., Yakubovskaya [/bib_ref] [bib_ref] Detection of low-fraction K-ras mutations in primary lung tumors using sensitive methods, Keohavong [/bib_ref]. The GTT to ATT mutation (codon 9) and GCT to ACT mutation (codon 11) found in patient 1 had not been reported previously and their biological significance in lung carcinogenesis is not known. Among the lung adenocarcinomas with high fraction mutations, 2 observations can be made. First, these mutations can be found in only the tumour tissue and not in the matched histologically normal tissue (Patients 3, 5, 7 and 8). The fact that they were detected at multiple sites in lung tumour tissue suggests that they may represent events occurring early in small precursor lesions before tumour progression. Secondly, some K-ras mutations are found not only in the tumour tissue but also, at a mutant fraction generally lower than that detected in the tumour tissue, in histologically normal tissues adjacent to as well as those distant from the tumour (Patients 2, 4 and 6). These results confirmed our previous study showing that genotypically identical K-ras mutations were detected in both the tumour tissue and, at a lower mutant fraction, in tumour-adjacent normal-appearing tissue in 4 of 8 cases of lung adenocarcinoma [bib_ref] K-ras mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients, Zhu [/bib_ref]. These mutations may represent early events, perhaps initiating events, in the development of these tumours. Furthermore, Patient 4 provides an intriguing example since only 1 mutation, a GGT to GTT, is present in the tumour tissue, but 2 mutations, a GGT to GTT and GGT to AGT, are present in the matched histologically normal tissue. In addition, while the GTT mutation was detected in all samples taken from 6 different sites of the histologically normal tissue, the GGT to AGT mutation was detected in only cell samples taken from a defined area of the normal-appearing tissue distant from the tumour. This observation is compatible with the existence of 2 overlapping fields with each a different mutated K-ras gene, and the field with the GTT mutation may contribute to the development of this tumour [bib_ref] Field cancerization in oral stratified epithelium, Slaughter [/bib_ref] [bib_ref] Field cancerization in the aerodigestive tract -its etiology, manifestation, and significance, Strong [/bib_ref]. It is, however, unclear why a field with a specific mutation would have a higher ability to develop into a tumour than one with another mutation type. Some studies suggested that the substitution of the wild-type codon 12 amino acid (glycine) with certain amino acids, including valine and arginine, in the H-ras gene may give rise to gene products with a more potent transforming ability than the wild-type or other amino acids [bib_ref] Biological properties of human c-H-ras genes mutated at codon 12, Seeburgh [/bib_ref] [bib_ref] Biological and Biochemical properties of human ras H genes mutated at codon..., Der [/bib_ref]. It will require an expanded study involving a larger number of lung cancer patients to determine whether our explanation for the observation in patient 4 can be supported. K-ras mutations were detected in histologically normal mucosal tissues adjacent to or distant from colorectal carcinoma [bib_ref] Aberrant crypts: putative preneoplastic foci in human colonic mucosa, Minna [/bib_ref] [bib_ref] Mutant K-ras in apparently normal mucosa of colorectal cancer patients: its potential..., Minamoto [/bib_ref] [bib_ref] K-ras mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients, Zhu [/bib_ref]. They have been also suggested to be preneoplastic events in pancreatic cancer [bib_ref] Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from..., Yanagisawa [/bib_ref] [bib_ref] Identification of K-ras mutations in pancreatic juice in the early diagnosis of..., Berthelemy [/bib_ref] [bib_ref] Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas..., Tada [/bib_ref] [bib_ref] ) K-ras mutations in the duodenal fluid of patients with pancreatic carcinoma, Wilentz [/bib_ref]. However, in lung cancer, there have been conflicting reports on K-ras mutations in non-neoplastic tissues. On the one hand, [bib_ref] Malignant activation of a K-ras oncogene in lung carcinoma but not in..., Santos [/bib_ref] investigated K-ras codon 12 mutation in lung tissues obtained from a patient with squamous cell carcinoma of the lung and reported that the mutation detected in lung carcinoma was absent from the normal bronchial and parenchymal tissues of that patient. The authors suggested that malignant activation of K-ras oncogene may be specifically associated with the development of a human neoplasm. [bib_ref] K-ras mutations are relatively late event in the pathogenesis of lung carcinomas, Sugio [/bib_ref] reported that K-ras mutations were absent in metaplasia and normal-appearing cells of the lung and were infrequent in dysplastic lesions, suggesting that these mutations are relatively late events in the pathogenesis of lung cancer. Likewise, [bib_ref] Codon 12 Ki-ras mutation in non-small-cell lung cancer: comparative evaluation in tumoural..., Urban [/bib_ref] did not detect any K-ras mutation in non-neoplastic peripheral bronchial or parenchymal tissues associated with lung tumours. Finally, [bib_ref] High frequency of K-ras mutations in normal appearing lung tissues and sputum..., Yakubovskaya [/bib_ref] detected K-ras mutations in 60% of normal-appearing lung tissue samples, 63% of tissue samples from tumour tissue, and 80% of samples of metastasis obtained from patients with nonsmall cell lung cancer. However, the fact these mutations were present at a low fraction suggests that they did not contribute to the development of lung cancer. On the other hand, the results of these studies are not in agreement with other published studies. For instance, K-ras mutations were identified in atypical alveolar hyperplasia (AAH), a potential precursor from which lung adenocarcinoma arises [bib_ref] K-ras oncogene activation in atypical alveolar hyperplasia of the human lung, Westra [/bib_ref]. In another study, [bib_ref] Analysis of K-ras gene mutations in malignant and nonmalignant endobronchial tissue obtained..., Clements [/bib_ref] analysed bronchoscopy specimens obtained from lung cancer patients and showed that K-ras mutations were found in both malignant and nonmalignant tissues in 22.7% (5 of 22) of the patients and in only nonmalignant tissue in 9.1% (2 of 22) of the patients. Finally, a recent study by [bib_ref] Detection of codon 12 K-ras mutations in non-neoplastic mucosa from bronchial carina..., Urban [/bib_ref] showed that K-ras mutations were detected in both lung tumour and bronchial carina tissue in 21% (4 of 19) of the patients, while being present only in the bronchial carina but not in the tumour in 11% (2 of 19) of the patients. These latter studies and our present study showed that K-ras mutations can be frequently found in non-neoplastic lung tissues, including histologically normal tissues.
Questions remain about the usefulness of K-ras mutations as an early detection markers for lung cancer. Mutation in the K-ras gene is a well-established example of the association between environmental exposure (chiefly tobacco smoke) and human lung cancer, since mutations in the K-ras gene are found almost exclusively in lung tumours from smokers, while being rare in lung tumours from non-smokers [bib_ref] ras gene mutation as a prognostic marker in adenocarcinoma of the human..., Sugio [/bib_ref] [bib_ref] Detection of ras gene mutations in human lung cancer by single-stranded conformational..., Suzuki [/bib_ref] [bib_ref] Detection of K-ras mutations in lung carcinomas: Relationship to prognosis, Keohavong [/bib_ref] [bib_ref] Molecular staging of non-small cell lung cancer according to K-ras genotypes, Rosell [/bib_ref]. In theory, these mutations should be useful markers of exposure to carcinogens in tobacco smoke, and their detection in high-risk individuals without cancer would be an indication of damaged airway epithelium. The presence of these mutations in airways of ex-smokers might also be an indication of persistence of genetic damage in 'condemned epithelium'. The technique of laser capture microdissection as described here could be applied to mucosal biopsies taken by either white light or fluorescent bronchoscopy to determine if K-ras mutations are present in preneoplastic or histologically normal epithelium from the airways of individuals at high risk for lung cancer.
[fig] Figure 1: An example of tissue site sampling from a formalin-fixed and paraffin-embedded tissue section from one case of lung adenocarcinoma (see patient 4 in [/fig]
[table] Table 1: Summary of K-ras mutations in lung tissue sections from 10 cases of lung adenocarcinoma a [/table]
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How Safe Are Common Analgesics for the Treatment of Acute Pain for Children? A Systematic Review
Background. Fear of adverse events and occurrence of side effects are commonly cited by families and physicians as obstructive to appropriate use of pain medication in children. We examined evidence comparing the safety profiles of three groups of oral medications, acetaminophen, nonsteroidal anti-inflammatory drugs, and opioids, to manage acute nonsurgical pain in children (<18 years) treated in ambulatory settings. Methods. A comprehensive search was performed to July 2015, including review of national data registries. Two reviewers screened articles for inclusion, assessed methodological quality, and extracted data. Risks (incidence rates) were pooled using a random effects model. Results. Forty-four studies were included; 23 reported on adverse events. Based on limited current evidence, acetaminophen, ibuprofen, and opioids have similar nausea and vomiting profiles. Opioids have the greatest risk of central nervous system adverse events. Dual therapy with a nonopioid/opioid combination resulted in a lower risk of adverse events than opioids alone. Conclusions. Ibuprofen and acetaminophen have similar reported adverse effects and notably less adverse events than opioids. Dual therapy with a nonopioid/opioid combination confers a protective effect for adverse events over opioids alone. This research highlights challenges in assessing medication safety, including lack of more detailed information in registry data, and inconsistent reporting in trials.
# Introduction
Pain is the most common reason for seeking healthcare in the Western world [bib_ref] A prospective study of ED pain management practices and the patient's perspective, Tanabe [/bib_ref] [bib_ref] The high prevalence of pain in emergency medical care, Cordell [/bib_ref] [bib_ref] Pain in the emergency department with one-week follow-up of pain resolution, Johnston [/bib_ref]. Key organizations have voiced concern with our medical services' inability to provide appropriate analgesia for children's pain [bib_ref] Needle and dread: is it just a little poke? A call for..., Mcmurtry [/bib_ref] [bib_ref] Medicine AAoPCoPEMaSoAaP: relief of pain and anxiety in pediatric patients in emergency..., Zempsky [/bib_ref] [bib_ref] American pain society recommendations for improving the quality of acute and cancer..., Gordon [/bib_ref] [bib_ref] Clinical policy: evidence-based approach to pharmacologic agents used in pediatric sedation and..., Mace [/bib_ref]. Inadequate pain treatment can have significant detrimental effects [bib_ref] Pain, plasticity, and premature birth: a prescription for permanent suffering?, Anand [/bib_ref] [bib_ref] Current status of pain management in children, Howard [/bib_ref] [bib_ref] Consequences of inadequate analgesia during painful procedures in children, Weisman [/bib_ref] [bib_ref] Pain in children and adolescents: prevalence, impact on daily life, and parents'..., Haraldstad [/bib_ref]. Still, fear of adverse events (AEs) is commonly cited by both families and physicians as an obstruction to appropriate use of analgesic medication in childhood [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref] [bib_ref] Outpatient pediatric pain management practices for fractures, Drendel [/bib_ref].
AEs are a major health problem for affected children, their family, and society [bib_ref] Incidence of fatal adverse drug reactions: a population based study, Wester [/bib_ref] [bib_ref] Children's deaths linked with postsurgical codeine, Voelker [/bib_ref] [bib_ref] Pediatric adverse drug events in the outpatient setting: an 11-year national analysis, Bourgeois [/bib_ref]. Surprisingly, comprehensive drug safety in children remains understudied, despite emerging evidence that AEs are frequent and are commonly cited 2 Pain Research and Management as a reason for terminating prescribed therapy [bib_ref] Incidence of fatal adverse drug reactions: a population based study, Wester [/bib_ref] [bib_ref] Children's deaths linked with postsurgical codeine, Voelker [/bib_ref]. A systematic review of AEs in hospitalized pediatric patients reported an overall incidence rate of 9% [bib_ref] The incidence and nature of in-hospital adverse events: a systematic review, De Vries [/bib_ref]. Furthermore, up to 30-50% of pediatric analgesic users will experience at least one AE [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref]. Milder AEs (i.e., vomiting, sleepiness, and constipation) contribute to limitations in activity and function for children and negatively impact their caregivers' productivity and time off work [bib_ref] Outpatient pediatric pain management practices for fractures, Drendel [/bib_ref]. Importantly, AEs may discourage future use of analgesics, thereby exposing children to unnecessary pain and its resultant negative consequences.
Children's pain management varies greatly across North America [bib_ref] Outpatient pediatric pain management practices for fractures, Drendel [/bib_ref] [bib_ref] A randomized, controlled trial of acetaminophen, ibuprofen, and codeine for acute pain..., Clark [/bib_ref]. Recently, clinicians have been compelled to rapidly shift their prescribing practices due, in part, to concerns regarding the safety profile of oral opioids [bib_ref] Outpatient pediatric pain management practices for fractures, Drendel [/bib_ref] [bib_ref] Safety of codeine during breastfeeding: fetal morphine poisoning in the breastfed neonate..., Madadi [/bib_ref] [bib_ref] Drug dosing error with drops-severe clinical course of codeine intoxication in twins, Hermanns-Clausen [/bib_ref] [bib_ref] Pediatric pain practice variation amongst emergency doctors: a canadian survey, Ali [/bib_ref]. With the FDA boxed warning regarding codeine use in children post-ENT surgeryas well as the European Medicines Agency and Health Canada's advisory to avoid all codeine use in children less than 12 years of age [bib_ref] Restrictions on Use of Codeine for Pain Relief in Children-CMDh Endorses PRAC..., Agency [/bib_ref] , clinicians have reduced their use of codeine and now search for a suitable alternative oral opioid. A recent survey of North American physicians showed considerable variability in practice management by center, country, and specialty [bib_ref] Acute musculoskeletal pain treatment: a North American survey of practice variation, Kircher [/bib_ref]. The American Academy of Pediatrics' consensus statement on the assessment and management of pain in children recommends acetaminophen, ibuprofen, and opioids as the top three medication choices for the treatment of acute pain in children. These are also the top three most commonly used treatments in the emergency department for children with fracture pain [bib_ref] Outpatient pediatric pain management practices for fractures, Drendel [/bib_ref] [bib_ref] Pediatric pain practice variation amongst emergency doctors: a canadian survey, Ali [/bib_ref] [bib_ref] Acute musculoskeletal pain treatment: a North American survey of practice variation, Kircher [/bib_ref] [bib_ref] Ibuprofen provides analgesia equivalent to acetaminophencodeine in the treatment of acute pain..., Friday [/bib_ref] [bib_ref] Pediatric musculoskeletal pain management in the ED: a medical record review, Kircher [/bib_ref]. It stands to reason that clinicians (and certainly patients and their families) would prefer the drug that has the best safety profile. However, as noted in the 2014 report by the Council of Canadian Academies, available published literature is not properly synthesized to provide the data needed to make such treatment decisions.
This systematic review compares the safety profiles of three groups of oral medications, acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDS), and opioids, to manage acute, nonsurgical pain in children treated in ambulatory settings. While much has been written about the efficacy of these medications, to date, there has been no comprehensive synthesis of safety and in particular their comparative safety. Moreover, since emerging studies are suggesting equivalence (or close to equivalence) and clinical equipoise for many of the current pain management options [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref] [bib_ref] A randomized, controlled trial of acetaminophen, ibuprofen, and codeine for acute pain..., Clark [/bib_ref] [bib_ref] Acetaminophen versus acetaminophen with codeine after pediatric tonsillectomy, Moir [/bib_ref] [bib_ref] Myth: ibuprofen is superior to acetaminophen for the treatment of benign headaches..., Manzano [/bib_ref] , clinicians may make practical decisions driven by the perceived safety profile. In an effort to address known challenges in safety literature nomenclature [bib_ref] Core outcome domains and measures for pediatric acute and chronic/ recurrent pain..., Mcgrath [/bib_ref] , we attempted to capture a range of undesirable effects (e.g., side effects, adverse effects, adverse events, and adverse reactions). For the purposes of this manuscript, we use the term "adverse events" to represent all safety concerns captured by this variable nomenclature.
# Methods
We followed a protocol established a priori (available from authors) based on standards for conducting and reporting systematic reviews
## Study selection.
Search results were screened independently by two reviewers. Two reviewers then assessed the full text of all potentially relevant citations using a standard form with predefined eligibility criteria. Disagreements were resolved by consensus.
## Inclusion criteria.
We included primary studies of any design involving children (<18 years) with acute pain (pain related to injury or illness less than 3 months in duration) who were treated in an ambulatory setting (e.g., outpatient clinics, emergency). The ambulatory setting was chosen as it represents the most common location for presentation of children with acute injuries and illnesses requiring short-term analgesic use. Pain medications included acetaminophen, NSAIDS, and opioids. We included any study design and publication type. We excluded studies of surgically induced pain, as we felt these patients represented medically induced pain and included the possible influence of general or regional anesthesia.
## Data extraction.
One reviewer extracted data using a structured form; a second reviewer verified data for accuracy and completeness. We extracted study and patient characteristics; interventions (type, dose, route of administration, timing, and duration); care setting (e.g., emergency department, outpatient clinic, primary care, and others); AEs; and funding source.
We extracted AEs as reported in the studies and grouped them into nine categories: nausea, vomiting, other gastrointestinal symptoms, headache, drowsiness (includes sleepiness and tiredness), dizziness, other CNS symptoms, dermatological symptoms, and pulmonary symptoms. For each AE, we counted each event as if it corresponded to a unique individual. tool covering issues of how data on harms were defined, collected, and reported. Two reviewers independently assessed quality and resolved discrepancies through discussion.
# Assessment of methodological
## Data synthesis.
A description of the studies is provided in tables. We present a summary of AEs by treatment arm (i.e., intervention) for an overall picture of which interventions had a high risk of specific AEs. This enabled us to include data from both comparative and noncomparative studies. For each AE, risks (incidence rates) were pooled using a random effects model to obtain a summary estimate and 95% confidence interval (CI). Analyses were conducted using Review Manager 5.2 (Cochrane Collaboration, 2012).
# Results
Forty-four studies met our inclusion criteria; however, 21 did not report AEs [fig_ref] Figure 1: PRISMA flow diagram of study retrieval and selection [/fig_ref]. Therefore, we included 23 studies involving 2,300 patients: 17 randomized controlled trials, 2 nonrandomized controlled trials, 1 case report, 1 cross-sectional survey, 1 chart review, and 1 prospective cohort [fig_ref] Table 1: Characteristics of included studies [/fig_ref] [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref] [bib_ref] A randomized, controlled trial of acetaminophen, ibuprofen, and codeine for acute pain..., Clark [/bib_ref] [bib_ref] Ibuprofen provides analgesia equivalent to acetaminophencodeine in the treatment of acute pain..., Friday [/bib_ref] [bib_ref] Randomized, double-blind, multicenter, controlled trial of ibuprofen versus acetaminophen (paracetamol) and placebo..., Bertin [/bib_ref] [bib_ref] A randomized, double-blind, multicentre controlled trial of ibuprofen versus acetaminophen and placebo..., Bertin [/bib_ref] [bib_ref] Acute treatment of episodic childhood tension-type headache with flupirtine and paracetamol-a double-blind..., Pothmann [/bib_ref] [bib_ref] Nimesulide and acetaminophen for the treatment of juvenile migraine: a study for..., Soriani [/bib_ref] [bib_ref] Children's ibuprofen suspension for the acute treatment of pediatric migraine, Lewis [/bib_ref] [bib_ref] The effect of standard care, ibuprofen, and distraction on pain relief and..., Tanabe [/bib_ref] [bib_ref] Oral morphine administration for children's traumatic pain, Wille [/bib_ref] [bib_ref] Naproxen versus acetaminophen for therapy of soft tissue injuries to the ankle..., Cukiernik [/bib_ref] [bib_ref] Ibuprofen-induced papillary necrosis causing bilateral ureteric obstruction, Ismail [/bib_ref] [bib_ref] Effectiveness of oxycodone, ibuprofen, or the combination in the initial management of..., Koller [/bib_ref] [bib_ref] Oxycodone versus codeine for triage pain in children with suspected forearm fracture:..., Charney [/bib_ref] [bib_ref] Paracetamol versus ibuprofen: a randomized controlled trial of outpatient analgesia efficacy for..., Shepherd [/bib_ref] [bib_ref] Treatment of children with migraine in emergency departments: national practice variation study, Richer [/bib_ref] [bib_ref] A randomized, double-blind, placebo-controlled trial of paracetamol and ketoprofren lysine salt for..., Ruperto [/bib_ref] [bib_ref] Treatment of childhood migraine attacks with oral zolmitriptan and ibuprofen, Evers [/bib_ref] [bib_ref] Ibuprofen or acetaminophen for the acute treatment of migraine in children: a..., Hämäläinen [/bib_ref] [bib_ref] Outpatient pediatric pain management practices for fractures, Drendel [/bib_ref] [bib_ref] Sublingual ketorolac versus sublingual tramadol for moderate to severe post-traumatic bone pain..., Neri [/bib_ref] [bib_ref] Efficacy of an ibuprofen/codeine combination for pain management in children presenting to..., May [/bib_ref] [bib_ref] Oral administration of morphine versus ibuprofen to manage postfracture pain in children:..., Poonai [/bib_ref]. Studies were published between 1991 and 2014 (median year 2007) and were conducted in the United States ( = 7), Canada (5), France (3), Italy (3), and Germany (2) and one each in Finland, New Zealand, and the United Kingdom. Most studies did not report on sources of funding ( = 13), one specifically reported no funding [bib_ref] Paracetamol versus ibuprofen: a randomized controlled trial of outpatient analgesia efficacy for..., Shepherd [/bib_ref] , seven reported grant funding [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref] [bib_ref] A randomized, controlled trial of acetaminophen, ibuprofen, and codeine for acute pain..., Clark [/bib_ref] [bib_ref] The effect of standard care, ibuprofen, and distraction on pain relief and..., Tanabe [/bib_ref] [bib_ref] Naproxen versus acetaminophen for therapy of soft tissue injuries to the ankle..., Cukiernik [/bib_ref] [bib_ref] Effectiveness of oxycodone, ibuprofen, or the combination in the initial management of..., Koller [/bib_ref] [bib_ref] Treatment of children with migraine in emergency departments: national practice variation study, Richer [/bib_ref] [bib_ref] Efficacy of an ibuprofen/codeine combination for pain management in children presenting to..., May [/bib_ref] [bib_ref] Oral administration of morphine versus ibuprofen to manage postfracture pain in children:..., Poonai [/bib_ref] , and two reported funding from industry [bib_ref] A randomized, double-blind, placebo-controlled trial of paracetamol and ketoprofren lysine salt for..., Ruperto [/bib_ref] [bib_ref] Efficacy of an ibuprofen/codeine combination for pain management in children presenting to..., May [/bib_ref]. The median quality score was 8/14 (interquartile range 6 to 8) (Appendix B). Only 41% predefined harms while none defined serious or severe AEs. Sixty-nine percent included active data collection for AEs; 48% involved passive (six studies used both). Seventy-eight percent specified who collected AE data, and 57% provided information on their training and/or background. Forty-three percent specified the timing and frequency of AE collection; only 9% used a standard scale or checklist for harms collection. The majority (83%) specified that the reported harms encompassed all events (not a select sample). The majority specified the number of AEs in each study arm (90%), the number that withdrew or were lost to follow-up for each group (87%), and the number for each type of AE (78%). Few specified the type of analyses undertaken for harms data (35%).
Figures 2-4 present the risks of AEs for all pain medications and placebo (Appendix C provides detailed risk data).
The following summarizes the data by type of AE.
Gastrointestinal (GI) . Acetaminophen had a similar GI AE profile as NSAIDS. Opioids trended towards greater "other GI AEs," including constipation. Codeine monotherapy showed cumulatively more GI AEs than all other analgesics. NSAIDS and acetaminophen reported less than 10% rate of GI AEs. Opioid/nonopioid combinations had varying degrees of GI AEs associated with them; of note, oral morphine demonstrated the highest reported risk of nausea, followed by acetaminophen with codeine combination medication. Placebo-related AEs of nausea and vomiting were equal to or greater than that of some pain medications.
Central Nervous System (CNS) . Opioid monotherapy showed the highest risk of CNS AEs, with drowsiness/tiredness being noted in close to one-third of children receiving oxycodone or oral morphine and half of children receiving codeine. CNS symptoms of drowsiness and Dermatological and Pulmonary System [fig_ref] Figure 4: Risk [/fig_ref]. Opioid medications demonstrated a greater risk of dermatologic symptoms. Children receiving only codeine had almost double the risk of experiencing dermatologic manifestations compared to all other medications. Pulmonary AEs were rare. No children receiving a NSAID experienced bronchospasm.
## Registry data
We requested acetaminophen data from the U.S. Food and Drug Administration (FDA) through the U.S. FDA Adverse Event Reporting System. The report provided was a nonsearchable PDF comprising 7,523 pages with 17,806 reports of AEs for acetaminophen for all ages (i.e., adults and children). The team confirmed with the FDA that it was not possible to restrict the search results by age group or intention (i.e., nonintentional causes). Consequently, it was not feasible to include FDA data in this manuscript. Through Health Canada, we received reports of AEs among children and youth from the Canada Vigilance Adverse Reaction Online Database. We received a total of 625 reports for all the three classes of drugs. For acetaminophen, we identified 232 reports; 39 were relevant based on our inclusion criteria. The median age was 5 years (range 7 weeks to 18 years). The most common AEs were vomiting ( = 8), dermatological symptoms ( = 7), other GI symptoms ( = 5), and psychiatric effects ( = 4). The remaining AEs were reported for either one or two patients. For NSAIDS, we identified 209 reports; 69 were relevant. The median age was 7 years (range 3 months to 18 years). The most common AEs were allergic-type reactions ( = 14), headache ( = 4), vomiting ( = 4), nausea ( = 2), other GI symptoms ( = 4), dermatological ( = 3), and psychiatric effects ( = 3). For opioids, we identified 184 reports; 21 were relevant. The median age was 13 years (range 18 months to 18 years).
The most common AEs were other GI symptoms ( = 4), vomiting ( = 3), nausea ( = 3), and dermatological ( = 3).
Four deaths were reported in the Health Canada data [fig_ref] Table 2: Description of deaths reported in Health Canada's Vigilance Adverse Reaction Online Database [/fig_ref]. Case 1 was a 2-year-old who took morphine (route of administration unclear) and suffered respiratory failure. Case 2 was an 18-year-old who took acetaminophen, alone, as well as an acetaminophen/codeine combination. The route of administration for both was oral; no dosing information was provided. Case 3 was a 4-year-old who took an acetaminophen/codeine combination (oral; no dosing information). The patient was described as having a "medically important condition" but no further description was provided. Respiratory distress was plausible with codeine use. Case 4 was a 3-month-old, 4 kg child who was given acetaminophen. Reaction information included apnea, cyanosis, and respiratory depression, which are not typical AEs of acetaminophen. Drug levels were noted to be supratherapeutic but no further details were provided; a 4 kg weight likely suggests prematurity or chronic illness.
# Discussion
Each year, more than 50% of children use at least one medication [bib_ref] Medication use among children <12 years of age in the United States:..., Vernacchio [/bib_ref] , with acetaminophen and ibuprofen being the most common. There is an alarming paucity of research regarding the drug safety of common analgesics in children. Children's response to medications is often different from that of adults due to metabolism, ontogeny, and other age-related differences; as such, examination of safety data must be specific to children. Our study is one of the first to synthesize the currently available evidence and provides urgently needed safety information for clinicians treating children with acute pain in ambulatory settings.
Our study of the safety profiles of commonly used pain medications for children in the outpatient setting has demonstrated that (1) acetaminophen and ibuprofen have similar risk of nausea and vomiting, (2) opioids have the greatest risk of CNS AEs, and (3) dual therapy with a nonopioid/opioid medications results in a lower risk of AEs than opioids alone.
Our findings, specific to its use for acute, painful conditions, are consistent with previous work that has examined acetaminophen and ibuprofen in the context of its antipyretic use. A large randomized controlled trial (RCT) of ibuprofen and acetaminophen in outpatient practice showed the risk of hospitalization for gastrointestinal bleeding, renal failure, or anaphylaxis was not increased following short-term use of ibuprofen [bib_ref] An assessment of the safety of pediatric ibuprofen. A practitioner-based randomized clinical..., Lesko [/bib_ref]. A notable limitation of their study was the lack of information on less severe outcomes. A second large RCT of febrile infants (<2 years of age) receiving acetaminophen or ibuprofen demonstrated a 1.4% risk of serious AEs (defined as rate of admission for acute gastrointestinal bleeding, acute renal failure, anaphylaxis, Reye's syndrome, asthma, bronchiolitis, and vomiting/gastritis) [bib_ref] The safety of acetaminophen and ibuprofen among children younger than two years..., Lesko [/bib_ref]. A 2004 systematic review of acetaminophen and ibuprofen, consisting of 3 pain studies and 14 fever studies in children, concluded that single doses of ibuprofen and acetaminophen had similar safety profiles, when assessing "major" (e.g., abdominal pain, vomiting, and hypothermia) and "minor" (e.g., nausea, sweating, and cutaneous rash) AEs [bib_ref] Efficacy and safety of acetaminophen vs ibuprofen for treating children's pain or..., Perrott [/bib_ref]. Our study adds important and comprehensive information regarding AEs for healthy children with injury or illness-related pain. This group is medically different from febrile and infected children and, as such, merits separate consideration.
Determining the appropriate analgesic agent for a child with acute pain can be a complex decision, influenced by multiple factors including patient age and genomics, anticipated degree and duration of pain, ability to swallow pills, and preexisting medical problems. An understanding of the AE profile can aid in making an evidence-based, personalized choice that takes into account a child's individual circumstances.
We have determined that acetaminophen, traditionally considered one of the most widely studied, gastrointestinally "benign" pain medications (as compared to NSAIDs or opioids), has a similar GI side effect profile as ibuprofen, for children. As such, it would make clinical sense to use the pain medication that conferred the most clinical analgesic effect, even when GI side effects were of concern for a specific child. Limiting short-term ibuprofen use due to concern for GI AEs is not supported by current evidence. It is noteworthy that the placebo-related side effects of nausea and vomiting were equal to or greater than that of the various pain medications. This phenomenon has been previously welldescribed [bib_ref] The magnitude of nocebo effects in pain: a meta-analysis, Petersen [/bib_ref] and underscores the importance of measuring and comparing relative differences in side effect profiles, rather than only absolute values. Further, in the context of treating pain, one must be cautious to attribute all AEs to the pain medications used, as pain, itself, may plausibly have a causative role in some of the symptoms (e.g., nausea, agitation).
Oxycodone, oral morphine, and codeine monotherapy had the greatest risk of CNS side effects, with drowsiness/tiredness being noted in close to one-third of children receiving oxycodone or oral morphine and half of children receiving codeine. CNS symptoms of drowsiness and dizziness were notably higher for all opioid medications, when compared to nonopioid choices. This makes physiologic sense, as opioids have CNS depressant effects which NSAIDS and acetaminophen lack. A child who needs to avoid CNS symptoms (e.g., a school-aged child, during daytime hours) should likely be treated with ibuprofen or acetaminophen for mild-moderate pain, as they have a lower AE profile, as compared to low-mid dose opioids, a comparably potent analgesic agent [bib_ref] A randomized, controlled trial of acetaminophen, ibuprofen, and codeine for acute pain..., Clark [/bib_ref] [bib_ref] Ibuprofen provides analgesia equivalent to acetaminophencodeine in the treatment of acute pain..., Friday [/bib_ref] [bib_ref] Effectiveness of oxycodone, ibuprofen, or the combination in the initial management of..., Koller [/bib_ref]. Current evidence suggests that ibuprofen likely has superior analgesic potency for many clinical conditions including fractures, sprains, and postoperative pain [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref] [bib_ref] A randomized, controlled trial of acetaminophen, ibuprofen, and codeine for acute pain..., Clark [/bib_ref] [bib_ref] Oral administration of morphine versus ibuprofen to manage postfracture pain in children:..., Poonai [/bib_ref] [bib_ref] The magnitude of nocebo effects in pain: a meta-analysis, Petersen [/bib_ref] [bib_ref] Morphine or ibuprofen for post-tonsillectomy analgesia: a randomized trial, Kelly [/bib_ref] [bib_ref] A comparison of paracetamol, ibuprofen or their combination for pain relief following..., Gazal [/bib_ref] [bib_ref] Fracture pain relief for kids? Ibuprofen does it better, Morris [/bib_ref]. Further, knowing that its GI AE profile is comparable to acetaminophen, one would likely choose ibuprofen as first line therapy for most such pediatric acute pain conditions with mild to moderate pain. When escalating pain necessitates the use of oral opioids, dual therapy (with a nonopioid medication, such as ibuprofen or acetaminophen) appears to "protect" the child against many of the negative AEs associated with opioids, particularly CNS effects. This is likely due to decreased opioid dosage required to achieve analgesia. Opioid medications should be added to, rather than replace, these nonopioids when needed for moderate-severe pain, a strategy endorsed by the World Health Organization.
Opioid medications were associated with a greater risk of dermatologic symptoms, which is plausible given the histamine release phenomenon that can occur with their use [bib_ref] Histamine-releasing and allergenic properties of opioid analgesic drugs: resolving the two, Baldo [/bib_ref]. Children receiving only codeine had almost double the risk of experiencing dermatologic manifestations compared to all other medications. Of note, however, the confidence intervals for all opioid mono-and combination therapies overlapped significantly, with the notable exception of codeine monotherapy; children receiving only codeine had almost double the risk of experiencing dermatologic manifestations. Pulmonary side effects were rare. No children in the included studies receiving an NSAID experienced bronchospasm, a suggested risk for ibuprofen in some medical literature pertaining to fever management [bib_ref] The prevalence of ibuprofen-sensitive asthma in children: a randomized controlled bronchoprovocation challenge..., Debley [/bib_ref] [bib_ref] Ibuprofen and increased morbidity in children with asthma: fact or fiction?, Kauffman [/bib_ref].
Codeine monotherapy has cumulatively more gastrointestinal side effects than all other analgesics that were a part of this review, including other opioids. GI AEs associated with codeine monotherapy are poorly tolerated by children and may result in premature termination of therapy [bib_ref] A randomized clinical trial of ibuprofen versus acetaminophen with codeine for acute..., Drendel [/bib_ref] [bib_ref] Pain management of musculoskeletal injuries in children: current state and future directions, Ali [/bib_ref] and significant short-and long-term effects [bib_ref] Relief of pain and anxiety in pediatric patients in emergency medical systems, Fein [/bib_ref] [bib_ref] Consensus statement for the prevention and management of pain in the newborn, Anand [/bib_ref] [bib_ref] Clinical implications of unmanaged needle-insertion pain and distress in children, Kennedy [/bib_ref] due to untreated pain. This information, coupled with the recent FDA warningand other recommendations [bib_ref] Restrictions on Use of Codeine for Pain Relief in Children-CMDh Endorses PRAC..., Agency [/bib_ref] to avoid codeine use in children under 12 years of age, supports the general avoidance of codeine use in opioid-naïve children, except in the exceptional circumstance of an older child who has previously received codeine and tolerated it well.
This study has also led to some noteworthy observations regarding the use of national databases for medicationrelated research. We contacted the FDA through the Adverse Event Reporting System and received a report that was not usable for study purposes. We contacted Health Canada for data collected through the Canada Vigilance Adverse Reaction Online Database, and reports were readily available to us. While we were able to review them for relevance, it was impossible to draw conclusions regarding causality. As such, the results should be interpreted cautiously as there are several limitations: there is no denominator information to calculate incidence rates; reports are voluntarily submitted by consumers and nonphysician health professionals and lack details. We recommend that federal agencies consider modifying the manner in which medication-related AEs are reported and recorded. This would allow researchers and policy-makers to understand and use this information in a more meaningful way. Specifically, we suggest recording the treating clinicians' postulated interpretation of the cause of the AEs, creation of a searchable database, and clearer recording of the dose and frequency of the drugs used.
Given the inconsistent and often passive information collection of AEs in past studies, it is imperative that future trials of pain medications include a standardized and universal approach to the collection of AEs, in order to create a growing repository of such information, which can be resynthesized at a future date, and lead to even more robust conclusions regarding safety.
# Strengths and limitations
To our knowledge, this is the first clinician-friendly, comprehensive synthesis of AEs for the most commonly used pain medications for children. This is an important first step in addressing the lack of postmarket safety data for medications for children. Our unique style of presenting the data, based on AE rather than medication type, provides a more clinically meaningful way to interpret the results, as clinicians generally approach decision-making for patients from the perspective of "Which medication will provide the most pain relief with the least symptoms of concern for my patient?" This study has some limitations. The synthesis of information was dependent on accurate and explicit recording of AEs within the original studies. Many of the included studies were not designed to systematically collect data on adverse events. As such, our results are a reflection of what is available but may not exactly reflect the true postmarket safety of the included medications. Further, the number of studies and patients is small given the prevalent use of these agents in practice. In some cases, the rates were variable across studies with wide confidence intervals. This could be due to a number of factors (e.g., dosing); however, few studies and sparse data did not allow for subgroup analyses to delineate risks according to other factors. Data was particularly sparse for NSAIDs other than ibuprofen. Results and conclusions regarding ibuprofen should not be extrapolated to other NSAIDs that may have more potent adverse event profiles. Finally, we were not able to interpret the FDA and Health Canada data in a meaningful fashion.
# Conclusion
Based on the available evidence, ibuprofen and acetaminophen have similar reported adverse effects and notably less adverse events than opioids, for the initial treatment of acute pain in children in ambulatory settings. Dual therapy with a nonopioid/opioid combination confers a protective effect for AEs over opioids alone, suggesting that opioid medications should be added to, rather than replace, nonopioids when needed for moderate-severe pain. In order to allow for meaningful synthesis and evidence-informed care, it is imperative that future trials of pain medications include a standardized and universal approach to the collection of AEs and that ongoing national data registry monitoring be more detailed surrounding AEs (particularly causation).
[fig] Figure 1: PRISMA flow diagram of study retrieval and selection. [/fig]
[fig] Figure 4: Risk (incidence rate) of dermatological and pulmonary symptoms by medication. * Dermatological symptoms included itchiness, rash, and pruritus. Results are presented in lowest to highest risk order with placebo at the end (where data were available). [/fig]
[table] Table 1: Characteristics of included studies (in chronological order by date of publication). [/table]
[table] Table 2: Description of deaths reported in Health Canada's Vigilance Adverse Reaction Online Database. [/table]
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A Study on Solubilization of Poorly Soluble Drugs by Cyclodextrins and Micelles: Complexation and Binding Characteristics of Sulfamethoxazole and Trimethoprim
The present study is focused on the characterization of solubilization of poorly soluble drugs, that is, sulfamethoxazole (SMX) and trimethoprim (TMP) by cyclodextrins (α-, β-, and γ-CDs) and anionic surfactant sodium dodecyl sulfate (SDS). The phase solubility diagrams drawn from UV spectral measurements are of the A L type and indicate an enhancement of SMX and TMP solubility in the presence of CDs. Complex formation tendency of TMP with CDs followed the order: γ-CD > β-CD > α-C. However, the complex formation constant values, for SMX-CD system yielded the different affinity and follow the order: β-CD > γ-CD > α-CD. With taking into consideration of solubilization capacity of SDS micelles, it has been found that the solubility enhancement of TMP is much higher than that of SMX in the presence of SDS micelles. The binding constants of SMX and TMP obtained from the Benesi-Hildebrand equation are also confirmed by the estimated surface properties of SDS, employing the surface tension measurements. In order to elucidate the solubilization characteristics the surface tension measurements were also performed for nonionic surfactant Triton X-100. Polarity of the microenvironment and probable location of SMX and TMP were also discussed in the presence of various organic solvents.
# Introduction
Solubilization of poorly soluble drugs is one of the most important physicochemical properties for drug development since more than one-third of drugs are poorly water soluble or water insoluble. In order to prepare a liquid dosage formulation of these drugs, a solubilization technique is usually applied. The most commonly used techniques are pH adjustment, cosolvency, micellization, and complexation [bib_ref] Solubilization of monovalent weak electrolytes by micellization or complexation, He [/bib_ref] [bib_ref] Solubility principles and practices for parenteral drug dosage form development, Sweetana [/bib_ref] [bib_ref] Solubilization of flavopiridol by pH control combined with cosolvents, surfactants, or complexants, Li [/bib_ref]. Cyclodextrins (CDs) are widely used as complexing agents for lipophilic and amphiphilic substances and functional excipients that have gained widespread use and attention because of their ability to solubilize, and in some instances stabilize, poorly water-soluble drug candidates enabling both oral and parenteral formulation [bib_ref] Cyclodextrins-enabling excipients: their present and future use in pharmaceuticals, Thompson [/bib_ref] [bib_ref] Cyclodextrins in drug delivery: an updated review, Challa [/bib_ref] [bib_ref] Cyclodextrin-based pharmaceutics: past, present and future, Davis [/bib_ref] [bib_ref] Role of cyclodextrins in improving oral drug delivery, Loftsson [/bib_ref] [bib_ref] Cyclodextrins in topical drug formulations: theory and practice, Loftsson [/bib_ref]. CD molecules are peculiar cyclic oligomers of α-D-glucose; their shapes resemble truncated cones with primary and secondary hydroxyl groups located around their narrower and wider rim. The mostly used native cyclodextrins consist of 6, 7, and 8 glucose units, and, according to the number of monomers in the macrocycle, they are named as α, β, and γ-cyclodextrin, respectively. These molecules have a hydrophobic inner cavity, while the large number of hydroxyl groups on the outer surface makes them water soluble. Due to this special molecular structure, cyclodextrins are capable of forming inclusion complexes with many drugs and other compounds by taking up a lipophilic guest molecule (or its hydrophobic part) of the appropriate size into the cavity [bib_ref] Cyclodextrins: their future in drug formulation and delivery, Stella [/bib_ref] [bib_ref] Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization, Loftsson [/bib_ref]. The chemical structures of CDs are shown in [fig_ref] Figure 1: Chemical structures of α-CD, β-CD, γ-CD, SDS, TMP, and SMX [/fig_ref]. The ability of the CDs to form inclusion complexes with a variety of organic compounds is based on their ability to provide a hydrophobic cavity in aqueous solution for a hydrophobic guest molecule or hydrophobic moieties in the guest molecule. Moreover CDs have been used as a model [bib_ref] Solubility principles and practices for parenteral drug dosage form development, Sweetana [/bib_ref] The Scientific World Journal for proteins and enzymes. The pharmaceutical interest in CDs extends to enhance the solubility, chemical stability, and bioavailability of poorly soluble drugs to reduce the toxicity and control the rate of release of some drugs [bib_ref] Study on inclusion complex of cyclodextrin with methyl xanthine derivatives by fluorimetry, Wei [/bib_ref]. Complexation of water-insoluble or poorly soluble drugs with native or modified cyclodextrins has been studied by a large number of workers [bib_ref] Ternary systems of naproxen with hydroxypropyl-β-cyclodextrin and aminoacids, Mura [/bib_ref] [bib_ref] Co-solubilization of poorly soluble drugs by micellization and complexation, Rao [/bib_ref] [bib_ref] Complexation of morin with three kinds of cyclodextrin. A thermodynamic and reactivity..., Jullian [/bib_ref] [bib_ref] Comparative study of the inclusion complexation of pizotifen and ketotifen with native..., Omari [/bib_ref] , and some of the reports generated on this topic have been reviewed [bib_ref] Cyclodextrins and their uses: a review, Martin Del Vale [/bib_ref] [bib_ref] Cyclodextrins and their pharmaceutical applications, Loftsson [/bib_ref] [bib_ref] Cyclodextrins and the biopharmaceutics classification system of drugs, Loftsson [/bib_ref] [bib_ref] Pharmaceutical applications of cyclodextrins: basic science and product development, Loftsson [/bib_ref].
Micellar solubilization is also a powerful alternative for dissolving hydrophobic drugs in aqueous environments. Because of its amphiphilic nature, micelles are known to play a vital role in many processes of interest in both fundamental and applied sciences. Micellar system can solubilize poorly soluble drugs increasing their bioavailability, and they can be used as a model system for biomembrane, as well as drug carriers in numerous drug delivery and drug targeting systems. Micelles are known to have an anisotropic water distribution within their structure. In other words, the water concentration decreases from the surface towards the core of the micelle, with a completely hydrophobic (water-excluded) core. Consequently, the spatial position of a solubilized drug in a micelle will depend on its polarity: nonpolar molecules will be solubilized in the micellar core, and substances with intermediate polarity will be distributed along the surfactant molecules in certain intermediate positions. Surfactants such as sodium dodecyl sulfate (SDS) are used as excipients and usually added to the formulation to facilitate the preparation, patient acceptability, and functioning of the dosage form [bib_ref] Micellar solubilization of drugs, Rangel-Yagui [/bib_ref]. The SDS molecule contains a 12-carbon-saturated alkyl chain bound to a negatively charged sulfonate head (OSO 3 − ), and therefore, it is structurally comparable to many biosurfactants for which it is also used as a model. In aqueous medium, SDS [fig_ref] Figure 1: Chemical structures of α-CD, β-CD, γ-CD, SDS, TMP, and SMX [/fig_ref] forms micelles which mimic biological membrane system as a model of globular protein and capable of solubilizing hydrophobic molecules resulting in a high local concentration. From this point of view, the interactions of drug with surfactants provide an insight into more complex biological processes such as the passage of drugs through cell membranes. The fundamental event in the interaction of drugs with biological tissues at the molecular level is their binding to membranes. A number of studies have been dedicated to drug/surfactant interactions [bib_ref] Interaction of chlorpromazine and trifluoperazine with anionic sodium dodecyl sulfate (SDS) micelles:..., Caetano [/bib_ref] [bib_ref] Study of valsartan interaction with micelles as a model system for biomembranes, Čudina [/bib_ref] [bib_ref] Solution behaviour of rivanol in micellar environments, Göktürk [/bib_ref] [bib_ref] Interaction of epirubicin HCl with surfactants: effect of NaCl and glucose, Erdinç [/bib_ref] [bib_ref] Spectral studies on the molecular interaction of anticancer drug mitoxantrone with CTAB..., Enache [/bib_ref]. This is an important issue because it relates to the mechanism of drug action. Therefore, the study of surfactant micelles and their role in pharmacy is of paramount importance, especially with respect to their ability of solubilizing hydrophobic drugs [bib_ref] Co-solubilization of poorly soluble drugs by micellization and complexation, Rao [/bib_ref] [bib_ref] Relationship between Polysorbate 80 solubilization descriptors and octanol-water partition coefficients of drugs, Alvarez-Núñez [/bib_ref] [bib_ref] Surfactants in pharmaceutical products and systems, Corrigan [/bib_ref] [bib_ref] The effects of surfactants on the dissolution profiles of poorly water-soluble acidic..., Park [/bib_ref] [bib_ref] Combined effect of SLS and (SBE)7M-β-CD on the solubilization of NSC-639829, Yang [/bib_ref] [bib_ref] Solubility of nonsteroidal anti-inflammatory drugs (NSAIDs) in aqueous solutions of non-ionic surfactants, Ullah [/bib_ref]. Despite the large number of articles devoted to the interaction of drugs by surfactants or cyclodextrins, the studies that combine spectroscopy with surface tension measurements under identical experimental conditions are still missing. The aim of this work is to improve the effects of different types of excipients such as surfactant and cyclodextrins used in pharmaceutical applications on the solubilization and interactions of poorly soluble drugs in aqueous media. To more precisely analyze and evaluate interactions of drugs in different media, surface tension data are compared for the SDS and CDs in the presence of SMX and TMP in aqueous media. The dependence of SMX and TMP absorption spectra on the SDS and CDs concentration leads to interesting correlations with the surface tension measurements in terms of type of solubilization. For example, the solubilization of drug may not only involve incorporation into the micellar interior or cyclodextrins cavity but may also be substantially due to adsorption at the micelle-water interface or outer surface of cyclodextrins. Therefore, in this work, poorly soluble drugs sulfamethoxazole (SMX) and trimethoprim (TMP) were chosen as model to study their solubilization characteristics in the presence of SDS micelles and CDs (α, β, and γ-CD) in aqueous media at 298 K. The chemical structures of SMX and TMP are shown in [fig_ref] Figure 1: Chemical structures of α-CD, β-CD, γ-CD, SDS, TMP, and SMX [/fig_ref]. SMX and TMP possess poor solubility in water; that is, S SMX = 600 mg L −1 (≈ 2.368 × 10 −3 M) and S TMP = 400 mg L −1 (≈ 1.377 × 10 −3 M). The combination antibacterial product containing SMX and TMP in a fixed 5 : 1 ratio is well known as Co-trimoxazole. Keeping in mind the scope of the study on solubilization of poorly soluble drugs SMX and TMP by SDS micelles and α-, β-, and γ-CDs, the present study is divided into four sections. In the first section, the interaction of fixed concentration of SMX and TMP (4.0 × 10 −5 M) with SDS and CDs is discussed using the Benesi-Hildebrand equation to determine the binding constants of SMX and TMP in different environments. The concentration of 4.0 × 10 −5 M was chosen as a common concentration to study interaction of two drugs with SDS and CDs to compare their binding tendency. At this concentration both SMX and TMP are freely soluble in water. The changes in surface tension of SDS and CDs in the presence of SMX and TMP are addressed in the second section to determine the surface excess of SDS by using the Gibbs adsorption equation. In the third section, the performance and effect of SDS micelles and CDs on the solubilization of SMX and TMP at their water insoluble concentrations range are compared and discussed using phase solubility diagrams. Finally, in order to gain further insight about the probable localization of poorly soluble drugs into the SDS and CDs, it has been monitored the variation of SMX and TMP absorption spectra in the presence of various organic solvents. Effect of medium polarity on the solubilization mechanism is also discussed in the present study.
# Material and methods
# Materials.
All the experiments were performed with analytical-grade chemicals and solvents. Trimethoprim [2, 4-diamino-5-(3' , 4' , 5'-trimethoxybenzyl pyrimidine)] (C 14 H 18 N 4 O 3 ) and sulfomethoxazole [4-amino-N-(5-methylisoxazol-3-yl)-benzenesulfonamide] (C 10 H 11 N 3 O 3 S) were purchased from Sigma (Germany), respectively. α-, β-, and γ-cyclodextrins were obtained from Sigma-Aldrich. Sodium dodecyl sulfate and Triton X-100 were Sigma (Germany) products. Doubly distilled conductivity water was used for solution preparation.
## Surface tension measurements.
Surface tension of aqueous solutions of CDs and surfactant in the absence and presence of fixed concentrations of SMX and TMP were measured at 298 K by computer-controlled Wilhelmy plate method in order to determine maximum surface excess concentration of surfactants and CDs in the presence of each drug.
## Solubility measurements.
Phase-solubility studies of SMX and TMP in aqueous solutions of CDs and SDS were carried out according to the Higuchi-Connors procedure [bib_ref] Phase-solubility techniques, Higuchi [/bib_ref]. Various amounts of SDS and CDs were generally dissolved in distilled water, and excess amounts of SMX and TMP were loaded in glass vials. The vials were shaken in a temperature-controlled room at 298 K for 24 h to achieve the equilibrium. Appropriate aliquots were then withdrawn and filtered appropriately diluted with distilled water, and the total concentration of the drugs in the filtrate was analyzed by UV absorbance spectrum. The absorbances at 274 nm for TMP and 265 nm for SMX were measured, in order to determine the concentration of the dissolved drugs.
## 4
The Scientific World Journal
# Theoretical background
## Determination of binding constants of drugs with cds and sds micelles.
The binding constants of drugs to micelle and/or cyclodextrins can be quantitatively determined using the Benesi-Hildebrand equation [bib_ref] A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons, Benesi [/bib_ref] in the following modified form [bib_ref] Solution behaviour of rivanol in micellar environments, Göktürk [/bib_ref] [bib_ref] Experimental and theoretical studies on the inclusion complexation of β-cyclodextrin with phenothiazine..., Guo [/bib_ref] [bib_ref] Spectral studies of safranin-O in different surfactant solutions, Göktürk [/bib_ref] :
[formula] [D]l A − A 0 = 1 Δε + 1 K b [C](Δε) ,( 1 ) [/formula]
## Determination of surface excess amount and minimum
Area per Molecule. On the basis of a plot of the surface tension, γ, as a function of the equilibrium concentrations of SDS and CDs in the presence of fixed concentrations of SMX and TMP, the changes of surface properties, namely, maximum surface excess concentration (Γ max ) and minimum area per molecule (A min ), can be determined by application of the Gibbs adsorption isotherm:
[formula] Γ max = − 1 RT dγ d ln C ,( 2 ) [/formula]
where R is the gas constant, T is the temperature in Kelvin, and C is the concentration of SDS or CD. Changes in minimum surface area per molecule can be obtained from the maximum surface excess concentration at the air-solution interface, A (Å 2 molecule −1 ), and were evaluated from
[formula] A = 1 N A Γ max ,( 3 ) [/formula]
where N A is the Avogadro constant.
## Drug solubilization using cyclodextrins.
Phase solubility diagrams were analyzed to obtain estimates of the complex formation constants of soluble complexes described earlier [bib_ref] Phase-solubility techniques, Higuchi [/bib_ref]. The linear part of the experimental solubility isotherms suggests that in these equilibria CDs-SMX or CDs-TMP associates of 1 : 1 molar ratio are dominantly formed. The K 1:1 stability constants for associates of 1 : 1 molar ratio have been calculated using the approach offered by Iga et al. [bib_ref] New direct calculation of K(1:1) and K(1:2) complexation constants using solubility method, Iga [/bib_ref] :
[formula] K 1:1 = slope S 0 1 − slope ,( 4 ) [/formula]
where S 0 is the solubility of the drugs at 298 K in the absence of cyclodextrins and slope means the corresponding slope of phase-solubility diagrams, that is, the slope of the drug molar concentration versus CDs molar concentration graph.
## Drug solubilization using surfactant.
The solubility of a drug in the presence of a surfactant alone can be expressed by considering the two-phase model wherein it is assumed that the micellization is seen only above the critical micellar concentration (CMC) and that the monomer concentration is constant irrespective of the total surfactant concentration above the CMC. For such a system, the total solubility of the drug in the presence of the surfactant (C S ) is given by the following equation [bib_ref] Combined effect of SLS and (SBE)7M-β-CD on the solubilization of NSC-639829, Yang [/bib_ref] [bib_ref] Solubilization behavior of poorly soluble drugs with combined use of Gelucire 44/14..., Kawakami [/bib_ref] :
[formula] S T = K M (C S − C CMC ) + S 0 .(5) [/formula]
Here, K M , C S , and C CMC are the solubilizing capacity of micelles, surfactant concentration, and critical micelle concentration, respectively. Solubilizing capacity of micelles (K M ) determined from the slope of the solubilization curve will be expressed by M −1 in this paper.
# Results and discussion
## Interactions of smx and tmp with sds, α-, β-, and
γ-CDs. SMX is an acidic compound, and the spectrum undergoes a hypsochromic shift with increasing pH, corresponding to the loss of a proton from the -SO 2 -NH-group, implying the existence of electron-withdrawing groups in the SMX anion [bib_ref] Photosensitizing activity of the anti-bacterial drugs sulfamethoxazole and trimethoprim, Zhou [/bib_ref] [bib_ref] Physicochemical studies on the binding of chemicals with proteins. I. The binding..., Nakagaki [/bib_ref]. At pH values above 5.6, SMX exists predominantly as anionic species; at pH values between 1.7 and 5.6, SMX is uncharged, while at, pH values below 1.7, it is positively charged [bib_ref] Pharmaceutical retention mechanisms by nanofiltration membranes, Nghiem [/bib_ref]. In our experimental conditions, the medium pH is above 5.7 where more stable SMX anions exist. TMP is a basic compound, and a proton is associated with the NH 2 substituents in acidic solutions, but the spectrum shows a bathochromic shift as the pH increased [bib_ref] Photosensitizing activity of the anti-bacterial drugs sulfamethoxazole and trimethoprim, Zhou [/bib_ref]. The basic drug TMP and acidic drug SMX having the structures shown in [fig_ref] Figure 1: Chemical structures of α-CD, β-CD, γ-CD, SDS, TMP, and SMX [/fig_ref] between cationic drug TMP and anionic SDS molecules due to the electrostatic interactions. The increase in absorbance values with increasing surfactant concentrations above the CMC indicates association of TMP molecules with SDS micelles. As more drug molecules are incorporated to micelles, the absorbance values of λ max reaches a limiting value and becomes almost constant; that is, the amount of solubilized TMP reaches saturation. The CMC value of SDS in the presence of TMP was determined spectrophotometrically and employed surface tension measurements as described previously [bib_ref] Spectral studies of safranin-O in different surfactant solutions, Göktürk [/bib_ref]. A good agreement was found between two methods, and the CMC of SDS was determined to be 4 mM in the presence of 4.0 × 10 −5 M TMP. The absorption spectra of SMX in the presence of various concentrations of SDS are shown in. At the concentrations below and above the CMC (8.0 mM), no spectral changes were observed, and the absorbance of SMX remained almost constant in the presence of SDS. As seen inthere is a slight increase in absorbance, that is, only a very weak interaction occurred between SMX and SDS micelles due to the electrostatic repulsion. Under working conditions, SMX bears a net negative charge and SMX anion is dominant form. The spectral behaviour of [fig_ref] Table 1: K 1 [/fig_ref]. It has been found that the interaction between cationic TMP and anionic SDS micelles is the strongest because of the opposite charge of the components as expected. The lack of interaction of SMX with SDS supports this expectation, and the binding constant of SMX in the case of SDS micelles could not be calculated. The K b values of SMX and TMP presented in [fig_ref] Table 1: K 1 [/fig_ref] for the interaction of three types of CDs indicated the different affinities which follow the order as β-CD > γ-CD > α-CD in the case of SMX and γ-CD > β-CD > α-CD in the case of TMP. Before discussing on the different binding tendency of drugs to CDs as well as SDS micelles, it is worth mentioning the data obtained from surface tension measurements. In order to get a better insight in the mechanism of interaction of SMX and TMP with SDS and CDs, a thorough study of the surface tension change of SDS and CDs was performed in the presence of 4.0 × 10 −5 M SMX and TMP. In this study, once the interfacial behaviours of SDS and CDs were analyzed in the absence of the drugs. α-, β-, and γ-CD did not show [bib_ref] Cyclodextrins and their pharmaceutical applications, Loftsson [/bib_ref] [bib_ref] Pharmaceutical applications of methylated cyclodextrin derivatives, Uekama [/bib_ref]. Since the same behaviour was observed for the surface tension measurements at various concentrations of SDS and CDs in the presence of 4.0 × 10 −5 M SMX and TMP, only the results for TMP are shown inas a representative figure. As seen in, no variation of the surface tension was observed for the CDs studied even with the increase in concentration of CD; on the contrary, the acting of SDS is different. The influences of SMX and TMP on surface properties of SDS are shown in, and the Gibbs equation, the quantities of adsorbing SDS and the apparent area of adsorbed SDS were determined in the absence and presence of SMX and TMP. The slope of surface tension (γ) versus ln concentration is proportional to the surface excess (Γ max ) as apparent from (2).summaries the corresponding Γ max and A min values. As seen inthe surface tension values of SDS in the presence of TMP are lower than those in the absence of TMP. On the contrary, the surface tension values of SDS in the presence of SMX is little higher than those in the absence of SMX. The data inshow that Γ max value of SDS increased and A min decreased in the presence of SMX while Γ max value of SDS decreased and A min increased in the presence of TMP. These findings support that electrostatic forces play an important role on the interaction between the drugs and SDS. The presence of TMP decreases the repulsion among head groups of SDS, and more TMP molecules can be adsorbed at the interface which is also confirmed by the higher value of A min ; that is, TMP increased the adsorption of SDS at the interface. The higher the Γ max value and the lower the A min value for SDS in the presence of SMX also explains that the electrostatic repulsion forces take place in the interaction of anionic SMX and anionic SDS molecules compared to a pure SDS solutions. In order to gain better insight into the phenomena taking place at the interface, the surface tension measurements were also performed for nonionic surfactant Tritonx-100 (TX-100) in the presence of SMX and TMP at 298 K. The variation of surface tension as a function of TX-100 concentration in the absence and presence of 4.0 × 10 −5 M SMX and TMP is also illustrated in, and the data obtained from surface tension measurements presented inin order to compare with the behaviour of SMX and TMP in the presence of ionic and nonionic micellar environments. It is also clearly seen that the surface tension values of TX-100 did not vary significantly as in the case of SDS with the addition of SMX and TMP; that is, the smaller increase of surface tension values of TX-100 was observed. Γ max value of TX-100 increased, and A min decreased in the presence of TMP as seen in. However, both Γ max and A min values of TX-100 remained almost the same as in the presence of SMX. It can be said that electrostatic forces take place in binding of SMX and TMP onto anionic SDS micelles, whereas hydrophobic interaction plays the main role in binding of SMX and TMP to nonionic TX-100 micelles. There is a reasonable agreement with the value obtained from spectrophotometric measurements due to the lack of interaction between SDS and SMX; that is, electrostatic repulsion forces overcome hydrophobic interaction.shows the solubility change of poorly soluble model drugs SMX and TMP as a function of SDS concentration. As seen in, the plots of drug solubility against surfactant concentrations can be divided into two straight lines; one for below the CMC (8 mM) and the other above it. Above the CMC solubility of drugs increased with the increase in SDS concentration and good linearity was observed between the surfactant concentration and the solubility. The solubilization capacity K M was determined to be 71.30 and 326.95 M −1 for SMX and TMP, respectively. These results obtained from (4) and given in [fig_ref] Table 1: K 1 [/fig_ref] are compatible with the results of interaction studies discussed in the previous section. The solubility of SMX and TMP in the aqueous SDS solutions increased linearly with increase of the SDS concentration. Solubilization effect of SDS on TMP is stronger than that of SMX. This behaviour is a consequence of the electrostatic interactions between the negatively charged surfactant SDS and cationic drug TMP. As indicated in the interaction studies, the addition of TMP caused a decrease in the repulsive forces between the head groups of SDS molecules, contributing to the micellization process, and thus the CMC value of SDS decreased from 8 mM to 4 mM. This is also supported by the influence of SMX and TMP on the surface properties of SDS based on surface tension measurements.
## Micellar solubilization of smx and tmp by sds and cds.
The Scientific World Journal 9and 7(c) show the solubilization data of two model drugs SMX and TMP as a function of the CD concentrations, respectively. The solubility profile showed an increase in their solubility when the concentrations of CDs increase. The 1 : 1 drug/CD complexes are typical of association where a single drug molecule is in the cavity of a CD, with a stability constant (K 1:1 ) for the equilibrium between the free and associated species. As seen inand 7(c), the solubility of SMX and TMP increased linearly as a function of CD concentration, and phase solubility studies revealed increase in solubility of the drug upon cyclodextrin addition, showing A L type of graph with slope less than one indicating formation of 1 : 1 stochiometry inclusion complex. The corresponding stability (or complex formation) constants are listed in [bib_ref] Pharmaceutical retention mechanisms by nanofiltration membranes, Nghiem [/bib_ref]. It is clearly seen that the stability of the inclusion complexes is in the different order for SMX and TMP. The stability constant K 1:1 values for the TMP/CD system followed the order γ-CD > β-CD > α-CD. However, the different trend was observed in the case of SMX/CD system and followed the order β-CD > γ-CD > α-CD. Host complexation is primarily determined by the tightness of the fit, that is, by the size and shape matching between the guest and the CD cavity. CDs are designated α, β, or γ corresponding to 6, 7, or 8 glucopyranose units, with cavity diameters of 4.7-5.3, 6.0-6.5, and 7.5-8.3Å, respectively [bib_ref] Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation, Irie [/bib_ref]. Based on these dimensions α-CD can typically complex low-molecularweight molecules or compounds with aliphatic side chains, β-CD will complex aromatics and heterocycles and γ-CD can accommodate larger molecules such as macrocycles and steroids. If the guest is the imperfect size, it will not fit properly into the cyclodextrins cavity and leads to a decrease in the host-guest interactions as well as the association constants [bib_ref] Influence of the cyclodextrin size cavity in the complexation of tetrahydroharmane, Velasco [/bib_ref]. The different ability of CDs to associate with TMP seems to be related with the fitting of the substrate into the CD cavity. The TMP molecule is approximately 7.39Å in width and 11.972Å in length [bib_ref] Removal of emerging contaminants of industrial origin by NF/RO-a pilot scale study, Dolar [/bib_ref]. Consequently, the internal diameters of the α-CD and β-CD cavities are too small for inclusion of the TMP molecules. The internal diameter of γ-CD cavity is suitable for accommodation of the TMP molecule. The relatively high K 1:1 value for complexation of TMP with γ-CD indicates the existence of a better geometric fit than that with the αor β-CD cavities. However, the different trend was observed in the case of SMX for the inclusion complex formation based on the dimensions of CDs. As data in [fig_ref] Table 1: K 1 [/fig_ref] show, α-CD behaves similarly to γ-CD, but its ability to associate with SMX is relatively smaller.
## Complexation of smx and tmp by cds.
One of the most interesting results of the present study is the lack of complexation of SMX with γ-CD. However, the capability of β-CD to form complexes with SMX seems to be stronger than the inclusion complex formation tendency of SMX with αand γ-CD. The reported molecular dimension of the SMX molecule is approximately 5.6Å in width and 12.9Å in length according to Comerton et al. [bib_ref] The rejection of endocrine disrupting and pharmaceutically active compounds by NF and..., Comerton [/bib_ref].
The weak inclusion complex formation of SMX with α-CD can be explained with the smaller internal diameter cavity of α-CD. Thus, SMX molecule should be tightly packed into the γ-CD cavity. Although prediction of compound solubilization by CDs continues to be highly empirical, various historical observations permit several general statements. The formation of an inclusion complex with cyclodextrins is also classically caused by interactions such as hydrogen bonding with the OH groups at the periphery of the cavity, van der Waals interactions, and hydrophobic effects [bib_ref] (S)-2-Hydroxypropyl-β-cyclodextrin, a new chiral stationary phase for reversed-phase liquid chromatography, Stalcup [/bib_ref]. Hydrophobic interactions should play an important role for the stronger inclusion complex formation of SMX with β-CD as compared with that of αand γ-CD. When we compare the aqueous solubility of α-(14.5%, w/v), β-(1.85%, w/v), and γ-CD (23.2%, w/v) in water [bib_ref] Cyclodextrins and the biopharmaceutics classification system of drugs, Loftsson [/bib_ref] , it can be resulted that γ-CD is the most soluble whereas β-CD is the least soluble in water; that is, β-CD is more hydrophobic than γ-CD. The interaction studies performed for the fixed concentration of SMX supports the behaviour of SMX in the presence of β-CD. The maximum absorption spectra of SMX shifted from 265 nm to 270 nm in the presence of β-CD while the maximum absorption spectra of SMX did not change significantly in the presence of αand γ-CD. Comparison of different shifts in the presence of these organic solvents indicates the order of the polarity of the media follow the trend; water > Gly > PG > EG > EOH. This follows the same increasing order of λ max of TMP. The λ max of TMP at 274 nm in water shifted to 273, 281, 289, and 290 nm in the presence of Gly, PG, EG, and EOH, respectively. Our data showed that, upon going from the more hydrophilic to the more hydrophobic environment, TMP underwent a red shift in the absorption maximum. The red shift is also a clear indication of the tendency of TMP to less polar environment. The λ max values given in [fig_ref] Table 1: K 1 [/fig_ref] support these findings in the presence of SDS and CDs. The parallelism between polarity of the medium and higher micellar binding constant demonstrates significantly that hydrophobic interactions play an important role in solubilization of TMP by SDS micelles beside electrostatic interactions. The small blue shift in the presence of Gly also supports the behaviour of TMP in the presence of SDS; that is, Gly is more hydrophilic than the other solvents studied. It can be said that TMP is located in the interfacial region of SDS micelles while in the case of CDs TMP prefers to locate in the hydrophobic region depending on the cavity size. As seen in [fig_ref] Table 1: K 1 [/fig_ref] , in contrast with TMP, the absorption maxima of SMX is red shifted by 5 nm in β-CD, whereas blue shifted 1-3 nm in αand γ-CD compared with those in aqueous medium. These observed shifts show that SMX is transferred from a highly polar phase (H 2 O) to a less polar phase. This is supported with the red shift (from 265 to 272 nm) obtained when Gly, PG, EG, and EOH were used as solvent instead of water. As seen inno significant shifts of the λ max of SMX were observed with the change of medium polarity. The higher binding constant of SMX determined in the presence of β-CD confirms the more binding tendency of SMX due to more hydrophobic character of β-CD. The inclusion complex formation of SMX with β-CD might be explained with only a part of the SMX molecule accommodated in the β-CD cavity and leaving the rest of the molecule protruding into water taking into consideration molecular geometry. The spectral behaviour of SMX in the presence of organic solvents indicates that SMX is willing to transfer more hydrophobic region. This is confirmed that the electrostatic repulsion forces are dominant on the interaction between SMX and SDS in aqueous media; that is, no significant shift was observed in the spectrum of SMX in the presence of SDS. The surface excess and molecular occupied area obtained from surface tension measurements also confirm these results (see S1 and S2 in Supplementary Material available online at doi:10.1100/2012/718791).
## Probable location of tmp and smx: effect of medium
# Conclusions
In this work the solubilizing ability of three different CDs (α-, β-, and γ-CD) and SDS were compared for two poorly soluble drugs, that is, SMX and TMP based on spectrophotometric and surface tension measurements. An extensive study on the solubilization characteristics of SMX and TMP in different environmental conditions has been presented at 298 K. To obtain more information about the nature and properties of the solubilization of the drugs by different structures for the fixed concentration of the drugs, as a model system for biomembranes, interactions of SMX and TMP with SDS and CDs were studied separately. The aqueous solubility of poorly soluble drugs can considerably be improved by micellar solubilization. The increases of the aqueous solubility of SMX and TMP in the presence of α-, β-, and γ-CD indicate that they are able to form inclusion complexes with different manner. Stability or equilibrium constants of the drug-CD complexes are important since this is an index of changes in physicochemical properties of a compound upon inclusion. Solubility of drugs in various media and the binding constants of drugs into CDs and SDS micelles were found to depend on drug characteristics as well. The solubilization of TMP with different CDs revealed that complex stability is affected by the geometric fit with CD. However, the higher tendency of SMX to complex with β-CD in aqueous solution is governed by the hydrophobic effect. Changes in surface properties of surfactants determined by surface tension measurements in the presence of drugs are in good agreement with the binding constants determined spectrophotometrically. Therefore, it is important to study the change in surface properties to understand the action mechanism of the drug especially in the case of surfactants. Another interesting point is that the different tendency was observed in the absorption maxima of SMX and TMP in the presence of various organic solvents. The λ max values of SMX and TMP in the presence of CDs or SDS and organic solvents are in conformity with the localization of SMX and TMP for complexation and solubilization manner. As a conclusion, the presented results should be of special interest in the pharmaceutical researches in drug delivery systems.
[fig] 2. 2: Instruments. The spectroscopic measurements were performed on a double-beam UV-Vis spectrophotometer (Shimadzu UV-2100 S) equipped with a matched pair [/fig]
[fig] Figure 1: Chemical structures of α-CD, β-CD, γ-CD, SDS, TMP, and SMX. of 10.0 mm path length in a water-jacketed thermostatic cell holder. [/fig]
[fig] 2. 3 1: Spectrophotometric Measurements. Changes in absorbance of SMX at 265 nm and TMP at 274 nm were observed and determined linear between the concentrations of 1.0 × 10 −5 -1.0 × 10 −4 M (R 2 = 0.9989). Absorption spectra of SMX and TMP in aqueous solution with a varying wide concentration range of SDS, α-, β-, and γ-CDs have been recorded at 298 K (±0.1), keeping the concentration of drugs (4.0 × 10 −5 M) fixed in each one. Binding constants were determined from the absorbances of a series of solutions containing a fixed concentration of drugs and increasing concentration of SDS, α-, β-, and γ-CDs. [/fig]
[fig] Figure 2: exhibit the maximum absorption bands at 274 and 265 nm, respectively. The molar extinction coefficients (ε 0 ) of TMP at 274 nm and SMX at 265 nm were calculated as 6.21 × 10 3 and 15.17 × 10 3 mol −1 dm 3 cm −1 in the concentration range of 1.0 × 10 −5 -1.0 × 10 −4 mol dm −3 of the drugs at 298 K, respectively. The linear relation between absorbance and drug concentration (R 2 : 0.9998) indicates the validity of Beer's Law. The effect of anionic surfactant SDS, at the concentrations varied from 1.0 × 10 −5 to 4.0 × 10 −2 M (from premicellar to micellar region), on the absorption spectrum of SMX and TMP at fixed concentration of 4.0 × 10 −5 M was studied. The change of absorption spectrum of TMP in the presence of various concentration of SDS was shown in Figure 2(a). TMP exists in cationic form in aqueous solution. As seen in Figure 2(a) the absorbance decreased with a small blue shift with the increase in SDS concentration up to 4 mM. It was observed that the increase in absorbance with the increase in SDS concentrations above the CMC, λ max of TMP at 274 nm shifted to 270 nm. This observed blue shift and the increase in absorbance are a clear indication that TMP incorporate to SDS micelles. Figure 2(b) shows the relation between the absorbance of TMP and the various concentrations of SDS. A decrease in the absorbance observed at the concentrations below the CMC indicates the molecular (a) Visible absorption spectra of TMP (4.0 × 10 −5 M) at various concentrations of SDS at 298 K: (1) no SDS, (2) 2 mM, (3) 4 mM, (4) 8 mM, (5) 10 mM, (6) 20 mM SDS. (b) The absorbance change of 4.0 × 10 −5 M TMP (below and above the CMC) with the concentration of SDS. [/fig]
[fig] Figure 3: (a) Visible absorption spectra of SMX (4.0 × 10 −5 M) at various concentrations of SDS at 298 K: (1) no SDS, (2) 2 mM, (3) 4 mM, (4) 8 mM, (5) 10 mM, (6) 20 mM SDS. (b) The absorbance change of 4.0 × 10 −5 M SMX (below and above the CMC) with the concentration of SDS. 4.0 × 10 −5 M TMP and SMX in aqueous solutions containing different concentration of CDs were studied. Figures 4(a) and 4(b) show the relation between the absorbance of TMP and SMX with the various concentrations of CDs, respectively. The increase in absorbance values with increasing CDs concentrations indicates association of drugs with CDs. Changes of absorption spectra observed for SMX and TMP in the presence of CDs were analogical, so in the present work only the figures are shown for the most effective CD on the complexation tendency of SMX and TMP (Figures 5(a) and5(b)). A similar behavior, that is, a progressive enhancement in absorbance with the increase of CD concentration, was observed for all CDs.Figure 5(a)shows UV spectra of TMP in the presence of various concentrations of γ-CD. No shift was observed in the λ max of TMP at 274 nm when complexes are formed with α-, β-, and γ-CD. A similar behavior, that is, a progressive enhancement in absorbance of SMX, was also observed for SMX in the presence of various concentrations of β-CD and shown inFigure 5(b). From the spectral measurements, the binding constant values (K b ) related to the extent of drug-SDS and/or drug-CDs interactions were calculated using the Benesi-Hildebrand equation and shown in [/fig]
[fig] Figure 4: (a) The absorbance change of 4.0 × 10 −5 M TMP with the concentrations of CDs. (b) The absorbance change of 4.0 × 10 −5 M SMX with the concentrations of CDs. [/fig]
[fig] Figure 5: (a) Visible absorption spectra of TMP (4.0 × 10 −5 M) at various concentrations of γ-CD at 298 K: (1) no γ-CD, (2) 1 mM, (3) 2 mM, (4) 4 mM, (5) 6 mM, (6) 8 mM, (7) 10 mM γ-CD. (b) Visible absorption spectra of SMX (4.0×10 −5 M) at various concentrations of β-CD at 298 K: ↑ (1) no γ-CD, mM γ-CD. any surface property: the measured surface tensions of their solutions varied in the range of 71.6-72.0 mN/m only. As already reported in the literature native α-, β-, and γ-CD were not surface active [/fig]
[fig] Figure 6: (a) Surface tension results for SDS and CDs in the presence of 4.0×10 −5 M TMP at 298 K. (b) Surface tension versus ln (concentration) plots for SDS in the absence ( ) and presence of 4.0 × 10 −5 M SMX ( ) and TMP(+) at 298 K. (c) Surface tension versus ln (concentration) plots for TX100 in the absence ( ) and presence of 4.0 × 10 −5 M SMX ( ) and TMP(+) at 298 K. [/fig]
[fig] Figure 7: (a) Solubility curves of SMX and TMP as a function of SDS concentration at 298 K. (b) Phase solubility diagrams of SMX-CD systems at 298 K. (c) Phase solubility diagrams of TMP-CD systems at 298 K. [/fig]
[fig] Figure 8: (a) Variation of visible absorption spectra of SMX (4.0 × 10 −5 M) in the absence and presence of various solvents at 298 K: (1) in water, (2) Gly, (3) PG, (4) EOH, (5) EG. (b) Variation of visible absorption spectra of TMP (4.0 × 10 −5 M) in the absence and presence of various solvents at 298 K: (1) in water, (2) Gly, (3) PG, (4) EOH, (5) EG. [/fig]
[table] Table 1: K 1:1 and K M values for SMX and TMP in the presence of SDS and CDs at 298 K. K b and λ max (nm) values for fixed concentration of SMX and TMP (4.0 × 10 −5 M) obtained in SDS and CDs. [/table]
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Interprofessional education to implement patient falls education in hospitals: Lessons learned
Aim:The aim of this study was to design, deliver and evaluate an interprofessional education programme for healthcare professionals on how to implement a modified version of the safe recovery programme to prevent falls in hospitalized patients.Design: Mixed methods design incorporating pre-and post education surveys and individual semi-structured interviews.Methods: Thirty-four health professional participants attended a 1-h face-to-face or Zoom® interprofessional education session to learn how to deliver an evidence-based patient falls prevention education strategy, the modified Safe Recovery Programme.Results: A 1-hour education session was insufficient to build full confidence to deliver the Safe Recovery Programme. There was no statistically significant change in participant views on interprofessional collaboration. Participants recommended prior consultation and preparation before delivery of IPE, with additional opportunities for discussion and feedback during implementation with patients. The findings highlight the importance of interprofessional education for evidence-based interventions in hospitals. Health professionals value education that is timely, interactive, realistic and engaging.
## | introduc ti on
Despite a wide range of interventions for preventing falls in hospitals and care facilities, falls in healthcare organizations remain a serious problem worldwide [bib_ref] Effectiveness of patient-centered interventions on falls in the acute care setting compared..., Avanecean [/bib_ref] [bib_ref] Interventions for preventing falls in older people in care facilities and hospitals, Cameron [/bib_ref] [bib_ref] Divesting from a scored hospital fall risk assessment tool (FRAT): A cluster..., Morris [/bib_ref]. Patient education is an integral part of falls prevention and has been found to have a positive effect on the risk of hospital falls [bib_ref] Falls after hospital discharge: A randomized clinical trial of individualized multi-modal falls..., Hill [/bib_ref]. Falls prevention education for patients aims to increase their understanding of falls and falls risk, as well as empowering them with falls prevention strategies.
## | backg rou n d
The Safe Recovery Programme (SRP) is an individualized patient education programme based on the principles of health behaviour change and has been found to be effective in reducing falls rates in hospitals [bib_ref] Patient education to prevent falls among older hospital inpatients: A randomized controlled..., Haines [/bib_ref]. The SRP has four stages aimed at empowering patients to improve their safety in hospital: (i) assessing risks; (ii) goal setting; (iii) reviewing goals; and (iv) follow-up [bib_ref] Patient education to prevent falls among older hospital inpatients: A randomized controlled..., Haines [/bib_ref].
Patients have been found to prefer individualized education as well as consistent and standardized information from all clinical staff [bib_ref] Patient perspectives on hospital falls prevention education, Heng [/bib_ref]. Allied health professionals and nurses play a key role in providing patient education [bib_ref] It promoted a positive culture around falls prevention': Staff response to a..., Hill [/bib_ref] [bib_ref] Education interventions for health professionals on falls prevention in health care settings:..., Shaw [/bib_ref] , making it essential to educate them on how to do this effectively. Education programmes for healthcare professionals that embed evidence-based practice as well as evidence-based teaching and learning methods enhance quality patient care [bib_ref] Evidence-based practice education for healthcare professions: An expert view, Lehane [/bib_ref]. High-quality, interactive education programmes for healthcare professionals are known to influence implementation of falls prevention strategies in a hospital setting . Effective implementation of falls prevention interventions necessitates the creation of a positive learning environment that increases receptivity to a new intervention .
Interprofessional education (IPE) offers opportunities for participants from different professional groups to learn with, from and about each otherto improve collaborative practice and patient-centred care. To optimize falls prevention education to patients and ensure the content and delivery of patient education is consistent across professions, education for healthcare professionals should reflect the importance of an interprofessional approach [bib_ref] Falls prevention education: Interprofessional training to enhance collaborative practice, Mckenzie [/bib_ref] [bib_ref] An updated synthesis of review evidence of interprofessional education, Reeves [/bib_ref] [bib_ref] Physical and occupational therapy practice improvement following interprofessional evidence-based falls prevention training, Wheeler [/bib_ref]. Biggs' 3P model for educational design has been expanded to a 4P approach (Planning, Presage, Process and Product). See . The 4P model offers a comprehensive systems-based theoretical model of learning and allows for a greater understanding of the different factors that impact educational delivery and implementation [bib_ref] Exploring an IPE faculty development program using the 3-P model, Baker [/bib_ref].
The aim of this study was to design, deliver and evaluate an IPE programme for healthcare professionals on how to implement a modified version of the SRP to prevent falls in hospitalized patients.
The intervention from this study supported a trial that involved healthcare professionals delivering the modified SRP to patients in an acute hospital setting. We sought to investigate whether an IPE programme develops healthcare professionals': (i) motivation and confidence in facilitating an interactive patient education intervention on falls prevention, (ii) appreciation of the value and role F I G U R E 1 4P model of educational design of other health professionals in falls prevention, (iii) knowledge and capability for interprofessional collaboration.
## | me thods
## | study design
The study employed a mixed methods pre-and post questionnaire design followed by semi-structured telephone interviews, to triangulate the data from different approaches. Sequential exploratory design was used with the interview data building on the survey results.
## | sample and study settings
The study took place on the medical wards of an Australian private acute hospital. All allied health professionals and nurses working on the intervention ward were eligible and invited to participate via email. On the day of the IPE intervention, consenting participants completed a PICF, pre-test and post-test surveys.
## | intervention
Training was delivered either face to face or via a video conferencing system, Zoom®. Participants received a 1-h IPE programme, which was the most time available for busy clinicians to attend. The programme educated participants on the latest evidence on patient education for falls prevention, how to implement the modified SRP and how to achieve effective interprofessional collaborative practice. A mixture of interactive teaching methods was used including small group discussion on participants' current falls prevention education to patients, a small group critical thinking activity on the barriers and facilitators to delivering falls prevention education to patients, and content delivery on the latest evidence on patient education and its role in falls prevention in hospitals. Three pre-recorded vignettes using simulated participants demonstrated delivery of the modified SRP. Laminated scripts of the modified SRP were provided for participants to use during the education intervention and instructions for implementation (see Supplementary Material). Multiple copies were also available on the hospital ward throughout implementation of the intervention. The PowerPoint presentation of the education was made available for those staff unable to attend training and to supplement learning for staff who attended.
## | ethics approval
Research Ethics Committee approval was obtained from La Trobe University's Health and Engineering College Human Ethics Sub-Committee (HEC21023).
## | data collection and instruments
A pre-test, post-test method of data collection was used. A commercial online survey software program, Q ualtrics (Q ualtrics Provo, Utah, USA), was used for online data collection. Prior to receiving the IPE programme, consenting participants responded to a survey to measure their attitudes and perceptions towards evidence-based falls prevention education and interprofessional collaborative practice. The survey on evidence-based falls education
## | qualitative data
Interviews were transcribed verbatim, transferred into Excel and analysed thematically [bib_ref] Qualitative research in psychology using thematic analysis in psychology using thematic analysis..., Braun [/bib_ref]. One researcher developed initial descriptive themes and subthemes using the relevant aspects TA B L E 2 Views on interprofessional collaboration pre-and posteducation (1 = Poor to 5 = Excellent)
## | re sults
## Ta b l e 5
Qualitative themes based on the 3P approach to IPL
## Themes
## Perceptions of learning experience
## Enhancing factors (what was done well) limiting factors (suggestions for improvement) presage components: the context
Relationship with stakeholders: Consultation and pre-course preparation Consultation and explanation required prior to initial education session to facilitate on-boarding. Regular "lead in" updates during ward handover. "…I think the best way to go about introducing it, probably would be… an initial onboarding orientation to the facility that's very clear, 'This is how we do it here, this is the model that we take as our approach', and then providing ongoing refreshment in-services." (P3) Initial session needs to be followed up with at least one or more refresher sessions. "…doing one -the in-service and then maybe two or three weeks later doing a refresher in-service, might be helpful just to say, 'OK, we've been doing this for two weeks now, just let's quickly recap how are you finding it, do you need support?' I think that would probably be a good way to do it." (P3)
Deliver as on-ward in-service training outside of working shift. "I think it's probably cheaper to have us stay for half an hour, than to pay for X number of falls per year. So, having the support of staff staying a little bit…I would stay an extra half an hour to enable other staff on my ward to attend an in-service." (P3)
Learning environment:
Online versus face to face
The ability to complete the training online. "Look, doing shift work … doing the Zoom ones actually worked a lot better, I think. It is something that corona has given to the world is the ability to actually do this, whereas people were resistant to that before." (P7)
Face-to-face delivery to allow full interaction of clinicians. "I think it would have been more valuable as an in-person thing, but that's just because the way that I learn, I like face-to-face learning… I was just sitting in my lounge room, not as engaged as I probably could have been." (P3)
Face-to-face training "I mean, all in all, the education was thorough enough that I was able to get what I needed to out of it, to then implement it to the best of my ability on the ward." (P3) Accessibility of training online followed by interactive face-to-face discussion "I would do regular discussion groups, but short. I would have the bulk of the information done online, and then I would have, maybe after they've completed that, a 10, 15 minute discussion, very quick, and then a week or two weeks, another little discussion, how you going, what can we improve on?" (P5)
The learners: Interprofessional collaboration
Existing intra and interprofessional communication further reinforced "I think behaviour that helped was everyone being involved. I mean, we were all educated about it, around about the same time, so we all sort of -I guess we were on the same page, which I think was more or less a protective factor in terms of implementing the work." (P3)
No real change in intra and interprofessional collaboration posttraining "'I didn't have a lot of discussions about this programme, but allied health is always discussing with us nurses about the mobility and falls risk and so forth of the patient. I didn't really have the discussions with them about it. I didn't notice it [change in communication], so I think it's maybe stayed the same." (P5)
## Process: the education programme
Content: Evidencebased practice
Increased confidence and motivation to implement "…for me, being able to be involved in something like this, as well as I guess, putting into practice having evidence-based care, as a clinician that's personally important. I think the development of evidence-based care is essential to nursing. I mean, it's the only way we get anywhere really. So, it was good, I suppose, knowing that that's what it was." (P3)
Already confident the care given was evidence-based therefore no impact "I didn't have any concerns about what I was doing in the past, because I was using evidence to the best of my knowledge, so I wasn't using this any more than the things I'd done in the past before." (P1)
reminders during ward handover to foster engagement and facilitate assimilation with the new patient education approach.
## | management support
## Time available for education
Participants agreed that a 1-h session on its own was not long enough and an extended initial education session would have been more beneficial. Additionally, participants perceived that the initial session needed to be followed up with one or more refresher sessions to reinforce the process.
## | learning environment
## Themes
## Perceptions of learning experience
## Enhancing factors (what was done well) limiting factors (suggestions for improvement)
## Teaching approaches and activities
Simulation videos demonstrating the intervention "…yeah, it was like a real-life scenario, hearing it actually out loud and sort of being role played, yeah, it showed how the conversation can flow and it showed how it can work."
Training scenarios need to be more realistic, for example hearing difficulties, language barriers "…with the role plays …a little bit less towards the perfect patient and a little bit more towards the ones with the kind of barriers we have." (P7)
Make session more interactive, for example increased opportunity for role play amongst nurses so they could practice the script and potential scenarios "I think [the] education can be improved with including more interactive sessions, like let nurses role-play on themselves when you cover the programmes." (P2)
Demonstration of intervention with actual patients, for example researcher delivers education and staff provided with the opportunity to observe "I'd probably just sort of say, 'Hey, I'm going to go in and have a chat with Mrs Jones about this. Come along and just have a listen.'...it's the sort of thing that's best -I think it's best seeing in person" (P7)
Teaching methods Demonstration of ways to approach two different patients More interactive sessions, for example small group discussion following viewing of the videos. "I think education can be improved with including more interactive sessions, like let nurses role-play on themselves when you cover the programmes. Like, instead of working, a lot of videos." (P2)
Provision of ongoing support and feedback from research leads "…maybe two in-services are the best way to do it. As in, 'OK, this is the evidence behind it, this is what we want you to do', and then the in-service being, 'This is an example of how to do it', and so they're giving us an opportunity to have a go at doing it, maybe role-playing or something, and giving feedback …so we're a bit more confident that we're …delivering the education the right way." (P3) Consistent discussion and reinforcement required during follow-up sessions "…for the videos that we watch online and then have a 15-minute discussion, would've been better." (P5)
## Educational resources
Accessibility of cognitive aids such as scripts. "…we had the laminated sheets pretty much everywhere, I think. For a while we had them stuck to our handover boards. Yeah, so they were very readily available, and it did give you the confidence of, you know, oh, that's right, we'll go in and just have that chat with them." (P7)
Access to online learning outside of education sessions. I think if I'd known that I was able to go back and review it, that would have been helpful for me, just to cement my understanding of it. (P3) TA B L E 5 (Continued)
## | the learners
Interprofessional collaboration
There was no statistically significant difference between participants' views on interprofessional collaboration before and after the education intervention . Whilst one participant commented that following the education session existing intraprofessional (within profession) and interprofessional (between professions) communication had been further reinforced, another claimed that there had been no change as they already worked collaboratively.
## | process components: the education programme
Participants' views on the education programme are presented in. Mean scores ranged from 3.71 to 4.04 for questions related to the content of the education.
## | content: evidence-based practice
Mean scores were mostly above 4.00 for participants' views on evidence-based education for falls prevention . The only statistically significant increase (p < .05) was participants feeling more prepared to educate patients on falls prevention posteducation. The only question that scored below a mean of 4.00 both preand post-education was participants believing they could overcome the barriers to implementing patient education of falls prevention.
One participant reported increased confidence and motivation to implement the intervention knowing that the care they were providing was evidence-based. However, another commented that they were already confident that the care they were giving was evidencebased and therefore the education did not influence their practice.
## | teaching approaches and activities
One participant found the simulation videos demonstrating the intervention to be beneficial. Conversely, another felt that the scenarios needed to be more realistic to the patients they would be delivering the education to, such as demonstrating the intervention applied to patients with, for example hearing difficulties, language barriers, or cognitive barriers.
# | teaching methods
It was recommended that demonstration of the intervention with actual patients on the ward would have been beneficial, providing participants with the opportunity to observe how the education should be delivered. Another suggestion was to make the education session more interactive, for example using a role-play scenario, which would allow participants to practice delivering the script before educating their patients. Some participants felt that a one-off education session was not enough and there should be ongoing support and feedback from clinical leads on the ward throughout implementation. Others suggested there should be follow-up sessions to allow for further discussion and additional reinforcement of the implementation of the intervention.
## | educational resources
Many participants appreciated the availability and accessibility of cognitive aids on the ward such as the patient education scripts, which served as reminders to conduct the SRP intervention. Others proposed that the videos of the education session be obtainable for access outside of the education session, suggesting that they were not aware that these resources were available.
## | discuss ion
This hospital-based study highlights important factors influencing the implementation of an interprofessional education programme to educate patients on falls prevention. Recommended strategies to overcome barriers are summarized in . Application of the 4P model optimized the evaluation and reporting of the education intervention.
## | presage components: the context
The findings suggest that a pre-requisite for implementing an effective education intervention is building relationships with health professionals and clinical managers [bib_ref] Fostering implementation of health services research findings into practice: A consolidated framework..., Damschroder [/bib_ref]. It is essential to continuously build the case for change [bib_ref] Factors influencing the implementation of a hospital-wide intervention to promote professionalism and..., Mckenzie [/bib_ref] and promote the benefits of patient education in falls prevention. Regular and effective communication should be provided throughout implementation to ensure sustained staff engagement and enthusiasm [bib_ref] The effectiveness of research implementation strategies for promoting evidence-informed policy and management..., Sarkies [/bib_ref]. Leadership engagement has been identified as being essential for implementation of research findings into practice [bib_ref] Fostering implementation of health services research findings into practice: A consolidated framework..., Damschroder [/bib_ref]. Other research in falls prevention education identifies the need for clinical leaders to regularly connect with their staff and support them during implementation . One way of achieving this could be to consider appointing local ward-based champions to disseminate the key messages, assist frontline health professionals with implementation, and act as a point of contact between the health professionals who are delivering the education and the project leaders [bib_ref] Barriers and enablers to the implementation of the 6-PACK falls prevention program:..., Ayton [/bib_ref].
Patient education has been found to be the single most important evidence-based strategy for falls prevention, which demonstrates that healthcare organizations need to place greater value in educating healthcare professionals to undertake this task. In our study, some participants asserted that the initial education was insufficient in length. Previous research on the implementation of evidence-based guidelines for falls prevention demonstrates that longer training fosters clinicians' confidence increases satisfaction and prepares them to more effectively educate others . Additionally, our findings indicated that a one-off education session may not equip staff with the necessary skills to translate knowledge into practice, which accords with other research in falls prevention education . Previous research has found that whilst allied health professionals know the importance of evidence for changing their practice, they often feel ill-equipped or undersupported to implement this [bib_ref] How do health professionals prioritize clinical areas for implementation of evidence into..., Wenzel [/bib_ref]. Participants expressed a desire for additional refresher sessions, including consistent discussion on the topic and continual reinforcement. Regular interaction and small group interactive training sessions have been found to be necessary factors in clinical guideline implementation.
There were several challenges delivering the IPE both online and concurrent with face-to-face delivery, which may partly explain why those attending via online education felt less engaged.
These findings demonstrate the need to deliver the education separately to ensure that each participant is fully engaged and personally involved. This allows for greater responsiveness to participants' learning needs.
## | process: the education programme
Our education intervention did not significantly change participants' views on evidence-based practice. This may be because they felt the organization already fostered a culture where the use of evidence was valued, and participants perceived that the care they were delivering was already evidence-based.and the effective translation of evidence-based practice [bib_ref] Strategies for teaching evidence-based practice in nursing education: A thematic literature review, Horntvedt [/bib_ref]. With more time available for training, teaching strategies such as problem-based learning, group discussions and critical thinking through case-based learning could be employed.
Participants appreciated the availability of resources related to the implementation of the patient education such as laminated signposts and the education script. However, some participants were unaware that recordings of the education session were available for further review. The availability of educational materials such as the audio-visual recording needs to be made more explicit and reminders provided in ward handover. Healthcare professionals also need to have easy access to digital technologies such as tablets onwards to be able to revise the education intervention whenever they required.
## | con clus ion
Educating healthcare professionals to prevent hospital falls is an important activity warranting dedicated time and resources. This study showed that a single education session is not enough for lasting changes in health professional knowledge and patient falls prevention behaviours. The implementation sciences literature, coupled with these findings, reinforce the need for co-production of falls education, involving the patient and interprofessional team. Developing and evaluating health professional education programmes using the 4P model of education design, ensures all elements of the teaching context, student approaches to learning and the outcomes of learning are considered.
## Auth o r co ntr i b uti o n s
Louise Shaw: Conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writingoriginal draft, and writing-review and editing. Debra Kiegaldie:
Conceptualization, methodology, validation, formal analysis, investigation, resources, and writing-review and editing. Hazel Heng:
Conceptualization, writing-reviewing and editing. Meg E. Morris:
Funding acquisition, project administration, and writing-review and editing, supervision.
## Ack n owled g em ents
We thank the representatives from our partner organizations who have contributed to the development and implementation of this study, particularly the clinicians who participated and the managers and consumers at the hospitals. We also thank Dr. Michael Esler for his statistical analysis. This study aligns with the Australian Government's national initiative of preventing falls amongst older people (Australian Commission on Safety and Quality in Health Care, 2019).
## Co n fli c t o f i nte r e s t
The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of this paper.
## Data ava i l a b i l i t y s tat e m e n t
Data from the study are available from the corresponding author on reasonable request.
## E th i c s s tatem ent
[table] Table 3: Following the IPE programme and implementation of the SRP patient education trial (4 weeks), individual telephone interviews were conducted with a sample of consenting participants and a representative from hospital management(Table 1), where they elaborated on the IPE and interprofessional collaborative practice strategies used during the SRP trial. The demographic make-up of the participants was characterized by simple frequency statistics. Participant responses to Likert scale survey TA B L E 1 Participant characteristics and demographics [/table]
[table] Table 1: presents participant demographics. Tables 2-4 present the results with pertinent quantitative data integrated into the Presage and Process components of the 4P model. Table 5 outlines the qualitative data according to the themes of Presage and Process and describes participants' views of the education intervention according to enhancing and limiting factors.Some participants recommended that in the weeks preceding implementation of the intervention, there should be induction and orientation. One participant suggested this could be achieved by regular TA B L E 4 Views on evidence-based education for falls prevention (1 = strongly disagree to 5 = strongly agree)It is important to me that the education I deliver to patients on falls prevention is based on the best available evidence [/table]
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Associaton of Retinol Binding Protein 4 (RBP4) Levels With Hyperuricemia: A Cross-Sectional Study in a Chinese Population
Background: There are few studies on predictive biomarkers for hyperuricemia, and the predictive value of these biomarkers tends to be poor. Additionally, no reports have described the predictive value of retinol binding protein 4 (RBP4) for hyperuricemia.Purpose: This study was performed to evaluate the value of RBP4 for predicting the risk of hyperuricemia in a general population, determine whether RBP4 could be used alone or in combination with other factors to predict the risk of hyperuricemia in the general population, and establish an optimum predictive model.Methods:We conducted a population-based cross-sectional survey in 2018, involving a questionnaire, physical examination, and laboratory testing. We enrolled 2303 individuals by stratified random sampling, and 2075 were included in the data analysis after applying the eligibility criteria.Results: Serum RBP4 level had a highly significant association with hyperuricemia (P<0.001). After adjusting for potential confounders, logistic regression indicated that the risk of hyperuricemia was highest in the highest RBP4 quartile (odds ratio: 7.9, 95% confidence interval [CI]: 4.18-14.84, compared to the lowest quartile). The area under the receiver operating characteristic (ROC) curve (AUC) for RBP4 was 0.749 (95% CI: 0.725-0.774, P<0.001), which was higher than that for all the other predictors assessed. The optimum model for predicting hyperuricemia in the general population consisted of RBP4, sex (male), body mass index, serum creatinine, high-sensitivity C-reactive protein, fasting blood glucose, insulin, and alcohol consumption. The AUC was 0.804 (95% CI: 0.782-0.826, P<0.001).Conclusions: RBP4 is strongly associated with hyperuricemia, and its predictive value was higher than that of traditional predictors.
# Introduction
The global prevalence of hyperuricemia has increased rapidly in the past few decades [bib_ref] Gout and Hyperuricaemia: A Worldwide Health Issue of, Gamala [/bib_ref] [bib_ref] One Year in Review 2018: Gout, Punzi [/bib_ref] ; it is 14.6-20% in the US (4) and 13. in China, and it is more common (25.5%) in southern China [bib_ref] Association Between Eating Away From Home and Hyperuricemia: A Population-Based Nationwide Cross-Sectional..., Liu [/bib_ref] [bib_ref] Risk Factors for the Development of Hyperuricemia: A STROBE-Compliant Cross-Sectional and Longitudinal..., Ni [/bib_ref] [bib_ref] Regional and Temporal Trends of Hyperuricemia Epidemics in Mainland China From 2000..., Li [/bib_ref]. Our 2012 epidemiological survey in Zhuhai city showed that the prevalence of hyperuricemia reached a staggering 32.4%, which is the highest for any location assessed in China [bib_ref] An Epidemiological Study on the Prevalence of Hyperuricemia and its Relationship to..., Shao [/bib_ref]. In addition to causing gout, numerous studies have shown that hyperuricemia increases the risk of metabolic syndrome (MetS) [bib_ref] Uric Acid in Metabolic Syndrome: From an Innocent Bystander to a Central..., Kanbay [/bib_ref] , chronic kidney disease [bib_ref] The Case for Uric Acid-Lowering Treatment in Patients With Hyperuricaemia and CKD, Sato [/bib_ref] , acute kidney injury [bib_ref] Hyperuricemia and Contrast-Induced Acute Kidney Injury: A Systematic Review and Meta-Analysis, Zuo [/bib_ref] , hypertension [bib_ref] Effect of Uric Acid-Lowering Therapy on Blood Pressure: Systematic Review and Meta-Analysis, Qu [/bib_ref] , cardiovascular disease [bib_ref] Expert Consensus for the Diagnosis and Treatment of Patient With Hyperuricemia and..., Borghi [/bib_ref] , and cerebral infarction [bib_ref] Higher Serum Uric Acid May Contribute to Cerebral Infarction in Patients With..., Du [/bib_ref].
Unfortunately, studies on the risk and predictors of hyperuricemia are rare. Although some groups have reported on factors (age, body mass index , waist circumference , triglycerides [TG], etc.) [bib_ref] Prevalence of Hyperuricemia Among Chinese Adults: A National Cross-Sectional Survey Using Multistage,..., Liu [/bib_ref] that influence hyperuricemia, few studies have focused on individual biomarkers (visceral adiposity index [bib_ref] Positively Increased Visceral Adiposity Index in Hyperuricemia Free of Metabolic Syndrome, Gu [/bib_ref] , TG-glucose index [bib_ref] Usefulness of Triglyceride-Glucose Index for Estimating Hyperuricemia Risk: Insights From a General..., Shi [/bib_ref] , etc.) or models for predicting hyperuricemia [bib_ref] Body Mass Index, and PPARgamma Polymorphism are Good Indicators in Hyperuricemia Prediction..., Lee [/bib_ref] [bib_ref] Incidence and Simple Prediction Model of Hyperuricemia for Urban Han Chinese Adults:..., Cao [/bib_ref] [bib_ref] Exploration of Machine Learning for Hyperuricemia Prediction Models Based on Basic Health..., Lee [/bib_ref]. Additionally, most reported predictors have poor performance or are not suitable for large-scale clinical use because of issues such as cost and complexity.
Retinol binding protein-4 (RBP4) was recently recognized as a type of adipokine [bib_ref] The Metabolic Role of Retinol Binding Protein 4: An Update, Christou [/bib_ref]. Several small-sample studies have reported an association between RBP4 and hyperuricemia in patients with diabetes, chronic kidney disease, and MetS, but none of them focused on the general adult population [bib_ref] Levels of Retinol-Binding Protein 4 and Uric Acid in Patients With Type..., Chen [/bib_ref] [bib_ref] Relationship Between Plasma Adiponectin, Retinol-Binding Protein 4 and Uric Acid in Hypertensive..., Park [/bib_ref]. To our knowledge, there are no studies on the predictive value of RBP4 for hyperuricemia.
This large-scale study aimed to explore the association between RBP4 and hyperuricemia in the general population, and to evaluate whether RBP4 can predict hyperuricemia. We also hope that in the absence of serum uric acid results, abnormally elevated levels of RBP4 may suggest the importance of further serum uric acid testing.
# Methods
## Study population
A cross-sectional survey of the general population in Wanzai town in Zhuhai city, China, was conducted in 2018. Using stratified random sampling, 2303 adults who were permanent residents of Wanzai town were initially enrolled in the study [1054 in the first visit in 2018 and another 1249 later in 2018 (the second visit was the follow-up visit of a similar epidemiological survey that we conducted in 2012 in Wanzai town)]. We followed the same sampling procedures described in previous reports on our 2012 epidemiological survey [bib_ref] Association of Insulin Resistance With Chronic Kidney Disease in non-Diabetic Subjects With..., Chen [/bib_ref] [bib_ref] Central Obesity, C-Reactive Protein and Chronic Kidney Disease: A Community-Based Cross-Sectional Study..., Chen [/bib_ref] [bib_ref] Insulin Resistance and Metabolic Syndrome in Normal-Weight Individuals, Chen [/bib_ref] [bib_ref] Metabolic Syndrome and Chronic Kidney Disease in a Southern Chinese Population, Li [/bib_ref] [bib_ref] Association Between Lipid Ratios and Insulin Resistance in a Chinese Population, Zhang [/bib_ref].
The exclusion criteria were as follows: (1) treatment with uric acid-lowering drugs or drugs that affect uric acid in the last 6 months, (2) severe liver or kidney damage, (3) acute cardiovascular or cerebrovascular diseases, and (4) pregnancy or breastfeeding.
After applying the eligibility criteria, 2075 individuals were included in the final analysis.
## Ethics approval
The study was approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University and was conducted in accordance with the Declaration of Helsinki. All subjects provided written informed consent at recruitment.
## Data collection
Data on sociodemographic factors (age, sex, and education level), lifestyle (physical inactivity [physical activity <1 time/week], current smoking, and current alcohol consumption [≥1 time/ week]), medical history (hypertension, diabetes, coronary heart disease [CHD], and stroke), and medication use were collected using a questionnaire. The detailed design of this study (including the urine specimen collection) was the same as in our previous epidemiological survey conducted in 2012 [bib_ref] Association of Insulin Resistance With Chronic Kidney Disease in non-Diabetic Subjects With..., Chen [/bib_ref] [bib_ref] Central Obesity, C-Reactive Protein and Chronic Kidney Disease: A Community-Based Cross-Sectional Study..., Chen [/bib_ref] [bib_ref] Insulin Resistance and Metabolic Syndrome in Normal-Weight Individuals, Chen [/bib_ref] [bib_ref] Metabolic Syndrome and Chronic Kidney Disease in a Southern Chinese Population, Li [/bib_ref] [bib_ref] Association Between Lipid Ratios and Insulin Resistance in a Chinese Population, Zhang [/bib_ref].
Physical examinations were performed to collect data on blood pressure (systolic blood pressure [SBP] and diastolic blood pressure [DBP]), weight and height (which were used to calculate BMI), and WC. BMI was used to define obesity according to Chinese obesity criteria. Normal weight was defined as BMI<24 kg/m 2 . Obesity was defined as BMI >=28 kg/m 2 , BMI 24-28 kg/m 2 was overweight.
Blood specimens were collected after overnight fasting, stored at 2-8°C immediately after collection, and transported to the Central Laboratory of the Third Affiliated Hospital of Southern Medical University within 3 hours (31). RBP4 levels were assessed using an immunoturbidimetric method (Shanghai Beijia Biochemical Reagent Company, Shanghai,China). Hyperuricemia was defined as ≥420 mmol/L (7 mg/dL) in males and ≥360 µmol/L (6 mg/dL) in females [bib_ref] Prevalence of Gout and Hyperuricemia in the US General Population: The National..., Zhu [/bib_ref]. The following parameters were also measured(apparatus: Roche Cobas c501, Penzberg, Germany): TG, high-density lipoprotein (HDL), low-density lipoprotein (LDL), fasting blood glucose (FBG), insulin, homeostasis model assessment of insulin resistance (HOMA-IR, defined as: (FBG×insulin)/22.5), serum creatinine (SCr), cystatin C, high-sensitivity C-reactive protein (hsCRP), interleukin (IL)-6, serum uric acid, and estimated glomerular filtration rate (eGFR, defined according to CKD-EPIscr formula). Urinary albumin-to-creatinine ratio (ACR), Nacetyl-b-D-glucosaminidase (NAG), and b2 microglobulin (b2MG) were also assessed.
# Statistical analysis
Continuous variables with a normal distribution are presented as the mean and standard deviation, while those with a non-normal distribution are presented as the median and interquartile range. Categorical variables are presented as frequencies and Abbreviations: ACR, albumin-to-creatinine ratio; b2MG, b2 microglobulin; BMI, body mass index; CHD, coronary heart disease; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; HDL, highdensity lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; hs-CRP, high sensitivity C-reactive protein; IL-6, interleukin 6; LDL, low-density lipoprotein; NAG, N-acetyl-b-D-glucosaminidase; RBP4, retinol binding protein 4; SBP, systolic blood pressure; SCr, serum creatinine; TG, triglycerides; WC, waist circumference. percentages. Continuous variables were compared between groups using independent-samples t tests or analyses of variance (for normally distributed variables) or Mann-Whitney U tests (for non-normally distributed variables). Categorical variables were compared between groups using c 2 or Fisher's exact tests.
Six binary logistic regression models (stepwise conditional), with hyperuricemia as the independent variable and RBP4 quartile as the independent variable, were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). The regression models adjusted for the following covariates: (1) age and sex; (2) model 1 covariates plus hypertension, diabetes, CHD, education of high school or above, physical inactivity, current smoking, and current alcohol consumption; (3) model 2 covariates plus SBP, DBP, log TG, LDL, HDL, BMI, eGFR, FBG, and log insulin; (4) model 2 covariates plus SBP, DBP, LDL, HDL, BMI, eGFR, and log HOMA-IR; (5) model 4 covariates plus log hs-CRP, and log IL-6; and (6) model 5 covariates plus log NAG and log ACR. SCr is strongly correlated with eGFR, so it was not included in the regression models.
The area under the receiver operating characteristic (ROC) curve (AUC) was used to assess the predictive value of RBP4, other predictors, and the following three predictive models for hyperuricemia: (1) RBP4, sex, BMI, SCr, log hs-CRP, log insulin, log HOMA-IR, and current alcohol consumption; (2) RBP4, sex, BMI, SCr, log hs-CRP, and FBG; and (3) RBP4, sex, BMI, SCr, log hs-CRP, FBG, log insulin, and current alcohol consumption. Youden's index (sensitivity + specificity -1) was used to select the optimum cutoff value of RBP4. The reciprocals of HDL and eGFR were used due to their negative associations with serum uric acid level.
Statistical analyses were performed in SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). A two-sided P value <0.05 was considered statistically significant.
# Results
The baseline characteristics of the participants are shown in [fig_ref] TABLE 1 |: Participant baseline characteristics [/fig_ref]. Those in the hyperuricemia group were more likely to be male and older compared to those in the non-hyperuricemia group, and they had higher rates of hypertension, diabetes, CHD, alcohol consumption, and physical inactivity (P<0.05). In addition, there were higher values of SBP, DBP, WC, BMI, TG, LDL, FBG, insulin, HOMA-IR, SCr, cystatin C, NAG, hs-CRP, IL-6, and RBP4 in the hyperuricemia group compared to the non-hyperuricemia group (P<0.05), but lower values of HDL, eGFR, and serum uric acid (P<0.001).
The participants were divided into quartiles based on RBP4. Those in the fourth quartile were more likely to be male and older compared to the participants in the first quartile, and they were less educated and had higher rates of hypertension, diabetes, smoking, alcohol consumption, and physical inactivity (P<0.05). The values of SBP, DBP, WC, BMI, TG, LDL, FBG, insulin, HOMA-IR, serum uric acid, SCr, cystatin C, NAG, hs-CRP, IL-6, and ACR were higher and the values of HDL and eGFR were lower in the fourth quartile compared to the first quartile [fig_ref] TABLE 2 |: Participant baseline characteristics by RBP4 quartile [/fig_ref].
The prevalence of hyperuricemia gradually increased from the first to the fourth RBP4 quartile from 5.0% to 58.2% (7.5% to 61.7% in males and 4.4% to 55% in females) (all P<0.001, [fig_ref] TABLE 3 |: Prevalence of hyperuricemia by RBP4 quartile [/fig_ref].
The associations between RBP4 and hyperuricemia, according to six multivariate binary logistic regression analyses, are shown in [fig_ref] TABLE 4 |: Association between RBP4 and hyperuricemia according to logistic regression [/fig_ref]. RBP4 and hyperuricemia were positively associated in all six models. In the final model (model 6), the OR comparing quartile 4 of RBP4 with quartile 1 was 7.90 (95% CI: 4.18-14.84; P < 0.001).
The AUC for RBP4 predicting hyperuricemia was 0.749 (95% CI: 0.725-0.774, P<0.001) and Youden's index was 0.36, with an optimum cutoff of 54.5 mg/L. Its predictive performance was better than the performances of SCr, cystatin C, eGFR, TG, WC, BMI, insulin, HOMA-IR, HDL, FBG, hs-CRP, and SBP [fig_ref] FIGURE 1 |: Receiver operating characteristic [/fig_ref].
The best prediction model was model 3, which involved RBP4 and other variables related to uric acid (sex, BMI, SCr, hs-CRP, FBG, insulin, and current alcohol consumption) in a binary logistic regression model (AUC: 0.804, 95% CI: 0.782-0.826, Youden's index: 0.36, P<0.001). The predictive power of model 1 (AUC: 0.803) and model 2 (AUC: 0.797) were close to that of model 3 [fig_ref] TABLE 5 |: Predictive value of RBP4 and other indicators for hyperuricemia in the general... [/fig_ref] ; [fig_ref] FIGURE 1 |: Receiver operating characteristic [/fig_ref]. The best predictive model for males was composed of RBP4, SCr, and BMI (AUC: 0.782, 95% CI: 0.749-0.815, Youden's index: 0.424, P<0.001). The best predictive model for females was composed of RBP4, SCr, hypertension, log insulin, and log hs-CRP (AUC: 0.824, 95% CI: 0.796-0.852, Youden's index: 0.510, P<0.001).
# Discussion
Using population-based data on southern Chinese adults collected in a single-center cross-sectional epidemiological survey, we found that RBP4 had a highly significant association with hyperuricemia (P<0.001). More importantly, RBP4 was a good predictor of hyperuricemia; indeed, it performed better than traditional predictors. We also explored the predictive value of RBP4 combined with routinely assessed clinical factors that are related to hyperuricemia. To our knowledge, this is the first study to investigate the predictive value of RBP4 for hyperuricemia.
Several groups have reported individual predictors or predictive models for hyperuricemia [bib_ref] Body Mass Index, and PPARgamma Polymorphism are Good Indicators in Hyperuricemia Prediction..., Lee [/bib_ref] [bib_ref] Incidence and Simple Prediction Model of Hyperuricemia for Urban Han Chinese Adults:..., Cao [/bib_ref] [bib_ref] Exploration of Machine Learning for Hyperuricemia Prediction Models Based on Basic Health..., Lee [/bib_ref]. Lee MF et al. [bib_ref] Body Mass Index, and PPARgamma Polymorphism are Good Indicators in Hyperuricemia Prediction..., Lee [/bib_ref] reported that sex, BMI, and peroxisome proliferatoractivated receptor (PPAR)-g polymorphism are good predictors of hyperuricemia. Cao et al. [bib_ref] Incidence and Simple Prediction Model of Hyperuricemia for Urban Han Chinese Adults:..., Cao [/bib_ref] developed sex-specific prediction models (the predictors for males were age, SBP, BMI, and blood uric acid, and the predictors for females were SBP, BMI, TG, and blood uric acid) for hyperuricemia. Lee S et al. [bib_ref] Exploration of Machine Learning for Hyperuricemia Prediction Models Based on Basic Health..., Lee [/bib_ref] developed a machine learning model (involving basic healthcare checkup test results) for predicting hyperuricemia. In addition, various novel blood lipid indicators such as the visceral adiposity index [bib_ref] Positively Increased Visceral Adiposity Index in Hyperuricemia Free of Metabolic Syndrome, Gu [/bib_ref] , TG-glucose index [bib_ref] Usefulness of Triglyceride-Glucose Index for Estimating Hyperuricemia Risk: Insights From a General..., Shi [/bib_ref] , lipid accumulation products [bib_ref] Visceral Adipose Accumulation Increased the Risk of Hyperuricemia Among Middle-Aged and Elderly..., Huang [/bib_ref] , and the cardiometabolic index [bib_ref] Body Adiposity Index, Lipid Accumulation Product, and Cardiometabolic Index Reveal the Contribution..., Wang [/bib_ref] have been independently associated with hyperuricemia. However, all these studies were limited to specific populations, and the results were not verified in other regions such as the southern China. RBP4, as a single factor, had a good performance in predicting the risk of hyperuricemia. The AUC for RBP4 in the general population was 0.749. This is better than previous traditional individual predictors and similar to the predictive model developed by , which had an AUC of 0.775. We also obtained a predictive model (model 3), involving RBP4 and traditional predictors, with a good AUC of 0.804. Notably, the predictive value of RBP4 was slightly better in females than males (AUC for RBP4: 0.738 vs. 0.756; AUC for predictive model 3: 0.782 vs. 0.824).
The conclusion of our investigation is consistent with two previous studies [bib_ref] Levels of Retinol-Binding Protein 4 and Uric Acid in Patients With Type..., Chen [/bib_ref] [bib_ref] Relationship Between Plasma Adiponectin, Retinol-Binding Protein 4 and Uric Acid in Hypertensive..., Park [/bib_ref]. Chen et al. were the first to report elevated serum RBP4 levels with increasing serum uric acid among 885 individuals with type 2 diabetes in Taiwan. Thereafter, Chan et al. [bib_ref] Relationship Between Plasma Adiponectin, Retinol-Binding Protein 4 and Uric Acid in Hypertensive..., Park [/bib_ref] reported that serum RBP4 was positively associated with uric acid in 26 subjects with hypertension and MetS.
Our study has the following strengths. First, the participants were randomly sampled from the general population and covered all ages of adults, so the sample is widely representative. Second, 2075 people were included in the analysis, far exceeding the sample sizes of previous studies. Data are shown as mean±standard deviation, median (interquartile range), or frequency (percentage). ACR, urinary albumin-to-creatinine ratio; b2MG, b2 microglobulin; BMI, body mass index; CHD, coronary heart disease; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; LDL, low-density lipoprotein; NAG, N-acetyl-b-D-glucosaminidase; RBP4, retinol binding protein 4; SBP, systolic blood pressure; TG, triglycerides; WC, waist circumference.
Third, strict eligibility criteria were adopted. For example, we excluded individuals who were treated with uric acid-lowering drugs and those with a history of diseases or medications that might affect the RBP4 level. Fourth, the association remained significant after adjustment. For example, after adjusting for 21 potential confounders, the final regression model still showed that the risk of hyperuricemia in the highest RBP4 quartile was still 7.9 times higher than that in the lowest quartile, indicating a significant independent association between RBP4 and hyperuricemia. Finally, this is the first study to evaluate the value of RBP4 for predicting the risk of hyperuricemia, and we found that RBP4 alone performed better than traditional
## 0.23
Data are shown as mean±standard deviation, median (interquartile range), or frequency (percentage). ★ vs. quartile 1, P < 0.05; ▲ vs. quartile 2, P < 0.05; ◆ vs. quartile 3, P < 0.05. predictors. This is also the most important finding of this study, as it indicates that RBP4 might be useful for predicting the risk of hyperuricemia and could be used in epidemiological research on hyperuricemia in general adult populations. Though direct assessment of uric acid should be prefered over measuring a surrogate, the association between RBP4 and uric acid could be diagnostically exploited in case uric acid values are not available. The mechanisms by which how RBP4 predicts hyperuricemia remain unclear. RBP4 is considered independently related to insulin resistance, which is implicated in the pathogenesis of hyperuricemia [bib_ref] Retinol-Binding Protein 4 and Insulin Resistance in Lean, Obese, and Diabetic Subjects, Graham [/bib_ref] [bib_ref] Serum Retinol-Binding Protein: A Link Between Obesity, Insulin Resistance, and Type 2..., Wolf [/bib_ref]. A recent study showed that RBP4 might be involved in hyperuricemia-induced insulin resistance by inhibiting IRS/PI3K/Akt phosphorylation [bib_ref] RBP4 Is Associated With Insulin Resistance in Hyperuricemia-Induced Rats and Patients With..., Liu [/bib_ref]. Even after adjusting for insulin level plus FBG, or HOMA-IR, RBP4 remained strongly associated with hyperuricemia. This indicates that RBP4 may be related to hyperuricemia through other mechanisms besides insulin resistance. This study also indicated that the mechanisms may not involve renal function. When we adjusted for all renal function indicators (including glomerular and renal tubular function; i.e., eGFR, ACR, and NAG) in regression model 6, the regression results showed that the independent association between RBP4 and hyperuricemia remained significant. We speculate that besides insulin resistance, RBP4 may have additional unique non-renal function mechanisms such as pro-inflammatory effects and direct effects on vascular smooth muscle and uric acid [bib_ref] The Metabolic Role of Retinol Binding Protein 4: An Update, Christou [/bib_ref] [bib_ref] Retinol Binding Protein-4 Levels and Non-Alcoholic Fatty Liver Disease: A Community-Based Cross-Sectional..., Chen [/bib_ref] [bib_ref] The Relationship Between Circulating Irisin, Retinol Binding Protein-4, Adiponectin and Inflammatory Mediators..., Tabak [/bib_ref]. Further basic research on the mechanisms is needed in the future. Our results should be considered in the context of several limitations. First, the participants were all Han Chinese adults from Zhuhai city, and the results may not be generalizable to other ethnicities. In addition, this was a single-center study and therefore inevitably limited regarding the sample size; large multicenter studies are needed to verify the conclusions. Furthermore, the study was cross-sectional, and the underlying mechanisms were not explored in depth. Cohort or case-control studies and basic research on the underlying mechanisms should be performed to verify our findings.
# Conclusions
This study found that RBP4 is significantly positively associated with hyperuricemia in adults and has good predictive value for the condition. Clinically, it can be used alone or in combination with other traditional indicators to predict the risk of hyperuricemia.
# Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
# Ethics statement
The studies involving human participants were reviewed and approved by Ethics Committee of the Third Affiliated Hospital of Southern Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
# Author contributions
G-BH, HZ, and C-XX participated in the research design, performance of the research, data analysis, and manuscript writing. X-FS, QZ, and X-SK participated in the research design and data analysis. P-CG, X-LL, JN, H-SC, and HX participated in the performance of the research and data analysis. All authors were involved in revising the manuscript and approved the final version.
[fig] FIGURE 1 |: Receiver operating characteristic (ROC) curves of RBP4 and other indicators for predicting hyperuricemia in the general population. [/fig]
[table] TABLE 1 |: Participant baseline characteristics. [/table]
[table] TABLE 2 |: Participant baseline characteristics by RBP4 quartile. [/table]
[table] TABLE 3 |: Prevalence of hyperuricemia by RBP4 quartile . [/table]
[table] TABLE 4 |: Association between RBP4 and hyperuricemia according to logistic regression. covariates plus medical history (hypertension, diabetes, and CHD), education of high school or above, physical inactivity, current smoking, and current alcohol consumption.Regression model 3: adjusted for model 2 covariates plus SBP, DBP, log TG, LDL, HDL, BMI, eGFR, FBG, and log insulin. Regression model 4: adjusted for model 2 covariates plus SBP, DBP, log TG, LDL, HDL, BMI, eGFR, and log HOMA-IR. Regression model 5: adjusted for model 4 covariates plus log hs-CRP, and log IL-6. Regression model 6: adjusted for model 5 covariates plus log NAG and log ACR. [/table]
[table] TABLE 5 |: Predictive value of RBP4 and other indicators for hyperuricemia in the general population.Predictive model 1: RBP4, sex, BMI, SCr, log hs-CRP, log insulin, log HOMA-IR, and current alcohol consumption. Predictive model 2: RBP4, sex, BMI, SCr, log hs-CRP, and FBG. Predictive model 3: RBP4, sex, BMI, SCr, log hs-CRP, FBG, log insulin, and current alcohol consumption. [/table]
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Ambient Particulate Matter Air Pollution in Mpererwe District, Kampala, Uganda: A Pilot Study
Air quality in Kampala, the capital of Uganda, has deteriorated significantly in the past two decades. We made spot measurements in Mpererwe district for airborne particulate matter PM 2.5 (fine particles) and coarse particles. PM was collected on Teflon-membrane filters and analyzed for mass, 51 elements, 3 anions, and 5 cations. Both fine and coarse particle concentrations were above 100 g/m 3 in all the samples collected. Markers for crustal/soil (e.g., Si and Al) were the most abundant in the PM 2.5 fraction, followed by primary combustion products from biomass burning and incinerator emissions (e.g., K and Cl). Over 90% of the measured PM 2.5 mass can be explained by crustal species (41% and 59%) and carbonaceous aerosol (33%-55%). Crustal elements dominated the coarse particles collected from Kampala. The results of this pilot study are indicative of unhealthy air and suggest that exposure to ambient air in Kampala may increase the burden of environmentally induced cardiovascular, metabolic, and respiratory diseases including infections. Greater awareness and more extensive research are required to confirm our findings, to identify personal exposure and pollution sources, and to develop air quality management plans and policies to protect public health.
# Introduction
Air pollution is an increasing problem in urban dwellings worldwide. The World Health Organization (WHO) ranks indoor air pollution (from biomass fuel combustion) and urban outdoor air pollution 10th and 14th, respectively, among 19 leading risk factors for global mortality. There is indisputable epidemiologic evidence that air pollution exposure in humans increases not only the risk of respiratory conditions such as asthma and respiratory tract infections but also cardiovascular disease and metabolic diseases such as diabetes mellitus. In the developing world, air pollution presents a particular threat to the health of populations in cities that are growing in a fast, poorly planned, and unregulated manner [bib_ref] Evaluation of emissions and air quality in megacities, Gurjar [/bib_ref] [bib_ref] Environmental health in China: progress towards clean air and safe water, Zhang [/bib_ref]. This is the case in Kampala, Uganda, where air quality has deteriorated significantly during the past two decades in parallel with the fast-paced economic development. Kampala is the capital of Uganda in East Africa, a country with a rapidly increasing population and expanding economic growth. With a growing middle class, Uganda, and specifically Kampala, has seen fast expanding vehicular traffic comprised of imported second hand and reconditioned cars, aging, exhaust-unregulated cars, trucks, buses, and motorcycles. Furthermore, the national economic strategy gives priority to the industrial and manufacturing sectors and has promoted and realized a rise in cottage and other larger industries within the city. In addition to the ever growing road traffic and unregulated industrial emissions, waste burning by individuals to manage uncollected waste, also contributes to the deterioration of air quality in Kampala.
However, reports on air pollution in Kampala are primarily anecdotal [bib_ref] Kampala chokes on pollution, Lanyero [/bib_ref] ]. An ongoing study shows an increase of ischemic heart disease from virtually absent up to the end of the 1990s to more than 10% of all admissions on the Mulago University Hospital cardiology ward in 2011. As part of an effort to preliminarily characterize airborne particulate matter (PM) within Kampala, we made spot measurements of ambient PM in a typical bustling, low-income, mixed commercial, and residential section of the city (Mpererwe District). This paper provides a summary of the findings.
# Methods
To characterize the content of air pollution PM, we made spot measurements in Mpererwe District, Kawempe South Division, at Kalerwe Market, 80 meters east from Gayaza Kampala Road in Kampala City [fig_ref] Figure 1: Map showing Kampala with detail of the sampling area [/fig_ref]. Employing a Sioutas Personal Cascade Sampler (SKC Inc., Eighty Four, PA, USA) with a flow rate of 9 liters per minute, we collected fine particles (PM 2.5 , or particles with aerodynamic diameters less than 2.5 m) and coarse particles (PM 7-2.5 ) (this fraction contains total suspended particles with aerodynamic diameters greater than 2.5 m) during two 24-hour sampling periods (6:30 p.m. to 6:30 p.m., Local Standard Time [LST]) between December 31st, 2012, and January 1st, 2013, and between January 2nd, 2013, and January 3rd, 2013. PM were collected on Teflon-membrane filters (37 mm, 2 mm pore, Pallflex Gelman Scientific, Ann Arbor, MI, USA). The personal PM monitor as well as a Leland Legacy Pump (SKC Inc., Eighty Four, PA, USA) were installed 3 meters above ground level, on the outside wall of a single story structure under a 50 cm protruding metal sheet roof. PM 2.5 and PM coarse samples were removed from the monitors at the end of each sampling period, stored in plastic petri dishes, sealed and transferred to the USA for further analysis. Filters were equilibrated under a temperature (21.5 ± 1.5 ∘ C) and relative humidity (35 ± 5%) controlled environment before gravimetric analysis following the US Environmental Protection Agency (EPA) protocol at the Environmental and Occupational Health Sciences Institute (EOHSI) in New Jersey. PM 2.5 samples were submitted for elemental analysis by X-ray fluorescence (XRF) including 51 elements from Na to U (see [fig_ref] Table 1: Concentrations of all measured PM 2 [/fig_ref] and [bib_ref] X-ray fluorescence analysis of ambient air samples, Watson [/bib_ref]. Three anions (Cl − , NO 3 − , and SO [bib_ref] Evaluation of emissions and air quality in megacities, Gurjar [/bib_ref] 2− ) and five cations (NH + , Na + , Mg 2+ , Ca 2+ , and K + ) were measured for PM 2.5 by ion chromatography (IC) [bib_ref] Ion chromatography in elemental analysis of airborne particles, Chow [/bib_ref]. Particle loadings from the personal PM monitor are centered on a slit, so only 18 elements were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS).
# Results
Based on the PM mass, fine and coarse particle concentrations were 104.9 g/m 3 and 132.7 g/m 3 for the sample from December 31, 2012 and 103.7 g/m 3 and 208.1 g/m 3 for the sample from January 2, 2013, respectively. PM 2.5 mass concentrations and chemical compositions are listed in, which includes typical marker species representing major PM 2.5 sources, such as soil (Al, Si, Ca, Fe, and Ti), oil combustion (V and Ni), biomass combustion (K + ), traffic (Pb and Zn), incinerator (Cl), and secondary PM (SO 4 2− ). A full list of all measured PM 2.5 species is in [fig_ref] Table 1: Concentrations of all measured PM 2 [/fig_ref]. The relative abundance of major PM 2.5 species is similar across the two sampling days. Markers for crustal/soil are the most abundant, followed by primary combustion sources, such as biomass burning and incinerator emissions. Elevated water-soluble to total potassium ratio (K + /K = 0.91) for both PM 2.5 samples confirms an abundance of biomass burning during the sampling period [bib_ref] Descriptive analysis of PM2.5 and PM10 at regionally representative locations during SJVAQS/AUSPEX, Chow [/bib_ref]. An exploratory PM 2.5 mass reconstruction was conducted based on major species measured, that is, SO [bib_ref] Evaluation of emissions and air quality in megacities, Gurjar [/bib_ref] 2− , NO 3 − , Al, Si, Ca, Fe, and Ti, and closing the mass balance by assuming the remaining mass was contributed by carbon species [bib_ref] Retained nitrate, hydrated sulfates, and carbonaceous mass in federal reference method fine..., Frank [/bib_ref]. illustrates the percent contributions of SO [bib_ref] Evaluation of emissions and air quality in megacities, Gurjar [/bib_ref] 2− , NO 3 − , crustal sources, and carbon. Over 90% of measured PM 2.5 mass can be explained by crustal species and carbonaceous aerosol.shows the elemental concentrations measured for PM coarse samples. Abundant (>200 ng/m 3 ) crustal species (e.g., Al, Ca, and Fe) are found with large sample-to-sample variations.
The meteorological conditions during the sampling period are presented in [fig_ref] Table 4: Meteorological conditions during sampling [/fig_ref]. Mean temperatures across the four sampling dates ranged from 21.7 ∘ C to 24.4 ∘ C, with dew point temperatures between 18.9 and 20.0 ∘ C and mean wind speeds between 6.4 and 8.1 Km/hr.
# Discussion
To the best of our knowledge, there is no known data/sample collection and public information available on type and concentrations of particulate matter in Kampala's air and studies of the associated human health impacts are extremely limited. There is clear evidence that air pollution in Kampala has increased in the last decade [fig_ref] Figure 3: Views of the center of Kampala on a clear day [/fig_ref] and that awareness about this problem is mounting [bib_ref] Kampala chokes on pollution, Lanyero [/bib_ref]. The limited findings of the current study clearly support these observations and indicate that exposure to ambient air in Kampala may raise public health concerns such as the burden of cardiovascular and respiratory diseases. PM 2.5 mass concentrations observed [bib_ref] Metal concentration of PM2.5 and PM10 particles and seasonal Journal of Environmental..., Kulshrestha [/bib_ref]. In a measurement campaign, [bib_ref] Chemical composition of PM2.5 and PM10 in Mexico City during winter 1997, Chow [/bib_ref] reported that the mean and the maximum PM 2.5 mass concentrations in Mexico City were 35.2 and 183.7 g/m 3 , respectively [bib_ref] Chemical composition of PM2.5 and PM10 in Mexico City during winter 1997, Chow [/bib_ref]. It should be pointed out that the average concentrations from world cities mentioned above and in : Twenty-four-hour average PM 2.5 compositions of major components based on mass reconstruction, where fine soil = 2.2Al + 2.49Si + 1.63Ca + 2.42Fe + 1.94Ti [bib_ref] Examining the relationship between atmospheric aerosols and light extinction at Mount Rainier..., Malm [/bib_ref]. Ammonium (NH 4 + ) concentrations are low, so only sulfate and nitrate are included. The remaining PM 2.5 mass is assumed to be carbon. the literature were the monthly, seasonal, or annual average of measured daily PM 2.5 concentrations. Therefore, caution needs to be exercised when comparing our results and the data presented in the literature.
Coarse particles collected in Kampala were primarily composed of crustal elements (i.e., Al, Ca, and Fe), indicating soil dust as the major source. Unlike PM 2.5 , coarse particles are formed in the atmosphere through mechanical forces, for example, the resuspension of road dust. Indeed, many unpaved road sections around the sampling area were observed by the authors. Other activities, such as agriculture, can also contribute to airborne coarse particles. These findings warrant further studies to examine the air pollutant source contributions.
Various anthropogenic sources appear to contribute to the elevated PM 2.5 levels in Kampala. In 2011, the Uganda Revenue Authority (URA) estimated there were 635,656 vehicles in Uganda and about 50% of those were operating in greater Kampala [bib_ref] 500, 000 cars hit national roads in 20 years, Ogwang [/bib_ref]. Between 4,000 and 5,000 vehicles enter the country monthly from various countries [bib_ref] Radioactive Japanese cars on the market, Obore [/bib_ref]. These vehicles are mostly second-hand and purchased reconditioned from Japan. To date, there are no age restrictions and requirements for emission restrictions in place that exist in industrialized countries [bib_ref] Energy profile of Uganda, Saundry [/bib_ref]. According to the Uganda National Environment Management Authority (NEMA), all the resulting daily traffic jams result not only in loss of valuable health and time but also lead to a loss of about 500 6 Journal of Environmental and Public Health million Uganda Shillings (about US $200,000) in burnt fuel; that is, about 140,000 liters of fuel are burnt by idling cars every day [bib_ref] 500, 000 cars hit national roads in 20 years, Ogwang [/bib_ref].
Furthermore, the Bank of Uganda estimates that by 2003 there were 800,000 small scale enterprises in the entire country of Uganda, providing employment and income generation opportunities to low income sectors of the economy, but at the same time, potentially contributing to air pollution. In addition, there is no mandatory garbage collecting system in Kampala, Uganda. The majority of inhabitants has been and continues to simply burn their waste which includes a lot of plastics and results in toxic smoke.
The increased source emissions of PM 2.5 may result from the rapid urbanization. The National Development Plan 2010-2015 estimates that Uganda's population could double to nearly 61 million in about 22 years. The city has spread from the original 7 hills it sat on at independence in 1962, to approximately 50 now. City planners at the time planned urban infrastructure for approximately 500,000 residents. Today, Kampala has approximately 1,819,200 inhabitants (extrapolated from a population of 1,420,200 in 2008 and a population of 1,659,600 on June 16th 2011) and an additional estimated 2.5 million commuters from outside populating the roads of the city every day. Ever since independence no major infrastructural changes were initiated to accommodate this increase. The City's governing bodies are unable to cope with this massive population influx and the resulting consequences such as overcrowding, uncollected garbage, increased individual motor-vehicle traffic in absence of an organized public transport system, mostly on unpaved roads and uncontrolled informal markets, and small industries.
Given the evidence of abundant vehicle engine emissions and biomass burning in the PM samples from this study, future studies will have to include analysis of organic and elemental carbon as well as carbon speciation to further understand the source attribution and its potential health impacts. The current study is limited by a small sample size, limited PM speciation, assessment of PM at one location only, and by lack of seasonal assessments. The results of this pilot study are indicative of unhealthy air in Kampala and suggest that a large number of people may be threatened by cerebro-and cardiovascular as well as respiratory tract diseases and even diabetes mellitus resulting from air pollution exposure. Greater awareness and more extensive research on air pollution in Kampala are required to confirm our findings, to identify pollution sources that contribute to personal exposure, and to develop air quality management plans and policies to improve public health.
# Disclosure
Melody Corry, who has M.S. and cand. Ph.D. degrees deceased five weeks before manuscript submission.
[fig] Figure 1: Map showing Kampala with detail of the sampling area. Sampling location indicated with red triangle. Source: Google maps (https://maps.google.com/maps?hl=en&tab=wl). [/fig]
[fig] Figure 3: Views of the center of Kampala on a clear day (top) and a day with extensive air pollution (bottom). [/fig]
[table] Table 1: Concentrations of all measured PM 2.5 species (ng/m 3 ). [/table]
[table] Table 4: Meteorological conditions during sampling. [/table]
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Clinical safety of combined therapy of immune checkpoint inhibitors and Viscum album L. therapy in patients with advanced or metastatic cancer
Background: Despite improvement of tumour response rates in patients with progressive and metastatic cancer, immune checkpoint inhibitors (ICM) induce toxicities in cancer patients. Viscum album L. (VA, mistletoe) extracts are applied as add-on cancer therapy especially in German speaking countries and within integrative and anthroposophical concepts with the goal to improve quality of life. The primary objective of this pilot observational cohort study was to determine the rate of adverse events (AE) related to ICM therapy with and without VA in patients with advanced or metastatic cancer in a certified Cancer Center.Methods: ICM or combined ICM/VA therapies were applied in patients with progressive or metastatic cancer. AE rates of both therapy groups were compared.Results: A total of sixteen cancer patients were treated with ICM: nivolumab (75%), ipilimumab (19%) or pembrolizumab (6%). The median age of the study population was 64 years (IQR 57.8; 69.3); 44% were male. Of the sixteen patients receiving ICM, nine patients received additional VA (56%; ICM/VA group) and seven did not (44%; ICM group). No statistically significant differences were seen between groups with respect to AE-rates (67% ICM/VA versus 71% ICM). Adjusted multivariate regression analysis revealed that concomitant application of VA did not alter the AE rate in ICM treated patients. 85% of AEs were expected ICM reactions. No AEs of grade 3 or greater were documented for the total study cohort.Conclusions: This is the first study evaluating the clinical safety profile of ICM in combination with VA in patients with advanced or metastatic cancer. The overall AE rate of the study cohort is comparable to AE rates of ICM treatment in the literature. Our data indicate a first impression that concomitant VA application may not alter ICM-induced AE rates. However, the nature of this study does not allow excluding possible immunological interactions between ICM and VA. Further prospective trials in larger study cohorts should focus on the assessment of safety aspects, clinical efficacy and health related quality of life in patients with combined ICM/VA therapy. Trial registration: DRKS00013335, retrospectively registered (November 27th, 2017) at the German Clinical Trials Register (www.drks.de).
# Background
Cancer cells are able to gain control over a number of inhibitory pathways that are important for controlling immune responses [bib_ref] High immunosuppressive burden in cancer patients: a major hurdle for cancer immunotherapy, Kalathil [/bib_ref] [bib_ref] Programmed death-1 immune checkpoint blockade in the treatment of hematological malignancies, Tsirigotis [/bib_ref]. By overexpressing programmed cell death protein ligands (PD-Ls) that bind to the immune checkpoint receptor programmed cell death protein 1 (PD-1), solid tumour cells can modulate T-cell activation in inflammatory cascades. Uncontrolled activation of the PD-1 receptor by cancer cells leads to anergy of antigen-specific T-cells and therefore diminishes their anti-tumour effectiveness. Blockade of the PD-1/ PDL-1 pathway with immune checkpoint mononclonal antibodies (ICM) nivolumab (Obdivo®) and pembrolizumab (Keytruda®) can help to improve lung cancer treatment as shown by several clinical trials [bib_ref] Nivolumab for advanced nonsmall cell lung cancer: an evaluation of a phase..., Ulmeanu [/bib_ref] [bib_ref] Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer, Borghaei [/bib_ref] [bib_ref] Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer, Brahmer [/bib_ref] [bib_ref] Pembrolizumab for the treatment of non-small cell lung cancer, Lim [/bib_ref].
Another immune checkpoint is the cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) which is expressed on activated T-cells that modulates peripheral Tcell expansion after antigen presentation [bib_ref] CTLA-4 and their ligands: who does what and to whom?, Sansom [/bib_ref]. By inhibiting CTLA-4, CTLs are (re-)activated and able to sufficiently help to reduce tumour burden. Clinical phase III studies with CTLA-4 ICM ipilimumab (Yervoy®) in metastatic melanoma have shown superiority in survival over tumour vaccination [bib_ref] Improved survival with ipilimumab in patients with metastatic melanoma, Hodi [/bib_ref] [bib_ref] Combined Nivolumab and Ipilimumab or monotherapy in untreated melanoma, Larkin [/bib_ref] and a survival benefit in combination with chemotherapy versus chemotherapy alone [bib_ref] Ipilimumab plus dacarbazine for previously untreated metastatic melanoma, Robert [/bib_ref].
Regarding their toxicity profile, PD-1/PDL-1 and CTLA-4 immune checkpoint blockade has resulted in mild, severe and even fatal adverse events (AEs) [bib_ref] Management of immune-related adverse events and kinetics of response with ipilimumab, Weber [/bib_ref] [bib_ref] Toxicity profile of approved anti-PD-1 monoclonal antibodies in solid tumors: a systematic..., Costa [/bib_ref]. Grade 3-4 AE rates of up to 12% due to PD-1/PDL1 blockade [bib_ref] Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer, Brahmer [/bib_ref] [bib_ref] Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4..., Weber [/bib_ref] [bib_ref] Pembrolizumab versus Ipilimumab in advanced melanoma, Robert [/bib_ref] and up to 18% due to CTLA-4 blockade [bib_ref] Improved survival with ipilimumab in patients with metastatic melanoma, Hodi [/bib_ref] [bib_ref] Toxicities of immunotherapy for the practitioner, Weber [/bib_ref] have been reported in cancer patients. Despite improved tumour response rates, the combination of anti-PD-1/PDL-1 and anti-CTLA-4 therapies also seems to potentiate grade 3-4 toxicities in cancer patients [bib_ref] Combined Nivolumab and Ipilimumab or monotherapy in untreated melanoma, Larkin [/bib_ref] [bib_ref] Nivolumab and ipilimumab versus ipilimumab in untreated melanoma, Postow [/bib_ref]. In addition, potentiation in toxicities has already been seen with ICM in combination with chemotherapy [bib_ref] Nivolumab in combination with platinum-based doublet chemotherapy for first-line treatment of advanced..., Rizvi [/bib_ref]. Due to their new and elevated toxicity profile it is crucial to carefully monitor AEs related to ICM, especially in combination with other antineoplastic agents [bib_ref] Management of toxicities of immune checkpoint inhibitors, Spain [/bib_ref]. Thus, the next era of immunotherapy will involve the search for safe combinatory anti-cancer agents which do not interfere with the PD-1/PDL-1 or CTLA-4 immunomodulatory mode of action and do not potentiate related toxicities.
Viscum album L. (VA), known as mistletoe, has a long traditional herbal history. It has effectively been utilized as an add-on therapy of cancer treatment in Europe, especially in German speaking countries and within integrative and anthroposophical concepts with the goal to improve quality of life [bib_ref] Mistletoe therapy in oncology, Horneber [/bib_ref] [bib_ref] Viscum Album [L.] extract therapy in patients with locally advanced or metastatic..., Troger [/bib_ref] [bib_ref] Survival of cancer patients treated with mistletoe extract (Iscador): a systematic literature..., Ostermann [/bib_ref] [bib_ref] Retrolective studies on the survival of cancer patients treated with mistletoe extracts:..., Ostermann [/bib_ref] [bib_ref] Efficacy and safety of long-term complementary treatment with standardized European mistletoe extract..., Bock [/bib_ref]. Integrative therapies, comprising a systematic approach towards complementary alongside conventional therapies, intend to reduce physical and emotional symptoms and improve healthrelated quality of life (HRQL) in cancer patients [bib_ref] Complementary therapies and integrative medicine in lung cancer: diagnosis and management of..., Deng [/bib_ref].
The field of integrative oncology is growing combining patient-centred integrative and conventional therapies in the management of cancer diseases that are safe and effective [bib_ref] Clinical practice guidelines on the use of integrative therapies as supportive care..., Greenlee [/bib_ref]. The quality of published clinical VA studies has been criticized to vary considerably and according to a Cochrane review "more high quality, independent clinical research is needed" [bib_ref] Mistletoe therapy in oncology, Horneber [/bib_ref]. The survival benefit of VA in cancer is still a matter of controversial discussion [bib_ref] Viscum Album [L.] extract therapy in patients with locally advanced or metastatic..., Troger [/bib_ref] [bib_ref] Survival of cancer patients treated with mistletoe extract (Iscador): a systematic literature..., Ostermann [/bib_ref] [bib_ref] Complementary therapies and integrative medicine in lung cancer: diagnosis and management of..., Deng [/bib_ref] [bib_ref] Health services research of integrative oncology in palliative care of patients with..., Axtner [/bib_ref] [bib_ref] Evidence-based clinical practice guidelines for integrative oncology: complementary therapies and botanicals, Deng [/bib_ref] mostly due to risks of bias and the heterogeneous quality of studies published so far. With regards to adverse effects the safety profile of VA has indicated that subcutaneous (s.c.) and intravenous (i.v.) applications of VA in cancer were safe with no serious AEs [bib_ref] Safety of intravenous application of mistletoe (Viscum Album L.) preparations in oncology:..., Steele [/bib_ref] [bib_ref] Adverse drug reactions and expected effects to therapy with subcutaneous mistletoe extracts..., Steele [/bib_ref] [bib_ref] Use and safety of intratumoral application of European mistletoe (Viscum Album L)..., Steele [/bib_ref]. A meta-analysis of clinical studies that applied VA Iscador preparations concluded that it might have beneficial effects in psychosomatic self-regulation in addition to short-term quality of life-improving effects in cancer patients. The experimentally assessed mode of action of VA comprises induction of apoptosis, inhibition of cell proliferation and angiogenesis of the tumour cells as well as anti-inflammatory and immunomodulatory effects [bib_ref] Suppressive effect of a standardized mistletoe extract on the expression of activatory..., Lee [/bib_ref] [bib_ref] Immunologic effector mechanisms of a standardized mistletoe extract on the function of..., Heinzerling [/bib_ref] [bib_ref] Influence of mistletoe lectins and cytokines induced by them on cell proliferation..., Thies [/bib_ref] [bib_ref] Interaction of a standardized mistletoe (Viscum Album) preparation with antitumor effects of..., Weissenstein [/bib_ref]. Thus, the immunomodulatory nature of VA raises the question of whether there are immunologic interactions between VA and inhibitors of the PD-1/PDL-1 or CTLA-4 pathways that might lead to increased toxicity for cancer patients. On the other hand recent results of our group revealed that the adverse event rate of targeted therapy was significantly reduced in cancer patients when VA was concomitantly applied. This is in line with the observation that VA is applied in integrative oncology concepts concomitantly to adjuvant antineoplastic treatment to improve tolerability of chemotherapy-induced toxicity [bib_ref] Efficacy and safety of long-term complementary treatment with standardized European mistletoe extract..., Bock [/bib_ref] [bib_ref] Mistletoe as complementary treatment in patients with advanced non-small-cell lung cancer treated..., Bar-Sela [/bib_ref] [bib_ref] Immune modulation using mistletoe (Viscum Album L.) extracts Iscador, Bussing [/bib_ref]. We hypothesize that addition of VA therapy in the present study would either diminish or stabilize the known side effects of antineoplastic ICM treatment.
# Methods
## Study design
Safety and efficacy of anti-PD-1/PDL-1 and CTL-A4 ICM nivolumab, ipilimumab and pembrolizumab with or without concomitant VA therapy were examined in a pilot observational open cohort study. The primary outcome of the study was to investigate the occurrence of AEs during treatment with ICM with and without VA to assess the question of whether additional VA influences the AE rate in ICM treated patients with advanced or metastatic cancer. The secondary outcome was the explorative comparison of the disease response rate and overall survival in ICM and ICM/VA treated patients.
## Description of study participants
The Hospital Havelhöhe (GKH) is a certified Cancer Centre demonstrating highest quality controlled cancer treatment and outcome for three organ classes (lung, breast and colorectal cancer) in accordance with certification guidelines of the Deutsche Krebsgesellschaft (DKG; German Cancer Association) and the Deutsche Krebshilfe (DKH; German Cancer Aid). Patients with advanced or metastatic cancer receiving ICM with or without concomitant VA therapy and being part of the Network Oncology (NO) registry were enrolled in the study between October 2013 and May 2016 . Dates of first diagnosis for advanced and metastasized disease ranged from August 2008 until September 2015.
The following patients were included: patients who were 18 years or older and of either gender with advanced or metastatic cancer, having given written informed consent, being registered in the NO and who received ICM with or without concomitant VA therapy. Patients were excluded if they had any of the following co-morbidities: active auto-immune diseases, symptomatic interstitial lung diseases, non-treated brain metastases, primary central nervous system-melanoma, human immune deficiency virus, hepatitis B, or hepatitis C. Written informed consent has been obtained. The Network Oncology (NO) registry study has been approved by the ethical committee of the Medical Association Berlin (Eth-27/10).
ICM treatment was i.v. applied according to the Summary of Product Characteristics (SmPC). VA therapy was applied s.c. according to SmPC. Off-label i.v. application was performed in individual cases. The rationale for VA application in patients of the current study was the improvement of HRQL and self-regulation in cancer patients by meliorating cancer and therapy related symptoms. I.v. or s.c. VA was administered at the discretion of the physician. Patients who agreed to the combined treatment were allocated to the ICM/VA group. The others were allocated to the ICM group.
## Sample size determination
A study cohort consisting of a total of twelve patients (ICM group: three patients, ICM-VA group: nine patients) would be needed to detect a significant increase in the relevant (immunologic) side effect with 80% power using an alpha value of 0.05. The expected mean difference (0. [bib_ref] Viscum Album [L.] extract therapy in patients with locally advanced or metastatic..., Troger [/bib_ref]
## Data source and assessment
Demographic data as well as information on diagnosis, co-morbidities and previous treatment regimen were retrieved from the NO registry. NO is an accredited clinical registry of hospitals and out-patient practitioners specialised in IO [bib_ref] Network oncology (NO)-a clinical cancer register for health services research and the..., Schad [/bib_ref]. Retrieved data included application procedures of ICM and VA therapy and related AEs. Date of death and last contact date were retrieved from the NO registry. Complete response, partial response (PR), progressive disease (PD) and stable disease (SD) were assessed according to revised RECIST guidelines, Version 1.1 for solid tumours [bib_ref] New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1), Eisenhauer [/bib_ref].
AEs were classified as preferred terms according to the Medical Dictionary for Regulatory Activities (Med-DRA®) Version 15.0 and grouped by System Organ Classes (SOC). AEs, serious AEs, adverse drug reaction (ADR) were designated according to ICH guidelines topic E2A. Accordingly, an AE is "any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment". An ADR is defined as "a response to a drug which is noxious and unintended and which occurs at doses normally used in man for prophylaxis, diagnosis, or therapy of disease or for modification of physiological function". In terms of severity, AEs were evaluated according to the Common Terminology Criteria for AEs (CTCAE) v4.03and designated as serious or non-serious according to ICH guidelines.
VA-or ICM-related AEs were immediately reported by physicians. If VA and ICM were given on the same day, VA was administered as the first infusion, before ICM treatment, thus, acute ADRs were attributable to VA treatment.
# Statistical methods
Univariate two-sided Fisher's exact test was performed to detect differences in AE rates and tumour response rates between ICM and ICM/VA group. Multivariate regression analyses with binary outcome of experienced Flow chart of the study population. ICM, immune checkpoint monoclonal antibody; VA, Viscum album L., n, number of patients AE (yes/no) were performed to identify safety associated factors in ICM treated patients adjusting for age (in years), gender (male/female), VA treatment during ICM (yes/no), previous treatment with platinum-based chemotherapy (yes/no), with targeted therapy (yes/no), and with VA treatment (yes/no) and number of ICMcycles greater than ten (yes/no). Continuous variables were described as median with interquartile range (IQR); categorical variables were summarised as frequencies and percentages. Data distributions were inspected graphically using box plots and histograms and were arithmetically examined for skewness. Stepwise backward variable selection with Akaike information criterion was performed for consideration of parameters within regression models. P-values <0.05 were considered to be significant. All statistical analyses were performed using the software R (Version 3.3.0).
# Results
## Baseline characteristics
Sixteen cancer patients were treated with PD-1/PDL-1 or CTLA-4 ICM at GKH between October 2013 and May 2016. [fig_ref] Table 1: Baseline characteristics of patients receiving ICM [/fig_ref] shows the baseline characteristics of the patients. Of the sixteen patients 44% were male. The median age was 64 years (IQR: 57.8-69.3 years). Eleven patients had lung cancer (69%), four patients had a malignant melanoma (25%) and one patient had a pleural mesothelioma (6%). In twelve patients M1c-stage metastases were detected (75%). Predominant co-morbidities were respiratory disorders (10 patients, 62.5%), followed by blood & lymphatic disorders (6 patients, 37.3%), and cardiac disease disorders (5 patients, 31.3%), data not shown. Three of the ten patients with respiratory disorders had chronic obstructive pulmonary disease (COPD). Fourteen patients showed history of VA treatment (88%) (data not shown). Seven patients were pre-treated with at least two, and four patients with three or more anti-neoplastic systemic therapies including chemotherapy or targeted therapy. 50% of all patients received previous treatment with platinum-based chemotherapy, with the most frequently applied combination being carboplatin/ vinorelbine (38%, n = 3), followed by cisplatin/vinorelbine and cisplatin monotherapy (each 25%, n = 2; data not shown). 13% (n = 2) of the study population were previously treated with targeted therapy (i.e. erlotinib or vemurafenib).
## Icm therapy and combined icm/va therapy
Sixteen patients were treated with ICM therapy: twelve patients with nivolumab (75%), three patients with ipilimumab (19%), and one with pembrolizumab (6%). Of the sixteen patients, nine (56%) ICM-treated patients received VA extracts during the same interval while seven (44%) received no VA treatment during ICM treatment [fig_ref] Table 2: Dosage and application form of concomitant VA treatment during ICM therapy [/fig_ref]. Of the ICM/VA treated patients, two received s.c. VA (22.3%), five s.c VA. (55.6%), and two patients received both i.v. and s.c. VA (22.3%). The median duration of VA therapy was 84 days with a range of 1-196 days of treatment. The median duration of ICM treatment was also 84 days with a range of 31-196 days (data not shown). One patient received percutaneous radiation for the treatment of metastases during the last day of ICM treatment (lung cancer). None of the sixteen patients were treated with chemotherapy or underwent surgery during ICM therapy.
Clinical safety of ICM versus combined ICM/VA treatment [fig_ref] Table 3: Frequency of AEs during treatment with ICM with or without VA therapy [/fig_ref] comprises all AEs that were documented during ICM or combined ICM/VA treatment. Each patient having experienced AEs has been designated with a letter (a-l; j was omitted). The total AE-frequency in the current study was 68.8% with eleven patients experiencing at least one AE. Two ADRs of moderate nature occurred in the study population. No grade 3 and 4 AEs (CTC, version 4.03), no serious AEs, and serious adverse reactions (ICH)were documented for the total study cohort. The most frequent (>10%) AEs in the current study were malaise (18.8%), pyrexia (12.5%), bronchitis (12.5%), and skin reaction (12.5%, [fig_ref] Table 3: Frequency of AEs during treatment with ICM with or without VA therapy [/fig_ref].
With regards to treatment groups, five patients experienced at least one AE (71.4%) in the ICM group. One patient experienced one moderate ADR (14.3%), nausea, which was documented to be related to systemic ICM treatment. Further, in one patient (14.3%) ICM therapy (nivolumab) was interrupted for seven days due to bronchopneumonia, and symptoms were treated according to SmPC until disappearance. ICM therapy was then continued without re-appearance of pneumonia. Two patients that received ICM treatment experienced immune-related AEs (bronchitis: 14.3%, rash: 14.3%).
In comparison, of the nine patients treated with combined ICM/VA therapy, three did not experience any AE (33%). Six patients experienced at least one AE (66.7%), a rate which was 4% lower than the AE rate seen in the ICM group (not significant, p > 0.99). One patient treated with ICM/VA experienced one ADR (11%), nausea and vomiting. In this case the severity of the ADR was moderate and was indicated to be related to s.c. Abnobaviscum application. The VA therapy was discontinued and the patient recovered. Here the symptom lasted for one day only. In the ICM/VA group two patients experienced immune-related AEs (bronchitis: 11.1%, rash: 11.1%). Adjusted multivariate regression analysis revealed no significant association between ICM/VA application and AE rate (OR: 1.467, 95%CI: 0.183-11.693 p = 0.720). Furthermore, we could show by adjusted multivariate analysis that the following variables, greater cycle-numbers (>10) of either ICM treatment alone or combined ICM/ VA treatment, previous treatment with platinum-based chemotherapy, with targeted therapy or with VA were not associated with occurrence of AEs in the total patient cohort (data not shown).
## Clinical efficacy of icm versus combined icm/va treatment
From sixteen patients nine patients showed progressive disease (56.3%), four showed stable disease (25.0%) and three patients showed partial disease response (18.8%, [fig_ref] Table 4: Tumour response after treatment with ICM with or without VA therapy [/fig_ref]. In regards to treatment groups, progressive disease was observed in five patients treated with ICM (71.7%) and four patients (44.4%) treated with ICM/VA. In each treatment group two patients had stable disease (ICM group 28.6% versus ICM/VA group 22.2%). Partial response was only seen in patients treated with combined ICM/VA therapy (n = 3, 33.3%). No significant differences were detected between the groups. In the subgroup showing partial response, combined ICM/VA treatment resulted in stabilized size and number of metastases in one patient and size reduction of metastases and lymph nodes in two patients. In the cases of size reduction, one patient received nivolumab in combination with increasing doses of 20 to 60 mg i.v. Abnobaviscum and one patient received nivolumab in combination with 0.2 mg s.c. Abnobaviscum preparations.
The Kaplan-Meier curves for overall survival (OS) are shown in [fig_ref] Figure 2: Kaplan-Meier curves by treatment arm [/fig_ref] for a subgroup of eleven patients with advanced or metastasized lung cancer treated either with nivolumab (ICM) or nivolumab and VA (ICM/VA). Disease characteristics revealed squamous (45.5%), nonsquamous (36.4%) and not specified NSCLC (18.2%). The median survival for all stage III/IV NSCLC patients combined was 28 months (95% CI: 27, NA), with a range of 13-43 months. By the end of the observational period five patients (31.3% of the subgroup) have died from cancer.
# Discussion
The results of the present study reveal that the overall AE rate (69%) experienced by the advanced or metastatic cancer study group was within the range or lower than the reported rate for AEs for nivolumab (60-78%), pembrolizumab (71-82%) and combination of ipilimumab/ nivolumab (91-100%) [bib_ref] Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies, Naidoo [/bib_ref]. Importantly, no grade 3 or 4 AEs according to CTC, no serious AEs as well as no serious adverse reactions according to ICHwere documented for the total study cohort. With regards to treatment groups, concomitant application of VA extracts did not alter the AE rate in ICM treated patients. Furthermore, neither s.c. nor off-label utilization of i.v. mistletoe application increased the AE rate in ICM treated patients in the current study. These results are in line with published tolerability of VA during chemotherapy and safety of i.v. and s.c. VA therapy [bib_ref] Safety of intravenous application of mistletoe (Viscum Album L.) preparations in oncology:..., Steele [/bib_ref] [bib_ref] Adverse drug reactions and expected effects to therapy with subcutaneous mistletoe extracts..., Steele [/bib_ref].
According to SmPCs the most frequently occurring published AEs (>10%) during therapy with ICM are fatigue, reduced appetite, nausea, diarrhoea, skin reactions, pruritus, vomiting, abdominal pain, urticaria, arthralgia, and malaise. The present study resembles this picture with respect to skin reactions and malaise as most frequent AEs.
With respect to immune-related (ir) adverse events, only ir-bronchitis (12.5%) and ir-skin reactions (12.5%) were documented in the whole present cohort. Recent literature reveals that approximately 25% of patients treated with ICM monotherapy experienced immunerelated rash (all-grade). Whereas we observed an immune-related rash rate to be less than the published rates, the ir-bronchitis frequency in the present study cohort seems to be a log higher compared to literature: according to the SmPC of nivolumab, bronchitis rates are rather uncommon (up to 1 in 100 patients) and irpneumonitis rates account for around 3% of treated patients. However, in both bronchitis cases, the patients already showed signs of pulmonary comorbidity. These were a cough in one patient and pneumonia in the other. The immune-related symptoms resolved without the necessity of hospitalization, of radiologic, endoscopic, or operative intervention, of permanent discontinuation of treatment or application of high-dose corticosteroids. Interestingly, the observed ir-rash frequency in the total study cohort was one to two logs higher than the published ir-rash frequency of intravenous (2%) or subcutaneous (0.4%) VA treatment in cancer patients [bib_ref] Safety of intravenous application of mistletoe (Viscum Album L.) preparations in oncology:..., Steele [/bib_ref] [bib_ref] Adverse drug reactions and expected effects to therapy with subcutaneous mistletoe extracts..., Steele [/bib_ref]. However, as ir-rash frequencies are comparable between the ICM-VA and ICM group in the present study we assume that these immune-related skin reactions rather derive from ICM treatment itself. With regards to comparability of irAEs between both groups we further assume that the irAE rate was not altered due to additional VA treatment. Most AEs (85%) observed in the present study were expected AEs according to ICM SmPC (e.g. pyrexia and malaise); however, unexpected AEs were chronic bronchitis, decubitus ulcer and marasmus seen in two patients. The chronic bronchitis may have been related to the COPD status in one patient. Marasmus and decubitus ulcer were seen in the other patient that was treated with 180 mg Ipilimumab and 200 mg i.v. Helixor P and may be explained by the patient's reduced general condition described by being bedridden, experiencing obstipation, reduced appetite and needing to be taken care of.
Overall survival analysis was performed but due to the small numbers of patients, no definitive conclusions can be drawn from these data for a comparison of beneficial effects on survival. The present results regarding tumour progression status are, to our knowledge, the first preliminary clinical data for combined VA/ICM treatment. At the date of analysis, none of the study subjects had achieved complete disease response. While complete response has been reported in ICM studies (0.7% for nivolumab and 2-3% for pembrolizumab, we suspect that the absence of patients showing complete response in our study is related to the small sample size. The disease response rates in the present study were in line with published ICM data. Interestingly, partial remission was seen only in patients treated with the combined VA/ICM therapy regimen, therefore the possibility of synergistic antineoplastic effects between VA and ICM cannot be excluded. However, no final conclusions are to be drawn regarding the influence of combined treatment on disease response in the present study due to limited sample size.
Besides inhibiting proliferation and inducing apoptosis [bib_ref] Interaction of a standardized mistletoe (Viscum Album) preparation with antitumor effects of..., Weissenstein [/bib_ref] VA compounds lectins and lipophilic VA triterpenes have been shown to possess immunomodulatory properties in vitro [bib_ref] Enhanced dendritic cell maturation by the B-chain of Korean mistletoe lectin (KML-B),..., Kim [/bib_ref] [bib_ref] Plant lectin derived from Viscum Album induces cytokine gene expression and protein..., Hostanska [/bib_ref] and in vivo [bib_ref] Immunological response to mistletoe (Viscum Album L.) in cancer patients: a four-case..., Gardin [/bib_ref] , inhibit proliferation and induce apoptosis in breast carcinoma cells in vitro. VA lectins (VAL) increase expression of co-stimulatory molecules in blood derived dendritic cells in a Toll-like receptor 4 dependent way [bib_ref] Enhanced dendritic cell maturation by the B-chain of Korean mistletoe lectin (KML-B),..., Kim [/bib_ref]. They increase the percentage of Human Leukocyte Antigen-antigen-D-Related-positive T lymphocytes in vitro [bib_ref] Enhanced dendritic cell maturation by the B-chain of Korean mistletoe lectin (KML-B),..., Kim [/bib_ref]. Incubation of peripheral blood mononuclear cells with VAL enhances the expression and secretion of a plethora of cytokines [bib_ref] Plant lectin derived from Viscum Album induces cytokine gene expression and protein..., Hostanska [/bib_ref]. In vivo studies revealed IL-12 dependent activation of natural killer cells by VAL [bib_ref] Effect of a recombinant lectin, Viscum Album agglutinin on the secretion of..., Hajto [/bib_ref]. These immunomodulatory properties, in addition to the associated sound safety profiles [bib_ref] Safety of intravenous application of mistletoe (Viscum Album L.) preparations in oncology:..., Steele [/bib_ref] [bib_ref] Adverse drug reactions and expected effects to therapy with subcutaneous mistletoe extracts..., Steele [/bib_ref] , observed improvements in HRQL and self-regulation [bib_ref] Mistletoe as complementary treatment in patients with advanced non-small-cell lung cancer treated..., Bar-Sela [/bib_ref] [bib_ref] Targeting inflammation in cancerrelated-fatigue: a rationale for mistletoe therapy as supportive care..., Bock [/bib_ref] [bib_ref] Quality of life in breast cancer patients during chemotherapy and concurrent therapy..., Eisenbraun [/bib_ref] make VA extracts a potentially attractive add-on therapy in antineoplastic concepts. Clinical trials assessing the addition of VA applications to conventional treatment regimens have shown that VA can reduce chemotherapy ADRs and improve tolerability of chemotherapy [bib_ref] Efficacy and safety of long-term complementary treatment with standardized European mistletoe extract..., Bock [/bib_ref] [bib_ref] Mistletoe as complementary treatment in patients with advanced non-small-cell lung cancer treated..., Bar-Sela [/bib_ref]. The present study did not show significant reduction in cancer or ICM-related AEs by additional VA treatment, possibly due to the small number of patients enrolled and the limited observation duration. Furthermore, due to the nature of the current study, long-term side effects could not be determined. Nevertheless, this pilot study provides a first impression on the safety aspects of concomitant VA/ICM treatment in patients with advanced or metastatic cancer. Nevertheless, relevant pharmacological interferences between the known immunomodulatory mechanisms of VA and ICM therapy need to be investigated.
# Conclusions
The present pilot study gives a first insight into application of new generation immuno-oncological treatment with the natural immune-stimulant VA in patients with advanced or metastatic cancer. The overall AE rate for ICM treatment observed in the current study was in line with previously published studies. Furthermore, our data indicate a first impression that i.v. and s.c. applications of VA extracts during therapy with ICM may not alter ICM-induced AE rate, and that immune-related AE frequency was balanced in the ICM and ICM/VA group. However, the nature of the present study does not allow excluding possible immunological interactions between ICM and VA. In the light of newly approved cancer indications in the field of immuno-oncology, further studies in larger patient cohorts and respective experimental designs are warranted to assess immunological interactions between these two immunological active substances, clinical efficacy and HRQL in cancer patients receiving combined ICM/VA therapy.
[fig] Figure 2: Kaplan-Meier curves by treatment arm; advanced or metastasized lung cancer. Kaplan-Meier curves for survival by treatment arm during total observation period (55 months, 4.6 years) and median survival in months for patients with advanced or metastasized lung cancer. ICM, immune checkpoint inhibitor nivolumab, ICM-VA, therapy with immune checkpoint inhibitors and concomitant Viscum album L.; CI, confidence interval. *based on log rank test [/fig]
[table] Table 1: Baseline characteristics of patients receiving ICM [/table]
[table] Table 2: Dosage and application form of concomitant VA treatment during ICM therapy [/table]
[table] Table 3: Frequency of AEs during treatment with ICM with or without VA therapy [/table]
[table] Table 4: Tumour response after treatment with ICM with or without VA therapy [/table]
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Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics
Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We also discuss the effects of the NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems. We end our review discussing the limitations and challenges that should guide the quest for an efficient extraction method that can be integrated in a POC-Dx system.
# Introduction
Nucleic acid extraction (NAE) is one of the most pivotal steps in molecular biology, being routinely used in many areas of the biological and medical sciences, as this procedure marks a starting point in any molecular diagnostic kit [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref]. This crucial procedure has been known for over a century and has developed substantially over the last decades. However, some progress still has to be achieved so that NAE protocols leave the laboratory settings into the "real world" of point-of-care diagnostics (POC-Dx).
Nowadays, it is known that intracellular nucleic acids (NAs) may be broadly categorized as genomic (or chromosomal), plasmids, and different types of RNAs. Although RNAs possess uracil while DNAs present thymine [bib_ref] Why does DNA contain thymine and RNA uracil?, Lesk [/bib_ref] , nucleic acids exhibit similar basic biochemical properties but might have quite distinct tridimensional structures (genomic, plasmid, tRNA, mRNA, rRNA, etc). However, despite the structural differences, the most commonly used methods described in the present text can be applied to DNA in its many organizational formats (chromosomal, plasmid, etc.), as well as RNA and its multidimensional formats (mRNA, rRNA, tRNA, miRNA, etc.) with minor modifications [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref] [bib_ref] Methods for extracting genomic DNA from whole blood samples: current perspectives, Chacon-Cortes [/bib_ref] [bib_ref] Nucleic acid extraction techniques and application to the microchip, Price [/bib_ref].
NAE can be roughly divided into four steps, which can be modulated depending on the sample and downstream applications: (i) cell disruption; (ii) removal of membrane lipids, proteins, and other nucleic acids, (iii) nucleic acid purification/binding from bulk; and (iv) nucleic acid concentration [bib_ref] Mechanical/physical methods of cell distribution and tissue homogenization, Goldberg [/bib_ref].
Cell disruption or disintegration can be achieved by physical and/or chemical methods, whose main aim is to disrupt the cell wall and/or cellular membranes. Disruption methods are mainly based on properties of the sample and for this purpose a wide range of tools and approaches are used either alone or combined to achieve tissue/cell disruption. Lytic enzymes, chaotropic agents, and different types of detergents are the main components of chemical lysis, while mechanical method disrupts the cells by grinding, shearing, bead beating, and shocking [bib_ref] Guide to the Disruption of Biological Samples, Burden [/bib_ref]. It is interesting to note that if one technique does not yield good results, another might prove successful. Osmotic shock methods have yielded, in certain cases, better results than common NA purifications protocols such as phenol-chloroform extraction and bead beating [bib_ref] A novel procedure for total nucleic acid extractionfrom small numbers of eimeria..., Kaya [/bib_ref]. Not only is cell disruption important for DNA extraction, but it also plays a crucial role in the biopharmaceutical industry, as many recombinant proteins and other important constituents of the cell can be recovered through this process [bib_ref] Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells, Peternel [/bib_ref] [bib_ref] Isolation of cell-free bacterial inclusion bodies, Rodríguez-Carmona [/bib_ref] [bib_ref] A comparative study on effective cell disruption methods for lipid extraction from..., Prabakaran [/bib_ref]. Another approach for cell disruption is the use of different methods in combination. A good example is the case for enzymatic lysis, where many protocols use proteases to free the NA from its protective protein scaffold. Also, the inactivation of cellular nucleases that come free into solution in order to protect the new protein-free NA is crucial [bib_ref] Enzymatic lysis of microbial cells, Salazar [/bib_ref]. A combination of detergents and chaotropic salts in a single solution is used to solubilize cell wall and or cell membrane and inactivate intracellular nucleases [bib_ref] Comparison of the bile salts and sodium dodecyl sulfate stress responses in..., Flahaut [/bib_ref]. Mechanical disruption, on the other hand, makes use of force to extract out constituents of the cell. A classic example of grinding in biosciences is the use of mortar and pestle [bib_ref] Mechanical/physical methods of cell distribution and tissue homogenization, Goldberg [/bib_ref] , which is nowadays optimized with the use of liquid nitrogen (when allowed by the sample). Cells walls can also be disrupted by the shock waves created by rapid changes in pressure elicited by sonication or cavitation [bib_ref] Disruption and protein release by ultrasonication of yeast cells, Liu [/bib_ref] [bib_ref] Comparative study of fungal cell disruption-scope and limitations of the methods, Klimek-Ochab [/bib_ref] [bib_ref] Ultrasonic cavitation for disruption of microalgae, Greenly [/bib_ref]. Other mechanical tools available for cell disruption are shearing, which use a tangential force to make a hole in the cell [bib_ref] Mechanical/physical methods of cell distribution and tissue homogenization, Goldberg [/bib_ref] , and bead beating, which uses different glass or steel beads to rupture tough cell wall as mentioned by Bunge et al. [bib_ref] Mechanical disruption of Arthrobacter sp. DSM 3747 in stirred ball mills for..., Bunge [/bib_ref]. These processes are briefly summarized in NAE methods encompass extraction of both DNA and RNA but can be more broadly characterized into chemically driven or solid-phase methods; both contain the four steps mentioned above [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref] [bib_ref] Methods for extracting genomic DNA from whole blood samples: current perspectives, Chacon-Cortes [/bib_ref] [bib_ref] Nucleic acid extraction techniques and application to the microchip, Price [/bib_ref]. In the next sections, we will review the working principle of and/or rationale for the main methods used nowadays in the biological and medical sciences. Since molecular diagnostics rely heavily on techniques that start with NAE, we will also discuss some of the basic features of devices available for POC-Dx, culminating with the challenges and limitations of adapting NAE methods to point-of-care diagnostic tests.
# Chemically driven methods
These methods rely on biochemical properties of the cellular components to elicit the desired molecular separation and might exhibit preference or exclusivity in extracting DNA or RNA, depending on its intrinsic characteristics.
## Cesium chloride (cscl) gradient centrifugation with ethidium bromide (etbr).
This technique is mainly based on the phenomenon of buoyant and specific density. Ethidium bromide (EtBr) is an intercalating agent, thus reporting the location of the double-stranded DNA under UV-light and allowing the easy visual separation of the supercoiled BioMed Research International 3 and nonsupercoiled DNA molecules. The basic mechanism by which EtBr separates the two molecules is decreasing the buoyant density of comparatively linear molecules. After the ultracentrifugation, CsCl has to be dialyzed of the collected DNA. The method can be used to extract DNA from bacteria, although a large-scale culture is needed; this method can be used for purification of various forms of DNA, such as chromosomal, plasmid DNA, rDNA, or mitochondrial DNA [bib_ref] Rapid isolation of animal mitochondrial DNA in a small fixed-angle rotor at..., Carr [/bib_ref]. Being sensitive and provider of good yields of pure DNA, the method is laborious, time-consuming, and costly as compared to other purification protocols. Furthermore, EtBR can affect downstream applications, such as PCR, cloning, and DNA sequencing [bib_ref] Effects of ethidium bromide and SYBR(5) Green I on different polymerase chain..., Nath [/bib_ref]. There is concern about using EtBr, which is known to cause genotoxicity and frame shift mutations. For mice, nontoxic doses up to 50 mg/kg have been used, for cattle, up to 1 mg/kg of body weight. However, the concentration used in gel staining solutions (0.25-1 g/mL) is below the level of toxicity, even though care is suggested in handling EtBr [bib_ref] Effects of ethidium bromide and SYBR(5) Green I on different polymerase chain..., Nath [/bib_ref] [bib_ref] Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and..., Ohta [/bib_ref].
## Guanidinium thiocyanate-phenol-chloroform extraction.
A guanidinium thiocyanate-(GuSCN-) phenol-chloroform mixture allows for RNA extraction in a single-step procedure, as demonstrated by Chomczynski and Sacchi [bib_ref] Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Chomczynski [/bib_ref]. Prior to the development of guanidinium method, phenol extraction was normally used for extraction in a two-step, laborious process. The method was modified successively over time, starting from Ullrich et al. [bib_ref] Rat insulin genes: construction of plasmids containing the coding sequences, Ullrich [/bib_ref] who used guanidinium thiocyanate instead of guanidinium chloride for RNA isolation, followed later on by using GuSCN combined with extended hours of ultracentrifugation and a CsCl cushion. In order to enhance the quality of the final nucleic acid, the technique was improved by using guanidinium thiocyanate and phenol-chloroform with a shorter centrifugation time [bib_ref] The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something..., Chomczynski [/bib_ref]. Despite being less soluble in water than guanidine hydrochloride, another common salt of guanidine, GuSCN has stronger denaturing properties because both its ions are chaotropic.
The basic principle of the method is the separation of RNA from DNA and proteins after extraction with an acidic solution, which consists mainly of GuSCN, sodium acetate, phenol, and chloroform, followed by centrifugation. Total RNA remains in the upper aqueous phase, while most of DNA and proteins part remain either in the interphase or in the lower organic phase under acidic condition. Total RNA is then recovered through precipitation by isopropanol and can be used for subsequent process. The original method was carried out in mammalian tissue but, later on, it has been used for plants with some modification [bib_ref] A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques..., Meng [/bib_ref] , animals [bib_ref] Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Chirgwin [/bib_ref] , and cultured cell tissues as well [bib_ref] The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something..., Chomczynski [/bib_ref] [bib_ref] Isolation and characterization of RNA from snap-frozen tissues and cultured cells, Murnane [/bib_ref]. Optimum pH plays a critical role in the separation process as DNA partitions to the organic phase under acidic condition (pH [bib_ref] Methods for extracting genomic DNA from whole blood samples: current perspectives, Chacon-Cortes [/bib_ref] [bib_ref] Nucleic acid extraction techniques and application to the microchip, Price [/bib_ref] [bib_ref] Mechanical/physical methods of cell distribution and tissue homogenization, Goldberg [/bib_ref] or to the aqueous phase at neutral pH (pH 7-8). The main drawback of this method is that phenol and chloroform are both hazardous chemicals [bib_ref] The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something..., Chomczynski [/bib_ref]. This reagent is commercially available with different names, such as Sigma-Aldrich TRI Reagent5 and Thermo Fisher TRIzol5 Reagent. High purity and yield of the extracted NA are the hallmark of this procedure.
## Cetyltrimethylammonium bromide (ctab) extraction.
Cetyltrimethylammonium bromide extraction method is mainly used for plant samples and their parts, such as leaves, seeds, and grains. The method is used for various food samples as well. The basic composition of CTAB extraction buffer includes 2% CTAB at alkaline pH, but, like many other extraction protocols, CTAB has been modified according to the need of each sample [bib_ref] Detection of genetically modified crops and their derivatives: critical steps in sample..., Terry [/bib_ref]. CTAB works by precipitating nucleic acids and acidic polysaccharides in low ionic strength solutions, while proteins and neutral polysaccharides remain in solution. Next, the CTAB-nucleic acid precipitated complex is solubilized at high-salt concentrations, leaving the acid polysaccharides in the precipitate [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref]. During the precipitation and washing steps, CTAB method uses various organic solvents and alcohols such as phenol, chloroform, isoamyl alcohol, and mercaptoethanol. The main drawback of this procedure is that it is time-consuming and makes use of toxic chemicals like phenol and chloroform. Moreover, CTAB extracted DNA requires further purification to avoid inhibition of PCR analyzes [bib_ref] Influence of DNA extraction methods, PCR inhibitors and quantification methods on realtime..., Demeke [/bib_ref].
## Chelex5
Extraction. Chelex (Bio-Rad Laboratories, CA, USA) is a chelating resin frequently used in the field of forensics for DNA extraction from various sources, such as hair, blood stain cards, and buccal swabs [bib_ref] Chelex 100 as a medium for simple extraction of DNA for PCR-based..., Walsh [/bib_ref]. According to [bib_ref] Chelex 100 as a medium for simple extraction of DNA for PCR-based..., Walsh [/bib_ref] , boiling in the presence of Chelex can increase the signal during PCR amplification of relatively minor amount of DNA, possibly by inhibiting DNA degradation by chelating metal ions which cause DNA breakdown at high temperature and lower ionic conditions. Chelex is a styrene divinylbenzene copolymer containing paired iminodiacetate ions, which are used as chelators for polyvalent metal ions [bib_ref] A comparison of methods for forensic DNA extraction: Chelex-1005 and the QIAGEN..., Phillips [/bib_ref]. This technique is interesting as it is quick, has few manipulating steps, and does not use hazardous chemicals such as phenol/chloroform. Its main drawback is the inability to efficiently remove PCR inhibitors from complex samples due to the lack of purification steps [bib_ref] PCR inhibitors-occurrence, properties and removal, Schrader [/bib_ref]. This method is also not suitable for restriction fragment length polymorphism (RFLP) analyses, because exposure of DNA to the high temperature and alkalinity of this protocol results in denaturation and breakage of DNA.
## Alkaline extraction.
Alkaline extraction method is dedicated to plasmid DNA isolation, described by Bimboim and Doly [bib_ref] A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Bimboim [/bib_ref]. The basic principle of this method is selective alkaline denaturation of high molecular weight chromosomal DNA, while covalently bond circular plasmid DNA remains intact. After neutralization, chromosomal DNA renatures and makes an insoluble precipitate, while plasmid DNA remains in the supernatant. This method is useful for both small and large DNA plasmids [bib_ref] A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Bimboim [/bib_ref].
The method involves harvesting the bacteria of interest from culture media and exposing them to alkaline solution (consisting basically of SDS and NaOH). SDS act as detergent to lyse the cells and denature proteins, while alkaline condition denatures genomic DNA, plasmid DNA, and proteins. Potassium acetate (pH 5.2) addition neutralizes the mixture and results in renaturation of plasmid as well as genomic DNA. Further addition of ethanol (or : Summary of advantages and disadvantages of the main NAE methods. GuSCN, guanidine thiocyanate; CsCl, cesium chloride; EtBr, ethidium bromide; CTAB, cetyltrimethylammonium bromide.
# Method
Advantage Disadvantage Reference (1) GuSCN-phenol-chloroform extraction High purity and yield of DNA or RNA Hazardous chemicals [bib_ref] Effects of ethidium bromide and SYBR(5) Green I on different polymerase chain..., Nath [/bib_ref] (2) Alkaline extraction Fastest, reliable, and relatively easy procedure Medium purity and fragmentation of genomic DNA [bib_ref] Rat insulin genes: construction of plasmids containing the coding sequences, Ullrich [/bib_ref] (3) CsCl gradient centrifugation with EtBr
High purity and yield of DNA or RNA Laborious, costly and time consuming, [bib_ref] A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques..., Meng [/bib_ref] [bib_ref] Isolation and characterization of RNA from snap-frozen tissues and cultured cells, Murnane [/bib_ref] (4) Oligo(dT) cellulose chromatography Fast protocol, good yield of mRNA recovery Purification bias for mRNAs [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref] (5) Chelex5 extraction Quick and simple protocol; no use of hazardous chemicals Low purity of nucleic acids [bib_ref] PCR inhibitors-occurrence, properties and removal, Schrader [/bib_ref] [bib_ref] A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Bimboim [/bib_ref] (6) CTAB extraction Efficient method for plant and other "hard to lyse" samples Laborious, time-consuming; use of hazardous chemicalsisopropanol) precipitates genomic DNA, while plasmid DNA can be collected from the supernatant after a short 2-minute centrifugation. This technique is considered one of the fastest, most reliable, and relatively easy ways to obtain plasmid DNA from cells. Vigorous mixing during lysis and neutralization phases can cause fragmentation of genomic DNA, resulting in contamination with plasmid supernatant. The purified DNA is suitable for less sensitive applications. For more sensitive applications, a purifying step is needed, usually with spin columns.
## Purification of poly(a)+ rna by oligo(dt)-cellulose
Chromatography. Most of eukaryotic mRNA molecules possess a polyadenylated (polyA) tail of about 250 nucleotides at 3 end. This provides foundation for a simple and easy way of RNA extraction through chromatographic techniques. The basic mechanism of this method is that poly(A) RNA hybridizes with an oligo(dT)-cellulose matrix, under highsalt conditions. Eukaryotic mRNAs have a diverse range in terms of size (from 0.5 kb to over 20 kb) and abundance (from fewer than 15 copies to over 20,000 copies per cell) [bib_ref] Isolation of mRNA by affinity chromatography, Bryant [/bib_ref]. Polyadenylated RNA with minimum 20 residues has the ability to attach to the oligo(dT)-cellulose matrix, which usually consists of 10-20 nucleotides. After washing out all the nonpolyadenylated RNAs, a low-salt buffer is used to disrupt the oligo(dT)-poly(A) bond, resulting in the elution of poly(A) RNAs. The selection for poly(A) RNA can be made in column or batch chromatography [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref] , being fast and yielding good RNA recovery. Its drawback resides in the fact that the method selects only mRNAs and naturally excludes important biological information present in other RNAs, such as miRNAs, rRNAs, and tRNAs. summarizes the main advantages and disadvantages of the chemically driven methods discussed here.
## Solid-phase nucleic acid extraction
Solid-phase extraction (SPE) is one of the most efficient NAE techniques available in the market [bib_ref] DNA, RNA, and protein extraction: the past and the present, Tan [/bib_ref] [bib_ref] Nucleic acid extraction techniques and application to the microchip, Price [/bib_ref]. It is based on liquid and stationary phases, which selectively separate the target analyte from the solution based on specific hydrophobic, polar, and/or ionic properties of both solute and sorbent. The chemistry between sorbent and analyte of interest is the basis of this technique, while "weak" chemical interactions such as van der Waals forces (nonpolar interactions), dipoledipole interactions (polar interactions), and hydrogen bonding determine the retention mechanism in SPE.
SPE methods can be divided into normal/regular SPE, reverse SPE, and ion exchange SPE. Every sorbent used in SPE has unique characteristics, which give rise to a solution for a specific problem involved in extraction methods. A good example is acetonitrile, which decreases the polarity of the solution and decreases the interaction of DNA molecules with the stationary phase. Normally, reverse SPE uses polar/moderately mobile phase, nonpolar stationary phase, and semi-or nonpolar analytes, while normal SPE consists of semi-to nonpolar mobile phase, polar stationary phase, and polar analytes. On the other hand, ion exchange SPE is based on electrostatic interaction of both sorbent and the analyte of interest [bib_ref] Solid Phase Extraction Technique-Trends, Biziuk [/bib_ref].
Solid-phase microextraction (SPME) is a relatively new development in solid-phase extraction technique, introduced in 1990s by [bib_ref] Solid phase microextraction with thermal desorption using fused silica optical fibers, Arthur [/bib_ref] , being useful for various analytes including liquid, gaseous, and solid matrices [bib_ref] Recent developments and applications of solid phase microextraction (SPME) in food and..., Merkle [/bib_ref]. Two important steps are involved in SPME: (i) partitioning of analytes on fiber-coated extraction phase and (ii) handing over extract to separating instrument like gas chromatography where desorption takes place. SPME is a rapid and easy to use technique and have good detection limit (parts per trillion) for specific compounds [bib_ref] Trace determination of volatile sulfur compounds by solid-phase microextraction and GC-MS, Nielsen [/bib_ref]. Drawbacks of SPME include difficulty in analyzing high molecular weight compounds, sample carryover, and the eventual shortage of commercially available stationary phases.
## Silica matrices.
In 1979, it was found that silicates have high binding affinity for DNA under alkaline conditions and increased salt concentration [bib_ref] Preparative and analytical purification of DNA from agarose, Vogelstein [/bib_ref]. Silica matrices have revolutionized NAE procedures for both commercial as well as research purposes. Efficient and selective binding of NA to silica matrices is the hallmark of this fast and robust NA purification procedure [bib_ref] maxXbond: First regeneration system for DNA binding silica matrices, Esser [/bib_ref]. Silica matrices consist of silica material, in the form of either gel or glass particle (i.e., glass microfibers) [bib_ref] Nucleic acid purification on silica gel and glass mixture, Padhye [/bib_ref]. The mechanism involved in this technique is the affinity between negatively charged NA and positively charged silica material, resulting in selective binding of nucleic acids to the silica matrices, while the rest of the cell components and other chemicals are washed out. Silica surface is covered by positive ions, which enhances the binding of negatively charged DNA. As a final step, NA can be eluted from silica matrix by any hyposmotic solution, such as nuclease-free water or buffers such as alkaline Tris-EDTA. For RNA extraction, chaotropic agents have a second and very important task in denaturing RNases [bib_ref] RNA isolation from yeast using silica matrices, Mutin [/bib_ref]. Many modifications have been made to the original procedure, such as introduction of hydrated silica matrix and microchipbased silica SPE [bib_ref] Rapid, automated nuleic acid probe assays using silicon microstructures for nucleic acid..., Christel [/bib_ref]. In this technique, it is also noteworthy the role played by sodium ions in attracting the negatively charged oxygen present in nucleic acid's phosphate group and helping NA become insoluble because of the phenomenon known as "salting out" in the presence of high-salt conditions and acidic pH [bib_ref] Methods for extracting genomic DNA from whole blood samples: current perspectives, Chacon-Cortes [/bib_ref]. This technique provides high-purity DNA, is easy to perform, and also is able to reproduce quantitatively as well as qualitatively. Downside of this technique is being unable to recover small fragments DNA efficiently, as small fragments binds tightly with the silica matrix.
## Glass particles.
Glass particles, whether in powder as chromatography stationary phase or in microbeads form, have also been used for extraction of nucleic acids. Chaotropic salts are used to release the NA and allow binding to common silicate glass, flint glass, and borosilicate glass (arranged as glass fiber filters). The basic principle of binding of NA to glass particles is based on the adsorption affinity of the components present in the mixture (DNA, proteins, etc.) to the stationary phase of chromatography column (glass particles). Polar compounds such as DNA have a high tendency of attachment to polar stationary phase under specific ionic strength [bib_ref] Nucleic acid purification on silica gel and glass mixture, Padhye [/bib_ref].
## Diatomaceous earth.
Diatomaceous earth (DE), alternatively known as kieselguhr or diatomite, is mainly composed of silica 80% to 90% (sometimes up to 95%), alumina 2% to 4%, and hematite 0.5% to 2%. First described by Boom et al. [bib_ref] Rapid and simple method for purification of nucleic acids, Boom [/bib_ref] , this procedure is mainly based on the binding of NA to a solid phase (such as DE) in the presence of chaotropic agents, followed by elution in water or low-salt buffer. NA binds to the silica present in DE, following the same principles of binding to silica matrices. This procedure has the advantage of reduced pipetting error, shorter protocol time, and less number of steps for sample preparation, being used for plasmid as well as for single or double-stranded nucleic acids [bib_ref] Isolation of RNA and DNA fragments using diatomaceous earth, Koo [/bib_ref]. However, this technique is not routinely used because of comparably high cost.
## Magnetic beads-based nucleic acid
Purification. Magnetic beads technology is one of the emerging strategies for extracting RNA and genomic, plasmid, and mitochondrial DNA. The technique involves the separation of nucleic acids from complex mixtures via complementary hybridization [bib_ref] Magnetic bead-based solid phase for selective extraction of genomic DNA, Archer [/bib_ref]. In recent years, functionalized magnetic particle or beads have been coupled to suitable buffers systems for a rapid and efficient extraction procedure [bib_ref] A magnetic bead-based DNA extraction and purification microfluidic device, Azimi [/bib_ref]. The lack of centrifugation steps that can produce shear forces and cause breaking of nucleic acids is thought to better maintain intact longer fragments from genomic DNA. Usually, it is enough to apply a magnet to the side of a vessel or tube containing the sample mixed with the functionalized magnetic beads and exclusively aggregate the target particles near the vessel wall. The positive aspect of this technique is avoiding centrifugation steps as well as providing an alternative way for automation of extraction procedures from a large number of samples. The extraction technique can be used in batch processes with a multitude of samples (blood, tissues, and others) and is relatively easy to execute, being one of the best choices for automation, high-throughput applications, and high sample processivity [bib_ref] Magnetic particles for the separation and purification of nucleic acids, Berensmeier [/bib_ref] [bib_ref] Protein purification using magnetic adsorbent particles, Franzreb [/bib_ref]. This method is also suitable for using in low technological environments because it is virtually equipment-free.
# Anion exchange material.
Just like silica matrices, anion exchange resins are also widely used in DNA and RNA extraction. Unlike silicate negative charge, anion exchange resin makes use of the positively charged diethylaminoethyl cellulose (DEAE) to attract the negatively charged phosphate of nucleic acid. So, pH and salt concentration are the important aspects determining the binding or elution of NA to the anion exchange resin. Anion exchange has the advantage of extracting very pure DNA as compared to silica and the ability to reuse the resin upon renaturation. However, this method used high-salt concentration in the elution step, thus requiring desalting for downstream applications.
3.6. Cellulose Matrix. Absorbent cellulose-based paper is an interesting matrix for nucleic acids purification and storage. Cellulose is a polymer of glucose and therefore highly hydroxylated, producing a polar attraction strong enough to bind nuclei acids under specific chemical conditions. The commercially available FTA paper (Whatman Inc., USA) or fast technology for analysis is an interesting example of how to purify and store DNA using cellulose. FTA cards are simply a thick layer of paper, embedded with a proprietary mix of buffers, detergents, and chelating agents, such as the ubiquitous Tris pH 8, SDS, and EDTA. Once cells are spotted onto the paper, the detergent lyses the membranes and EDTA chelates metal ions that are cofactors to nucleases, also preventing the growth of contaminating organisms [bib_ref] Collecting, archiving and processing DNA from wildlife samples using FTA5 databasing paper, Smith [/bib_ref] [bib_ref] Solid medium and method for DNA storage, Burgoyne [/bib_ref]. Hence, when dried, nucleic acids are relatively well protected from the environment, especially due to the unavailability of water molecules. In fact, DNA extraction from dried blood spotting has been successfully used for PCR-mediated human diagnostics for more than 20 years [bib_ref] Diagnosis of vertical HIV-1 transmission using the polymerase chain reaction and dried..., Cassol [/bib_ref] [bib_ref] Dried blood spots collected on filter paper: an international resource for the..., Cassol [/bib_ref] [bib_ref] Rapid DNA extraction from dried blood spots on filter paper: potential applications..., Choi [/bib_ref].
Although FTA cards have many advantages regarding the easiness of use and storage, processing them to extract good yields of nucleic acids might be more complicated than expected, especially in diluted samples [bib_ref] Buccal DNA collection: comparison of buccal swabs with FTA cards, Milne [/bib_ref]. [fig_ref] Table 3: Summary of the advantages and disadvantages of solid-phase extraction methods [/fig_ref] summarizes the main advantages and disadvantages of most commonly used solid-phase extraction methods. [fig_ref] Table 4: Examples of commercially available kits applying each extraction method and typical yields... [/fig_ref] gives examples of commercially available kits using the methods described herein, as well as giving typical yields for NA extraction.
## Devices used in extraction methods
## Spin columns.
The binding element in spin-column systems is usually composed of glass particles or powder, silica matrices, diatomaceous earth, and ion exchange carriers. Nucleic acid binding is thus optimized with specific buffer solutions and extremely precise pH and salt concentrations [bib_ref] Methods for extracting genomic DNA from whole blood samples: current perspectives, Chacon-Cortes [/bib_ref]. Column-based NAE is one of the best techniques among the options available, playing a vital role in ion exchange methods, as it provides a robust stationary phase for a rapid and reliable buffer exchange and thus NAE. This method is fast and reproducible, and its main drawback is the need for a small centrifuge as equipment requirement.
## 4.2.
Beads or Magnetic Beads. Magnetic particle or beads are the first option to eliminate centrifuge-dependent steps in the extraction process. Magnetic beads make use of different ligands such as antibodies, antigens, oligonucleotides, or aptamers, which bind specifically to its target in sample. The first magnetic particle used for extraction consisted of an iron-oxide core covered by functional carboxylic group, which then binds DNA or RNA. Since then, many modifications have been made using different surface functional group, such as sulphate, amino, and hydroxyl groups [bib_ref] Comparison of three magnetic bead surface functionalities for RNA extraction and detection, Adams [/bib_ref]. Besides these functional groups, preactivated magnetic beads with different functional groups are available such as tosyl or epoxy groups [bib_ref] Use of magnetic beads in selection and detection of biotoxin aptamers by..., Bruno [/bib_ref] [bib_ref] Strategies for electrochemical detection in immunochemistry, Centi [/bib_ref]. Magnetic beads activated (2) Beads or magnetic beads DNA, RNA Simple to use; high automation potential; equipment-free process
Labor intensive [bib_ref] Use of magnetic beads in selection and detection of biotoxin aptamers by..., Bruno [/bib_ref] (3) Automation (liquid handling robots) DNA, RNA Precise manipulation of sample and reagents, reducing losses and cost
High cost [bib_ref] A 384-well solid-phase extraction for LC/MS/MS determination of methotrexate and its 7-hydroxy..., Rule [/bib_ref] (4) Microfluidics and "lab-on-a-chip" cartridges DNA, RNA Sensitive and specific Incompatibility of common NAE chemicals [bib_ref] Rapid molecular detection of tuberculosis and rifampin resistance, Boehme [/bib_ref] with protein A, protein G, or streptavidin are commercially available. Magnetic bead separation presents many benefits over centrifuge-dependent extraction process by allowing an equipment-free process. Equipment-free separation of NA is also possible with nonmagnetic beads, where the beads are trapped inside plastic bubble pipets [bib_ref] Electricity-free, sequential nucleic acid and protein isolation, Pawlowski [/bib_ref]. [bib_ref] Laboratory automation: trajectory, technology, and tactics, Markin [/bib_ref]. To fulfill this demand, various automated devices have been developed and introduced in the market. The most successful examples are the automated liquid handling robots, which are routinely used in many life science and clinical analysis laboratories for dispensing precise amount of sample, reagents, or other liquids to designated containers. Because of this technology, it is now possible to handle many samples simultaneously with precision and rapidity. In addition, barcode readers are an integral part of such equipment, allowing for easy traceability of samples and results. Fully automated NAE protocols have been developed for such equipment, using either solid-phase or magnetic beads methods [bib_ref] A 384-well solid-phase extraction for LC/MS/MS determination of methotrexate and its 7-hydroxy..., Rule [/bib_ref]. However, high sample processivity is a positive aspect of automation while maintaining the sensitivity can be compromised, as low-copy NA targets might be lost [bib_ref] DNA/RNA preparation for molecular detection, Thatcher [/bib_ref]. Small versions of these robots are available and could be useful in laboratory settings with minimal infrastructure. Liquid handling robots certainly have a niche in life sciences and clinical laboratories, but not as POC devices.
## Automation (liquid handling robots). the increase in growth of diagnostic tests and patient numbers highlights the need for automation in life sciences
## Microfluidics and "lab-on-a-chip" cartridges.
Nucleic acid-based detection (NAT) is preferable compared to immunoassay-based detection because of sensitivity and specificity, but NAT-based diagnosis requires complex infrastructure, sophisticated machinery, and trained personnel. To overcome this hurdle, microfluidic chips have been designed and produced, carrying, on inner chambers, all necessary reagents for molecular based tests as a part of POC-Dx strategy. Usually, microfluidic chips (or "lab-on-a-chip" cartridges) rely on solid phase extraction for NA isolation, in the form of either membranes or beads [bib_ref] Preparative and analytical purification of DNA from agarose, Vogelstein [/bib_ref] [bib_ref] Microfluidic devices for nucleic acid (NA) isolation, isothermal NA amplification, and real-time..., Mauk [/bib_ref].
The union of automation with the need for miniaturization in POC devices led to the development of cartridges that perform one or several biological reactions in a closed container. These reactions comprise most of the current molecular biology methods, such as NAE, amplification, and identification, as well as serological signatures analyses. One of the greatest examples of a microfluidic cartridge, although not POC, is the milestone related to diagnosis of Mycobacterium tuberculosis (Mtb) achieved by the platform GeneXpert MTB/RIF [bib_ref] Rapid molecular detection of tuberculosis and rifampin resistance, Boehme [/bib_ref]. Specific cells/spores are selected through filtration followed by a lysing step (sonification). Microtubes, pumps, and rotary drives transfer liquids into the specific cartridge chambers where washing, purification, and concentration of nucleic acids take place. The next step is the movement of the extracted NA to a reaction chamber where real-time PCR happens [bib_ref] Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays..., Dineva [/bib_ref]. A recent systematic metaanalysis study reviewed hundreds of papers concluded that GeneXpert was the most cost-effective strategy for POC-Dx of Mtb, although its performance was evaluated solely in clinics and primary care centers [bib_ref] Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic..., Drobniewski [/bib_ref]. However, it is undisputed that GeneXpert is a breakthrough in NA testing.
The FilmArray 2.0 system (BioFire Diagnostics LLC, Salt Lake City, USA) is a multiplexed PCR system that incorporates specimen processing, NA amplification, and detection in a specialized pouch. Specific pouches are used to amplify different targets present in the sample, using Nested PCR, followed by real-time PCR with chemistrybased detection. The software then automatically generates identification reports using DNA melting analysis based on specific control reactions or a melting curve database of known sequences. [fig_ref] Table 5: Summary of available devices used in nucleic acid extraction protocols [/fig_ref] presents a summary of the devices available most commonly used in NAE protocols.
## Limitations for implementation of extraction protocols in portable devices
A major obstruction for the development of a complete and easy-to-use solution for POC-Dx is the integration of sample preparation protocols into the portable devices. Removing interferents and extracting the target molecules are no trivial task especially due to the vast differences among sample matrices as well as characteristics of the target analytes. While NAE protocols are well established in the laboratory and many advances have been made since the inception of microfluidic Dx devices, commercial availability of these devices is still rare [bib_ref] Microfluidic sample preparation for medical diagnostics, Cui [/bib_ref]. Excellent reviews are available discussing the technical difficulties as well as the obstacles for implementation and acceptance of new tests based on new technologies [bib_ref] Microfluidic sample preparation for medical diagnostics, Cui [/bib_ref] [bib_ref] The impact of new trends in POCTs for companion diagnostics, non-invasive testing..., Huckle [/bib_ref] [bib_ref] Advances in addressing technical challenges of pointof-care diagnostics in resource-limited settings, Wang [/bib_ref] [bib_ref] Exploring the barriers and facilitators to use of point of care tests..., Hardy [/bib_ref] [bib_ref] Opportunities and challenges for the application of microfluidic technologies in point-of-care veterinary..., Busin [/bib_ref]. Several organic chemicals routinely used in molecular biology can react with the plastic materials commonly used in POC cartridges/devices, which makes difficult for some polymers to sustain their initial mechanical and physicochemical properties. One of properties paramount to the performance characteristics of the plastic materials is chemical inertness, that is, the material to which the active substance of interest will be in contact with will not interact and generate undesirable products, generally classified as extractable or leachable [bib_ref] Evaluation of the chemical compatibility of plastic contact materials and pharmaceutical products;..., Jenke [/bib_ref]. Toxicological or functional studies often replace extraction and interaction studies, which would be necessary to determine the levels of extractable or leachable products under a given environmental condition. Such replacement is acceptable, although not ideal, because the biological assessment performed for toxicological studies should include basic extraction/interaction evaluations [bib_ref] Evaluation of the chemical compatibility of plastic contact materials and pharmaceutical products;..., Jenke [/bib_ref]. Studies of structural properties of glassy polymers such as the commonly used thermoplastics polycarbonate (PC) and polymethylmethacrylate (PMMA) correlate the polymer solubility when exposed to several solvents to the extent of stress cracking [bib_ref] The role of organic agents in the stress crazing and cracking of..., Bernier [/bib_ref]. An advantage of PMMA is its high optical transparency into the ultraviolet range, while PC offers a compatibility with a wider range of solvents and a higher glass transition temperature well suited to applications such as polymerase chain reaction for NA amplification [bib_ref] Micro fluidic device fabrication by thermoplastic hot-embossing, Yang [/bib_ref]. However, neither of these is good enough to be used with the chemicals routinely used for NAE. For example, PMMA cannot be cleaned by strong solvents such as acetone or methanol, because these chemicals would significantly damage its surface and decrease transparency [bib_ref] Micro fluidic device fabrication by thermoplastic hot-embossing, Yang [/bib_ref].
Some chemicals have the potential to affect polymer's color, surface appearance, flexibility, and mass, generating extractable/leachable products that must be evaluated. These changes can happen due to several physicochemical reactions, such as (i) chemical interaction with polymer chain which can disturb their structure and result in depolymerization; (ii) physical interaction, that is, adsorption of chemicals into the plastics, which results in swelling and softening; or (iii) stress-associated cracking may happen due to the stresscracking agents, such as plasticizers, or adhesives used during the manufacturing of polymer parts, or even detergents or oils used during the molecular biology processes [bib_ref] Environmental stress cracking: a review, Robeson [/bib_ref].lists the effect of the chemicals most commonly used in NAE on the plastics most commonly used for microfabrication of microdevices. Alterations induced by any chemical, as minor as it seems, need to be thoroughly evaluated. In extreme cases, chemicals must be substituted, such as that of ethanol/isopropanol. Ethanol/isopropanol storage in cartridges is also problematic because of its volatility, flammability, and potential to leak. Such chemical properties make alcohols a substance highly regulated by the International Air Transport Association (IATA). Therefore, substitutes such as diethylene glycol monoethyl ether (DGME) and diethylene glycol monoethyl ether acetate (DGMEA), must be tested and validated.
Finally yet importantly, there is concern about the volume of sample needed to obtain a meaningful results [bib_ref] A microfluidic platform for precision small-volume sample processing and its use to..., Fong [/bib_ref]. Because the volume of buffers and, therefore, of harsh chemicals used for cell lysis is directly proportional to the volume of the sample, POC-Dx tests are most useful in illness where the pathogen is present in higher counts, such as virus and most bacterial infections. Parasitic infections, however, present a challenge to POC-Dx because parasite loads can get very close to the limits of detection of the techniques used [bib_ref] Malaria detection using inertial microfluidics, Warkiani [/bib_ref] , thus greatly affecting the availability of target NA in the BioMed Research International 9 sample. The volume of the reagents is also important to assure proper mixing of solutions without the common laboratory instruments because small volumes are easier to homogenize [bib_ref] Applications of microfluidics in chemical biology, Weibel [/bib_ref].
## Challenges for implementation in poc diagnostic tests
Lessons learned from previous attempts in developing diagnostic tests have taught us that availability of the best possible POC-Dx test is not enough. Its implementation is also very important and often underestimated, since only few diseases have a validated POC-Dx, such as HIV or malaria [bib_ref] Low-cost tools for diagnosing and monitoring HIV infection in low-resource settings, Wu [/bib_ref]. Implementation should be considered during the development phase of the POC-Dx, so that end-users are identified, their level of experience is assessed, and the developing test is used at the right lab tier [bib_ref] Advances in addressing technical challenges of pointof-care diagnostics in resource-limited settings, Wang [/bib_ref]. The major features of POC tests are described by the WHO acronym ASSURED: affordable, sensitive, specific, user-friendly, equipment-free, and deliverable to end-users [bib_ref] Low-cost tools for diagnosing and monitoring HIV infection in low-resource settings, Wu [/bib_ref]. The main idea is to provide low cost and timely effective healthcare to the patient and quick decision making for healthcare providers. One platform which seems to have the potential to meet the ASSURED criteria is microfluidic paper-based analytical devices ( PADs), which provide a robust, affordable, equipment-free, and multiplex facility [bib_ref] Diagnostics for the developing world: microfluidic paperbased analytical devices, Martinez [/bib_ref] [bib_ref] Toward practical application of paper-based microfluidics for medical diagnostics: state-of-the-art and challenges, Yamada [/bib_ref] [bib_ref] Advances in paper-based pointof-care diagnostics, Hu [/bib_ref]. Paper-based devices are abundant, either directly operating or directing the biochemical, serological, or nucleic acid reactions [bib_ref] Toward practical application of paper-based microfluidics for medical diagnostics: state-of-the-art and challenges, Yamada [/bib_ref] [bib_ref] Advances in paper-based pointof-care diagnostics, Hu [/bib_ref]. Because they are easily manipulated to attach recognition molecules (antibodies, enzymes, proteins, nucleic acids, etc.), PADs devices have been very successful in several areas of biological research, such as biochemical analysis of blood or urine, detection of pathogen's nucleic acids, detection of drugs, or environmental contamination. PADs can also be designed for direct sensing the target molecule by using nanotechnologies, such as microelectromechanical systems, field effector transistors, or nanocantilevers. However, since describing each of the available PADs is beyond the scope of our review, the reader is directed to other available texts on the subject [bib_ref] Toward practical application of paper-based microfluidics for medical diagnostics: state-of-the-art and challenges, Yamada [/bib_ref] [bib_ref] Advances in paper-based pointof-care diagnostics, Hu [/bib_ref] [bib_ref] A Review on Microfluidic Paper-Based Analytical Devices for Glucose Detection, Liu [/bib_ref]. Unlike protein or metabolite-based POC tests, one of the major challenges for nucleic acid-based POC tests is the need to consolidate three distinct protocol procedures into a single device: (1) nucleic acid extraction; (2) amplification; and (3) detection. Development of a nucleic-based testing device that is specific, sensitive, portable, and relatively easy to operate has presented several challenges that have been elegantly reviewed elsewhere [bib_ref] Microfluidic sample preparation for medical diagnostics, Cui [/bib_ref]. Below we discuss the challenges strictly related to NAE for POC-Dx.
Development of an ideal NAE method for POC is impaired by many factors and researchers are still in quest for a suitable solution. At present, solid-phase extraction [bib_ref] Microfluidic sample preparation: cell lysis and nucleic acid purification, Kim [/bib_ref] and magnetic beads [bib_ref] A magnetic bead-based DNA extraction and purification microfluidic device, Azimi [/bib_ref] are the primary choices for NAE in POC-Dx devices. However, neither method is yet good enough for widespread implementation in POC-Dx methods. Solid-phase extraction depends on centrifugation, while magnetic beads require an external magnet source for mixing. In this aspect, magnetic beads are favored because implementation of magnetic stirring in POC-Dx devices is somewhat easier than implementation of separation through stationary membranes. Although both rely on the use of chaotropic reagents for lysing cells and releasing the NA from the scaffold and structural nucleic proteins, washing steps are more efficient in beads-based methods. The main challenges in implementing molecular biology-based systems in resource-constrained areas are the high cost of instrument and reagents, as well as lack of reliable infrastructure and continuous maintenance support and temperature maintenance devices [bib_ref] Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays..., Dineva [/bib_ref]. Proper disposal of biological waste generated by medical tests is also a concern, not to mention that some waste is chemical and requires special treatment before disposal (e.g., guanidine thiocyanate) [bib_ref] Advances in addressing technical challenges of pointof-care diagnostics in resource-limited settings, Wang [/bib_ref].
# Conclusion
After almost 150 years after the first successful isolation of DNA by Friedrich Miescher, nucleic acids are now central to obtaining biological information in areas as distinct as specimens' identification for conservational purposes to the realms of personalized medicine and pharmacogenomics. Protocols and devices used for NAE have evolved from thiocyanate-phenol-chloroform manual techniques to userfriendly column-technology and automated platforms, but no general gold-standard method has yet been established. This review analyzed the working principle of each available method, as well as their advantages and disadvantages. The take-home message is that each application has specific characteristics, which should then guide each researcher to the most suitable method.
Although molecular biology techniques are sensitive and accurate methods, they require a rather well established laboratory setting and expensive instruments, as well as skilled personnel to run the tests and analyze the results, which are not always available. In the last years, lab-onchip technology has brought the promise of taking the management of biological information where it is needed, such as low-resource settings, a doctor's clinic or a hospital patient bedside. However, although progress has been made, several obstacles still hamper the use of NAE protocols in POC-Dx tests, as it can be seen by the low number of products using lab-on-chip technology. Overcoming the challenges and limitations of NAE protocols will greatly increase the use of molecular biology techniques and thus increase the overall quality of life of the general population by providing access to better diagnostic tests.
## Conflicts of interest
The authors declare that there are no conflicts of interest regarding the publication of this article.
[table] Table 3: Summary of the advantages and disadvantages of solid-phase extraction methods. [/table]
[table] Table 4: Examples of commercially available kits applying each extraction method and typical yields for distinct samples. [/table]
[table] Table 5: Summary of available devices used in nucleic acid extraction protocols. [/table]
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Middle meningeal artery embolization for chronic subdural hematomas. A systematic review of the literature focused on indications, technical aspects, and future possible perspectives
Surgical Neurology International - 2022 - 13(94) | 1is is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.ABSTRACTBackground: Chronic subdural hematoma (CSDH) is one of the most common neurosurgical diseases that affect elderly and fragile patients and as a consequence, management can be challenging. Surgery represents the standard treatment; however, alternative options are under investigation. Middle meningeal artery (MMA) embolization is considered a minimally invasive treatment although with poor evidence. In this review, we tried to summarize the findings about MMA embolization as a treatment for a CSDH to provide a useful guidance for clinical practice and for future speculative aspects.Methods: Literature review on PubMed until March 2021 was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement. We conducted a research on PubMed with a various combinations of the keywords "CSDH" and "middle meningeal artery" and "embolization," "refractory subdural hematoma, " and then we reviewed the references of the relevant studies as additional source of eligible articles.Results:Among the 35 studies eligible for this review, 22 were case series, 11 were case reports, one was a technical note, and 1 was a randomized trial. A total of 746 patients were found in the literature. Failure rate of MMA embolization was between 3.9 and 8.9% of the cases according the indication to treat CSDH (upfront vs. after surgery).Conclusion: e global impression deriving from the data available and the literature is that MMA embolization is a safe procedure with very low complications and with a low failure rate, both when associated with surgery or in case of a standalone treatment.
# Introduction
Chronic subdural hematoma (CSDH) is one of the most common neurosurgical diseases characterized by the presence of an abnormal fluid collection in the subdural space made of blood and blood degradation compounds. [bib_ref] Pathophysiology of chronic subdural haematoma: Inflammation, angiogenesis and implications for pharmacotherapy, Edlmann [/bib_ref] CSDH develops as a separation of dural border cells layer that triggers a healing inflammatory response. In this setting, the inflammatory chain is associated with the release of angiogenetic cytokines. is reaction determines the development of a capillary network vascularized by the middle meningeal arteries (MMAs) which provide the blood flow to the dura mater. [bib_ref] Pathophysiology of chronic subdural haematoma: Inflammation, angiogenesis and implications for pharmacotherapy, Edlmann [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A series of 60..., Link [/bib_ref] [bib_ref] Chronic subdural hematoma in elderly patients: Is this disease benign, Uno [/bib_ref] [bib_ref] Chronic subdural hematoma, Yang [/bib_ref] e overall incidence of CSDH ranges from 1.72 to 127.1/100.000 inhabitants, depending on the reports published in the literature, and it increases with age with the higher peak in patients over 65 years of age. [bib_ref] Longterm health outcomes in survivors after chronic subdural haematoma, Moffatt [/bib_ref] [bib_ref] Chronic subdural hematoma incidence, complications, and financial impact, Rauhala [/bib_ref] For a subgroup of patients, a conservative approach may be proposed as first choice of treatment, but specific inclusion criteria for this "wait and see" management is far away to be clearly reported in literature.
Moreover, a conservative management, when indicated, is necessarily associated to a prolonged neuroradiological and clinical follow-up. [bib_ref] To drill or not to drill, that is the question: Nonsurgical treatment..., Scerrati [/bib_ref] [bib_ref] How to remove those bloody collections: Nonsurgical treatment options for chronic subdural..., Yun [/bib_ref] In case of symptomatic CSDH, the standard treatment of care is mainly represented by surgical evacuation with several technical options described. [bib_ref] Longterm health outcomes in survivors after chronic subdural haematoma, Moffatt [/bib_ref] [bib_ref] Chronic subdural hematoma in elderly patients: Is this disease benign, Uno [/bib_ref] Nevertheless, surgical management is often challenging since it is associated with a high recurrence rate, morbidity and mortality, [bib_ref] Development of the subdural hematoma in the elderly (SHE) score to predict..., Alford [/bib_ref] [bib_ref] Chronic subdural haematoma in the elderly--a North Wales experience, Asghar [/bib_ref] [bib_ref] Chronic subdural hematoma: A sentinel health event, Dumont [/bib_ref] [bib_ref] Chronic subdural hematoma in the elderly: Not a benign disease, Miranda [/bib_ref] [bib_ref] Chronic subdural haematoma in the elderly: Is it time for a new..., Shapey [/bib_ref] [bib_ref] Chronic subdural hematoma in elderly patients: Is this disease benign, Uno [/bib_ref] [bib_ref] Chronic subdural hematoma, Yang [/bib_ref] especially when patients may present with many age related comorbidities that can affect the postoperative outcome. [bib_ref] Chronic subdural hematoma in the elderly: Not a benign disease, Miranda [/bib_ref] [bib_ref] Chronic subdural hematoma in elderly patients: Is this disease benign, Uno [/bib_ref] An extreme variability is related to the clinical course after surgery such as postoperative/in hospital mortality reported [bib_ref] Dexamethasone therapy in symptomatic chronic subdural hematoma (DECSA-R): A retrospective evaluation of..., Miah [/bib_ref].5% and recurrence rates considered to be from 10% to 30% that may be connected to different prognostic factors such as age, comorbidities and use of anticoagulant or antiplatelet medications. [bib_ref] Influence of antithrombotic agents on recurrence rate and clinical outcome in patients..., Abboud [/bib_ref] [bib_ref] Development of the subdural hematoma in the elderly (SHE) score to predict..., Alford [/bib_ref] [bib_ref] Chronic subdural hematoma in the elderly: Not a benign disease, Miranda [/bib_ref] [bib_ref] Chronic subdural haematoma in the elderly: Is it time for a new..., Shapey [/bib_ref] [bib_ref] Chronic subdural hematoma in elderly patients: Is this disease benign, Uno [/bib_ref] [bib_ref] Chronic subdural hematoma, Yang [/bib_ref] e primary end-point of the surgical evacuation of CSDH is to release the intracranial hypertension and reduce the local mass effect. [bib_ref] Pathophysiology of chronic subdural haematoma: Inflammation, angiogenesis and implications for pharmacotherapy, Edlmann [/bib_ref] [bib_ref] Chronic subdural hematoma, Yang [/bib_ref] erefore, surgery is not meant to deal with the causes that lead to the CSDH formation, and this may justify the high failure rate.
As a consequence, starting from the pathophysiology of CSDH, several pharmacological approaches have been proposed to avoid surgery, when possible, like use of corticosteroids, tranexamic acid, or ACE-inhibitors [bib_ref] Statins as a medical adjunct in the surgical management of chronic subdural..., Guidry [/bib_ref] [bib_ref] To drill or not to drill, that is the question: Nonsurgical treatment..., Scerrati [/bib_ref] [bib_ref] How to remove those bloody collections: Nonsurgical treatment options for chronic subdural..., Yun [/bib_ref] without a clear evidence.
More recently, in addition to those strategies, an interventional approach under investigation is the devascularization of the external membrane of the CSDH through the embolization of the MMAs. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A series of 60..., Link [/bib_ref] [bib_ref] Dexamethasone therapy in symptomatic chronic subdural hematoma (DECSA-R): A retrospective evaluation of..., Miah [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: Meta-analysis and systematic review, Srivatsan [/bib_ref] [bib_ref] Application of intraoperative ultrasound in neurological surgery, Sun [/bib_ref] At present, the literature data seem more prone to suggest this endovascular treatment for recurrent CSDH and/or for hematoma with no clear surgical indication.
e aim of our work is to provide an updated review about the findings regarding the efficacy of
# Materials and methods
## Literature review
We reviewed the existing literature on PubMed until March 22, 2021, in English language, without restrictions about the paper publication status, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement. [bib_ref] Preferred reporting items for systematic reviews and metaanalyses: e PRISMA statement, Moher [/bib_ref] We conducted research on PubMed with a various combinations of the keywords "CSDH" and "middle meningeal artery" and "embolization" and "refractory subdural hematoma. " en we reviewed the references of the relevant studies as additional source of eligible articles. We decided to include works specifically focused only on CSDH and treatment with MMA embolization without limitation about the kind of work and to exclude pure reviews (without new cases) and editorials.
Afterward, all titles and abstracts were screened to exclude unrelated studies; this includes neurological conditions other than/causing other than CSDH (e.g., acute subdural hematomas, epidural hematomas, and tumor related CSDHs), studies about anatomy of MMA, studies without patients' follow-up (either clinical or radiological) and spinal hematomas. Some other studies could have successively been excluded after full-text article reading.
Data of the eligible works were obtained through careful analysis of full text by one author and checked by another.
After having analyzed all the methods of the studies, patients were also classified according to indication for MMA embolization into the following: 1. Standalone embolization. 2. Symptomatic CSDH (close to surgery to prevent recurrence or as prophylaxis for a recurrence). 3. Recurrent CSDH. 4. Recurrent after second surgery.
Case series with more than four patients and including a comparison with a control group were selected for further analyses and for comparison with our series of patients.
# Results
roughout literature searching on PubMed (MEDLINE) 340 articles were reported with several combinations of the keywords described in the "methods" section. Four articles were eligible for reviewing after reference check. As a consequence, a total of 344 articles were screened for the review. ree-hundred and nine articles were excluded on the basis of the inclusion criteria of the present review [ [fig_ref] Figure 1: e PRISMA flow chart of our systematic review from 2 [/fig_ref] ].
For each study, we extrapolated the indication for MMA embolization, the number of patients treated, the kind of study, the kind of surgery, the number of MMAs embolized, the number of treatment failures, the presence or absence of a control group, and the number of complications related to MMA embolization. All results are summarized in [fig_ref] Table 1: Cases reported in the literature [/fig_ref] ].
## General considerations
Among the 35 studies eligible for this review, 22 were case series, 11 were case reports, one was a technical note, and one was a randomized trial. Among the 22 case series, five presented comparisons with an historical group of patients treated in a conventional way.
Starting from year 2000 with the first report by Mandai et al. [bib_ref] Middle meningeal artery embolization for refractory chronic subdural hematoma. Case report, Mandai [/bib_ref] till 2018, a total of 13 works could be considered eligible for analysis in this review while an increase of publications has been observed starting from 2019 with 22 works published in the past 27 months.
A total of 746 patients have been described in literature till the date of the literature search. According to the published papers, we divided the studies reported in the literature into two groups; those that proposed MMA embolization only as a first treatment for a CSDH and those that proposed MMA embolization as an adjunctive treatment after surgical drainage. e last group included patients that underwent MMA embolization as prophylaxis for recurrence, patients that were treated with MMA embolization in case of CSDH recurrence and patients that underwent MMA embolization as a salvage option in case of multiple recurrences. is kind of subdivision is reported in [ [fig_ref] Table 1: Cases reported in the literature [/fig_ref] ].
## Mma embolization alone for csdh
MMA embolization was reported as a standalone treatment for a CSDH in 16/35 works and of these 16 studies, four were case reports. In these 16 works about standalone MMA embolization for CSDH treatment, a total of 309 patients (41.4% of total reported patients) were studied but some of them received a MMA embolization as a standalone treatment of a recurrent CSDH.
Indication for MMA embolization alone was different from one study to another [ [fig_ref] Table 2: Summary of indications for case series for upfront treatment of CSDH with... [/fig_ref] ]. In some cases, MMA embolization was performed as per patients' preferences. [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] In the majority of the patients reported in the works by Link and Ban, MMA embolization alone was performed after failure of a conservative management for mildly symptomatic patients. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A series of 60..., Link [/bib_ref] In four studies, MMA embolization was proposed in patients under anticoagulant or antiplatelets treatments in order to prevent risk of subdural collection enlargement or to avoid suspension of such medications. [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] [bib_ref] Embolization of the middle meningeal artery for refractory chronic subdural haematoma: Usefulness..., Hirai [/bib_ref] [bib_ref] Midterm follow-up of patients with middle meningeal artery embolization in intractable chronic..., Okuma [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma with high risk of..., Yajima [/bib_ref] In particular, in 2021, Entezami et al. proposed MMA embolization as an alternative therapy for the management of CSDHs as a day case procedure in the COVID pandemic scenario. [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] Two works did not report the criteria used for selecting patients eligible for MMA embolization but just reported the outcome (e.g. Joyce et al. 2020 or [bib_ref] DynaCT Enhancement of subdural membranes after middle meningeal artery embolization: Insights into..., Mureb [/bib_ref] or included patients with different indications. [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] [bib_ref] DynaCT Enhancement of subdural membranes after middle meningeal artery embolization: Insights into..., Mureb [/bib_ref] Surgical rescue or refilling of subdural collection was considered the criteria for the failure of MMA embolization in all papers. In case MMA embolization was performed as a standalone treatment, the failure rate was 8.9% (8 patients out of 90); while the remaining patients experienced in all cases a resolution of the subdural collection at the follow-up CT scans. is failure rate was calculated taking into account only those studies in which it was possible to correlate treatment failure and indication to treat with MMA embolization alone. In some studies, like the one by Catapano et al., it is not specified who failed treatment with MMA embolization (patients treated for asymptomatic CSDHs with MMA embolization alone or patients treated for a recurrent CSDH after surgery? [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] A comparison series of patients undergoing conventional treatment is reported only by Ban et al. in which no failure was associated with MMA embolization for these kinds of patients. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] Four works were case reports. ese studies included a total of 305 patients (41.4% of total reported patients). One study did not report the number of patients treated in a prophylactic way making some of the results partly uncompleted. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma with high risk of..., Yajima [/bib_ref] In general, patients were eligible for MMA embolization when they were considered high risk for CSDH recurrence but the way it was assessed differed among the studies. Okuma et al. in 2019 established a checklist for high risk CSDHs that were used to select patients at high risk and patients at low risk for recurrence. [bib_ref] Midterm follow-up of patients with middle meningeal artery embolization in intractable chronic..., Okuma [/bib_ref] In the study by Ban et al., 45 consecutive patients were enrolled and treated with CSDH surgical drainage and MMA embolization without any selection criteria for MMA embolization. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] In the series of 60 patients described by Link in 2018, 17 patients were treated after surgical evacuation of a CSDH. In their work they treated consecutively such patients but they did not report the motivations that lead to propose MMA embolization as prophylaxis. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A series of 60..., Link [/bib_ref] In the study by Shotar et al., MMA embolization was proposed as prophylaxis in those patients with at least one known risk factor for recurrence but this decision was under the personal experience of the medical staff involved in the treatment of a patient. [bib_ref] Middle meningeal artery embolization reduces the post-operative recurrence rate of at-risk chronic..., Shotar [/bib_ref] In the study by Joyce et al. in 2020, they reviewed a multi-institutional database but they did not specify the selection criteria for MMA embolization. [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] In patients enrolled in the clinical trial by Ng and colleagues, the randomization after surgery was the only criteria for MMA embolization after surgery and this is the only experience of this kind reported in literature. [bib_ref] Middle meningeal artery embolization as an adjuvant treatment to surgery for symptomatic..., Ng [/bib_ref] In 14/35 studies MMA embolization was proposed as an alternative treatment after surgery for recurrent CSDH to prevent a second surgery. Patients included in this group were 158 accounting for 21.2% of the total cases described in literature. ree of the studies in this group were case reports.
## Failure rates of mma embolization and procedural complications
Analyzing the 746 patients reported in the literature, 41 patients reported a failure of MMA embolization to prevent surgery or hematoma refilling with an overall failure rate of 5.5% while considering the series with more than 20 patients included, the failure rate remained quite the same at 5.6% with 28 patients on a total of 496 reported in 11 works [ [fig_ref] Table 3: Summary of the case series including more than 20 patients [/fig_ref] ]. In all the studies considered with more than 20 patients, MMA embolization was proposed with all the four indications but only one study enrolled patients with more than one CSDH recurrence. [bib_ref] Preferred reporting items for systematic reviews and metaanalyses: e PRISMA statement, Moher [/bib_ref] e majority of patients treated with MMA embolization were described in publications from year 2017; while before that year, the number of patients in case series was smaller. Considering the single indication to perform MMA embolization, the failure rate was different from one group to another. In studies dealing with MMA embolization with multiple indications, it was difficult to define which patients failed to respond to MMA embolization according to the indication. So far, in the following considerations, we included only those cases in which it was possible to determine the nature of the indication.
As reported above, failure rate after MMA embolization alone was 8.9% while in case of MMA performed as a recurrence prophylaxis after surgery, the failure rate was 3.9% (six patients out of 195). In case of MMA embolization performed after CSDH recurrence it was not possible to measure the failure rate since only the study by Link in 2018 was described a failure on a series of 6 patients; [bib_ref] Middle meningeal artery embolization for recurrent chronic subdural hematoma: A case series, Link [/bib_ref] while in other studies with overlapping indications for MMA embolization, it was not specified the number of failures after MMA embolization. Finally, in case of MMA embolization performed after second recurrence, failure rate was 2.9% (1 patient out of 35).
Complications of MMA embolization was reported in six cases with a consequent very low rate with a global rate of 0.8% on 746 patients. Treatment failure was considered as a separate complication. Complications encompassed one cerebral infarction, one seizure, one intermittent aphasia, one cerebrovascular complication, one cerebrovascular infarction, and one acute worsening of CSDH.
## Studies including a comparison group with conventional treatment
In the literature, five studies encompassed a comparison group. In five studies a comparison group made by an historical cohort of patients treated with conventional technique.
In total, the control groups of the five studies included 727 patients whose outcome was compared with 205 patients undergoing MMA embolization with heterogeneous indications.
Globally, patients that received MMA embolization showed a lower recurrence rate and required less surgical procedures. In fact, the failure rate of MMA embolization in preventing surgery was 3.9% (6 patients on 155) while the recurrence rate after conventional treatment in historical cohorts was 29.5% (214 patients on 727 patients). Most of the patients were treated with MMA embolization for recurrent hematomas while only 27 patients enrolled in the work by Ban et al. were treated with MMA embolization alone with no cases of failure. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref]
# Discussion
## Considerations about indications for mma embolization
From the analysis of the literature, we identified four indications for MMA embolization:
- To prevent surgery in patients with paucisymptomatic CSDHs, [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] [bib_ref] Embolization of middle meningeal artery for the treatment of headaches induced by..., Entezami [/bib_ref] [bib_ref] Upfront middle meningeal artery embolization for treatment of chronic subdural hematomas in..., Gomez-Paz [/bib_ref] [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A multi-center experience of..., Kan [/bib_ref] [bib_ref] Middle meningeal artery embolization for recurrent chronic subdural hematoma: A case series, Link [/bib_ref] [bib_ref] Midterm follow-up of patients with middle meningeal artery embolization in intractable chronic..., Okuma [/bib_ref] [bib_ref] Immediate development of dural arteriovenous fistula after middle meningeal artery embolization: First..., Piergallini [/bib_ref] [bib_ref] Transradial middle meningeal artery embolization for chronic subdural hematoma using Onyx: Case..., Rajah [/bib_ref] [bib_ref] Recurrence and coniglobus volumetric resolution of subacute and chronic subdural hematoma post-middle..., Tiwari [/bib_ref] [bib_ref] Recurrent bilateral chronic subdural hematoma after interventional embolization combined with drilling and..., Wang [/bib_ref] [bib_ref] Safety and effectiveness of embolization for chronic subdural hematoma: Systematic review and..., Waqas [/bib_ref] - As prophylaxis of a recurrence after a first surgery, [bib_ref] Middle meningeal artery embolization following burr hole in chronic subdural hematoma, Arham [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] [bib_ref] Embolization of middle meningeal artery for the treatment of headaches induced by..., Entezami [/bib_ref] [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] [bib_ref] Middle meningeal artery embolization for recurrent chronic subdural hematoma: A case series, Link [/bib_ref] [bib_ref] Middle meningeal artery embolization as an adjuvant treatment to surgery for symptomatic..., Ng [/bib_ref] [bib_ref] Midterm follow-up of patients with middle meningeal artery embolization in intractable chronic..., Okuma [/bib_ref] [bib_ref] Immediate development of dural arteriovenous fistula after middle meningeal artery embolization: First..., Piergallini [/bib_ref] [bib_ref] Transradial middle meningeal artery embolization for chronic subdural hematoma using Onyx: Case..., Rajah [/bib_ref] [bib_ref] Middle meningeal artery embolization reduces the post-operative recurrence rate of at-risk chronic..., Shotar [/bib_ref] [bib_ref] Dural arteriovenous fistula formation following bilateral middle meningeal artery embolization for the..., Shotar [/bib_ref] [bib_ref] Recurrent bilateral chronic subdural hematoma after interventional embolization combined with drilling and..., Wang [/bib_ref] - After a first recurrence to prevent further recurrence, [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] [bib_ref] Usefulness of embolization of the middle meningeal artery for refractory chronic subdural..., Hashimoto [/bib_ref] [bib_ref] Embolization of the middle meningeal artery for refractory chronic subdural haematoma: Usefulness..., Hirai [/bib_ref] [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A multi-center experience of..., Kan [/bib_ref] [bib_ref] Embolization therapy for refractory hemorrhage in patients with chronic subdural hematomas, Kim [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A series of 60..., Link [/bib_ref] [bib_ref] Middle meningeal artery embolization for recurrent chronic subdural hematoma: A case series, Link [/bib_ref] [bib_ref] Efficacy of middle meningeal artery embolization in the treatment of refractory chronic..., Mino [/bib_ref] [bib_ref] Transradial middle meningeal artery embolization for chronic subdural hematoma using Onyx: Case..., Rajah [/bib_ref] [bib_ref] Usefulness of middle meningeal embolization to prevent recurrent spontaneous chronic subdural hemorrhage, Sirh [/bib_ref] [bib_ref] Recurrence and coniglobus volumetric resolution of subacute and chronic subdural hematoma post-middle..., Tiwari [/bib_ref] [bib_ref] Safety and effectiveness of embolization for chronic subdural hematoma: Systematic review and..., Waqas [/bib_ref] [bib_ref] Organized chronic subdural hematoma treated with middle meningeal artery embolization and small..., Yokoya [/bib_ref] - As an adjunctive treatment in case of a second surgery. [bib_ref] Recurrence of a refractory chronic subdural hematoma after middle meningeal artery embolization..., Chihara [/bib_ref] [bib_ref] Usefulness of embolization of the middle meningeal artery for refractory chronic subdural..., Hashimoto [/bib_ref] [bib_ref] Middle meningeal artery embolization for refractory chronic subdural hematoma. Case report, Mandai [/bib_ref] [bib_ref] Hemispheric asymmetry of the arcuate fasciculus: a preliminary diffusion tensor tractography study..., Matsumoto [/bib_ref] [bib_ref] Efficacy of middle meningeal artery embolization in the treatment of refractory chronic..., Mino [/bib_ref] [bib_ref] Enhanced hematoma membrane on dynact images during middle meningeal artery embolization for..., Nakagawa [/bib_ref] [bib_ref] A case of neuromyelitis optica diagnosed with a chronic subdural hematoma, Tempaku [/bib_ref] e first scenario is completely different; after standalone MMA embolization the remaining subdural fluid collection may be at risk of progression due to its pro-inflammatory features. In the other scenarios with the combined treatment, surgical evacuation can decrease the pro-inflammatory and vasogenic content of the subdural fluid collection, while the vascularization of the parietal membrane of the CSDH itself is reduced by MMA embolization. [bib_ref] Pathophysiology of chronic subdural haematoma: Inflammation, angiogenesis and implications for pharmacotherapy, Edlmann [/bib_ref] [bib_ref] Review natural history of chronic subdural haematoma, Lee [/bib_ref] us, this pathophysiological difference may be related to the lower failure rate observed in CSDHs treated with combined procedures respect to the first scenario (3.9% vs. about 8.9%).
Standalone MMA embolization has been successfully achieved in all 27 cases reported by Ban et al. in 2017; [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] while a failure rate of 16% has been reported by Catapano et al. in 2020 but it may be due to the fact that the inclusion criteria were also extended to patients with midline shift more than 5 mm that could be more prone to become symptomatic and to consequently require surgical evacuation. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] Finally, the high rate of successful MMA embolization after first surgical evacuation or after recurrence should take in consideration a fraction of patients who could still have recovered completely after surgery even without MMA embolization and the low risk of further recurrences.
In fact, among a series of 372 patients treated for a CSDH, only 20 cases (5.4%) experienced a third recurrence that underwent MMA embolization as reported by Waqas et al. in 2017. [bib_ref] Safety and effectiveness of embolization for chronic subdural hematoma: Systematic review and..., Waqas [/bib_ref] is is summarized in [ [fig_ref] Table 1: Cases reported in the literature [/fig_ref] ] along with the literature how indication for MMA embolization has changed; being reported at the beginning as a salvage treatment [bib_ref] Usefulness of embolization of the middle meningeal artery for refractory chronic subdural..., Hashimoto [/bib_ref] [bib_ref] Middle meningeal artery embolization for refractory chronic subdural hematoma. Case report, Mandai [/bib_ref] [bib_ref] Hemispheric asymmetry of the arcuate fasciculus: a preliminary diffusion tensor tractography study..., Matsumoto [/bib_ref] [bib_ref] Enhanced hematoma membrane on dynact images during middle meningeal artery embolization for..., Nakagawa [/bib_ref] [bib_ref] A case of neuromyelitis optica diagnosed with a chronic subdural hematoma, Tempaku [/bib_ref] then as a treatment aimed at preventing recurrence, mainly for patients under anticoagulant or antiplatelet medications. [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A multi-center experience of..., Kan [/bib_ref] [bib_ref] Embolization therapy for refractory hemorrhage in patients with chronic subdural hematomas, Kim [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma with high risk of..., Yajima [/bib_ref] At present, MMA embolization represents the first therapeutic option in the majority of cases reported in literature. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] [bib_ref] Embolization of middle meningeal artery for the treatment of headaches induced by..., Entezami [/bib_ref] [bib_ref] Upfront middle meningeal artery embolization for treatment of chronic subdural hematomas in..., Gomez-Paz [/bib_ref] [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A multi-center experience of..., Kan [/bib_ref] [bib_ref] Middle meningeal artery embolization for recurrent chronic subdural hematoma: A case series, Link [/bib_ref] [bib_ref] Midterm follow-up of patients with middle meningeal artery embolization in intractable chronic..., Okuma [/bib_ref] [bib_ref] Immediate development of dural arteriovenous fistula after middle meningeal artery embolization: First..., Piergallini [/bib_ref] [bib_ref] Transradial middle meningeal artery embolization for chronic subdural hematoma using Onyx: Case..., Rajah [/bib_ref] [bib_ref] Recurrence and coniglobus volumetric resolution of subacute and chronic subdural hematoma post-middle..., Tiwari [/bib_ref] [bib_ref] Recurrent bilateral chronic subdural hematoma after interventional embolization combined with drilling and..., Wang [/bib_ref] [bib_ref] Safety and effectiveness of embolization for chronic subdural hematoma: Systematic review and..., Waqas [/bib_ref] Nevertheless, the eligibility of patients is not yet well established, or even left to the surgeon's judgment as reported by in 2020, [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: A multi-center experience of..., Kan [/bib_ref] or to patient's preference as reported by Entezami et al. in 2021. [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] us, the indication for a MMA embolization appears now to be more wide than strictly associated to the recurrence prevention although with such data collected in the context of different pathophysiological scenarios, is not correct to perform statistical comparisons among each group of patients.
## Number of mma embolization, number of patients treated and outcome
e total amount of the number of patients that underwent MMA embolization is not well reported in literature. Some authors provide the number of CSDH treated but not the number of patients, such as in the series reported by Santiago Gomez-Paz et al. or by Joyce et al., [bib_ref] Upfront middle meningeal artery embolization for treatment of chronic subdural hematomas in..., Gomez-Paz [/bib_ref] [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] where a bilateral blood collection is considered as 2 CSDH and not as one patient. Some authors reported indications that may overlap on the same patient so a bilateral hematoma could be operated just on one side but may be treated bilaterally with two different indications; one as upfront treatment and the other as a prophylactic treatment as reported, for example, by Sirh et al. in 2018. [bib_ref] Usefulness of middle meningeal embolization to prevent recurrent spontaneous chronic subdural hemorrhage, Sirh [/bib_ref] So far, this way of taking into account bilateral subdural collections and analyzing data may not have a clinical utility since in daily practice, patients with bilateral CSDHs represent a single entity to deal with and maybe they should be considered as well in studies on MMA embolization with separate analyses or in separate papers from unilateral CSDHs. is consideration is also in line with some authors that consider patients with bilateral collections as different kinds of patients due to the increased risk for recurrence than patients with unilateral CSDH. [bib_ref] e Danish chronic subdural hematoma study predicting recurrence of chronic subdural hematoma, Andersen-Ranberg [/bib_ref] [bib_ref] Bilateral chronic subdural hematoma: Unilateral or bilateral drainage?, Andersen-Ranberg [/bib_ref] In addition, the indication to proceed with the MMA embolization is not always well reported. Yajima et al.
included patients with an objectively inclusion criteria as a third recurrence, and a more subjective inclusion criteria as the risk of recurrence without differentiating the results for each of these two groups. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma with high risk of..., Yajima [/bib_ref] Joyce et al. reported a big series of 151 embolization of MMA for CSHD. e indication for MMA embolization was not stated but they reviewed a multiinstitutional database of patients in which were included both patients treated with standalone MMA embolization or after surgical evacuation. [bib_ref] Middle meningeal artery embolization treatment of nonacute subdural hematomas in the elderly:..., Joyce [/bib_ref] Moreover, in case of patients included for a standalone MMA embolization, there is no agreement about eligibility of candidates. While a poor symptom set seems to be generally reported as clinical inclusion criteria, conversely some neuroradiological data, like the midline shift, are considered exclusion criteria by Ban et al. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma, Ban [/bib_ref] [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] and inclusion criteria by Catapano et al. [bib_ref] Middle meningeal artery embolization for chronic subdural hematoma: An institutional technical analysis, Catapano [/bib_ref] Should we consider a standardized way for describing case series in view of future speculations?
e difficulty in systematically reviewing the data of the literature is the heterogeneity of data itself. e number of patients treated should be considered differently from the number of CSDHs treated and, in particular, bilateral CSDHs should be considered a category of patients separately due to their predisposition to develop a recurrent hematoma.
Patients should also be categorized by indication for MMA embolization given the different rate of procedure failure with regards to the indications for it and additionally the time between surgery and MMA embolization should be specified. is would allow to understand if there is a maximal length of time to perform a MMA embolization.
Moreover, patients under antiplatelet or anticoagulant medications could be considered for separate studies since they sometimes represent a clinical challenge (patients at risk for acute hemorrhages after surgery or at risk for complications related with drug discontinuation). In fact, contrary to what happens for surgical drainage, MMA embolization can be performed without suspending administration of prohemorrhagic drugs. Moreover, MMA embolization in addition to surgery may be of help in reducing the risk of CSDH recurrence and consequently, the complications related to discontinuation of anti-platelet or anticoagulant medications.
Although the great heterogeneity of the cases found in the literature, past experiences are of great importance in leading the neurosurgical community to trace a way to understand the efficacy and usefulness of invasive treatments. According to the experiences reported in literature, two points are of paramount importance in depicting future scenarios and they are related to the presence of the two main groups of patients with CSDH treated with MMA embolization; one group treated with standalone MMA embolization and the other treated after surgery.
First, from a pathophysiological point of view, the clear cut between these two groups is due to the presence of a residual CSDH collection that can reduce the efficacy of MMA embolization. Indirect evidence is related with the lower failure rate in case MMA embolization is performed soon after surgery. In this view, the neurosurgical community might look forward to better understand if some patients affected by a CSDH may have the opportunity to avoid a surgical drainage of the CSDH. A clear demonstration of such evidence would have a significant impact on social and economic costs since MMA embolization alone can be managed as a day-case procedure as proposed during the SARS-Cov2 pandemic. [bib_ref] Outpatient management of chronic expanding subdural hematomas with endovascular embolization to minimize..., Entezami [/bib_ref] Second, performing MMA embolization after surgery may lead to reduction of CSDH recurrence rate and may reduce the timing needed for CSDH resolution leading to a possible consequent reduction in in-hospital days, morbidity and mortality due to hospitalization. Finally, reduction in the number of surgeries required in case of recurrence may reduce the economic costs of the CSDH disease. Taken together, these considerations would lead to a global benefit to the community since CSDH incidence is expected to increase over the next years. [bib_ref] Review natural history of chronic subdural haematoma, Lee [/bib_ref] [bib_ref] Chronic subdural hematoma in the elderly: Not a benign disease, Miranda [/bib_ref] [bib_ref] Chronic subdural hematoma in elderly patients: Is this disease benign, Uno [/bib_ref] Starting from these considerations, two kind of randomized clinical trials would be needed: one comparing conventional surgery versus standalone MMA embolization for poorly symptomatic subdural collections and one comparing conventional surgery versus surgery plus MMA embolization for CSDHs. In July 2020, Ng et al. published the first clinical trial on CSDH treatment with MMA embolization. ey performed a randomized clinical trial allocating 21 patients for surgery alone and 25 patients for surgery + MMA embolization. ey published a preliminary report of their trial demonstrating that MMA embolization after surgery reduces the time needed for CSDH absorption. [bib_ref] Middle meningeal artery embolization as an adjuvant treatment to surgery for symptomatic..., Ng [/bib_ref] is study may represent a milestone in this field.
# Conclusion
In this review, we tried to summarize the findings about MMA embolization as a treatment for a CSDH in order to provide a useful guidance for the clinical practice and for future speculative aspects about alternative CSDH treatments. A significant limitation of our work is related with the lack of distinction in several works about the failure rates of MMA embolization according to the indication for which the treatment has been proposed. As a consequence, it is not possible to understand which patient failed to respond to MMA embolization (upfront treatment vs. postsurgical) and it is not possible to draw the exact number of patients but only the exact number of subdural collections that may affect the same patient or different patients. However, the global impression deriving from the data available and the literature is that in the near future, MMA embolization is a safe procedure with very low complications and with a low failure rate and will probably become one of the standards of care.
## Declaration of patient consent
Patient's consent not required as there are no patients in this study.
## Financial support and sponsorship
Nil.
## Conflicts of interest
ere are no conflicts of interest.
[fig] Figure 1: e PRISMA flow chart of our systematic review from 2.3% to [/fig]
[fig] In 8 / 34: studies, MMA embolization was proposed as a salvage treatment after second recurrence. e majority of the publications in this group were produced between the years 2000 and 2015; year after which the number of patients included in case series increased. In this scenario MMA embolization was performed on 35 patients (4.6% of the total number of patients) of which 18/35 were described between the year 2000 and the year 2015. [/fig]
[table] Table 1: Cases reported in the literature. [/table]
[table] Table 2: Summary of indications for case series for upfront treatment of CSDH with MMA embolization. [/table]
[table] Table 3: Summary of the case series including more than 20 patients. [/table]
|
Clinical Characteristics and Outcomes in Patients with COVID-19 and Cancer: a Systematic Review and Meta-analysis
Much of routine cancer care has been disrupted due to the perceived susceptibility to SARS-CoV-2 infection in cancer patients. Here, we systematically review the current evidence base pertaining to the prevalence, presentation and outcome of COVID-19 in cancer patients, in order to inform policy and practice going forwards. A keyword-structured systematic search was conducted on Pubmed, Cochrane, Embase and MedRxiv databases for studies reporting primary data on COVID-19 in cancer patients. Studies were critically appraised using the NIH National Heart, Lung and Blood Institute's quality assessment tool set. The pooled prevalence of cancer as a co-morbidity in patients with COVID-19 and pooled in-hospital mortality risk of COVID-19 in cancer patients were derived by randomeffects meta-analyses. In total, 110 studies from 10 countries were included. The pooled prevalence of cancer as a co-morbidity in hospitalised patients with COVID-19 was 2.6% (95% confidence interval 1.8%, 3.5%, I 2 : 92.0%). Specifically, 1.7% (95% confidence interval 1.3%, 2.3%, I 2 : 57.6.%) in China and 5.6% (95% confidence interval 4.5%, 6.7%, I 2 : 82.3%) in Western countries. Patients most commonly presented with non-specific symptoms of fever, dyspnoea and chest tightness in addition to decreased arterial oxygen saturation, ground glass opacities on computer tomography and non-specific changes in inflammatory markers. The pooled in-hospital mortality risk among patients with COVID-19 and cancer was 14.1% (95% confidence interval 9.1%, 19.8%, I 2 : 52.3%). We identified impeding questions that need to be answered to provide the foundation for an iterative review of the developing evidence base, and inform policy and practice going forwards. Analyses of the available data corroborate an unfavourable outcome of hospitalised patients with COVID-19 and cancer. Our findings encourage future studies to report detailed social, demographic and clinical characteristics of cancer patients, including performance status, primary cancer type and stage, as well as a history of anti-cancer therapeutic interventions.
# Introduction
Populations at risk of severe outcomes of COVID-19 are gradually being identified [bib_ref] Clinical course and risk factors for mortality of adult inpatients with COVID-19..., Zhou [/bib_ref] and thus far include people with diabetes, obesity and hypertension, as well as sociodemographic risk factors, such as male sex, ethnicity and smoking status .
Patients with cancer represent a population of particular interest and are not only vulnerable to the direct impacts of COVID-19 infection, but also to the effects of healthcare reprioritisation, with subsequent delays in cancer diagnosis and treatment. Over the course of the pandemic, the UK's National Institute for Health and Care Excellence (NICE) advised clinicians to weigh up the risk of delaying inhospital cancer care with the risk of nosocomial COVID-19 infection. However, there is still little robust data on the nature of the risks involved. Consequent post-pandemic surges in healthcare demands are expected to lead to decreased cancer survival rates.
Common immunosuppressive therapies are thought to increase vulnerability to severe outcomes of COVID-19 in cancer patients. Although recent studies of immunosuppressed patients indicate that outcomes may be less severe, larger studies of malignancy indicate an association with higher mortality rates [bib_ref] Clinical impact of COVID-19 on patients with cancer (CCC19): a cohort study, Kuderer [/bib_ref] [bib_ref] The UK Coronavirus Cancer Monitoring Project: protecting patients with cancer in the..., Anil [/bib_ref]. These risks probably vary across specific tumour types [bib_ref] Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China, Liang [/bib_ref] [bib_ref] SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in..., Yu [/bib_ref] and therapeutic approaches [bib_ref] Risk factors associated with acute respiratory distress syndrome and death in patients..., Wu [/bib_ref]. Given the heterogenous nature of malignancies, determining the impact of cancer and its treatment on the presentation and prognosis of COVID-19 remains an unmet challenge.
Here we collated evidence pertaining to the manifestation of COVID-19 in patients with active malignancy. Specifically, we considered:
(i) The prevalence of cancer in hospitalised patients with COVID-19; (ii) How COVID-19 presents in cancer patients; (iii) Whether patients with cancer and COVID-19 are at increased risk of mortality.
Cancer heterogeneity, including pathology, treatment and service-level determinants will probably influence COVID-19 outcomes. To meaningfully answer the questions above, we therefore considered demographic, social and healthcare factors, as well as primary cancer type and stage, and recent therapeutic management. Together, this study provides an overview of the evidence and quality of data currently available, to guide future reports of larger cohort studies with regards to the prevalence, presentation and outcome of patients with COVID-19 and cancer.
# Materials and methods
## Protocol
In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, the study protocol was prospectively registered on PROSPERO (CRD42020182103).
## Inclusion and exclusion criteria
Patients of any age, sex, nationality and healthcare setting were included. Active malignancy was defined as current malignant disease or treatment for malignancy within the last 12 months. All study types reporting primary clinical data or original analyses of COVID-19 in patients with an existing diagnosis of active cancer in the English language were eligible for inclusion. To account for the rapid evolution of the evidence base and to minimise the effects of publication bias, preprint literature was included. Exclusion criteria were protocol-only publications, commentaries, opinion pieces and systematic reviews without original statistical analyses.
## Sources and search strategy
We conducted searches in PubMed, EMBASE (OVID) and MedRxiv for studies published between 1 December 2019 and 24 April 2020 (see Appendix A). We subsequently manually searched the citation lists of all included studies for papers that were not identified on systematic database searches.
## Study selection
Two reviewers independently screened each title and abstract for eligibility, and an independent third reviewer resolved all disagreements. To ensure that cancer subgroups were not overlooked, abstracts without explicit mention of cancer patients were not discarded. Finally, we used the same approach to screen the full manuscripts of included studies.
## Data collection and quality assessment
We developed a standardised data extraction tool and used the NIH National Heart, Lung and Blood Institute's Study Quality Assessment tools to quality assess included papers. Included papers underwent single-reviewer data extraction and quality assessment, and a 30% random sample was checked by a second reviewer. Using the NIH National Heart, Lung and Blood Institute's Study Quality Assessment tools, each study was quality appraised by two reviewers and converted into 'high', 'moderate' and 'low' risk of bias ratings through consensus between three reviewers.
# Synthesis of results
We reviewed all papers with respect to the primary questions and conducted qualitative evidence characterisation for each question. To account for variations in cancer prevalence, we categorised studies by geography into China and Western countries. We used results from the geographical subgroups to infer qualitative interpretations. We calculated (i) the pooled prevalence of co-morbid cancer in hospitalised COVID-19 patients and (ii) the pooled inhospital mortality risk for patients with cancer and COVID-19. For the pooled analyses, we screened for studies that overlapped in terms of study site and recruitment window, in order to prevent duplication of data, i.e. double counting of the same patients within the quantitative synthesis. Where two or more studies were conducted at the same hospital with overlapping recruitment windows, the study with the longest recruitment window and/or the largest sample size of cancer patients was retained. Studies with inappropriate designs for the purposes of each pooled analysis were also excluded, i.e. studies deliberately selecting cancer patients or selecting on the basis of another co-morbidity/admission reason, for the pooled cancer prevalence analysis; studies with a short follow-up or not reporting death/discharge outcomes for the pooled mortality risk analysis; and case-control studies selecting patients on the basis of specific adverse outcomes such as death or intensive therapy unit admission for both analyses. Case series or reports, and studies including hospital staff or out-patients in their cohort were also excluded from both analyses. The pooled analyses were conducted using a random-effects model after the FreemaneTukey Double Arcsine Transformation of variances in the Metaprop package in Stata 14 [bib_ref] Metaprop: a Stata command to perform meta-analysis of binomial data, Nyaga [/bib_ref]. Here, exact or Clopper Pearson 95% confidence intervals were calculated for individual studies [bib_ref] The use of confidence or fiducial limits illustrated in the case of..., Clopper [/bib_ref]. To address heterogeneity between studies, we used random-effects models that incorporate the assumption that studies are estimating different, however related, effects. Specifically, the I 2 statistic was used to assess statistical heterogeneity.
# Results
## Study selection
We retrieved 4635 titles through a systematic search and identified 10 further studies through manual searches of the reference lists. After removing duplicate records, 805 titles were retained for title and abstract screening, of which 311 articles were selected for full-text screening. In total, 110 records were subsequently included in the qualitative synthesis (see Appendix B). Of these, 37 studies were used to calculate the pooled prevalence of cancer in hospitalised patients with COVID-19, 30 studies were used to characterise the presenting features of COVID-19 and 17 studies were used to calculate the pooled mortality risk (see Appendix C).
## Study characteristics
Eighty cohort studies, six cross-sectional studies, two case-control studies, one interventional trial, 10 case series and 11 case reports were included [fig_ref] Fig 1: Flowchart of included and excluded studies [/fig_ref]. Sixty-seven (60.9%) of the included studies were preprint copies. Given the geographical emergence and spread of COVID-19, most studies were from China (n ¼ 82), Italy (n ¼ 10) and the US (n ¼ 8). Two pairs of studies considered exactly the same cohorts [bib_ref] Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China, Liang [/bib_ref] [bib_ref] Risk factors associated with acute respiratory distress syndrome and death in patients..., Wu [/bib_ref] [bib_ref] Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in..., Yang [/bib_ref] [bib_ref] Comorbidity and its impact on 1590 patients with Covid-19 in China: a..., Guan [/bib_ref] , 31 studies had overlapping patient cohorts (study site and recruitment window) with possible duplication of included patients, two studies [bib_ref] Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China, Liang [/bib_ref] [bib_ref] Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in..., Wang [/bib_ref] described themselves as case series but were categorised as cohort studies, and one study described itself as a cross-sectional survey but was categorised as a case series. Due to the inherent selection and reporting biases of this study design, all case series and reports (n ¼ 21) were classified as high risk of bias. Eighty-one other papers were identified as having a high risk of bias, three a moderate risk of bias and only five a low risk of bias.
The characteristics of the study populations are outlined in [fig_ref] Table 1: Study characteristics [/fig_ref]. Most studies (n ¼ 93) described a general cohort of COVID-19 patients with coincidental inclusion of co-morbid cancer. Socio-demographically, 23 studies reported age, 25 reported gender, six reported socioeconomic status, four reported smoking status and two reported occupational status. Thirty studies distinguished cancer types, nine identified tumour size and/or metastases and three provided cancer stage. Twenty-three studies reported recent cancer treatment (chemotherapy, radiotherapy, immunotherapy or surgery), six described the aim of treatment (palliative, radical, maintenance), 12 referenced the time since treatment (current or <12 months) and only one paper reported on immune competence.
## Prevalence of cancer in hospitalised covid-19 patients
The pooled prevalence of active cancer in hospitalised patients with COVID-19 across 37 cohort studies was 2.6% (95% confidence interval 1.8%, 3.5%) [fig_ref] Fig 2: Fig 2 [/fig_ref]. The prevalence in China and Western countries was 1.7% (95% confidence interval 1.3%, 2.3%) ] and 5.6% (95% confidence interval 4.5%, 6.7%), respectively [46e51]. There was significant heterogeneity in the estimates for each group (P < 0.001), with I 2 ¼ 92.0% across all 10 countries, and specifically, 57.6% for China and 82.3% for Western countries.
## Presenting features of covid-19 in cancer patients
The clinical presentation of COVID-19 in hospitalised cancer patients across 30 studies is detailed in [fig_ref] Table 2: Clinical presentation of COVID-19 in cancer patients [/fig_ref]. Here, 16 (53.3%) were observational cohort studies, five were case series and nine were case reports. Patients most commonly presented with non-specific symptoms of fever, cough, dyspnoea, fatigue, myalgia, chest tightness and headache. Where computed tomography was carried out, abnormal changes (including ground glass opacities) were seen up to 6 days in advance of the clinical presentation. Laboratory findings included non-specific changes in inflammatory markers and decreased arterial oxygen saturations.
## In-hospital mortality risk for cancer patients with covid-19
Across 17 retrospective cohort studies, clinical outcomes were identified in 904 hospitalised patients with cancer and COVID-19 . The pooled in-hospital mortality risk was 14.1% (95% confidence interval 9.1%, 19.8%; [fig_ref] Fig 3: Proportion of deaths among hospitalised patients with cancer and COVID-19 [/fig_ref]. There was significant heterogeneity in the estimate (P ¼ 0.01) with I 2 ¼ 55.9%. Only six of 17 studies reported the outcome for all cancer patients at the end of the study observation window [bib_ref] SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in..., Yu [/bib_ref] [bib_ref] Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three..., Zhang [/bib_ref]. The median length of in-hospital observation was 7e31 days and 0e75% of the cancer patients remained hospitalised at the end of the study [bib_ref] SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in..., Yu [/bib_ref] [bib_ref] Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three..., Zhang [/bib_ref]. Furthermore, only eight studies specified the cancer type and severity and/or treatment during admission or within the past 12 months . Of these, only two studies reported both cancer type and treatment for all included patients [bib_ref] SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in..., Yu [/bib_ref] [bib_ref] Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three..., Zhang [/bib_ref]. Together, cancer types were reported for 276 patients and genitourinary, gastrointestinal and respiratory system cancers accounted for 91.6% of all reported cancers [bib_ref] Clinical course and risk factors for mortality of adult inpatients with COVID-19..., Zhou [/bib_ref] [bib_ref] The UK Coronavirus Cancer Monitoring Project: protecting patients with cancer in the..., Anil [/bib_ref] [bib_ref] SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in..., Yu [/bib_ref] [bib_ref] Clinical characteristics and prognosis in cancer patients with COVID-19: a single center's..., Ma [/bib_ref] [bib_ref] Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three..., Zhang [/bib_ref] [bib_ref] Do patients with cancer have a poorer prognosis of COVID-19? An experience..., Miyashita [/bib_ref] [bib_ref] Early appearance of coronavirus disease 2019 associated pulmonary infiltrates during daily radiotherapy..., Suppli [/bib_ref]. Oncological treatments were reported for 68 patients, and more than a third were treated with chemotherapy [bib_ref] SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in..., Yu [/bib_ref] [bib_ref] Clinical characteristics and prognosis in cancer patients with COVID-19: a single center's..., Ma [/bib_ref] [bib_ref] Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three..., Zhang [/bib_ref]. Across 14 case reports and series, 24 cancer patients with COVID-19 were identified . Nine died, three remained in hospital and 12 were discharged by the end of the study period. Of these, 23 had active cancer and one was in remission [bib_ref] A rapid fatal evolution of Coronavirus Disease-19 (COVID-19) in an advanced lung..., Bonomi [/bib_ref]. The average in-patient stay was 18 days. Cancer types included 20 solid organ cancers (lung, ovarian, cervical, endometrial, breast) and four haematological cancers [bib_ref] First case of COVID-19 in a patient with multiple myeloma successfully treated..., Zhang [/bib_ref] [bib_ref] COVID-19 in posttransplant patients-report of 2 cases, Huang [/bib_ref] [bib_ref] COVID-19 in a patient with chronic lymphocytic leukaemia, Zheng [/bib_ref] with different stages of severity. Although cancer treatments were non-homogenous, all patients received oxygen therapy, antibiotics, intensive care admission and mechanical ventilation for treatment of COVID-19 ].
# Discussion
We narratively synthesised results from 110 studies across 10 countries. Included studies were predominantly from China (n ¼ 82), Italy (n ¼ 10) and the USA (n ¼ 8), in line with the evolution of the pandemic.
## Prevalence of co-morbid cancer
From 37 studies, the overall prevalence of active cancer as a co-morbidity among hospitalised patients with COVID-19 was estimated to be 2e4%, with a higher prevalence seen in studies from Western settings (5e7%) compared with studies conducted in China (1e2%). These findings are unsurprising considering a recent study that reported a lower incidence of total cancer in China compared with Western countries such as the USA and UK [bib_ref] Current cancer situation in China: good or bad news from the 2018..., Feng [/bib_ref].
## Clinical presentation
Cancer patients with COVID-19 seem to most commonly present with non-specific symptoms of fever, dyspnoea and chest tightness, in addition to decreased arterial oxygen saturation, ground glass opacities on computed tomography and non-specific changes in inflammatory markers. One studyreported that cancer patients more commonly presented with confusion. Consistent with the presentation of COVID-19 in non-cancer patients [bib_ref] Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis, Rodriguez-Morales [/bib_ref] , laboratory findings in cancer patients showed non-specific changes in inflammatory markers, decreased arterial oxygen saturations and abnormal computed tomography scans. Where patients presented with lymphopenia, nonlocalising symptoms and disseminated infection, underlying immunosuppression must be considered.
## Mortality
The estimated in-hospital mortality risk for patients with COVID-19 and cancer is between 9 and 20% across all settings. The pooled mortality risk estimate of 14.1% among hospitalised patients with COVID-19 and cancer is at least five times higher than the reported mortality risk of COVID-19 in non-elderly patients without underlying predisposing conditions across Europe and America. These results corroborate a recently published systematic review and meta-analysisthat reported that malignancy, among age and other comorbidities, is associated with a greater risk of death from COVID-19 infection.
The findings in this review are in line with findings from four of the included studies that independently reported increased mortality risks in their respective cohorts. Specifically, two studies reported a higher risk of mortality in cancer patients with COVID-19 when adjusting for age and sex (hazard ratio 1.4; 95% confidence interval 1.0e2.0) [bib_ref] Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three..., Zhang [/bib_ref] and another reported a significantly higher relative risk of mortality in cancer patients under the age of 50 years compared with non-cancer patients in the same age group (relative risk 5.01; 95% confidence interval 1.55e16.2) [bib_ref] Do patients with cancer have a poorer prognosis of COVID-19? An experience..., Miyashita [/bib_ref]. Finally, cancer patients were found to have a higher composite risk of admission to an intensive care unit, invasive ventilation or death when adjusting for both age and smoking status (hazard ratio 3.501; 95% confidence interval 1.604e7.643) [bib_ref] Comorbidity and its impact on 1590 patients with Covid-19 in China: a..., Guan [/bib_ref].
# Methodological considerations
There are several important limitations inherent in the available evidence that restrict the inferences that can be drawn from these findings. Here, we first review the limitations of the available data and then address specific measures that should be undertaken to ensure that studies examining prevalence, risk and mortality differences are epidemiologically robust from the outset. Proportion of hospitalised COVID-19 patients with cancer. The pooled prevalence of active cancer in hospitalised patients with COVID-19 across 37 cohort studies was 2.6% (95% confidence interval 1.8%, 3.5%). In China and Western countries, the prevalence figures were 1.7% (95% confidence interval 1.3%, 2.3%) and 5.6% (95% confidence interval 4.5%, 6.7%), respectively.
All included studies accurately reported the prevalence of cancer in hospitalised patients with a confirmed diagnosis of COVID-19. Although this provides insight into the proportion of co-morbid cancer, it is not a measure of the risk of COVID-19 acquisition among people with cancer.
Notably, the need for hospitalisation may be inflated due to extensive clinical monitoring, a group coached to present early with infection or may have been an incidental finding [bib_ref] Do patients with cancer have a poorer prognosis of COVID-19? An experience..., Miyashita [/bib_ref]. Together, behavioural and healthcare factors such as self-surveillance, health literacy, access to healthcare and thresholds for testing or admission may lead to earlier presentation and over-representation of cancer patients within the COVID-19 cohort. Cancer patients with COVID-19 were largely identified coincidentally in cohort studies, where the primary objective was not to investigate the prevalence, clinical presentation or course of disease. Consequently, relevant confounders, including demographic, social and clinical characteristics, were not considered or reported. Most studies did not report the specific cancer type or treatment history for included cancer patients, nor did they outline the primary reason for hospital admission. This means that for certain patients with cancers requiring closer monitoring or more frequent contact with healthcare services, there may be a bias with regards to the detection of COVID-19 symptoms and consequent diagnosis. There may also be a bias towards detection of COVID-19 in patients whose disease or therapy causes symptoms similar to COVID-19, prompting higher rates of testing and, consequently, detection. For this review, only studies examining data from hospital in-patients were considered. However, none of the included studies specified the original reason for admission. Therefore, it remains uncertain whether included patients were originally admitted for reasons specific to their cancer diagnosis, such as elective admission for cancer treatment or emergency admission for cancer-related complications, or whether they were admitted due to illness caused primarily by COVID-19, which poses a significant challenge when interpreting the available data with regards to risk of infection. Furthermore, differences in study design, study population and risk of bias inevitably lead to heterogeneity between studies, and this is particularly present in combined pooled estimates of prevalence in the West and China. To address these heterogeneities, we used a randomeffects model that incorporates an assumption that studies are estimating different, yet related, effects. We also examined these regions separately and heterogeneity was lower when we did this, particularly for China, which shows that some of the heterogeneity is explained by betweencountry differences. Heterogeneity is often high in metaanalyses of proportions [78e81] and care must be taken if the results from the analysis are used for clinical decision making. Outcomes for 75%of patients in cohort studies remain unknown due to insufficient follow-up periods and the specific causes of mortality are not specified. Without cause of death information, it is not possible to distinguish between mortality caused by cancer or its associated treatment complications and mortality as a direct result of COVID-19; in cases where COVID-19 causes decompensation of underlying disease, even cause of death data may be unreliable. Excess all-cause mortality in cancer patients diagnosed with COVID-19 would provide the only robust metric of the risk posed by COVID-19 and requires routine setting-specific data and large sample sizes to ascertain.
## Development of future studies
Included studies were largely (92.7%) assessed as having a high potential of bias, and we uncovered significant limitations in the existing literature base, which must be addressed [fig_ref] Fig 4: Recommendations for future studies of COVID-19 in cancer patients [/fig_ref]. It is recommended that future studies include data on cancer type and treatment regimens of all cancer patients, as well as primary admission reason for all patients, with details on the causes of death if mortality data are presented. To estimate the relative risk of SARS-CoV-2 infection in cancer patients, the incidence of COVID-19 should be measured through longitudinal followup of cohorts of individuals with and without cancer. Additionally, sub-cohorts of specific cancer types and stages should be considered; ideally, without prior or current SARS-CoV-2 infection at baseline. Cohorts must be selected through probabilistic sampling methods to ensure they are balanced and representative, particularly with respect to other demographic and clinical variables. Follow-up duration should adequately allow for declining incidence, and inconsistency in follow-up duration should be addressed using personetime metrics. Studies must define, collect and present data on all potential confounding and interacting variables, such as those discussed under 'Methodological considerations', and adjust for these at the point of design or analysis. We further encourage future studies to report comprehensive social, demographic and clinic characteristics of cancer patients. Clinical characteristics of interests pertain specifically to performance status, co-morbidities, cancer type, cancer stage, treatment type, history and plan.
Future studies will need to reflect the changing epidemiology of COVID-19 with regards to their design and outcomes of interest. In light of the current resurgence, or 'second wave', with high community transmission nationally, community-based follow-up of known cancer patients should also be undertaken; specifically considering household and occupational exposures, as well as healthcare exposures, and linking this with hospital admission and clinical outcomes data. Aggregated findings from outbreak analyses based on household or institutional clusters may reveal insights regarding transmission risk for cancer patients compared with non-cancer contacts of COVID-19 cases. In order to examine mortality risks, it is essential to capture both in-hospital and out-of-hospital deaths. Routine mortality data, such as cause of death certification, should be utilised, which can be linked with both national COVID-19 surveillance data and healthcare datasets. When community transmission is effectively controlled, researchers should continue to monitor large cohorts of cancer patients over the course of treatment, with a focus on nosocomial transmission.
Going forward, ceilings of care for those receiving palliative versus curative therapy should be considered, and the effects of immunosuppressive chemotherapy versus targeted immunotherapy, radiotherapy, surgery or other types of treatment should be reported.
# Conclusions
The results from this review show that cancer constitutes a co-morbidity in 1e2% of hospitalised COVID-19 patients in China and 5e7% in Western countries. They present similarly clinically to non-cancer patients, and the preliminary evidence suggests there is an increased in-hospital mortality risk in patients with cancer and COVID-19. However, a comprehensive assessment of the qualities of the included studies show the need for longitudinal follow-up studies, where cohorts are selected through probabilistic sampling methods, and follow-up duration is extended to encompass clinical progression and outcome. In order to improve generalisability, granularity and understanding of heterogeneity, we further encourage future studies to report detailed social, demographic and clinic characteristics of cancer patients, including but not limited to, performance status, other co-morbidities, cancer type and stage, and treatment type, history and plan. We recommend that future studies include primary admission reason for all patients, with details on the causes of death if mortality data are presented. Finally, in light of the current 'second wave' of COVID-19 with high community transmission, it is essential to report both in-hospital and out-of-hospital deaths, and communitybased follow-up should be implemented.
[fig] Fig 1: Flowchart of included and excluded studies. Eighty cohort studies, six cross-sectional studies, two case-control studies, one interventional trial, 10 case series and 11 case reports were included. [/fig]
[fig] Fig 2: Fig 2. Proportion of hospitalised COVID-19 patients with cancer. The pooled prevalence of active cancer in hospitalised patients with COVID-19 across 37 cohort studies was 2.6% (95% confidence interval 1.8%, 3.5%). In China and Western countries, the prevalence figures were 1.7% (95% confidence interval 1.3%, 2.3%) and 5.6% (95% confidence interval 4.5%, 6.7%), respectively. [/fig]
[fig] Fig 3: Proportion of deaths among hospitalised patients with cancer and COVID-19. The pooled in-hospital mortality risk among patients with COVID-19 and cancer was 14.1% (95% confidence interval 9.1%, 19.8%). [/fig]
[fig] Fig 4: Recommendations for future studies of COVID-19 in cancer patients. [/fig]
[table] Table 1: Study characteristics [/table]
[table] Table 2: Clinical presentation of COVID-19 in cancer patients [/table]
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Olaparib in patients with metastatic castration-resistant prostate cancer with DNA repair gene aberrations (TOPARP-B): a multicentre, open-label, randomised, phase 2 trial
Background Metastatic castration-resistant prostate cancer is enriched in DNA damage response (DDR) gene aberrations. The TOPARP-B trial aims to prospectively validate the association between DDR gene aberrations and response to olaparib in metastatic castration-resistant prostate cancer.Methods In this open-label, investigator-initiated, randomised phase 2 trial following a selection (or pick-thewinner) design, we recruited participants from 17 UK hospitals. Men aged 18 years or older with progressing metastatic castration-resistant prostate cancer previously treated with one or two taxane chemotherapy regimens and with an Eastern Cooperative Oncology Group performance status of 2 or less had tumour biopsies tested with targeted sequencing. Patients with DDR gene aberrations were randomly assigned (1:1) by a computer-generated minimisation method, with balancing for circulating tumour cell count at screening, to receive 400 mg or 300 mg olaparib twice daily, given continuously in 4-week cycles until disease progression or unacceptable toxicity. Neither participants nor investigators were masked to dose allocation. The primary endpoint of confirmed response was defined as a composite of all patients presenting with any of the following outcomes: radiological objective response (as assessed by Response Evaluation Criteria in Solid Tumors 1.1), a decrease in prostate-specific antigen (PSA) of 50% or more (PSA50) from baseline, or conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to <5 cells per 7·5 mL blood). A confirmed response in a consecutive assessment after at least 4 weeks was required for each component. The primary analysis was done in the evaluable population. If at least 19 (43%) of 44 evaluable patients in a dose cohort responded, then the dose cohort would be considered successful. Safety was assessed in all patients who received at least one dose of olaparib. This trial is registered at ClinicalTrials.gov, NCT01682772. Recruitment for the trial has completed and follow-up is ongoing. Findings 711 patients consented for targeted screening between April 1, 2015, and Aug 30, 2018. 161 patients had DDR gene aberrations, 98 of whom were randomly assigned and treated (49 patients for each olaparib dose), with 92 evaluable for the primary endpoint (46 patients for each olaparib dose). Median follow-up was 24·8 months (IQR 16·7-35·9). Confirmed composite response was achieved in 25 (54·3%; 95% CI 39·0-69·1) of 46 evaluable patients in the 400 mg cohort, and 18 (39·1%; 25·1-54·6) of 46 evaluable patients in the 300 mg cohort. Radiological response was achieved in eight (24·2%; 11·1-42·3) of 33 evaluable patients in the 400 mg cohort and six (16·2%; 6·2-32·0) of 37 in the 300 mg cohort; PSA50 response was achieved in 17 (37·0%; 23·2-52·5) of 46 and 13 (30·2%; 17·2-46·1) of 43; and circulating tumour cell count conversion was achieved in 15 (53·6%; 33·9-72·5) of 28 and 13 (48·1%; 28·7-68·1) of 27. The most common grade 3-4 adverse event in both cohorts was anaemia (15 [31%] of 49 patients in the 300 mg cohort and 18 [37%] of 49 in the 400 mg cohort). 19 serious adverse reactions were reported in 13 patients. One death possibly related to treatment (myocardial infarction) occurred after 11 days of treatment in the 300 mg cohort.Interpretation Olaparib has antitumour activity against metastatic castration-resistant prostate cancer with DDR gene aberrations, supporting the implementation of genomic stratification of metastatic castration-resistant prostate cancer in clinical practice.
# Introduction
Molecular stratification for treatment is not currently the standard of care for metastatic prostate cancers despite evidence of substantial interpatient genomic hetero geneity. Most therapeutic strategies for advanced prostate cancers target androgen receptor signalling; taxane based chemotherapies and radiopharmaceuticals are also approved. [bib_ref] Prostate cancer, Attard [/bib_ref] Although these drugs have improved outcomes in the past decade, metastatic prostate cancer remains invariably fatal and new therapeutic strategies involving molecular stratification are urgently needed. Genomic studies of metastatic prostate cancer have identified a number of potentially actionable recurrent genomic aberrations, [bib_ref] The long tail of oncogenic drivers in prostate cancer, Armenia [/bib_ref] [bib_ref] Integrative clinical genomics of advanced prostate cancer, Robinson [/bib_ref] [bib_ref] Genomic hallmarks and structural variation in metastatic prostate cancer, Quigley [/bib_ref] including lossoffunction alterations in DNA repair genes in 20-25% of cases, such as defects in homologous recombinationmediated repair genes. [bib_ref] Integrative clinical genomics of advanced prostate cancer, Robinson [/bib_ref] Among these, germline or somatic alterations in BRCA2 are the most common, accounting for 6-12% of cases across studies. [bib_ref] The long tail of oncogenic drivers in prostate cancer, Armenia [/bib_ref] [bib_ref] Integrative clinical genomics of advanced prostate cancer, Robinson [/bib_ref] [bib_ref] Genomic hallmarks and structural variation in metastatic prostate cancer, Quigley [/bib_ref] These data underpin the evaluation of poly(ADPribose) polymerase (PARP) inhibitors in metastatic prostate cancer. [bib_ref] Targeting the DNA repair defect in BRCA mutant cells as a therapeutic..., Farmer [/bib_ref] [bib_ref] Specific killing of BRCA2 deficient tumours with inhibitors of poly(ADPribose) polymerase, Bryant [/bib_ref] Olaparib is an orally bioavailable inhibitor of the catalytic activity of PARP1 and PARP2, which have key roles in DNA damage response (DDR). Olaparib is approved for the treatment of advanced ovarian and breast cancers associated with germline BRCA1 or BRCA2 mutations. [bib_ref] Olaparib monotherapy in patients with advanced cancer and a germline BRCA1/2 mutation, Kaufman [/bib_ref] It is also approved as a maintenance therapy after response to platinumbased chemotherapy for ovarian cancer, indicating benefit from PARP inhibition beyond tumours with BRCA1/2 mutations. [bib_ref] Olaparib tablets as maintenance therapy in patients with platinumsensitive, relapsed ovarian cancer..., Pujadelauraine [/bib_ref] [bib_ref] Olaparib maintenance therapy in patients with platinumsensitive relapsed serous ovarian cancer: a..., Ledermann [/bib_ref] Furthermore, olaparib has antitumour activity in vitro and in vivo in models that are defective in other DDR proteins, including PALB2, ATM, FANCD2, RAD51, and RAD54, among others, although the magnitude of preclinical sensitisation varies between proteins, with BRCA2 loss being arguably the most potent sensitising event. [bib_ref] Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib, Murai [/bib_ref] [bib_ref] Trapping of PARP1 and PARP2 by clinical PARP inhibitors, Murai [/bib_ref] To evaluate the antitumour activity of olaparib against metastatic castrationresistant prostate cancer, we designed TOPARP, an adaptive programme of serial phase 2 clinical trials aimed at identifying predictive biomarkers for response to PARP inhibition in metastatic castrationresistant prostate cancer. In the first trial, TOPARPA, we identified an association between putatively deleterious DDR gene aberrations and res ponse to olaparib in 49 molecularly unselected patients. [bib_ref] DNArepair defects and olaparib in metastatic prostate cancer, Mateo [/bib_ref] In this Article, we present the results of TOPARPB, which was designed to validate the observed antitumour activity of olaparib in patients with metastatic castration resistant prostate cancer presenting with DDR gene aberrations.
# Methods
## Study design and participants
TOPARPB is a multicentre, openlabel, investigator initiated, randomised phase 2 trial. Patients were recruited from 17 UK hospitals (appendix p 2).
Patients with prostate cancer that had developed metastasis and castration resistance were first registered on the trial for molecular preselection by targeted next generation sequencing (NGS) of primary or metastatic prostate cancer biopsies. Eligible patients were men aged 18 years or older, with histologically confirmed prostate adeno carcinoma (metastatic and castrationresistant), and whose tumours had a putatively pathogenic mutation or homozygous deletion in a DDR gene that could be
## Research in context
Evidence before this study Trials for advanced prostate cancer have rarely pursued molecular stratification, and none of the drugs approved to date for metastatic prostate cancer care have a validated companion biomarker. Before starting this study, several genomic landscape studies were published describing an enrichment for aberrations in DNA repair genes in metastatic prostate cancers (studies identified in PubMed, searching for "prostate cancer", "genomics", and "biopsy", between Jan 1, 2010 and Nov 1, 2015, with no language restrictions). Preclinical and clinical studies identified in PubMed (searching for "cancer", "PARP", and "BRCA" or "DNA repair" between Jan 1, 2005 and July 1, 2019, with no language restrictions) have established a correlation between different DNA repair defects and sensitivity to PARP inhibition in different tumour types, leading to drug approvals in ovarian and breast cancer. In the TOPARP-A trial, we identified an association between somatic alterations in DNA repair genes and antitumour activity of olaparib in 49 patients with metastatic prostate cancer. Other clinical trials of PARP inhibitors in prostate cancer were identified on the ClinicalTrials.gov website, searching for "prostate cancer" and "PARP" for studies published from database inception to July 1, 2019, without language restriction.
## Added value of this study
To our knowledge, TOPARP-B is the first prospective clinical trial in a genomically defined population of patients with metastatic prostate cancer. TOPARP-B aimed to clinically qualify a predictive biomarker for treating metastatic prostate cancer. TOPARP-B also assessed different doses of olaparib, and correlated different genomic aberrations and antitumour activity. This study has confirmed the antitumour activity of olaparib against metastatic prostate cancer with defective DNA repair secondary to either germline or somatic gene inactivation.
## Implications of all the available evidence
Randomised phase 3 trials for DNA repair-defective prostate cancers are now ongoing based on the TOPARP data. Our results, if confirmed in registration studies, would support the implementation of tumour genomic testing in clinical practice for treatment stratification in advanced prostate cancer.
See Online for appendix associated with sensitivity to PARP inhibition as identified by NGS. Patients were required to have previously received at least one but no more than two taxanebased chemotherapy regimens, regardless of prior exposure to novel hormonal drugs. Other inclusion criteria included: documented prostate cancer progres sion at trial entry, defined by either rising prostate specific antigen (PSA) serum concentration (according to the Prostate Cancer Working Group 2 [PCWG2] criteria [bib_ref] Design and end points of clinical trials for patients with progressive prostate..., Scher [/bib_ref] or radiologically (according to modified Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1 14 or by bone scan as per PCWG2 criteria); a castrate testosterone concentration of less than 50 ng/dL; an Eastern Cooperative Oncology Group (ECOG) perfor mance status of 2 or less; and adequate organ function (including haemoglobin ≥9 g/dL after a protocol amend ment on March 15, 2018 [previously ≥10 g/dL], platelets ≥100 × 10⁹ per L, serum creatinine ≤1·5 times the institu tional upper limit of normal, and albumin >25 g/L). Patients previously treated with PARP inhibitors, platinum, cyclophosphamide, or mito xantrone were not eligible, nor were patients with known symptomatic brain metastasis or untreated spinal cord compressions. The baseline count for circulating tumour cells (CellSearch system; Menarini Silicon Biosystems, Castel Maggiore, Italy) had to be five cells per 7·5 mL blood or higher except in patients with radiologically measurable target lesions of 2 cm or more in diameter on the baseline CT scan and a PSA concentration of 2 ng/mL or higher on screening. The full eligibility criteria are in the appendix (pp 3-4). The complete study protocol is available in the appendix.
The study was approved by the London-Surrey Borders Research Ethics Committee (REC reference 11/LO/2019), and cosponsored by The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research (ICR), London, UK. The trial was done in accordance with the principles of good clinical practice and overseen by independent data monitoring and trial steering committees. A trial management group was responsible for the daytoday running of the trial. The Clinical Trials and Statistics Unit at ICR (ICRCTSU) had overall responsibility for trial coordination, monitoring, and analysis. Patients provided written informed consent before enrolment, both for the NGS prescreening and treatment stages.
## Randomisation and masking
Eligible patients were randomly allocated (1:1) to receive olaparib at 300 mg (approved dose for the tablet formulation in ovarian and breast cancer 15 ) or 400 mg twice a day. Randomisation was done centrally by the ICRCTSU via telephone. The allocation sequence was generated centrally by a computergenerated mini misation algorithm derived by the ICRCTSU, with circulating tumour cell count at screening (≥5 cells per 7·5 mL blood vs <5 cells per 7·5 mL blood) as a balancing factor. ICRCTSU staff involved in the randomisation were not involved in the clinical running of the trial or data collection. Neither participants nor clinicians were masked to dose allocation.
## Procedures
The targeted NGS of tumour samples was done at the Cancer Biomarkers Laboratory at ICR. DNA was extracted from formalinfixed and paraffinembedded (FFPE) tumour blocks with a DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Samples that passed quality control testing with an FFPE QC Kit (Illumina, San Diego, CA, USA) were used for library preparation with a customised panel (GeneRead DNAseq MixnMatch Panel V2; Qiagen) covering 113 genes; libraries were read with a MiSeq Sequencer (Illumina). Further details on the sample processing, quality control, bioinformatics pipelines, and panel design are available in the appendix (pp 5-7). [bib_ref] Gene copy number estimation from targeted nextgeneration sequencing of prostate cancer biopsies:..., Seed [/bib_ref] Patients previously known to have germline aberrations were eligible only on confirmatory tumour testing by NGS.
All patients received oral olaparib (300 mg or 400 mg, tablet formulation) twice daily continuously in 4week cycles until evidence of radiographic progression (based on RECIST 1.1 for soft tissue disease, or the appearance of ≥2 lesions on bone scan), unacceptable toxicity according to investigator review, or patient decision to discontinue. Discontinuation because of clinical progression was the decision of the treating physician; discontinuation based solely on rising PSA in the absence of radiographic or clinical progression was discouraged. Patients treated with 300 mg twice daily were offered the option of dose escalation to 400 mg twice daily on confirmation of radiographic progression, providing the escalation was considered to be clinically indicated by the treating physician and the patient had not previously required a dose reduction for management of toxicity.
Clinical assessments, including reviews of adverse events (according to the National Cancer Institute Common Terminology Criteria for Adverse Events [CTCAE] version 4.02) and ECOG performance status, physical examination, and routine blood tests (haematology and biochemistry), took place 2 weeks after the start of treatment, and then at the start of every new 4week cycle. Radiological assessments (CT and bone scans) were done every 12 weeks. Local radiological response assessments were used for the primary endpoint definition; all RECIST 1.1 responses were confirmed by central review by radiologists at ICR (AC and NT). Circulating tumour cell counts were measured every cycle for the first 12 weeks, and thereafter every 12 weeks. Circulating tumour cell counts were centrally analysed at the Cancer Biomarkers Laboratory at ICR (by PF, BE, and GF) and results were not made available to the treating physician. PSA serum measure ments were collected every cycle if available, and every 12 weeks at a minimum. Blood samples for correlative biomarker studies were taken every 4 weeks. Repeated tumour biopsies were optional, and pursued when feasible at baseline, after 1-4 weeks on therapy, and at the time of progression. Guidance on drug interruptions or dose reductions for CTCAE grade 3-4 haematological and nonhaematological toxicities were implemented as outlined in the protocol. Up to 42 days of temporary interruption of treatment was allowed prior to mandating permanent discontinuation.
## Outcomes
The primary endpoint was confirmed response, defined as a composite of any of the following outcomes: radiological objective response (as assessed by RECIST 1.1 [modified with PCWG2 recommendations], a decrease in PSA of 50% or more (PSA50) from baseline, and conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to <5 cells per 7·5 mL blood 17 ). To be judged a response confirmation in a second consecutive assessment at least 4 weeks later was required.
Secondary endpoints were: radiographic progression free survival, defined as the time from randomisation to first evidence of radiographic progression (according to RECIST 1.1 or bone scan as per PCWG2 criteria) or death; time to radiographic progression, defined as the time from randomisation to first evidence of radiographic progression; progressionfree survival, defined as the time from randomisation to radiographic progression, unequivocal clinical pro gression, or death; overall survival, defined as the time from randomisation to death from any cause; time to PSA progression, defined as a confirmed increase of 25% or more and an absolute increase of 2 ng/mL or more in PSA from the nadir (PCWG2); duration of PSA response, defined as the time from the first documented PSA decrease of 50% or greater to PSA progression; best percentage change in PSA from baseline while on treatment; percentage change in PSA from baseline at 12 weeks (or earlier if therapy was discontinued); proportion of patients with circulating tumour cell count conversion; and the safety and tolerability profile of olaparib.
A prespecified exploratory endpoint was response in patients in whom dose was escalated to 400 mg twice daily after progression on 300 mg twice daily. A pharma cokinetics substudy was planned but because of patients declining recruitment it was closed prematurely with no analyses pursued.
# Statistical analysis
This trial followed a selection (or pickthewinner) design. [bib_ref] Screened selection design for randomised phase II oncology trials: an example in..., Yap [/bib_ref] Each dose cohort was assessed independently for the primary endpoint. The sample size needed to show the minimum desired antitumour activity was determined on the basis of A'Hern's onestage design, with a response of 30% or less for the null hypothesis, and a response of more than 50% for the alternative hypothesis (onesided α level of 0·05 and a β level of 0·15). Following the A'Hern design, if at least 19 (43%) of 44 evaluable patients in a dose cohort responded, then the dose cohort would be considered successful. If the 400 mg twice daily dose cohort was deemed successful, the DDR biomarker identified in TOPARPA, in which all patients received 400 mg twice daily, would be considered validated as being predictive of response. In the event of both dose cohorts being successful, the pick thewinner selection strategy would include consideration of secondary end points. No formal interim analyses were planned.
For the primary endpoint, the evaluable population was defined as all randomly assigned patients who met all of the eligibility criteria and commenced trial treatment, unless they discontinued treatment prior to 12 weeks for reasons that were not related to the study drug or disease. Sensitivity analyses of the primary endpoint were done in the intentiontotreat (ITT) population (all randomly assigned patients) and per protocol population (all evaluable patients who received at least one cycle of olaparib and had no eligibility violations). A posthoc sensitivity analysis in patients with a circulating tumour cell count of five or more cells per 7·5 mL blood at baseline was done for comparison with TOPARPA results. All other efficacy analyses were done in the ITT population. Toxicity was analysed in all patients who received at least one dose of olaparib, and the worst grades of adverse events that occurred during treatment for each dose cohort are reported. Serious adverse events and deaths observed within 30 days of the last dose of study treatment were summarised by dose cohort, as well as the exposure to study drug and reasons for discon tinuation, dose modification or interruption, and treatment delay.
Analysis of the primary endpoint was triggered when all patients had completed at least 6 months of treat ment (in the absence of prior discontinuation). Evaluable patients who discontinued treatment prior to 12 weeks due to progression or toxicity and had no follow up assessments for the primary endpoint were considered nonresponders. Response is presented along with exact twosided 95% CIs. Percentage changes from baseline in PSA concentration and the sum of target lesions (RECIST 1.1) are represented in waterfall plots. Timetoevent endpoints are summarised by Kaplan Meier curves, and median times estimated with 95% CIs. For radiographic progressionfree survival and progressionfree survival, patients alive and without progression were censored at the last scheduled disease assessment during the study. For time to radiographic progression, patients who did not progress radiologically were censored at the last scheduled disease assessment during the study or date of death, whichever occurred first. Patients alive at the end of followup were censored for the analysis of overall survival. Landmark analyses *Non-mutually exclusive subgroups: one patient had BRCA1/2, CDK12, and other mutations, and two patients had both PALB2 and other mutations (included in each subgroup). †The BRCA2 K3226* variant is supposedly non-pathogenic [bib_ref] BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and..., Meeks [/bib_ref] and was therefore not considered sufficient for patients to be considered eligible; however, one patient with a BRCA2 K3226* variant was included because of evidence of concomitant loss of the contralateral allele.
were used to explore the association between circulating tumour cell conversion at 8 weeks and 12 weeks with radiographic progressionfree survival and overall survival. Additionally, exploratory subgroup analyses according to different genes of interest were preplanned for the efficacy endpoints. Five nonmutually exclusive subgroups were predefined: patients with alterations in BRCA1/2, ATM, CDK12, PALB2, and any other gene related to DDR or associated with PARP inhibitor sensitivity. Patients who had more than one DDR gene aberration were included in the analysis of all relevant subgroups.
The trial was not powered for headtohead direct comparisons of the two dose cohorts, and so tests to compare them were considered hypothesisgenerating (ie, χ² test to compare the proportion of patients with a response and logrank test to compare KaplanMeier curves). Statistical analyses were done with Stata software (version 15), on a snapshot of the data taken on July 5, 2019. The statistical analysis plan is available in the appendix.
This trial is registered with ClinicalTrials.gov, NCT01682772 and on the European Clinical Trials database, EudraCT 2011-000601-49.
## Role of the funding source
The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication. Data are median (IQR) or n (%). Percentages might not add up to 100% due to rounding. PSA=prostate-specific antigen. CTC=circulating tumour cell. RECIST=Response Evaluation Criteria in Solid Tumors. *More than one site could be reported. †Assessment of CTC count at screening not possible due to CTC kit shortage; the patient was allowed to be randomly assigned as he had RECIST 1·1 measurable disease; for randomisation CTC count was assumed to be <5 cells per 7·5 mL blood but the patient was unevaluable for CTC response. ‡Non-mutually exclusive subgroups: one patient in the 300 mg cohort had BRCA1/2, CDK12, and other mutations, and two patients in the 300 mg cohort had PALB2 and other mutations (in MSH2 and NBN, respectively). 98 patients with DDR gene aberrations were randomly assigned and treated in the two dose cohorts (49 patients in each cohort). At the time of the data snapshot, two patients remained on olaparib treatment. A greater number of participants were recruited than originally planned, at the recommendation of the independent data monitoring committee, to account for six participants (three in each cohort) who were deemed not evaluable (ineligible post randomisation) for the primary end point analyses. Median followup was 24·8 months (IQR 16·7-35·9).
# Results
## Between
The baseline characteristics of all patients assigned to a dose cohort are shown in table 1. All patients had previously received docetaxel, and 88 (90%) had also been treated with one or both of abiraterone acetate and enzalutamide prior to study entry. The distribution of gene aberration subgroups was largely similar between the two dose cohorts, except for CDK12 alterations (table 1). The composition of the prespecified gene aberration subgroups in the intentiontotreat popu lation are shown in. Baseline features of each gene aberration subgroup are summarised in the appendix (p 8).
92 patients (46 in each dose cohort) were evaluable for the primary endpoint. 70 (76%) patients were evaluable for the RECIST 1.1 response, 89 (97%) for PSA response, and 55 (60%) for circulating tumour cell conversion. A confirmed com posite response was observed in 25 (54·3%; 95% CI 39·0-69·1) of 46 patients in the 400 mg cohort and 18 (39·1%; 25·1-54·6) of 46 patients in the 300 mg cohort (p=0·14; table 2). Radiological response according to RECIST 1.1 was observed in eight (24·2%; 95% CI 11·1-42·3) of 33 evaluable patients in the 400 mg cohort and six (16·2%; 6·2-32·0) of 37 in the 300 mg cohort; PSA50 response was observed in 17 (37·0%; 23·2-52·5) of 46 and 13 (30·2%; 17·2-46·1) of 43, respectively; and circulating tumour cell count conversion was observed in 15 (53·6%; 33·9-72·5) of 28 and 13 (48·1%; 28·7-68·1) of 27, respectively. Based on the first 44 evaluable patients included in each cohort (as planned initially), 25 (57%) confirmed responses were recorded in the 400 mg cohort and 18 (41%) in the 300 mg cohort; thus, the predefined criteria for success was met for the 400 mg regimen but not for the 300 mg regimen.
When including in the analysis only the 55 evalu able patients with a circulating tumour cell count of ≥5 cells per 7·5 mL blood at baseline, confirmed composite response was observed in 17 (60·7%; 95% CI 40·6-78·5) of 28 evaluable patients in the 400 mg cohort and 13 (48·1%; 28·7-68·1) of 27 in the 300 mg cohort (appendix p 9). In keeping with previous reports, 17,20 circulating tumour cell conversions posttreatment were significantly associated with longer radiographic progressionfree survival and overall survival in landmark analyses (appendix p 15).
The best percentage change from baseline in PSA concentration and in the sum of target lesions in each patient in the intentiontotreat population are pre sented in [fig_ref] Figure 2: although five patients achieved CTC conversion [/fig_ref] and 2B. At the time of analysis, 45 (92%) of 49 patients on 400 mg olaparib and 46 (94%) of 49 patients on 300 mg olaparib had radio graphic progression or died; median radiographic progressionfree survival was 5·5 months (95% CI 4·4-8·3) in the 400 mg cohort and 5·6 months (3·7-7·7) in the 300 mg cohort (figure 2C). 39 (80%) patients on 400 mg and 38 (78%) patients on 300 mg had died, with a median overall survival of 14·3 months (9·7-18·9) in the 400 mg cohort and 10·1 months (9·0-17·7) in the 300 mg cohort. Further results on the secondary endpoints are sum marised in the appendix (pp [bib_ref] Gene copy number estimation from targeted nextgeneration sequencing of prostate cancer biopsies:..., Seed [/bib_ref] [bib_ref] Circulating tumor cell biomarker panel as an individuallevel surrogate for survival in..., Scher [/bib_ref] [bib_ref] Screened selection design for randomised phase II oncology trials: an example in..., Yap [/bib_ref] [fig_ref] Figure 2: although five patients achieved CTC conversion [/fig_ref]. A summary of treatment dose reductions, escalations (300 mg cohort), interruptions, and discontinuations in each dose cohort is presented in the appendix (p 10). Dose escalation from 300 mg to 400 mg was pursued in 11 patients. At the time of the data snapshot, ten had discontinued treatment: two due to adverse events and eight due to disease progression. These 11 patients were on treatment with 400 mg olaparib for a median of 7·8 weeks (IQR 3·7-10·4). None of these patients achieved a response after dose escalation.
The confirmed composite response, and response by individual components, for each of the predefined gene subgroups are shown in table 2. Further analysis of secondary endpoints per gene subgroup are presented in [fig_ref] Figure 3: Gene aberration subgroup, median [/fig_ref] and the appendix (pp [bib_ref] Trapping of PARP1 and PARP2 by clinical PARP inhibitors, Murai [/bib_ref] [bib_ref] DNArepair defects and olaparib in metastatic prostate cancer, Mateo [/bib_ref] [bib_ref] Design and end points of clinical trials for patients with progressive prostate..., Scher [/bib_ref] [bib_ref] New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1), Eisenhauer [/bib_ref] [bib_ref] BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and..., Meeks [/bib_ref]. The BRCA1/2 subgroup had the highest number of responses both for the composite endpoint of confirmed response and across all its component outcomes (table 2) and the longest median radiographic progressionfree survival [fig_ref] Figure 3: Gene aberration subgroup, median [/fig_ref] of all DDR gene aberration subgroups. Of the 32 patients in the BRCA1/2 subgroup, 13 had germline mutations in BRCA2, six somatic mutations in BRCA2, 11 homozygous deletions in BRCA2, and the remaining two cases had mutations in BRCA1 (one germline and one somatic). Ten patients in the BRCA1/2 subgroup (five allocated to 400 mg and five to 300 mg) remained on treatment for more than 1 year. 21 patients with suspected deleterious ATM aber rations were treated (table 1; one patient with homozygous deletion and the rest with germline or somatic mutations that are predicted to result in either truncation or missense mutations affecting the kinase domain), and 19 were evaluable for response (table 2). Details of each component of response in the evaluable patients with ATM aberrations are shown in the appendix (p 12).
No confirmed PSA50 or RECIST responses were observed in the 20 evaluable patients in the CDK12 subgroup [fig_ref] Table 2: Overall antitumour activity of olaparib in patients with DNA damage response gene... [/fig_ref] alteration; appendix p 13). Conversely, four of seven patients with PALB2 mutations responded to treatment [fig_ref] Table 2: Overall antitumour activity of olaparib in patients with DNA damage response gene... [/fig_ref]. 20 patients were evaluated in the subgroup with other gene alterations associated with DDR or PARP inhibitor sensitivity [fig_ref] Table 2: Overall antitumour activity of olaparib in patients with DNA damage response gene... [/fig_ref]. PSA50 responses were seen in two patients: one with a somatic nonsense mutation in FANCA and one with a CHEK2 mutation.
The safety population included all 98 patients treated [fig_ref] Table 3: Treatment-emergent adverse events [/fig_ref]. The tolerability profile was in line with what has been previously reported for olaparib and other PARP inhibitors. [bib_ref] The poly(ADPribose) polymerase inhibitor niraparib (MK4827) in BRCA mutation carriers and patients..., Sandhu [/bib_ref] [bib_ref] Phase I, doseescalation, twopart trial of the PARP inhibitor talazoparib in patients..., De Bono [/bib_ref] [bib_ref] Inhibition of poly(ADPribose) polymerase in tumors from BRCA mutation carriers, Fong [/bib_ref] The most common grade 3-4 adverse event in both cohorts was anaemia in the 300 mg cohort and 18 [37%] in the 400 mg cohort).
18 (37%) patients in the 400 mg cohort and six (12%) in the 300 mg cohort required at least one dose reduction (appendix p 10), with anaemia being the most common adverse event leading to dose reductions (two patients in the 300 mg cohort and nine in the 400 mg cohort). Eight patients who achieved a response on 400 mg continued to respond for more than 6 months after dose reduction to 300 mg or lower. Overall, 18 (19%) of the 98 patients were permanently discontinued from olaparib treatment due to adverse events (appendix p 10) The most common adverse events leading to discon tinuation were anaemia (two of five patients who discontinued on 400 mg and five of 13 on 300 mg) and fatigue (three on 400 mg and four on 300 mg).
107 serious adverse events were reported in 49 (50%) patients (300 mg cohort: 49 events in 22 patients; 400 mg cohort: 58 events in 24 patients) with 19 serious
(1) adverse reactions (possibly related to study drug; 11 in the 300 mg cohort and eight in the 400 mg cohort) in 13 patients (8 patients in the 300 mg cohort and 5 in the 400 mg cohort). The most common serious adverse reaction was anaemia (occurring in six patients in the 300 mg cohort and five patients in the 400 mg cohort). Four serious adverse reactions were considered suspected unexpected, two in each dose cohort group. In the 400 mg cohort, one patient had communityacquired pneumonia, and the same patient had atrial fibrillation with myocardial infarction (recovering from both events).
In the 300 mg cohort, one patient was diagnosed with myelodysplasia after 6·5 months of treatment, and developed acute myeloid leukaemia after olaparib discontinuation. One patient on 300 mg olaparib died due to a myocardial infarction [fig_ref] Table 3: Treatment-emergent adverse events [/fig_ref] , assessed as possibly drugrelated, after 11 days of treatment. All other deaths were unrelated to treatment (n=76; 70 due to disease and six due to other causes).
# Discussion
The TOPARPB trial has confirmed the antitumour activity of olaparib against metastatic castrationresistant prostate cancer with specific DDR gene aberrations. The number of composite responses observed in the cohort of patients who received 400 mg tablets of olaparib twice daily met the predefined criteria for success, validating the DDR biomarker identified in TOPARPA as being predictive of response. [bib_ref] DNArepair defects and olaparib in metastatic prostate cancer, Mateo [/bib_ref] Overall, the data suggest that both drug dose and the specific type of DDR gene aberration might influence antitumour activity, given that the composite response at the 300 mg regimen was lower and did not reach the predefined criteria for success. The antitumour activity observed varied considerably for different DDR gene aberrations, with the greatest antitumour activity seen in the subgroup with BRCA1/2 alterations. Despite randomisation, CDK12 aberrations were imbalanced between the cohorts, with an enrichment in the 300 mg cohort. This imbalance might explain, at least in part, the inferior composite response in the 300 mg cohort. [bib_ref] Genomic hallmarks and structural variation in metastatic prostate cancer, Quigley [/bib_ref] [bib_ref] Inactivation of CDK12 delineates a distinct immunogenic class of advanced prostate cancer, Wu [/bib_ref] The rationale to explore the two doses originated from prior clinical observations indicating a dose-response relationship for olaparib between 100 mg and 400 mg at twice daily dosing, although 400 mg has been associated with enhanced toxicity. [bib_ref] Oral poly(ADPribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations..., Tutt [/bib_ref] [bib_ref] Oral poly(ADPribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations..., Audeh [/bib_ref] In keeping with this finding, 37% patients at 400 mg had to reduce their dose to 300 mg, most commonly because of anaemia. All of these data would need to be considered when assessing the optimal dose of olaparib for prostate cancer treatment.
Our results support the implementation of routine genomic testing of metastatic prostate cancer, to detect DNA repair defects for targeting by PARP inhibition. In a previous study, we reported an enrichment of germline inherited mutations in DDR genes in metastatic prostate cancer, [bib_ref] Inherited DNArepair gene mutations in men with metastatic prostate cancer, Pritchard [/bib_ref] which has led to the recommendation of broad Antitumour activity was also observed in other DDR gene aberration subgroups. Responses in tumours with PALB2 mutations were frequent, although the low prevalence of these mutations means that further data are required to confirm these findings. Clinical qualifi cation of lowprevalence biomarkers is challenging in the pursuit of precision medicine approaches; the valid ation of genomic signatures [bib_ref] Inactivation of CDK12 delineates a distinct immunogenic class of advanced prostate cancer, Wu [/bib_ref] [bib_ref] Signatures of mutational processes in human cancer, Alexandrov [/bib_ref] or functional biomarkers 29 that identify tumours with defective homologous recombination, regardless of the mutated gene of origin, could help move the field forward, but such assays have not yet been validated in prostate cancer.
Conversely, germline and somatic ATM aberrations are common in metastatic prostate cancer; ATM functions as a cell cycle checkpoint, preventing cell cycle progression in the presence of DNA damage rather than directly mediating repair, unlike BRCA2 and PALB2. In the TOPARPA trial, five patients had ATM aberrations in tumour biopsies: two of these had a PSA response, and two more had circulating tumour cell conversion. Preliminary results suggest that rucaparib, another PARP inhibitor, results in few PSA decreases in patients with ATM aberrations.In TOPARPB, we treated 21 patients with suspected deleterious ATM aberrations: two achieved a RECIST or PSA response, and several others had circulating tumour cell count conversions following therapy. Circulating tumour cell count decreases seen in this subgroup were associated with increased duration on the trial, tumour shrinkage per RECIST, and a PSA decrease, as was the case for the overall TOPARPB population, with circulating tumour cell conversions robustly associating with increased radiographic progressionfree survival and overall survival. Overall, the data suggest that the antitumour activity of olaparib in metastatic castrationresistant prostate cancer with ATM loss is less than that for BRCAaltered tumours; nevertheless, a subset of patients with ATM-altered metastatic castrationresistant prostate cancer appear to derive benefit. However, detection of ATM alterations alone might be insufficient to identify these sensitive tumours. Further studies, as well as the study of rational drug combinations, are now needed to elucidate how to best evaluate and treat metastatic castrationresistant prostate cancer with ATM alter ations. Ongoing exploratory analyses from this trial will look to further characterise exceptional responses within each genedefined subgroup to optimise patient stratification.
We do acknowledge limitations to this study. Although the use of targeted NGS facilitates the clinical imple mentation of patient stratification, this method might be insufficient to capture complex aberrations resulting in PARP inhibitor sensitivity. Furthermore, because all patients in our study had DDR gene aberrations and received olaparib, we are not able to fully differentiate the predictive value versus the prognostic effect of the gene aberrations in terms of survival. Randomised trials including patients with and without the biomarkers will be more able to clinically qualify putative predictive biomarkers.
Nonetheless, the results from TOPARPB have overall driven the design and conduct of several registration trials of PARP inhibitors in metastatic castration resistant prostate cancer (NCT02987543, NCT02975934, and NCT03148795), which are likely to guide the clinical use of PARP inhibitors in metastatic prostate cancer in the future. Most of these studies aim to validate PARP inhibition as a precision medicine strategy for prostate cancers with DDR gene aberrations. Other studies, in parallel, are exploring the addition of PARP inhibitors to the standardofcare drugs targeting the androgen receptor (NCT03732820 and NCT03395197), on the basis of results from a phase 2 clinical trial indicating that a broader target population than just patients with gene aberrations might benefit from these drugs. [bib_ref] Olaparib combined with abiraterone in patients with metastatic castrationresistant prostate cancer: a..., Clarke [/bib_ref] In conclusion, the data from TOPARPB have confirmed the antitumour activity of olaparib against meta static prostate cancer with particular DDR gene aberrations. The high response observed in patients with metastatic castrationresistant prostate cancer with germline or somatic BRCA1/2 aberrations, and the durability of many of these responses, support the use of olaparib in this subpopulation. The antitumour activity observed against tumours with ATM, PALB2, FANCA, or CHEK2 aberrations suggest that PARP inhibitors might have a role as single drug therapies or in rational combinations against these other subtypes of metastatic prostate cancer, although further data are needed to precisely assess the clinical relevance of each of these different DDR gene aberrations in prostate cancer.
[fig] Figure 1: (A) Trial profile and (B) DDR gene alterations in the intention-to-treat population (n=98) DDR=DNA damage response. CTC=circulating tumour cell. [/fig]
[fig] Figure 2: although five patients achieved CTC conversion (including one with concomitant BRCA1/2 Best change from baseline in sum of target lesions (%) Antitumour activity by allocated dose cohort (intention-to-treat population) (A) Best percentage change from baseline in PSA during treatment. (B) Best percentage change from baseline in the sum of target lesions (Response Evaluation Criteria in Solid Tumors 1·1) during treatment. (C) Radiographic progression-free survival. (D) Swimmers plot of time on treatment for each patient, indicating periods of treatment interruptions, dose reductions, and, in the 300 mg cohort, dose escalations. Treatment periods of ≥6 months and ≥12 months are highlighted. PSA=prostate-specific antigen. PSA50=decrease in prostate-specific antigen of ≥50%. [/fig]
[fig] Figure 3: Gene aberration subgroup, median (95% CI) BRCA / , 8·3 months (5·5-13·0) ATM, 5·8 months (4·4−10·9) CDK , 2·9 months (2·6−7·5) PALB , 5·3 months (0·4−NE) Other, 2·8 months (2·6−4·3) All ITT, 5·5 months (Antitumour activity by gene aberration subgroup (intention-to-treat population, pooled 300 mg and 400 mg cohorts) (A) Maximum percentage change from baseline in PSA during treatment. (B) Maximum percentage change from baseline in the sum of target lesions (Response Evaluation Criteria in Solid Tumors 1·1) during treatment. (C) Radiographic progression-free survival. (D) Swimmers plot of time on treatment for each patient. ITT=intention-to-treat. NE=not estimable. PSA=prostate-specific antigen. PSA50=decrease in prostate-specific antigen of ≥50%. *Patients presenting with mutliple mutations are represented in a single subgroup. [/fig]
[table] Table 1: Baseline characteristics of patients in the intention-to-treat population 27%) had DDR gene aberrations on the basis of NGS. An oncoprint summarising all alterations detected during prescreening is presented in the (p 14). The most commonly detected DDR gene aberrations were mutations or homozygous deletions in BRCA2 (44 [7%] of the 592 patients), ATM (40 [7%]), and CDK12 (33 [6%]). [/table]
[table] Table 2: Overall antitumour activity of olaparib in patients with DNA damage response gene aberrations by dose cohort and gene subgroup treatment for each patient is represented in [/table]
[table] Table 3: Treatment-emergent adverse events [/table]
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Principles of and Advances in Immunoglobulin Replacement Therapy for Primary Immunodeficiency
## Historical perspective
Today it seems quite straightforward to give IgG to patients who have immune deficiencies involving decreased antibody production. It is interesting reflect that, although serum therapy was used in the early 1900s, that treatment generally involved the use of serum from convalescent patients or from horses immunized with specific bacteria or toxins [bib_ref] Serum therapy in therapeutics and medical science, Von Behring [/bib_ref]. Primary immune deficiency (PID) had not yet been recognized, and penicillin had not yet been discovered; high-titered serum was the only specific therapy for common infections such as pneumococcal pneumonia. Not until World War II did concentrates of human immune globulin became available for widespread use, and it was not until 1952 that Brutonpublished the first report of the use of immune serum globulin (ISG) in treating a patient who had PID. The studies of the Working Group on Hypogammaglobulinemia in the United Kingdom firmly established the benefit of regular ISG injections in the treatment of this family of illnesses and set the dosage at 25 mg/kg/week. Not until the early 1980s were preparations of IgG that could be safely given intravenously licensed in the United States. Since that time, more purified and better-tolerated IgG preparations have become available, and there has been widespread interest in subcutaneous rather than intravenous administration. The doses of IgG used in patients who have PID have escalated steadily, with increasingly ambitious goals for prevention of infection and end-organ damage. Unfortunately, there also have been a number of reminders that treatment with blood products carries real and potential risks, including transmission of bloodborne infections. More recently, the use of high-dose intravenous immunoglobulin (IGIV) for its anti-inflammatory effects in diseases such as Kawasaki syndrome and for its immunomodulatory effects in autoimmune diseases has increased the demand for this precious commodity. To simplify the discussion of products that usually are denoted by their route of administration (ie, intravenous , subcutaneous , or intramuscular , this article uses the inclusive abbreviation ''ISG'' to refer to all polyclonal human immune globulin preparations.
What is immune serum globulin?
Stringent safety standards, the desire to provide antibodies against a wide range of pathogens, and the need to produce products with consistent tolerability and efficacy have led to large-scale industrial production of IgG concentrates which may contain the antibodies from 40,000 to 50,000 units of plasma per batch. These goals may seem to be at odds with the desire to assure safety by using a limited number of well-characterized, usually related, plasma donors for individual patients. The decades since IGIV was introduced have witnessed the recognition of HIV as a bloodborne virus, an outbreak of hepatitis C transmission by IGIV and other blood products in the early 1990s, and increasing concerns about the transmission of prions, which cause spongiform encephalopathies. Therefore, reducing the risk of transmission of known as well as possibly emerging bloodborne diseases has become one of the most important considerations for government regulators (the Food and Drug Administration in the United States) and for the plasma fractionation and protein therapeutics industry. Multiple safety steps are used during the purification of therapeutic IgG concentrates from blood. These steps can be summarized as falling into four major categories, summarized in Box 1.
Most of the plasma used for production of therapeutic proteins such as ISG is obtained specifically for this purpose by plasmapheresis and is termed ''source'' plasma, although some plasma from donations of whole blood (recovered plasma) still is used as well. All products marketed in the United States must be made from plasma obtained in the United States. FDA regulations governing donor selection and plasma collection are available on the Internet. Donors must complete a questionnaire, undergo a physical examination, and have normal blood counts and liver function tests before use of their plasma is considered. Units of plasma are tested for serologic markers of known bloodborne diseases and are discarded if found positive. Plasma services use a ''quarantine'' or ''inventory hold'' procedure in which an individual unit, even if it tests negative for all know pathogens, is stored separately until the donor returns and provides another unit of plasma. Only when the second (and subsequent donations) also tests negative can a previously obtained unit be used. Computerized databases enable the tracking of all products derived even in part from any given plasma donation. Patients receiving blood-derived proteins are encouraged to keep careful records of the lot numbers and names of all products they receive. If a donor ever is determined to have been incubating an undetected, potentially bloodborne disease (eg, a slow-virus encephalopathy or emerging viruses such as hepatitis C in the early 1990s, or West Nile virus more recently), ''lookback'' programs can be activated so that any patient who received a product containing plasma from that donor can be identified, examined and tested, and treated if necessary.
The manufacturing processes of all ISG products currently marketed in the United States include at least one step of the cold-alcohol fractionation process developed by Cohn and his colleagues in the early days of World War II [bib_ref] The separation of the antibodies. isoagglutinins, prothrombin, plasmonogen and beta-lipoprotein into subfractions..., Oncley [/bib_ref]. Different manufacturers then use different purification steps, including differing column chromatography protocols, to produce final products that are highly enriched (O 95%) in IgG. Most products are essentially free from IgM, but all contain at least small amounts of IgA. The properties of ISG products currently marketed in the United States are summarized in [fig_ref] Table 1: Box 1 [/fig_ref]. All products currently available in the United States contain more than 95% IgG, and all contain all subclasses of IgG in approximately the same ratio of concentrations as in normal plasma. Testing for the spectrum and titer of multiple different specific antibodies in any given lot of a product is not required; federal standards mandate only that a minimal titer of antibody against measles virus be present. The current FDA guidelines for licensing IGIV preparations are available on the Internet. Similar standards have been applied for the licensure of ISG intended for administration by the intramuscular and subcutaneous routes. Most of the products currently marketed in the United States have been licensed while these guidelines have been in effect. IgG molecules in concentrated solutions tend to aggregate, which brings their crystallizable fragments (Fc) into close proximity. These Fc portions can activate complement and cross-link Fcg-receptors, leading to the production of mediators that cause adverse reactions during IgG infusions. All the currently available ISG products contain excipients such as amino acids or sugars that are included to minimize the formation of aggregates and preserve the IgG in its monomeric state. These excipients differ in different products, as listed in [fig_ref] Table 1: Box 1 [/fig_ref]. The use of products with certain excipients may be inadvisable in specific patients (eg, sucrose in patients at risk for renal damage, products containing proline in patients who have disorders of metabolism of that amino acid, products containing maltose in diabetic patients whose glucose monitors might give false readings because of that sugar). Many products are treated at low pH, and some are bottled at low pH as well. The buffering capacity of the low-pH solutions is limited, however, and low pH has not been problematic. None of the products currently marketed in the United States, even those intended for intramuscular or subcutaneous use, contain thimerosal or any other preservative. Some products require refrigerated storage, but others have been shown to have satisfactory stability at room temperature. All should be brought to room temperature before administration. The content of salt and the concentration of IgG itself vary as well, and not all products are licensed for administration by all routes. Thus, the selection of the most appropriate product must be individualized for each patient. Despite the use of large donor pools, the spectrum of antibodies, concentrations of certain specific antibodies, and the presence of trace amounts of other plasma proteins in different preparations also differ. These differences may lead to unpredictable and idiosyncratic differences in tolerance of different products by individual patients. Therefore, IgG products should not be considered as interchangeable generics. Physicians should be notified and may want to slow the rate of infusion, administer premedications, and/or give the IgG under close observation if a patient must be given a product he or she has not received previously.
## Minimizing the risk of bloodborne pathogen transmission
A dilemma arises in assuring the absence of bloodborne pathogens in IgG preparations, because the IgG itself may complex with and impair detection of viruses and other pathogens without actually inactivating them. Complexes of virus particles with bound IgG usually have chemical properties that differ from the viruses themselves. Thus, the presence of antibodies against a virus may cause partitioning of the virus-antibody complex away from the product during purification steps that would not remove the uncomplexed virus itself. This phenomenon probably contributed to the transmission of hepatitis C when plasma containing antibodies to that virus was excluded from pools used to manufacture ISG at a time when sensitive tests for the virus itself were not available. To avoid this problem, animal viruses and prions that have properties similar to important human pathogens but against which humans are not likely to have antibodies are ''spiked'' into units of donor plasma that then are run through scaled-down production steps to assess the ability of each step to remove and/or inactivate the virus [bib_ref] Virus validation studies of immunoglobulin preparations, Biesert [/bib_ref]. These viruses are listed in [fig_ref] Table 2: Viruses used to test efficacy of viral removal/inactivation steps during IgG preparation [/fig_ref] together with the human viruses they are intended to simulate. Note that significant removal of many intact viruses can be accomplished by the Cohn coldalcohol precipitation steps used for the initial purification of the IgGcontaining fraction of plasma. The two major targets for viral inactivation are the protein coat of nonenveloped viruses and the lipid envelope of enveloped viruses. In general, surface proteins of nonenveloped viruses are more sensitive to inactivation by low pH, proteolytic enzymes, and heat (pasteurization) than are the lipid and protein structures of enveloped viruses. Inactivation of enveloped viruses generally requires dissolution of the lipid envelope, which is accomplished in some products by treatment with fatty acids, fatty alcohols, or solvent/detergent combinations such as tri-(N-butyl) phosphate/Triton X-100 [bib_ref] Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses..., Horowitz [/bib_ref]. Many products also are processed by passage through filters with nanometer pore sizes that can remove many virus particles by size, regardless of their chemical characteristics. Assessing the need for IgG replacement or augmentation in any given patient usually requires an in-depth understanding of the patient's condition and underlying diagnosis. In patients who have confirmed diagnoses known to result in severe antibody deficiency, such as severe combined immunodeficiency, X-linked agammaglobulinemia, Wiskott-Aldrich syndrome, and hyper-IgM syndromes, the diagnosis alone may provide sufficient grounds to initiate IgG replacement therapy as soon as it is established. The pattern of infections, family history, physical examination, and flow cytometry usually will lead readily to a diagnosis that can be confirmed by molecular analysis of the patient's mutation [bib_ref] Practice parameter for the diagnosis and management of primary immunodeficiency, Bonilla [/bib_ref]. For patients who present with recurrent infections or with symptom complexes that may or may not be caused by infection, the decision to start IgG therapy should be based on laboratory data demonstrating the antibody deficiency and a deficient response to appropriate vaccines, as well as on evidence for increased morbidity caused by infection. The use of measurements of specific antibody titers and vaccine responses to help determine whether IgG supplementation may be indicated for a given patient has been discussed extensively elsewhere [bib_ref] Assessment and clinical interpretation of reduced IgG values, Agarwal [/bib_ref] [bib_ref] Assessment and clinical interpretation of polysaccharide antibody responses, Paris [/bib_ref] [bib_ref] Evaluation of antibody responses to protein antigens, Chouksey [/bib_ref] and is beyond the scope of this article. Guidelines for the diagnosis and management of PID and the use of IgG replacement in antibodydeficiency diseases have been promulgated by the Immune Deficiency Foundation and the Joint Council on Allergy, Asthma and Allergy in the United States (JCAAI) [bib_ref] Practice parameter for the diagnosis and management of primary immunodeficiency, Bonilla [/bib_ref] and presented at a meeting of the European Society for Immune Deficiency. A very helpful scheme abbreviated from the latter is reproduced in Box 2.
In many patients the antibody deficiency may be only transient. These patients include very low birth weight babies, infants with delayed development of the full spectrum of necessary humoral responses, older children and adults who have been given cytotoxic chemotherapy for cancer, and patients who have been given cytotoxic or immunosuppressive therapy for autoimmune disease and/or to prevent transplant rejection. Nearly all patients who receive hematopoietic stem cell transplants for severe combined should be re-evaluated periodically to determine if IgG replacement/augmentation is still necessary. Re-evaluation typically is performed after the patient has been off IgG therapy for at least several months and usually involves measuring the specific antibody response after administration of protein and polysaccharide vaccines. There are no commercially available preparations of enriched IgA or IgM, so patients who have isolated IgA deficiency generally are not considered candidates for IgG supplementation. Patients who have significant morbidity associated with low or absent IgA levels should be checked for specific IgG antibodies and vaccine responses, however, because these conditions may coexist as part of a broader immune deficiency. Because most laboratories use very broad ranges for ''normal'' IgG levels, patients who do not meet rigorous criteria for common variable hypogammaglobulinemia (also termed ''common variable immune deficiency'') still may benefit from IgG supplementation. Conversely, patients who have IgG levels below a laboratory's normal ranges for the age of the patient do not necessarily require IgG replacement therapy if they have satisfactory specific antibody concentrations and vaccine responses. Many patients who do not have antibody-deficiency diseases receive IGIV for its immunomodulatory and/or anti-inflammatory properties. The most common such condition for which IGIV is used in pediatrics is Kawasaki syndrome [bib_ref] Kawasaki disease, Newburger [/bib_ref]. IGIV also is used in toxigenic bacterial infections and in idiopathic thrombocytopenic purpura (ITP), Guillain-Barre syndrome, chronic idiopathic demyelinating polyneuropathy (multifocal motor neuropathy), and other autoimmune diseases. A recent evaluation of the evidence for the use of IGIV in different conditions is available elsewhere [bib_ref] Use of intravenous immunoglobulin in human disease: a review of evidence, Orange [/bib_ref] and is beyond the scope of this article.
## Alternatives to immune serum globulin therapy
Patients who have confirmed antibody deficiency may be maintained without IgG replacement for periods of time, using relative isolation and/ or prophylactic antibiotics. If exposed or potentially exposed to viral pathogens (eg, in a local outbreak of chickenpox or mumps or when traveling to a developing nation), such patients may be given hyperimmune (eg, Varicella-zoster immune globulin) or standard intramuscular ISG preparations. This approach may be satisfactory for patients who reasonably can be expected to overcome a developmental delay in antibody production or to recover from the effects of a treatment regimen that has been completed. This approach should not be considered a long-term strategy for patients who have clearly diagnosed PID disease. IGIV has been found to be helpful in certain HIV-infected infants, but with prenatal treatment reducing the maternal transmission of HIV and improved antiviral chemotherapy, this setting is not a major use of ISG. In a previous era, plasma, often from a parent or relative, was used for antibody replacement in children who had PID disease. This approach is rarely used in the United States, mainly because of the time and effort required to be sure that the donor plasma is free from any bloodborne pathogens (which maybe clinically unapparent) and the desire to provide a broad range of protective antibodies.
How to administer immune serum globulin: intravenously or subcutaneously?
Although individual doses of ISG may be given intramuscularly, the pain and risks associated with deep intramuscular injections limit the amount that can be administered this way. Routine therapy by the intramuscular route is rarely used now, although it was the mainstay of treatment for PID disease for more than 30 years. Most IgG replacement/augmentation regimens now employ intravenous or subcutaneous administration. In general, doses are in the range of 300 to 800 mg/kg per month (see next section). Intravenous infusions of IgG generally are well tolerated. The introduction of ISG preparations that could be given safely by the intravenous route in the early 1980s was a major advance in the care of patients who have PID disease. Obstacles that had to be overcome included stabilizing the IgG molecules so they would not aggregate in solution and purification to remove traces of proinflammatory molecules such as activators of the kallikrein-kinin and clotting cascades. As noted earlier, a major breakthrough was the recognition of the importance of including excipients such as amino acids and/or sugars in the final preparations. Most adverse reactions to IGIV are related to the rate of infusion. Patients who are naive to IgG replacement, who have had interruptions in their therapy, and/or who are actively or chronically infected have an increased risk of infusion-related adverse effects. These effects may be related, in part, to the formation of antigen-antibody complexes while the IgG is being given and/or the rapid release of lipopolysaccharide or other components of pathogens already present in the recipient. The risk of these reactions may be reduced by making sure patients are afebrile and that active infections are being treated with antibiotics before beginning IGIV therapy or giving any scheduled infusion. Some studies have shown that the incidence of reactions is increased when patients already receiving therapy are given a different brand of IGIV [bib_ref] Safety, efficacy and pharmacokinetics of Flebogamma 5% for replacement therapy in primary..., Berger [/bib_ref]. To minimize rate-related adverse effects, infusions should be started slowly, at rates not above 0.01 mL/kg/minute (equaling 0.5 mg/kg/minute of 5% solution or 1 mg/kg/minute of 10% solution). Vital signs should be checked frequently during IGIV infusions, particularly in naive patients. If the patient remains comfortable and stable, the rate of the infusion may be increased in a stepwise manner, usually at 15-to 30-minute intervals, up to the maximum tolerated by the patient. Most preparations are labeled for administration at a maximum rate of 0.08 mL/kg/minute (4 or 8 mg/kg/ minute of 5% or 10% solution, respectively). Infusion-related adverse reactions frequently mimic the signs of infection, including chills and even rigors, arthralgias and/or myalgias, and headache. Because most of these symptoms are related to the rate of infusion, slowing or temporarily stopping the infusion may allow the symptoms to subside; then the infusion can be resumed at the previously tolerated rate. If these precautions fail to prevent these symptoms, premedication with antipyretics, antihistamines, and/or corticosteroids may help ameliorate the symptoms. During prolonged infusions, such medications may be repeated if necessary. In some cases, patients report systemic reactions including back pain, chest tightness, and a feeling of anxiety or a sense of impending doom, frequently in association with flushing and tachycardia. This type of reaction may resemble anaphylaxis but usually does not involve IgE. Therefore, this type of reaction has been termed ''anaphylactoid.'' A key difference between the anaphylactoid reactions that accompany IGIV infusions and true IgE-mediated anaphylaxis is that the former usually are associated with hypertension, rather than hypotension, as would be expected in true anaphylaxis. True anaphylaxis may occur in patients receiving IGIV, particularly in those who are deficient in IgA but still have the capacity to produce IgE [bib_ref] Anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia. Detection of..., Burks [/bib_ref]. True anaphylaxis occurs very rarely but may be life threatening. Therefore, any practitioner or facility that administers IGIV should be equipped to treat anaphylaxis if it occurs. The risk of true anaphylaxis can be minimized by screening patients for complete IgA deficiency, by starting initial infusions extremely slowly, by using products with the lowest concentration of IgA, and by testing the patient for IgE-antibodies against IgA if this is a concern. Many IgA-deficient patients also are deficient in other immunoglobulin isotypes and/or in specific IgG antibodies against important pathogens. Such patients should not be denied ISG therapy because of the IgA deficiency, but caution should be used in its administration.
Headaches may occur during or after intravenous infusions and sometimes repeatedly follow the infusions by as much as 48 to 72 hours. These headaches may have the character of migraines and are more common in patients who suffer from migraines independently of their IGIV infusions. In rare cases, headaches following IGIV infusions may be accompanied by meningismus, and aseptic meningitis has been well documented. Headaches sometimes may be prevented by the use of corticosteroids as premedication or for a day or two following the infusion, or by the use of triptans or other migraine treatments. Additional, rare complications of IGIV therapy include transfusion-related acute lung injury, renal failure, and thromboses. A comprehensive review of the adverse effects and complications of IGIV infusions is available and should be read by everyone who administers this form of therapy [bib_ref] Risks associated with the use of intravenous immunoglobulin, Pierce [/bib_ref]. Quite often, patients who experience headaches or other adverse effects with one brand of IGIV may tolerate another with no problem. Therefore, switching products may be indicated to obviate the adverse effects. Conversely, because not all products are tolerated equally by a given patient, substitutions should be made carefully, and under supervision. Generally when switching products, slow infusion rates should be used, at least initially.
The pharmacokinetics of IGIV have been well described [bib_ref] The half-lives of IgG subclasses and specific antibodies in patients with primary..., Mankarious [/bib_ref]. By the end of an intravenous bolus of IgG, the IgG is mostly intravascular, and its concentration can be expected to rise by 100 to 200 mg/dL for every 100 mg/kg given. It thus is common for peak serum IgG concentrations to rise by as much as 1000 mg/dL following doses in the conventional replacement range (300-800 mg/kg). Over the next 48 to 72 hours, the IgG becomes distributed into the total extracellular fluid volume, and the serum concentration may drop by 25% to 40%. After this re-equilibration, IgG is catabolized with first-order kinetics and has a half-life of around 22 days. Currently marketed ISGs contain intact IgG molecules that have not undergone any chemical modification. Thus, the distribution and half-life of intravenously administered IgG is essentially identical to that of endogenously produced native IgG [bib_ref] Pharmacokinetics and tolerability of a new intravenous immunoglobulin preparation, IGIV-C, 10% (Gamunex,..., Ballow [/bib_ref] [bib_ref] US-PID-IGIV 10% -Study Group. Efficacy, safety and tolerability of a new 10%..., Church [/bib_ref] [bib_ref] Octagam 5%, an intravenous IgG product, is efficacious and well tolerated in..., Ochs [/bib_ref] [bib_ref] Product equivalence study comparing the tolerability, pharmacokinetics and pharmacodynamics of various human..., Andressen [/bib_ref]. For this reason, most intravenous regimens repeat doses at 21-to 28-day intervals. Depending on the dose and whether there is any endogenous IgG production at all, dosing intervals of 28 days or longer frequently leave patients with serum IgG concentrations in the range of 400 to 500 mg/dL, or even less, at the end of the dosing interval. Many patients report flulike symptoms, increased fatigue, and/or malaise when the trough serum IgG level falls so low, and they may have increased susceptibility to infection at that time. In that situation, the dose may be increased or the interval shortened. Higher doses and/or shorter intervals also will be necessary in patients who have gastrointestinal or renal protein loss and/or increased catabolism of IgG, as discussed later.
Although many patients tolerate long-term intravenous IgG replacement regimens with no problems, surveys show that a high proportion of patients describe the conditions under which they receive their infusions as inconvenient, because they must travel to a hospital or infusion center, miss school or work, and/or experience difficulty with intravenous access. Subcutaneous regimens offer freedom from those concerns. Bruton treated the first agammaglobulinemic patient to be reported with subcutaneous injections of ISG, but other investigators used intramuscular injections, which became the standard of care for nearly 3 decades. In the early 1980s, the use of small, battery-powered syringe driver pumps to give subcutaneous infusions of 16% ISG intended for intramuscular use over several hours was described [bib_ref] Immunoglobulin replacement by slow subcutaneous infusion, Berger [/bib_ref] [bib_ref] Subcutaneous Infusions of gammaglobulin in management of agammaglobulinemia, Ugazio [/bib_ref] [bib_ref] Home treatment in patients with antibody deficiency by slow subcutaneous infusion of..., Roord [/bib_ref]. This method of administration is free from the pain associated with the deep intramuscular injections and was much better tolerated by the patients, so higher doses could be administered routinely [bib_ref] High dose immunoglobulin replacement therapy during pregnancy, Berger [/bib_ref]. In general, 25-to 27-gauge needles extending 6 to 11 mm under the skin are inserted perpendicularly. A wide variety of special needles and infusion sets are now available specifically for IGSC therapy. Depending on the size of the patient, 5 to 20 mL are delivered per site, usually over 2 hours or so. Several sites may be used for each infusion of ISG, so weekly dosing is a common regimen. Frequently used sites include the abdomen, inner or anterior thighs, and the backs of the arms. In general any site at which one can ''pinch an inch'' is acceptable for subcutaneous infusions of 10 to 20 mL of ISG. A very nice illustration of potential subcutaneous infusion sites and good technique for inserting subcutaneous needles is available on-line from the nursing staff of the National Institutes of Health Clinical Center. The author and colleagues have found that the size of the patient and the time over which the infusion is given are important factors in determining the maximum volume that can be infused at any given site. Thus, some patients may give themselves 40 to 60 mL of ISG in a single site, over a period of time as long as 8 hours or more. Some patients prefer this type of regimen and take their IGSC while they sleep. In contrast, other patients may prefer to take small doses of IGSC on a daily basis (eg, by pushing 10 mL into a single site over several minutes). In a series of 20 patients using 15% or 16% ISG preparations reported from UH/Rainbow Babies & Children's Hospital, most of the regimens were within the parameters of 0.12 to 0.24 mL/kg/site/hour [bib_ref] Subcutaneous IgG replacement therapy with preparations currently available in the US for..., Chouksey [/bib_ref] Basically, once the monthly dose of IGSC has been calculated, a wide variety of schedules may be used to tailor the infusion regimen to the patient's preferences.
Although most physicians and patients in the United States adopted the intravenous route when preparations that could be administered intravenously became available, patients in other countries and those who experience severe adverse effects from intravenous preparations continue to use the subcutaneous route [bib_ref] Subcutaneous immunoglobulin replacement in patients with primary antibody deficiencies: safety and costs, Gardulf [/bib_ref] [bib_ref] Slow subcutaneous immunoglobulin therapy in a patient with reactions to IM immunoglobulin, Welch [/bib_ref]. Serious systemic adverse effects are rarely a problem with subcutaneous infusions; most studies have reported that less than 1% of infusions are associated with systemic adverse events [bib_ref] Subcutaneous immunoglobulin replacement in primary immunodeficiencies, Berger [/bib_ref]. Because the expertise and experience necessary to establish and maintain intravenous access are not required for subcutaneous administration, and because of the low risk of serious reactions, subcutaneous infusions usually are administered at home by the patients themselves or their parents or partners. The major disadvantage of subcutaneous infusions is the limitation on the volume of ISG that can be given at one time. Because the infusions can be given at home and/or while the patient is performing other activities, most patients readily adapt to weekly or more frequent infusions. The division of the monthly dose of ISG into weekly or even more frequent infusions and the slower absorption of IgG into the circulation from the subcutaneous site than from intravenous boluses tends to flatten out the curve of serum IgG concentration over time, as seen in [fig_ref] Figure 1: Serum IgG concentrations in a 34-year-old man who has X-linked agammaglobulinemia [/fig_ref]. Elimination of the high peaks and low troughs associated with intermittent intravenous boluses ameliorates most infusion-related adverse effects and also the feelings of malaise and fatigue associated with the low troughs. Subcutaneous infusions frequently are associated with local swelling, redness, and an itching or burning sensation, but these effects are rarely serious and usually subside over several hours. If the patient continues with subcutaneous infusions, these local reactions tend to lessen with time, but the reasons for this change are not known. Although many patients have used the subcutaneous route for more than 25 years, chronic changes, fibrosis, or lipodystrophy at the infusion sites have not been problematic. It is rare to be able to identify the site at which a subcutaneous IgG infusion was given more than 24 hours after the infusion has been completed. Because it is easier for the patients to adjust their infusion schedules to their school or work schedule, rather than vice versa, and because patients no longer have to travel to infusion centers or the hospital for their treatments, many patients find that home IGSC regimens increase their sense of independence and autonomy. In turn, this response results in improved quality of life experienced by many, but not all, patients [bib_ref] Subcutaneous immunoglobulin replacement in primary immunodeficiencies, Berger [/bib_ref] [bib_ref] Children and adults with primary antibody deficiencies gain quality of life by..., Gardulf [/bib_ref] [bib_ref] Health-related quality of life and treatment satisfaction in North American patients with..., Nicolay [/bib_ref] [bib_ref] Patients' attitude to subcutaneous immunoglobulin substitution as home therapy, Kittner [/bib_ref].
At present, only one ISG preparation marketed in the United States is licensed for subcutaneous administration. Small series and anecdotal reports, however, suggest that most preparations licensed for administration by other routes are well tolerated when administered by the subcutaneous route. Several preparations are marketed for subcutaneous use in Europe, and it is likely that additional products with concentrations as high as 16% or even 20% will be available in the United States in the next few years. A comparison of the volumes required to give comparable amounts of IgG by the subcutaneous and intravenous routes using currently available preparations is given in Box 3, along with some sample subcutaneous regimens.
## Box 3. comparison of intravenous and subcutaneous dosing
1. For a 20-kg 6-year-old receiving 500 mg/kg per month = 10 g IgG = 200 mL of 5% intravenous solution = 100 mL of 10% intravenous solution = 62.5 mL of 16% subcutaneous solution 2. In subcutaneous treatment regimens using the unit dose approach to deliver approximately 10 g IgG per month to a 20-kg 6-year-old child, the volume of 16% solution required = approximately 62.5 mL = 16 mL/week (one 10-mL and two 3-mL bottles of 16% solution) administered as one infusion into two or three sites = 2.56 g/week = 10.24 g/month = 10 mL (one 10-mL bottle of 16% solution) administered every fifth day (six times/month) into one or two sites = 9.6 g/month = 6 mL (two 3-mL bottles of 16% solution) administered every third day as one infusion into one site = 0.96 g/dose = 9.6 g/month 3. In subcutaneous regimens for a 30-kg-child receiving approximately 15 g/month, the volume of 16% solution required = approximately 94 mL = 10 mL/week (one 10-mL bottle) administered every third day into one or two sites = 16 g/month = 20 mL administered once a week into two or three sites, with one extra infusion per month = 16 g/month = 3 mL administered daily into one site over 15 minutes (''push'') = 14.4 g/month 4. In subcutaneous regimens for a 40-kg-child receiving approximately 20 g/month, the volume of 16% solution required = 125 mL = 30 mL/week administered into one or two sites once a week = 4.8 g/week = 19.2 g/month = 20 mL administered every fifth day (six times per month) into one site = 19.2 g/month = 16 mL administered every Saturday and Sunday into one or two sites = 20.5 g/month = 10 mL delivered every Monday, Wednesday, and Friday (weekends off) by push or into one site = 19.2 g/month
## How much to give
The efficacy of IgG replacement in patients who have PID disease has been clearly demonstrated in studies going back to the 1950s and 1960s. A recent survey documented the decrease in the incidence of pneumonia after patients who had PID disease were put on IGIV treatment [bib_ref] Efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with..., Busse [/bib_ref]. The doses of IgG given in standard replacement regimens has increased over time, as more convenient routes of administration have become practical. In classic studies in the early 1960s, the British Medical Research Council working group on hypogammaglobulinemia reported that 50 mg/kg/week was more effective than 25 mg/kg/week in preventing febrile episodes, otitis media, and pneumonia, but the differences did not seem to be of sufficient clinical importance to warrant widespread use of the higher dosage. Licensing studies of the first generation of intravenous products in the United States used doses in the range of 100 to 200 mg/kg/month [bib_ref] Intravenous immune globulin therapy in hypogammaglobulinemia, Pirofsky [/bib_ref] [bib_ref] Safety and patient acceptability of intravenous immune globulin in 10% maltose, Ochs [/bib_ref]. Studies by Roifman and his colleagues [bib_ref] High-dose versus low-dose intravenous immunoglobulin in hypogammaglobulinaemia and chronic lung disease, Roifman [/bib_ref] [bib_ref] Intravenous immune serum globulin replacement in hypogammaglobulinemia. A comparison of high-versus low-dose..., Gelfand [/bib_ref] beginning in the mid-1980s showed that patients receiving doses of 200 mg/kg/month did not achieve trough serum IgG levels higher than 500 mg/dL. In contrast, when the patients were crossed over to the arm that received 600 mg/kg/month, they did sustain trough levels above 500 mg/kg. In turn, the incidence of both major and minor infections was reduced greatly when the patients received the higher dose and maintained higher levels. Similar findings were presented by Roifman and colleagues [bib_ref] Comparison of the efficacy of IGIV-C, 10% (caprylate/chromatography) and IGIV-SD, 10% as..., Roifman [/bib_ref] from a more recent study in which patients were maintained on doses of IGIV selected by their physicians: patients receiving higher doses and maintaining higher serum trough IgG levels had fewer infections. A crossover study comparing higher IGIV doses (600 mg/kg/ month in adults and 800 mg/kg/month in children) with ''standard'' doses (300 mg/kg/month in adults and 400 mg/kg/month in children) showed statistically significant reductions in the incidence of infection and cumulative days of illness in the higher-dose groups [bib_ref] The effect of two different dosages of intravenous immunoglobulin on the incidence..., Eijkhout [/bib_ref]. Licensing studies of the current generation of IGIV products marketed in the United States have used doses in the range of 400 to 500 mg/kg/month, reflecting the dose the patient had been receiving before entering the study [bib_ref] Safety, efficacy and pharmacokinetics of Flebogamma 5% for replacement therapy in primary..., Berger [/bib_ref] [bib_ref] Pharmacokinetics and tolerability of a new intravenous immunoglobulin preparation, IGIV-C, 10% (Gamunex,..., Ballow [/bib_ref] [bib_ref] US-PID-IGIV 10% -Study Group. Efficacy, safety and tolerability of a new 10%..., Church [/bib_ref] [bib_ref] Octagam 5%, an intravenous IgG product, is efficacious and well tolerated in..., Ochs [/bib_ref] [bib_ref] Product equivalence study comparing the tolerability, pharmacokinetics and pharmacodynamics of various human..., Andressen [/bib_ref]. These regimens all resulted in an annual incidence of serious bacterial infections (meeting FDA definitions) of 0.1 infection/patient/year or less and an overall incidence of infection of two to four infections per patient per year. The Practice Parameters promulgated by the JCAAI call for maintaining trough serum IgG levels of 500 mg/dL as a useful guideline [bib_ref] Practice parameter for the diagnosis and management of primary immunodeficiency, Bonilla [/bib_ref]. This level typically requires doses in the range of 300 to 600 mg/kg/month. In patients who have gastrointestinal and/or renal protein loss, higher doses and/or shorter dosing intervals may be necessary to maintain trough levels above 500 mg/dL and keep the patient free from infection. Higher dosesdup to 800 mg/kg/month or even higherdare recommended for patients who have chronic lung and/or sinus disease [bib_ref] Practice parameter for the diagnosis and management of primary immunodeficiency, Bonilla [/bib_ref] [bib_ref] High-dose versus low-dose intravenous immunoglobulin in hypogammaglobulinaemia and chronic lung disease, Roifman [/bib_ref] [bib_ref] Intravenous immune serum globulin replacement in hypogammaglobulinemia. A comparison of high-versus low-dose..., Gelfand [/bib_ref] [bib_ref] Comparison of the efficacy of IGIV-C, 10% (caprylate/chromatography) and IGIV-SD, 10% as..., Roifman [/bib_ref] [bib_ref] The effect of two different dosages of intravenous immunoglobulin on the incidence..., Eijkhout [/bib_ref]. There have been several reports of progressive lung disease and dysfunction in patients who seem clinically free from pneumonia and who report few or no symptoms of chronic bronchitis or bronchiectasis [bib_ref] Pulmonary abnormalities in patients with primary hypogammaglobulienaemia, Kainulainen [/bib_ref] [bib_ref] Pulmonary complications of common variable immunodeficiency, Busse [/bib_ref]. These reports are consistent with the anecdotal experience of most immunologists who have large numbers of antibody-deficient patients in their practices. Close monitoring, which may include annual high-resolution CT scans of the chest, formal pulmonary function testing, and even bronchoscopy in some cases, is recommended for all patients who have these diseases. In the current era, most experts agree that prevention of pneumonia and serious infection is no longer sufficient indication that the patient's management is optimal. Efforts aimed at normalizing pulmonary function tests, maximizing the patients' ability to participate in a full range of activities, and preventing progressive loss of lung function seem warranted [bib_ref] Goals of therapy in antibody deficiency syndromes, Berger [/bib_ref]. Preventing and/or arresting chronic lung and sinus disease in patients who have PID disease frequently requires IgG replacement doses higher than 750 mg/kg/month, particularly in patients who already have chronic infection and structural damage before therapy is started. Such patients usually benefit by comprehensive approaches, including intense antibiotic therapy, bronchodilators and/or inhaled corticosteroids, mucolytics, and physical therapy, to improve pulmonary toilet and/or sinus surgery. Patients who have selective or lacunar antibody deficiencies and who do not have severe hypogammaglobulinemia per se still require full doses of ISG replacement to maintain adequate titers of the specific antibodies they cannot produce on their own. Similarly, antibody-deficient patients who have high IgG levels caused by polyclonal B-cell activation, as occurs in systemic lupus erythematosus or HIV infection, and patients who have monoclonal gammopathies actually may have elevated serum IgG levels but still need full doses of ISG replacement to provide the spectrum of antibodies necessary for protection against the pathogens to which they may be exposed.
Some evidence suggests that the bioavailability of IgG is decreased is when it is given subcutaneously instead of intravenously [bib_ref] Safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency..., Ochs [/bib_ref]. The tendency toward higher trough levels with weekly subcutaneous treatment may counterbalance any decreased efficacy caused by tissue degradation of the IgG administered by the subcutaneous route, however. Data that would allow determination of the effects of maintaining higher trough levels by fractionating the cumulative dose that otherwise would be given by a single monthly intravenous bolus are sparse. In particular, the relative importance of the high peaks achieved with the intermittent boluses is unclear. Two major efficacy studies of the single ISG product currently licensed for subcutaneous administration in the United States have been performed. In the United States pivotal trial, a dose increase of 37% above the previous intravenous dose was used to meet the FDA requirement that the area under the curve of serum IgG concentration versus time be the same for both routes of therapy [bib_ref] Safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency..., Ochs [/bib_ref]. In contrast, a study performed contemporaneously in Europe and Brazil used one fourth of the previous monthly intravenous dose as the weekly subcutaneous dose. In both studies, equal rates of infection were obtained: 0.04 serious bacterial infections per patient per year, and 4.4 infections per patient per year overall. The range of subcutaneous doses spanned 34 to 352 mg/kg/week in the United States study and 51 to 147 mg/kg/week in the European/Brazil study. These dose ranges and results are quite consistent with those used in licensing trials of intravenous products in the United States in the past few years.
## Monitoring therapy
Most patients who have PID disease require IgG replacement therapy for life. It therefore is extremely important to monitor them closely to be sure that (1) the treatment itself is associated with as few adverse events and as little interference with normal activity as possible; (2) the treatment regimen is adequate and maintains control of acute infections as well as chronic complications of the underlying disease; (3) patients do not acquire any bloodborne infections or other long-term complications of their therapy.
## Monitoring during infusions
Before beginning any infusion, particularly an intravenous infusion, the patient should be reassessed. It is important to note any changes in medications and signs/symptoms of chronic or acute infection. Adverse effects occurring for up to 72 hours after the previous infusion should be noted. Changes in risk factors for adverse effects also should be noted. For example, starting oral contraceptives or increased cigarette smoking may increase the risk of thrombotic complications of IGIV infusions. Many patients who have PID disease and chronic bronchitis/bronchiectasis have some degree of reversible airway obstruction. If increased secretions and/or bronchospasm are present, bronchodilators may be helpful before or during the infusion. The author and colleagues find it useful to measure and record expiratory flow rates with an office spirometer or even a simple peak flow meter to help with this assessment and to document a patient's status over time. Having the airways as open as possible at the beginning of the infusion may help prevent serious problems if the patient experiences bronchospasm/chest tightness during the infusion. If patients are or recently have been febrile, it usually is helpful to pretreat with antipyretics. If there are signs/symptoms of acute bacterial infection, it may be desirable to defer the infusion for a few days while initiating antibiotic therapy, to avoid shaking chills and severe myalgias/headache during the infusion. The IgG may be needed to help resolve the infection, however, so this delay must be done with caution. Patients who have intercurrent acute gastroenteritis may benefit from antispasmodic or antiemetic treatment before or during the infusion. Some patients may require antipyretics, antiemetics, and/or antimigraine premedication on a routine basis, and some also may require corticosteroids. The author and colleagues often have the latter type of patient take a dose of steroids orally several hours before the infusion is initiated. The patient's hydration status should be assessed carefully, and it usually is a good idea to record the patient's weight at the time of each infusion to allow comparisons. It is possible that dehydration may increase the risk of renal complications, hyperviscosity, and thromboses. It may be prudent to give patients intravenous fluids before actually starting the IGIV to minimize these risks. Conversely, patients who have or are at risk for congestive heart failure may require deferring the fluid/salt/protein load of an infusion if they have recently gained weight, have increased dyspnea and/or rales on chest examination, and/or have increased peripheral edema. Patients who have congestive heart failure and/or fluid retention from other causes may benefit from the administration of diuretics before, during, or following their infusions. Obviously, monitoring such patients closely during the infusion for signs of dyspnea/fluid retention is important.
Before the infusion begins, vital signs should be recorded. It may be important to be sure that the patient has time to relax and adjusted to the ambient conditions before recording the ''baseline'' vital signs to avoid the phenomenon of ''white coat hypertension'' that then might lead to a misinterpretation of decreased blood pressure as the patient relaxes once the intravenous has been placed and the infusion is running. If the patient is comfortable or sleeping, and particularly if the pulse has decreased rather than increased, blood pressure drops of 10% or 20% or even more can be expected and do not necessarily mean that shock is imminent, especially if the patient is not flushed or having dyspnea. Intravenous infusions usually are started at 0.01 mL/kg/minute, and the rates are increased or doubled at 15-to 30-minute intervals to a maximum of 0.08 or 1.0 mL/kg/minute in most cases. Stepwise increases in rates should be made only if the patient is tolerating the infusion well. Therefore vital signs and the patient's condition should be recorded before and 5 to 10 minutes after each rate change. The maximum rate tolerated by the patient at previous infusions should be exceeded only with caution, and it is important to realize that stepwise increases in rates and the maximum infusion rate may vary when the same patient is given different products. Therefore, when any patient must switch products, close monitoring of the patient's condition and vital signs is necessary, because the infusion rates and/or intervals between rate increases may require adjustment.
Subcutaneous infusions of ISG very rarely are associated with systemic adverse effects or significant changes in vital signs. Inadvertent intravascular administration should be avoided by checking to be sure there is no blood return from the needle before actually starting the ISG infusion. Because with most subcutaneous regimens the total monthly dose is fractionated into four or more individual doses, because the IgG is given more slowly, and because adsorption from the subcutaneous site into the circulation is slower than with intravenous infusions, this route may be preferred in patients at risk from cardiovascular, thrombotic and/or renal complications.
Many patients who complain of severe headaches during or following intravenous ISG infusions have less severe problems after subcutaneous infusions, but migraines still may occur, and medication may be required for up to 48 hours after the infusion has been completed. Vital signs usually are not monitored repeatedly during subcutaneous infusions, but the infusion site should be observed by the patient a few times during the infusion. Most subcutaneous infusions are accompanied by swelling with or without redness, and many patients report local pruritus or a burning sensation. These symptoms may be obviated by treatment with antihistamines before or during the infusion. Pretreatment with corticosteroids or antiemetics is rarely needed. One area of special concern with subcutaneous infusions is the risk of cellulitis or other local infection at the infusion sites. Because most patients have swelling and redness, and the injected fluid may lead to a feeling of fluctuance, distinguishing this manifestation from infection sometimes can be difficult. Application of warm compresses or gentle massage may increase local circulation and help dissipate the infused product. In general, most local reactions to IGSC infusions subside within hours after the infusion is completed. Any site at which redness, swelling, or warmth is increasing with time after the infusion should be considered potentially infected and observed very closely or examined by a professional. Patients who self-infuse at home always should be able to contact a physician or nurse on-call. In some cases it may be helpful to ask the patient to mark the size of any local reaction with a pen, so that potential enlargement can be tracked objectively. Although most home-care services and physicians in the United States prescribe preloaded epinephrine injectors to be kept at home, that practice is no longer followed in the United Kingdom.
## Monitoring adequacy of dose and control of disease
Selection of the appropriate dose has been discussed in a previous section. Selection of the initial dose and monitoring its adequacy over time should be individualized. Certainly freedom from acute bacterial infections would be a readily agreed-upon goal, but many patients who have chronic bronchitis or bronchiectasis and some who seem to have reactive airways actually may be experiencing progressive subclinical lung disease. Similarly, patients may have progressive erosive sinus disease but may have learned to ''live with'' the symptoms. Monthly measurements of the white blood cell count, sedimentation rate, and C-reactive protein often are used to assess the presence of subclinical infection. In turn, these data are used to adjust ISG and antibiotic therapy. Frequent sputum cultures and physical examinations also may be useful. Although the practice parameters of the JCAAI suggest that 500 mg/dL is a suitable target serum IgG level, patients should be treated more according to their clinical condition than to achieve any designated level. Nevertheless, monitoring serum IgG levels at routine intervals is important for several reasons. They may serve as markers for adequacy of therapy and enable comparison of one regimen with another. Thus, in patients who experience exacerbations of underlying infection and/or chronic nonspecific symptoms when the IgG trough level falls below a certain value during treatment with intermittent IGIV infusions, that value may serve a target for subcutaneous therapy. Monitoring the serum IgG level also helps assess whether a patient is having increased gastrointestinal or renal protein loss and requires a higher dose or shorter dosing interval to maintain protection against infections. In patients who do not have severe hypogammaglobulinemia per se and/or who actually have elevated serum IgG levels because of monoclonal gammopathy or nonspecific polyclonal B-cell activation, monitoring the trough levels of specific antibody titers (eg, against pneumococcal polysaccharides) may be preferable to using the total IgG level for monitoring the adequacy of therapy.
Besides control of infection, it is important to remember that patients who have common variable immunodeficiency and some other PID diseases are at greater risk for developing malignancies and autoimmunity. Thus, part of the routine monitoring of all such patients, even if they are free from infection, should include careful review of the interval history and physical examination. In addition, selected patients may require radiographic and/or radionuclide studies at regular intervals. In many cases, the immunologist sees the patient much more frequently than any other physician. Sometimes the immunologist, by default, becomes the principal caregiver for the patient and must be diligent to be sure that routine health screening/maintenance is not neglected by the patient's primary care provider, if there is one. Clear communication is required to make sure that developmental and lead screenings are being performed on young children and that monitoring of serum lipids and other risk factors for cardiovascular disease, common malignancies (ie, mammograms, stool guaiac testing, prostate examinations, among others), and bone density is being performed as appropriate in adult patients. Bone density monitoring may be particularly important in postmenopausal women and even in some men who chronically require oral or high-dose inhaled corticosteroids.
## Monitoring for complications of therapy with blood products
Because all ISG products are prepared from large pools of plasma, and there are always threats of emerging diseases and of lapses in standard safety procedures, patients should be monitored for any sign of development of a potential bloodborne disease, including spongiform encephalopathies. Although there have been reports of chronic, slowly progressive neurodegenerative disease in patients receiving IGIV therapy for PID disease, it is not clear whether these conditions might represent chronic viral infections of the central nervous system or other complications of the PID disease per se [bib_ref] Progressive neurodegeneration in patients with primary immunodeficiency disease on IVIG treatment, Ziegner [/bib_ref]. Transient Coombs' positivity has been reported with certain IGIV preparations, but clinically significant hemolytic anemia is rare as a complication of IGIV therapy [bib_ref] Risks associated with the use of intravenous immunoglobulin, Pierce [/bib_ref]. Patients who have PID disease, especially those who have common variable immunodeficiency and hyper-IgM syndromes, also may develop hematologic and/or hepatic abnormalities resulting from their underlying disease or treatment with other medications. Obviously it is important to follow renal function in patients receiving repeated therapeutic infusions of fluid and protein. Careful initial characterization of the patient's baseline, including documentation of hematologic, hepatic, and renal function, and developmental screening thus are essential. In patients who do have some antibody production, tests for exposure to Epstein-Barr virus and cytomegalovirus, as well as HIV and viral hepatidies should be documented before therapy is begun because the presence of passively acquired antibody may make this documentation more difficult. Documentation of a patient's baseline virologic status may have important clinical and medicolegal implications in the future. To monitor for complications of therapy, the complete blood cell count, hepatic and renal function tests, and urinalysis should be repeated every 6 to 12 months. To monitor for bloodborne and other infectious diseases, nucleic acid tests such as polymerase chain reaction or reverse transcription polymerase chain reaction, when available, are preferred to serologic screening tests, because patients who have PID disease generally are antibody deficient and may not produce antibody as expected after exposure to a pathogen. These tests probably should be repeated once a year.
## Hyperimmune and other specific immune globulins
Besides standard preparations of polyclonal ISG, several special hyperimmune globulins are available. These are listed and described in detail elsewhere. In general, hyperimmune globulins are prepared from the plasma of individuals who have been exposed accidentally (eg, to snake or other venoms), are convalescing from specific infections (eg, chickenpox), or whose plasma has been found on testing to contain high titers of certain desirable antibodies. For a few antigens, plasma is drawn from persons specifically immunized against unusual but potentially important pathogens such as vaccinia or from healthy normal donors repeatedly immunized with common vaccines such as tetanus toxoid. Most pediatricians are familiar with Varicella zoster immune globulin and with the use of anti-Rh(D) to prevent alloimmunization of mothers. Guidelines for plasma donations from which these products are produced and the steps used to assure that they are free from bloodborne pathogens are the same as for standard polyclonal ISG products, as discussed previously.
## Unanswered questions
Although great progress has been made since the painful intramuscular injections of ISG in the 1950s and 1960s, many questions about the characteristics and uses of ISG remain unanswered. Important issues about the mechanisms of action of polyclonal IgG in inflammatory and autoimmune diseases remain, and it is not clear to what extent specifically modified or monoclonal preparations might obviate the use of polyclonal IgG for those conditions. Reports describing the spectrum of antibodies and the appropriate uses of ISG for prevention and/or management of influenza are scarce, and it is even less clear what will be done if new pandemic strains emerge. As the population of plasma donors shifts from those who have recovered from natural infection with measles, mumps, and similar viruses in childhood to those who were immunized and never had the wild-type disease, antibody levels in ISG preparations are changing. Concerns have been raised about how well the protection of antibody-deficient individuals will be maintained. On the other hand, a steadily increasing number of new vaccines has been introduced in recent years, now including meningococcal conjugate vaccine and human papilloma virus vaccine. Studies of the antibody content and efficacy of standard ISG preparations in preventing those infections are not available. In addition, with the experience of rapid, worldwide transmission of severe acute respiratory syndrome and West Nile virus infection in recent years, the threat of emerging diseases is no longer an exotic science fiction scenario. Thus, production of ISG and other bloodderived products and optimal care of patients who have PID disease will continue to evolve.
## Summary
Advances in the large-scale production of polyclonal ISG preparations during the last 2 decades have greatly improved the management of patients who have PID disease. The continued development of products with improved safety and tolerability profiles has allowed treatment to focus on quality of life and long-term freedom from the complications of PID disease rather than just on freedom from severe acute infections and survival. Currently available ISG preparations allow routine therapy by a variety of routes and regimens that can be tailored to suit any individual patient. Continued vigilance is required, however, because problems with emerging diseases and the costs and availability of ISG are likely to present continuing challenges.
[fig] Box 2: Guidelines for IgG replacement/augmentation presented at the 2006 meeting of the European Society for Immune Deficiency When is IgG replacement/supplementation indicated? IgG < 200 mg/dL: All patients IgG 200-500 mg/dL: If a specific antibody deficiency is identified and frequent infections are documented. IgG > 500: If a specific antibody deficiency identified and severe/ recurrent infections are documented immunodeficiency require IgG supplementation for at least a year. Many recipients of stem cell transplants have poor B-cell engraftment or function and require replacement therapy for life. Patients, particularly children under 5 years of age, who do not have confirmed genetic diagnoses of PID disease [/fig]
[fig] Figure 1: Serum IgG concentrations in a 34-year-old man who has X-linked agammaglobulinemia. (Left panel) The diamonds and blue line indicate serum IgG concentrations at various times after a single intravenous infusion of 30 g (406 mg/kg). The black line indicates the average daily IgG level calculated by interpolation from points indicated by diamonds. Mean AE SEM ¼ 850.6 AE 43 mg/dL. (Right panel) Serum IgG concentrations in the same patient receiving 12 g 16% IgG every 7 days. Mean AE SEM ¼ 816 AE 16 mg/dL. (From Berger M. Subcutaneous immunoglobulin replacement in primary immunodeficiencies. Clin Immunol 2004;112:1-7; with permission.) [/fig]
[table] Table 1: Box 1. Steps used to minimize risk of transmission of bloodborne diseases by immune serum globulinSpecific steps for viral inactivation/removal Cold ethanol precipitation and depth filtration Heat (pasteurization at 60 C) Low pH Treatment with pepsin or other proteases Fatty alcohol/fatty acid treatment Solvent/detergent treatment Nanofiltration Record keeping/recall notification Properties of polyclonal immune serum globulin products currently marketed in the United States [/table]
[table] Table 2: Viruses used to test efficacy of viral removal/inactivation steps during IgG preparation [/table]
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Focused attention meditation changes the boundary and configuration of functional networks in the brain
Research has shown that focused attention meditation not only improves our cognitive and motivational functioning (e.g., attention, mental health), it influences the way our brain networks [e.g., default mode network (DMN), fronto-parietal network (FPN), and sensory-motor network (SMN)] function and operate. However, surprisingly little attention has been paid to the possibility that meditation alters the architecture (composition) of these functional brain networks. Here, using a single-case experimental design with intensive longitudinal data, we examined the effect of mediation practice on intra-individual changes in the composition of whole-brain networks. The results showed that meditation (1) changed the community size (with a number of regions in the FPN being merged into the DMN after meditation) and (2) led to instability in the community allegiance of the regions in the FPN. These results suggest that, in addition to altering specific functional connectivity, meditation leads to reconfiguration of whole-brain network architecture. The reconfiguration of community architecture in the brain provides fruitful information about the neural mechanisms of meditation.Meditation is a practice aimed to enhance one's core psychological capacities, such as attentional and emotional self-regulation 1 . In several styles of practice, focused attention meditation involves sustaining attention to present-moment experiences without emotional reaction and judgment and has been found to produce significant beneficial outcomes, such as stress reduction 2 and improvements in attention processing 3 .Past research indicates that the meditation is primarily related to three brain networks: the fronto-parietal network (FPN), sensory-motor network (SMN), and default mode network (DMN) 1 . The FPN mainly consists of the rostro-and dorso-lateral prefrontal cortex (PFC), anterior insula, dorsal anterior cingulate cortex (ACC), and anterior inferior parietal lobule; all of these brain areas are critical for cognitive control functions, such as regulation of attention and emotion 4-7 . Especially in the early stages of long-term practice, this meditation increases activation of FPN regions[8][9][10][11], which is consistent with the general observation that focusing on the present moment requires effortful attentional control.Focused attention meditation also alters sensory experiences through the SMN 12,13 , consisting of motor cortices, primary somatosensory cortex, and insula. In a previous study, these brain areas showed reduced activation in a four-day meditation when beginners meditated in the presence of noxious stimulation causing pain 14 . This change in brain activity may be associated with enhanced body awareness, as the meditation requires individuals to focus on a body part or internal experiences, such as breathing 15 .The DMN, mainly consisting of the anterior medial PFC, posterior cingulate cortex (PCC), and posterior inferior parietal lobule, is a network implicated in supporting spontaneous thoughts and self-referential processing[16][17][18]. Because sustained attention on an anchoring object (e.g., one's breath) needs to detect distraction such as task-irrelevant thoughts, disengage attention from the distraction, and redirect attention on the object, the DMN is expected to be suppressed during meditation. In fact, the medial PFC and PCC showed less activity during meditation, and functional connectivity between the PCC, dorsal ACC, and dorso-lateral PFC was stronger in meditators compared to meditation-naive controls 19 . These results indicate that the meditation may increase cognitive control over the DMN functioning 19 .
Although previous work has provided various insights into how meditation influences the functional network of the brain, there are two critical limitations in the current literature. First, the brain networks were defined a priori in previous studies, precluding the possibility that meditation practice can alter the architecture of the primary brain networks themselves (i.e. FPN, SMN, and DMN). Because recent studies have shown that meditation can change functional connectivity across brain regions [bib_ref] Mind wandering and attention during focused meditation: a fine-grained temporal analysis of..., Hasenkamp [/bib_ref] [bib_ref] Meditation experience is associated with differences in default mode network activity and..., Brewer [/bib_ref] [bib_ref] Impact of mindfulness-based stress reduction training on intrinsic brain connectivity, Kilpatrick [/bib_ref] [bib_ref] Impact of meditation training on the default mode network during a restful..., Taylor [/bib_ref] , the whole-brain composition of the FPN, SMN, and DMN may be altered as a consequence of meditation.
Second, most of the previous research has employed a one-shot pre-post or nonmeditator-meditator comparison design [bib_ref] Attention training and attention state training, Tang [/bib_ref] [bib_ref] Neural correlates of mindfulness meditation-related anxiety relief, Zeidan [/bib_ref] , and compared the conditions after aggregating the data across heterogeneous participants. This inter-individual aggregation approach is useful to examine the effects of meditation averaged across participants. However, given the large individual differences in the whole-brain functional connectivity patterns [bib_ref] Personalized brain network models for assessing structure-function relationships, Bansal [/bib_ref] [bib_ref] Individual variability in functional connectivity architecture of the human brain, Mueller [/bib_ref] , there is danger that the approach potentially masks important intra-individual changes in the composition of the brain networks (e.g., some participant-specific network architectures may be canceled out by inter-individual aggregation). Therefore, adopting a design that allows us to focus on the intra-individual change may provide novel insights into how meditation alters the architecture of the brain networks.
The current research aims to expand our understanding of meditation by addressing these two critical issues. For that purpose, we will examine the effects of meditation using a single-case experimental design with intensive longitudinal data. Single-case experimental designs have a long tradition in psychology ; , and in later years, they have been applied to intensive longitudinal data (for a systematic review, see . Single-case experimental designs are effective in reliably detecting intra-individual changes in outcome variables in response to intervention [bib_ref] Single-case experimental designs: a systematic review of published research and current standards, Smith [/bib_ref]. However, this design has rarely been implemented in neuroimaging studies (for an exception without experimental manipulation, see . Based on this design, we scanned a single participant repeatedly over a long period of time (65 days), employing the meditation intermittently, and examined whether and how the whole-brain composition of the FPN, SMN, and DMN were altered over this period of meditation practice.
# Methods
The study was conducted in accordance with the principles of the Helsinki declaration.
Participant. The participant (author S.K.) is a right-handed Asian male, aged 28 years and had no experience of meditation practice at the onset of the study. The participant was healthy with no history of neuropsychiatric disorders. The study was approved by the research ethics committees of the University of Reading, UK (UREC 16/28).
The participant underwent 65 scanning sessions, each on a different day between June 15th 2016 and November 11th 2016. The scanning time (between 9am and 5 pm) varied unsystematically across days. Data acquisition was not completed on 6 out of the 65 days due to technical issues and data from an additional day was excluded due to excessive head movement (> 3.0 mm between adjacent scan slices). As a result, only data from 58 days were used in the following analyses. On each day, the participant underwent a resting-state fMRI scan with eyes open for 10 min and completed two sets of questionnaires carried out for a separate study. On 18 out of the 58 days [fig_ref] Figure 1: Properties of the ROIs that changes across days [/fig_ref] , the participant underwent a 15-min session of the meditation practice within half an hour before the scanning session. Note that we started the intervention 64 days after the first scanning session and the assignment of the intervention condition was randomized since then. During the meditation, the participant was instructed to focus on his breathing, specifically on sensations of the breath through the nostrils, and to redirect attention from spontaneously occurring thoughts to breathing when he realized his mind was wandered [bib_ref] Mind wandering and attention during focused meditation: a fine-grained temporal analysis of..., Hasenkamp [/bib_ref]. Before the data collection, the participant studied the meditation several times from an auditory instruction developed by a professional trainer , in revision) so that he did not need the instruction for each practice. In the following text, the "meditation condition" refers to the days on which scanning followed the meditation practice. The "no-meditation condition" refers to the days on which there was no meditation practice prior to scanning. The mean of recording days (i.e. days elapsed from the first scanning session) of the meditation condition and no-meditation condition were 23.7 and 42.3, respectively. Data acquisition. MR images were acquired using a Siemens 3.0-T Trio scanner equipped with a 32-channel head coil at the Centre for Integrative Neuroscience and Neurodynamics of the University of Reading. The resting-state fMRI data were obtained using a single-shot, gradient-echo echo-planar imaging (EPI) sequence. Sequence parameters were as follows: repetition time/echo time (TR/TE) = 2500/30 ms, slice thickness = 3.5 mm, field of view (FoV) = 256 mm, flip angle (FA) = 90°, data matrix = 64 × 64, in-plane resolution = 3.5 × 3.5 mm, 46 slices, 10 min scan length. Four dummy scans were discarded to remove the impact of magnetization instability. A high-resolution (spatial resolution: 1 mm 3 ) structural image was also acquired on the first day using a T1-weighted magnetization prepared rapid-acquisition gradient echo (MP-RAGE) pulse sequence. Data preprocessing. All preprocessing steps were performed using the Data Processing Assistant for Resting-State fMRI Advanced Edition (DPARSFA) [bib_ref] DPARSF: a MATLAB toolbox for "pipeline" data analysis of resting-state fMRI, Chao-Gan [/bib_ref] , which runs on Statistical Parametric Mapping 8 (SPM8) and the Resting-State fMRI Data Analysis Toolkit (REST) [bib_ref] REST: a toolkit for resting-state functional magnetic resonance imaging data processing, Song [/bib_ref]. Data preprocessing included the following steps: realignment of all functional images using a six-parameter rigid body transformation (T x = 0.04 ± 0.03 mm, T y = 0.30 ± 0.14 mm, T z = 0.21 ± 0.13 mm, R x = 0.27 ± 0.13°, R y = 0.06 ± 0.05°, R z = 0.10 ± 0.05°); slice-timing correction to the middle slice of each volume; co-registration of the structural image (T1-weighted MPRAGE) to the mean functional image using a rigid-body transformation; segmentation of the transformed structural image into the gray matter, white matter, and cerebrospinal fluid (CSF); nuisance covariate regression of six head motion parameters, average white matter signals, CSF signals, and the global signal in native space; spa-Scientific Reports | (2020) 10:18426 | https://doi.org/10.1038/s41598-020-75396-9
www.nature.com/scientificreports/ tial normalization of the functional images to the Montreal Neurological Institute (MNI) stereotactic standard space; spatial smoothing of the functional images with a 6-mm full-width at half-maximum (FWHM) Gaussian kernel using the Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) toolbox 33 ; band-pass filtering (0.01-0.10 Hz) to reduce low-frequency drift and high-frequency physiological noise. The relationship between the framewise displacement [bib_ref] Spurious but systematic correlations in functional connectivity MRI networks arise from subject..., Power [/bib_ref] The rows and columns correspond to the days and ROIs, respectively. While the majority of the ROIs were classified to the same community, the ROIs in fronto-parietal network were often categorized as part of the default mode network. The four communities were labeled the visual network (colored in purple), sensory-motor network (SMN; green), fronto-parietal network (FPN; blue), and default mode network (DMN; red). A white stripe describes a day in which a ROI belonged to more than one community and could not be assigned a community label.
Scientific Reports | (2020) 10:18426 | https://doi.org/10.1038/s41598-020-75396-9
www.nature.com/scientificreports/ Generalized Louvain method. The original Louvain method approximately maximizes the objective function (modularity) to partition the nodes in the given static network into communities. Communities are determined such that there are many edges or connections within each community and relatively few edges between communities [bib_ref] Fast unfolding of communities in large networks, Blondel [/bib_ref]. The generalized Louvain method considers the edges across multiple inter-dependent days and optimizes the generalized modularity instead of separately optimizing the modularity for each day. In the present study, a day represents the static functional connectivity on one day. The strength of the connections between days and the spatial resolution parameter were set as default (i.e., ω = 1) and the resolution parameter (i.e., γ = 1). We ran the algorithm 100 times and selected the community architecture yielding the largest generalized modularity value. As shown in the Results section, this procedure found four communities that are comparable with the communities in previous research Community labeling. First, we represented each community i (i = 1, 2, 3, 4) by its core members. The core members of the ith community were defined by the ROIs whose dominant community, that is the community to which the ROI belonged for the largest number of days under the given condition, was the ith community under both conditions. The relative overlap between the ith community and a community in the template communities defined by Yeo et al. [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref] , N j (j = 1, 2, …, 7), was defined as V (Ci∩Nj) / 7 l=1 V (Ci∩Nj) , where C i is the set of voxels belonging to a core member of the ith community, N j is interpreted as the set of voxels belonging to mask N j , and V (C i ∩N j ) represents the number of voxels that belong to both C i and N j . We then labeled each community i according to the community in the template that exhibited the largest overlap with the ith community.
Calculation and statistical significance test of community size. We defined the community size for a day t as the number of regions included in the time-dependent community on day t, and tested the difference of average community size across the conditions. We also explored potential changes in the community size over time by including days as an additional independent variable. More specifically, we performed a general linear model in which practice condition (meditation condition vs. no-meditation condition; a categorical variable), community (visual network, SMN, FPN, and DMN; a categorical variable), and day (t: 1,2, …, 58; a continuous variable) were included as independent variables. We also included the interactions of these independent variables.
Calculation and statistical significance test of community coherence. We defined the similarity of each community i (i = 1, 2, 3, 4) between day t 1 and day t 2 by the Jaccard index, i.e.
[formula] J (X,Y ) = |X ∩ Y |/|X ∪ Y | , [/formula]
where X is the set of nodes in community i on day t 1 and Y is the set of nodes in community i on day t 2 . Jaccard index J (X,Y ) ranges between 0 and 1. One obtains = J (X,Y ) 1 if and only if X and Y are exactly the same, and = J (X,Y ) 0 if X and Y do not share any ROIs. For each of the four communities, we calculated the similarity between all pairs of 58 days, obtaining a 58 × 58 similarity matrix.
Given the similarity matrix for a community, we compared the coherence of the community within and across the practice conditions. Specifically, we used a permutation test, which is commonly used for testing the significance of single-subject research [bib_ref] Validity of randomization tests for one-subject experiments, Edgington [/bib_ref]. The permutation test consists of the following three steps. In the first step, we classified the pairs of days into two groups. The congruent group contained the pairs of days which both belonged to the meditation condition or the pair of days which both belonged to the no-meditation condition. In contrast, the incongruent group contained the pairs of days from the different conditions (i.e. one from the meditation condition and the other from the no-meditation condition). Because there were 18 meditation days and 40 no-meditation days, congruent and incongruent groups contained 933 and 720 pairs of days, respectively. In the second step, we computed the coherence of the community, which is a Welch's t-value, by comparing the averaged similarity value between the congruent and incongruent groups. We then randomized the days by reassigning 18 uniformly randomly selected days to the fictive meditation condition and the remaining 40 days to the fictive no-meditation condition, and calculated the coherence for the randomized data. We repeated this procedure 10,000 times to obtain the null distribution of the coherence for the randomized labeling. In the third step, we assessed the probability of obtaining the coherence calculated on the basis of the true labeling of the days (i.e., meditation condition or no-meditation condition) or more extreme coherence under the null model in which the meditation condition was randomly assigned to individual days.
Calculation and statistical significance test of flexibility. We defined the flexibility of a ROI under each condition using the inverse participation ratio (IPR) [bib_ref] Statistical properties of randomly broken objects and of multivalley structures in disordered..., Derrida [/bib_ref]. The IPR of a ROI under a condition is defined as where I represents the number of days (no-meditation condition, 40; meditation condition, 18), and I i represents the number of days in which the ROI belonged to community i [fig_ref] Figure 1: Properties of the ROIs that changes across days [/fig_ref]. The IPR is equal to 0.75, which is the largest possible value, when a ROI belongs to all the communities with the same probability. In this case, the ROI is the most flexible in terms of the community membership. The IPR is equal to 0, which is the smallest possible value, when the ROI belongs to the same single community in all the days. In this case, the ROI is the least flexible. To investigate whether the meditation affects the community-wide flexibility of ROIs, for each community, we applied a paired sample t-test to test the mean difference in the flexibility between the two conditions. In this analysis, we defined each community i by its core members, i.e., the ROIs that belonged to community i as the dominant community under both conditions.
[formula] IPR = 1 − 4 i=1 I i I 2 , [/formula]
# Results
Similarity of the functional connectivity across time. To examine variability of the functional connectivity over time, we calculated the correlation of functional connectivity between days [fig_ref] Figure 1: Properties of the ROIs that changes across days [/fig_ref]. The correlation value ranged from 0.429 to 0.780, suggesting that the functional connectivity of a single person varies on a daily basis. This result is consistent with previous longitudinal scanning data from a single participant [bib_ref] Long-term neural and physiological phenotyping of a single human, Poldrack [/bib_ref].
Finding community architecture. To detect the community architecture in the time-varying functional connectivity across the 58 days, we applied a generalized Louvain method [fig_ref] Figure 1: Properties of the ROIs that changes across days [/fig_ref]. To label the four communities detected in the current study, we assessed how these communities overlapped with the template communities defined by Yeo et al. [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref]. As the result, we labeled the four communities as visual network (80.8% overlap), SMN (50.3% overlap), FPN (45.3% overlap), and DMN (58.4% overlap) (diagonal in [fig_ref] Community size: The analysis showed a significant main effect of community [/fig_ref].
Metrics that quantify the changes in community architecture. We examined the effect of meditation practice on intra-individual changes in the composition of the whole-brain networks with three metrics: community size, community coherence, and flexibility. www.nature.com/scientificreports/ three-way interaction, we examined the interaction between the meditation condition and day separately for each community. The interaction between the meditation condition and the day was significant for the FPN (F (1, 54) = 7.77, p = 0.007) and DMN (F (1, 54) = 9.46, p = 0.003) but not for the visual network (F (1, 54) = 3.52, p = 0.066) and SMN (F (1, 54) = 0.40, p = 0.527). In the FPN, the day effect was significantly positive in the meditation condition (F (1, 16) = 6.06, p = 0.026) and not in the no-meditation condition [bib_ref] The neuroscience of mindfulness meditation, Tang [/bib_ref] [bib_ref] Graph analysis of functional brain networks: practical issues in translational neuroscience, Fallani [/bib_ref] = 0.25, p = 0.617) indicating that the community size of the FPN increased as the meditation practice progressed. In the DMN, on the other hand, the day effect was significantly negative in the meditation condition (F (1, 16) = 6.98, p = 0.018) and not in the nomeditation condition [bib_ref] The neuroscience of mindfulness meditation, Tang [/bib_ref] [bib_ref] Graph analysis of functional brain networks: practical issues in translational neuroscience, Fallani [/bib_ref] = 0.73, p = 0.400), indicating that the community size of the DMN decreased as the meditation practice progressed. [fig_ref] Figure 2: The change of dominant community affiliation between the no-meditation condition [/fig_ref] shows whether the ROIs stayed in the same community or changed to a different dominant community between the two conditions (i.e. represents the main community that a ROI belonged to under each condition). The on-diagonal brains show the ROIs that stayed in the same dominant community across the two conditions. The off-diagonal brains show the ROIs that belonged to different dominant communities between the two conditions. Consistent with the results, the figure shows that a large number of ROIs (i.e. 21 ROIs) that belonged to the FPN in the no-meditation condition shifted to the DMN in the meditation condition (row 3, column 4; other community pairs ≤ 11 ROIs). The shift in the dominant community affiliation between the conditions is quantitatively depicted in [fig_ref] Community size: The analysis showed a significant main effect of community [/fig_ref] Coherence of community composition. The community size is one way of examining the change in the composition of the brain network across the two conditions. In fact, even if the relative community size is the same between the two conditions, the constituent ROIs of each community may be substantially different in the two conditions. Therefore, for each community, we examined the extent to which the community as identified by the set of ROIs comprising it is stable within each of the two conditions (community coherence). The results of the permutation test are shown in [fig_ref] Community size: The analysis showed a significant main effect of community [/fig_ref]. For the DMN, the coherence of the community architecture within the same condition was larger than the coherence between the no-meditation condition and meditation condition (DMN, p = 0.006), which remained significant after correction of the false discovery rate (FDR = 0.05). This result indicates that the meditation practice has changed the community composition (i.e., the set of ROIs composing the community) in the DMN, which is a direct consequence of our findings on the community size because a change in the community size implies a decrease in the coherence value. For the other communities, there was no difference in the coherence (visual, p = 0.137; SMN, p = 0.030; FPN, p = 0.028), which implies that the sets of ROIs composing these network communities were not influenced by the meditation at a significant level.
Flexibility of community allegiance. To assess experience-related changes in community allegiance of a ROI, we defined the flexibility of a ROI under each condition using the IPR (Derrida and Flyvbjerg 45 ). The change in flexibility between meditation condition and no-meditation condition for individual ROIs belonging to each community (i.e. visual network, SMN, FPN, and DMN) is shown in [fig_ref] Community size: The analysis showed a significant main effect of community [/fig_ref]. Positive values mean that flexibility of the ROI increased as a consequence of the meditation. One-sample t-tests of the difference in flexibility between the two conditions for each community revealed that the meditation significantly enhanced flexibility of the ROIs in the FPN [fig_ref] Community size: The analysis showed a significant main effect of community [/fig_ref] ; mean = 0.17 ± 0.13, t = 7.334, p < 0.001). Other communities did not show a significant difference in flexibility of the ROIs (|mean| ≦ 0.02, SD ≧ 0.10, |t| ≦ 0.802). These results suggest that the meditation increases the flexibility of the FPN community, but not the visual network, SMN, or DMN. [fig_ref] Table 1: Summary of the meditation effect [/fig_ref] summarizes the results.
# Discussion
Previous studies have provided evidence that focused attention meditation changes activation and connectivity patterns between specific brain regions [bib_ref] Mind wandering and attention during focused meditation: a fine-grained temporal analysis of..., Hasenkamp [/bib_ref] [bib_ref] Meditation experience is associated with differences in default mode network activity and..., Brewer [/bib_ref] [bib_ref] Impact of mindfulness-based stress reduction training on intrinsic brain connectivity, Kilpatrick [/bib_ref] [bib_ref] Impact of meditation training on the default mode network during a restful..., Taylor [/bib_ref] [bib_ref] Attention training and attention state training, Tang [/bib_ref] [bib_ref] Neural correlates of mindfulness meditation-related anxiety relief, Zeidan [/bib_ref]. Extending on this line of research, we employed a whole-brain graph theoretic analysis with a single-case experimental design using intensive longitudinal data to reveal that the meditation provokes the reconfiguration of the community architecture of the whole-brain functional network.
We found that the size of the FPN decreased and that of the DMN increased as a consequence of the meditation, although their size tended to return to the default size in the later period of experiment. The former result is consistent with the previous research in which experienced meditators showed increased functional connectivity of the PCC with the dorsal ACC and dorso-lateral PFC both during rest and meditation [bib_ref] Meditation experience is associated with differences in default mode network activity and..., Brewer [/bib_ref] compared with novice meditators. The research proposed that the composition of these brain networks might have changed over time and become a new "default mode" that can be observed during meditation as well as during the resting state. The current observation that some ROIs shift from the FPN to the DMN after the meditation partially supports their hypothesis. Considering such consistency, the return of the community size in the later period might reflect insufficient effect of the practice because of habituation-derived lack of concentration.
The FPN also showed enhanced flexibility under the meditation condition. A previous study suggested that enhanced flexibility in the FPN may reflect an (initial) learning process of a task [bib_ref] Dynamic reconfiguration of human brain networks during learning, Bassett [/bib_ref]. Accordingly, although the previous study used a different task with a different time scale, i.e., Bassett et al. [bib_ref] The modular and integrative functional architecture of the human brain, Bertolero [/bib_ref] observed the change in the flexibility within a few hours of motor-task training, the increased flexibility in the FPN in the current study may indicate that some form of learning process is operative in the meditation (e.g., how to control breathing, attention, bodily sensations etc.). Also, previous research proposed that the ROIs in the FPN integrate and modulate other networks in response to varying task demands [bib_ref] The modular and integrative functional architecture of the human brain, Bertolero [/bib_ref] [bib_ref] The diverse club, Bertolero [/bib_ref] [bib_ref] A mechanistic model of connector hubs, modularity and cognition, Bertolero [/bib_ref]. These findings are consistent with the observations from the current study; the FPN's enhanced flexibility as well as its integration into the DMN under the meditation condition [fig_ref] Community size: The analysis showed a significant main effect of community [/fig_ref] www.nature.com/scientificreports/ relationship between the reorganization and reconnection of the FPN and the DMN, and the positive effects of meditation such as improved concentration or emotion regulation. The present study demonstrated the value of a single-case experimental design with intensive longitudinal data. It allows us to detect intra-individual changes in the whole-brain network composition without being influenced by the large heterogeneity of individuals' brain functional networks [bib_ref] Individual variability in functional connectivity architecture of the human brain, Mueller [/bib_ref]. Previous studies on meditation heavily relied on the expert-beginner and/or pre-post comparison design 1 . Future research should be encouraged to adopt the single-case research design more frequently to seek further insights into intra-individual changes in patterns of brain networks as a consequence of meditation. One obvious limitation of the current research design is that the data were collected from a single participant, which makes it impossible to examine potential individual differences in our findings. However, although research in cognitive neuroscience typically collects data from multiple participants, for the majority of studies, their main focus is on the aggregated pattern of the brain activation/connectivity (but see person-centered research [bib_ref] Personalized brain network models for assessing structure-function relationships, Bansal [/bib_ref] , and individual differences have been typically treated as random noise (sampling error). Therefore, in our view, this limitation is superseded by the strength of the current design: sensitivity to the nuanced intra-individual changes in brain signals and functional connectivity. Nevertheless, the potential of the current intensive longitudinal design would be considerably improved by data obtained from multiple participants in future studies. Another limitation of the present study is that the participant performed meditation practice only for three months, which is considerably shorter than the . Coherence of the community within and across conditions. Coherence is a Welch's t-value that represents the difference in averaged similarity value of a community architecture between two groups, i.e., the congruent and incongruent group. The congruent group contained pairs of days that both belonged to the meditation condition (MC) or the no-meditation condition (NoMC). The incongruent group contained the pairs of days from the different conditions (i.e. one from the MC and the other from the NoMC). The figure shows null distributions of coherence (distributions of coherence for permutated data); vertical lines represent the t-values observed with the true labeling and their corresponding p-values representing the probability of obtaining such observed t-values (or more extreme t-values) in the null distribution. Significant effect was found only in the DMN after FDR correction, which would be a direct consequence of our findings on the community size because a change in the community size implies a decrease in the coherence value. www.nature.com/scientificreports/ www.nature.com/scientificreports/ previous studies with experts (e.g., more than one year of regular practice) [bib_ref] Mind wandering and attention during focused meditation: a fine-grained temporal analysis of..., Hasenkamp [/bib_ref]. In addition, due to the fact that the intervention was planned after the scanning session had started, meditation practice was embedded in the latter half of the period. This design issue makes it difficult to completely distinguish the exact effect of mindfulness from the general time course effect, although there is little evidence that repeated scans would change the nature of functional connectivity networks [bib_ref] Long-term neural and physiological phenotyping of a single human, Poldrack [/bib_ref]. Future research should collect data for a more prolonged period of time to examine how the progress of practice induces long-term changes in the community architecture.
## Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Received: 27 January 2020; Accepted: 14 October 2020 www.nature.com/scientificreports/
[fig] Figure 1: Properties of the ROIs that changes across days. (A) Similarity (correlation) between the functional networks for each pair of days. The correlation value ranged from 0.429 to 0.780, suggesting that the functional connectivity of a single person varies on a daily basis. The color code represents the value of the correlation coefficient. MC refers to the meditation condition. There were 18 out of 58 days in the MC. (B) Time-dependent community architecture. [/fig]
[fig] Community size: The analysis showed a significant main effect of community (F(3, 216) = 147.7, p < 0.001) while we observed no significant main effect for the condition (F (1, 216) = 0.01, p = 0.907) nor for the day (F (1, 216) = 0.00, p = 0.999). These main effects were qualified by a significant interaction between the community and the condition (F (3, 216) = 3.03, p = 0.030), suggesting that meditation changed the community size differently across the communities. To explore which communities showed the differential effect, we took all the possible pairs of communities (e.g., FPN and SMN) and examined whether the effect of meditation on community size was different between the paired communities. The results showed that meditation condition only significantly interacted with the FPN and the DMN (F (1, 112) = 4.54, p = 0.035; others, F (1, 112) ≤ 2.64, p ≥ 0.107). The significant interaction indicates that meditation increased the community size of the DMN while it decreased the size of the FPN, although post-hoc analysis did not show statistically significant simple main effects of meditation condition either for the FPN (F (1, 56) = 2.317, p = 0.134) or the DMN (F (1, 56) = 2.515, p = 0.118). We also observed a significant interaction across the community, condition, and day (F (3, 216) = 9.89, p < 0.001). To unpack the [/fig]
[fig] Figure 2: The change of dominant community affiliation between the no-meditation condition (NoMC) and the meditation condition (MC). (A) The shift of the dominant community allegiance of ROIs between the conditions. The schematic pictures of the brain on the diagonal show the ROIs that belonged to the same community with the highest probability across the two conditions. Those off the diagonal show the ROIs that belonged to different communities in the two conditions. (B) Quantitative description of dominant community shift across the conditions. While the ROIs were generally classified to the same community in both the MC and the NoMC, a large number of ROIs (i.e. 21 ROIs) that belonged to the FPN in the NoMC shifted to the DMN in the MC (row 3, column 4; other community pairs ≤ 11 ROIs).Scientific Reports | (2020) 10:18426 | https://doi.org/10.1038/s41598-020-75396-9 [/fig]
[fig] Figure 4: Changes in flexibility between two conditions. (A) Histograms for each community show the distribution of the values of ROIs that belonged to the corresponding community in both conditions. (B) Change in flexibility rendered on the cortical surface. Positive values mean the flexibility was higher in the meditation condition (MC) than the no meditation condition (NoMC). The ROIs that showed higher flexibility in the MC were in fronto-parietal network (FPN), whereas that showed higher flexibility in NoMC scattered in networks. Scientific Reports | (2020) 10:18426 | https://doi.org/10.1038/s41598-020-75396-9 [/fig]
[table] Table 1: Summary of the meditation effect. n.s. indicates not significant. MC meditation condition, NoMC no-meditation condition. Scientific Reports | (2020) 10:18426 | https://doi.org/10.1038/s41598-020-75396-9 [/table]
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A systematic review of the effect of infrastructural interventions to promote cycling: strengthening causal inference from observational data
Background: Previous reviews have suggested that infrastructural interventions can be effective in promoting cycling. Given inherent methodological complexities in the evaluation of such changes, it is important to understand whether study results obtained depend on the study design and methods used, and to describe the implications of the methods used for causality. The aims of this systematic review were to summarize the effects obtained in studies that used a wide range of study designs to assess the effects of infrastructural interventions on cycling and physical activity, and whether the effects varied by study design, data collection methods, or statistical approaches. Methods: Six databases were searched for studies that evaluated infrastructural interventions to promote cycling in adult populations, such as the opening of cycling lanes, or the expansion of a city-wide cycling network. Controlled and uncontrolled studies that presented data before and after the intervention were included. No language or date restrictions were applied. Data was extracted for any outcome presented (e.g. bikes counted on the new infrastructure, making a bike trip, cycling frequency, cycling duration), and for any purpose of cycling (e.g. total cycling, recreational cycling, cycling for commuting). Data for physical activity outcomes and equity effects was extracted, and quality assessment was conducted following previous methodologies and the UK Medical Research Council guidance on natural experiments. The PROGRESS-Plus framework was used to describe the impact on subgroups of the population. Studies were categorized by outcome, i.e. changes in cycling behavior, or usage of the cycling infrastructure. The relative change was calculated to derive a common outcome across various metrics and cycling purposes. The median relative change was presented to evaluate whether effects differed by methodological aspects.(Continued on next page)
# Background
Promoting physical activity is one of the key strategies to combat the burden of many chronic diseases. Cycling can contribute to meeting the recommended daily physical activity levels. A metaanalysis including 187,000 individuals and 2.1 million person-years showed that 2.5 h per week of cycling at moderate intensity was associated with a 10% lower mortality risk, independent of overall levels of physical activity. In addition to this, a Danish study found that those who cycled and, those who started cycling after the age of 50 years had a lower risk of coronary heart disease and developing diabetes than those who did not cycle. Modelling studies have also showed that the population health benefits of cycling outweigh the negative risks, such as exposure to air pollution and traffic accidents. This indicates that promoting cycling can result in populationlevel health benefits.
Providing an infrastructure that supports the needs of cyclists has been considered as an important strategy to encourage more cycling in cities. However, designing studies to evaluate such infrastructural interventions is challenging. Although randomized controlled trials (RCTs) are regarded as the gold-standard for estimating causal effects of health interventions, to our knowledge no studies exist that used the RCT design to assess the impact of infrastructural interventions on cycling. This is not surprising, as changes in the built environment are often beyond control of the researcher and therefore difficult to randomize. Other analytical techniques are required to evaluate these so-called "natural experiments", in which variation in accessibility to new cycling infrastructure is used to assign intervention and control groups.
Two recent systematic reviews have been completed which examine the impact of infrastructure on levels of cycling. Both reported that cycling increased following the introduction of new infrastructure, or upgrading of existing infrastructure. However, both reviews also noted that the methods in the included studies may have affected the study findings. Stappers and colleaguesnoted variable quality in study designs across studies examining impacts on physical activity, active transport and sedentary behavior. They suggest that more refined designs may decrease the possibility of detecting intervention effects. Panter and colleaguesfocused only on studies assessing walking and cycling, and examined the evidence for the effectiveness and mechanism of interventions. They found that higher quality studies were more likely to report intervention effects for cycling. Taken together, differences in methods may have impacted the overall conclusion (no changes vs positive changes), or the magnitude of the finding (small changes vs large changes). Ignoring methodological differences may wrongly lead to the conclusion that some interventions were more effective than others.
The current review builds on the main finding of previous reviews that interventions in the built environment may affect cycling. We focused on the methodological approaches undertaken to evaluate the effects of infrastructural interventions. Both reviews did not quantitatively summarize the findings, thereby leaving the question unanswered if the magnitude of the findings changed when using different methodology. One review was unable to capture relevant literature published outside of health-related journals. The research questions are likely to be different between health researchers and transportation researchers, potentially leading to differences in study designs and findings.
Focusing on whether different methodological approaches produce different results, and assessing the strengths and limitations of different methods for causality, will provide greater understanding about the implications of findings from research and their utility for policy makers and practitioners. Therefore, the aims of this systematic review were to summarize the effects of infrastructural interventions on cycling and physical activity in the population, and to evaluate whether the effects varied by study design, data collection methods, or statistical approaches.
# Methods
The protocol of this study was registered in March 2018 at PROSPERO (CRD42018091079). Our systematic literature search followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.
## Search strategy
Various electronic databases (Embase.com, Medline Ovid, Web of Science, PsycINFO Ovid, CINAHL EBS-COhost, Google scholar) were searched for literature published until February 2018 for any studies assessing infrastructural projects to promote cycling. We updated the initial search until June 2019 to additionally include most recent publications. Search terms for the different databases can be found in Additional file 1. Search terms were constructed of 3 parts, including synonyms for cycling infrastructure to identify exposures, synonyms for cycling behavior, active transport, physical activity and lifestyle changes to identify outcomes, and a term that excluded conference abstracts, letter to the editors, notes and editorials. No restrictions were made on language. Database searches were supplemented with searches of reference lists of included studies and key review papers.
## Study selection and inclusion criteria
All titles and abstracts identified during the initial search were screened for inclusion by two independent researchers (FJMM, NB). Additional articles identified through the updated search were screened by a single author (FJMM). After screening titles and abstracts, fulltext articles were screened according to predefined criteria. Articles obtained in full-text were reassessed for inclusion by the first two authors (FJMM, JP), and discrepancies were resolved after discussion with a third researcher (FJvL). Eligibility criteria included: 1) a study evaluating an infrastructural intervention to promote cycling, 2) any measure of cycling as outcome, 3) cycling measured before and after the intervention, and 4) reporting on a general adult population aged 16 years and above. Examples of interventions include the opening of cycling lanes, the installation of a city-wide cycling network, and the improvement of existing cycling infrastructure. We included papers that evaluated the same intervention, but reported on different outcomes or used different datasets or methods to collect outcome data. Controlled and uncontrolled studies were included to allow for a large variety of study designs. Studies were classified as controlled studies if data was collected in a different population that was selected based on comparable individual or neighborhood characteristics, and if similar data collection methods were used. We also classified studies as controlled studies if a comparison was made within the study population between people who lived closer to an intervention and those who lived further away. Studies that presented city-or area-wide cycling trends as a comparison were considered uncontrolled, as the data collection methods used in routine monitoring surveys often differed from that used in the intervention group, and population characteristics often differed between areas.
Studies that evaluated the introduction of cycling infrastructure together with other environmental components were included (i.e. bike parking, showers, rental bikes), as long as the main goal of the intervention was to promote cycling. Environmental interventions that did not change the cycling infrastructure were excluded. We specifically aimed to study population-based approaches to change health behaviors, and therefore excluded infrastructural interventions that were part of a combined intervention with behavioral components targeting the behavior of individuals (i.e. cycling courses, safety lessons, or other approaches that target individual behaviors). Studies that included media campaigns along the intervention were included, as long as they aimed to target the population as a whole.
We excluded opinion articles, qualitative evaluations without quantitative assessment, studies retrospectively collecting data on cycling, and studies not directly linked to an infrastructural intervention. We also excluded studies in which the presented outcome measure was not specified for cycling, like active travel which combined walking and cycling together, or modal shifts where the shift in mode was not specified.
## Data extraction
From the included studies, one researcher extracted data (FJMM) using a standardized data extraction form, and a second reviewer (JP) verified a 20% sample of the extracted data. The extracted data included publication details, description of the intervention, study design, data collection methods, analytical methodology, and study results.
Ideally, we would have extracted a single outcome related to cycling per study. However, most studies did not specify a primary outcome of cycling. Therefore, we extracted all cycling outcomes presented from the maximally adjusted model with the longest exposure time. We extracted all outcomes for various purposes of cycling (e.g. total cycling, recreational cycling, cycling for commuting), and all outcomes for various metrics of cycling (e.g. bike count data, cycling frequency, cycling duration). If the outcome was assessed in multiple populations or at multiple locations, we extracted the average change in cycling that was presented by the authors. If no summary measure was presented, we calculated an unweighted average effect. Some studies stratified the population by exposure status, and evaluated a possible exposure-outcome relationship by distance from home to the intervention or usage of the intervention. All available information was extracted for these studies and included in the descriptive part of the review. However, including all strata-specific outcomes in the quantitative analyses would mean that studies with multiple strata would have a much greater contribution to the findings than studies without stratification. Therefore, we only used the results from the group most likely to use the intervention in the quantitative summary (e.g. smallest distance or largest potential usage). We noted that various metrics were used for expressing data relevant to cycling. We distinguished outcomes that evaluated cycling behavior (e.g. making a bike trip, cycling frequency, cycling duration) from those that evaluated usage of cycling infrastructure (e.g. bikes counted in the city, bikes counted on the new infrastructure). We extracted data on both absolute change (no fixed unit, can refer to various metrics) and relative change (expressed as percentage change over time) in cycling between before and after measurements, and attempted to calculate outcomes for both where possible. We used a similar framework presented by Goodmanto compute measures of absolute and relative change. Outcomes expressed as ratios were interpreted as relative changes. For uncontrolled studies, the relative change was computed by dividing the absolute change by the baseline level of cycling in the study sample. For controlled studies, we first computed the relative change in the intervention and control group separately. Subsequently, the calculated relative change in the intervention group was divided by the calculated relative change in the control group. Likewise, to obtain an absolute change when only relative changes were presented, we multiplied the relative change by the baseline estimate in the study sample as a whole for uncontrolled studies, and by the baseline estimate in the control group for controlled studies. Examples of the data extracted and how outcomes were calculated are presented in Additional file 2. Authors were contacted if only the direction of the association was presented. For each study we extracted data on statistical tests performed, and if significant results were found (P < 0.05). However, we focused on directions of the association rather than significance, since a substantive part of the studies did not test for significant changes in cycling outcomes that were of interest for this review.
We extracted data on the methodological quality, and on all design elements and additional analyses that may have supported causal inference following previous methodologies. The quality items described by Ogilvie et al.were extracted, which used the criteria from the Community Guide of the US Task Force on Community Preventive Services to assess study design, and criteria developed for the Effective Public Health Practice Project in Hamilton, Ontario to score five items related to the quality of the research performed. The five items included representativeness, comparability, credibility of data collection instruments, retention, and attributability of the effect to the intervention. The original instrument also assessed randomization, but this was not assessed as the allocation to the intervention and comparison group was not under control of the researcher. In addition, we extracted the results from additional analyses that may support causal inference identified by the UK Medical Research Council guidance on natural experiments, including multiple comparison groups, the inclusion of a neutral outcome that is not expected to change as a consequence of the new cycling infrastructure, and the use of complementing research methodologies.
The PROGRESS-Plus framework was used to describe the impact of the infrastructural interventions on subgroups of the population. The PROGRESS-Plus framework considers nine factors for which differences in effect may occur: 1) place of residence, 2) race, ethnicity, culture, language, 3) occupation, 4) gender, sex, 5) religion, 6) education, 7) socioeconomic status, 8) social capital, and 9) the 'Plus'-factor that could be other characteristics associated with social disadvantage. In our study we considered age, health status or BMI, bike ownership, and car ownership as Plus-factors, since these factors may have been relevant determinants of disadvantage given the context of the intervention.
## Data synthesis
We provided a descriptive narrative synthesis of studies. There was no possibility to quantitatively summarize the results, because of the large variety of outcome metrics and purposes of cycling presented, the lack of a primary outcome, and the lack of a common outcome across studies. Therefore, we presented the median relative change for the umbrella-termscycling behavior and infrastructure usage for all studies, and by study design (controlled vs uncontrolled; exposure time ≥ 1 year vs < 1 year), data collection methods (objective vs subjective), and analytical approaches (tested vs not tested). We did not present units for the median relative change because it can refer to various metrics. For example, an increase in cycling behavior of 30% could refer to an increase in the proportion of cyclists, cycling frequency, or cycling duration. An overview of studies with baseline characteristics or performed adjusted analyses by any of the PROGRESS-Plus factors was presented. We provided a descriptive narrative synthesis for the studies that formally tested for differential effects on PROGRESS-Plus factors.
# Results
## Study characteristics
From the 3542 potential records, 125 full-text articles were screened and this resulted in 31 studies (29 interventions) from 11 countries that met the eligibility criteria. The major reason for exclusion of full-text articles is presented in Additional file 3.presents the characteristics of included studies categorized by the outcome of interest. Twenty studies presented data on cycling behavior, and 16 studies assessed usage of the cycling infrastructure. All infrastructural interventions were conducted in urban areas in high-income countries. The interventions were very diverse in terms of design and scale, ranging from the introduction of a cycling bridge, single or multiple cycle paths or lanes, or a city-wide cycling network. Six studies (5 interventions) described issues related to data collection due to delays in the construction work, resulting in shorter follow-up periods than planned. In addition to this, three studies (2 interventions) mentioned that the intervention was not fully completed within the study time frame. Most studies used a similar analytical approach by comparing a single estimate before the intervention with a single estimate after the intervention, with or without comparing it to changes in a control group. One study used a fixed-effects approach to evaluate the withinperson change over time, and three studies tested if there was a significant interaction between the intervention and time. One study conducted an interrupted time series analyses, whereby the date of the opening of the cycling track was used to set the time of interruption. exposure time, method of assessment, and whether significance was tested. In general, studies reporting behavioral outcomes found smaller changes than studies presenting usage of the infrastructure. Larger changes were also found for studies that tested for statistical significance and studies that used subjective measurement methods (such as surveys and direct observations of cyclists), compared to studies that did not perform statistical tests, and used objective measurement methods (such as GPS and accelerometers, and automatic counting stations). Additional file 4:provides further details of the number of studies which assessed cycling behavior or usage of the infrastructure for cycling, and whether these were in favor of the intervention or not. Twenty studies presented data on 52 cycling behavior outcomes. All but two, found an increase in cycling for at least 1 outcome, and 73% (38/52) of all outcomes presented were in favor of the intervention. A total of 36 cycling behavior outcomes were used to quantitatively summarize the results. Together, studies found a median relative increase in cycling behavior (median relative change: 23%; range: − 21 to 262%). Changes in cycling did not essentially differ between controlled and uncontrolled studies. Studies with an exposure time shorter than 1 year found smaller changes when compared to those using a longer exposure time. Studies that used objective measures to assess cycling behavior found smaller changes than those that used self-reported measures, and studies that did not test for statistical significance found smaller changes than those that did.
# Study results
Seven studies evaluated changes in physical activity patterns following cycling infrastructure interventions. Brown et al. showed that among cyclists, cycling time on intervention streets increased by 7 min/week and on other streets increased by 6 min/week. Daily energy expenditure increased in the study population by 0.19 kcal/ min, which translates into 275 kcal/day. Goodman et al. found that living 1 km closer to the intervention increased cycling for recreation by 3 min/week, and total physical activity by 13 min/week. There was no evidence that compensation of physical activity behaviors took place, since physical activity excluding walking and cycling was not associated with the intervention. Burbidge et al. did not find changes in total physical activity time, but the number physical activity episodes seemed to have declined by 0.2 trips/day following the introduction of cycling infrastructure. The other four studies did not find evidence that the introduction of cycling infrastructure affected physical activity.
Usage of the infrastructure was presented in 16 studies with 21 outcomes, and all were in favor of the intervention (median relative change: 62%; range: 4 to 438%). Changes for infrastructure usage were smaller for studies that were uncontrolled, studies with longer exposure time, studies using automatic counters or GPS tracking information, and studies that did not test for statistical significance (Additional file 4:. None of the studies was a randomized experiment, therefore randomization was not applicable for any of the studies and was not shown.
b
None of the studies presented data for neutral outcomes that were hypothesized to be unaffected by the new infrastructure designed to promote cycling, therefore this parameter was not shown. c A = controlled before-after study; B = uncontrolled study with at least two before and two after data points; C = uncontrolled study with only 1 before and after data point Quality assessmentpresents information on the quality of the studies. Nine out of twenty studies evaluating the impact of cycling infrastructure on cycling behavior presented data on participation, and nine on representativeness. Participation ranged between 2 and 49% for those that presented information. Thirteen studies collected data twice on the same individual, and retention ranged between 41 and 79%. Most studies used surveys to collect data, but the exact methodology and validity of the question items was often not reported.
When considering the quality of the studies for causal inference, studies reported that other changes in the physical and social environment might have affected or biased their results. Issues reported were the economic crisis, the rising cost of car transport, social marketing campaigns, and other infrastructural improvements during the same period. Authors were often unable to account for these and this could indicate that the changes observed could be partly attributable to other factors. Another problem mentioned is a spill-over effect, indicating that people from control areas might have used the facilities, which may have resulted in an underestimation of the effect. Some studies used multiple groups to test robustness of the findings by using different comparisons group or applying different cut-off values to define exposure or outcome. Some studies presented data for city-or nation-wide cycling trends, or historical time trends. None of the studies included a neutral outcome which was hypothesized to be unaffected by the new infrastructure designed to promote cycling, thereby functioning as a control measure that captures time trends in transportation or physical activity behaviors. Complementing methodologies performed were surveys among residents or employees, intercept surveys among infrastructure users, surveys among new residents who moved into the study area, and bike counts in the study area.
Sixteen studies presented data on usage of the infrastructure. Five studies used automatic counting stations or mobile app data to objectively measure cyclist movements for periods between 5 months and 3 years. Others monitored the number of cyclist on selected hours and days using observation techniques. Issues that authors reported that may have partly contributed to the increase in infrastructure usage were tunneling of existing riders to the new infrastructure, other infrastructural changes, traffic conditions, rising cost of car transport, weather conditions and seasonality, demographic changes, social marketing, and changing methodology to collect data. One study indicated that improvements made to the cycling infrastructure could have been a consequence of high cycling levels in specific areas.
Some studies presented data for city-or nation-wide cycling trends, or historical time trends. Additional methodologies included surveys among residentsor employees, survey among infrastructure users, and data collected on cycling behavior.shows that studies assessing cycling behavior collected information on population characteristics more often than those assessing usage, thereby potentially providing insights in the population under study and characteristics of those engaging in cycling, and allowing a comparison of intervention and control groups according to baseline characteristics. The items that were most often used by behavioral studies to describe the population at baseline were age (75%), gender (70%) and a measure of socio-economic status (SES) (50%). Only three studies tested for differential effects on cycling by population subgroups. Aldred et al. did not find any differential effects by demographic and socio-economic characteristics. showed that the change in cycling behavior was larger if there was no car in the household. showed that the increase in cyclists was larger among females than males.
## Equity effects
# Discussion
We identified 31 studies that assessed the effect of infrastructural interventions on cycling in adult populations. All were conducted in urban areas in high-income countries. Most of the evaluations found effects in favor of the intervention, showing that the number of cyclists using the facilities increased, and to a lesser extent that cycling behavior increased. Studies that collected behavioral data more often provided insights in characteristics of people engaging in cycling as compared to studies that reported bike counts. Seven studies reported on physical activity levels, and findings were mixed. Only three studies tested for equity effects, therefore we cannot draw any conclusions as to whether some population subgroups benefitted more than others. We provided data on relative changes that indicates the magnitude of the findings. We acknowledge that in context where only few people use a bike, large relative changes may result in only small population-health benefits. However, due to the large variety in outcomes used we could not further summarize the results.
Our findings suggest that the approach and the specific methods did provide different results. Previous reviews have indicated that this might be the case, but our synthesis of studies exclusively focusing on cycling according to the method used, provides more evidence of this. This review built on earlier findings by including studies with various study designs and published in health-related and transportation-related journals. Furthermore, we quantitatively summarized the findings to assess whether the magnitude of the change in cycling differed across study design. In the following three sections we describe the implications of the study design, data collection methods and statistical approaches for the study findings.
## Study design and implications for causal inference
An important aspect of study design is the choice of outcome. In this review we categorized outcomes broadly into those that assessed cycling behavior and infrastructure usage. We found that studies on behavioral outcomes found smaller relative changes than studies presenting usage of the infrastructure. If researchers are interested in outcomes relevant for population health, it is recommended that outcomes are framed around the duration and frequency of cycling, as these measures can be directly linked to health impacts. Assessing the proportion of cyclists in a population or the numbers using a route may be a good alternative. If researchers are interested in understanding usage, count data may be used to measure the number of cyclists on the new infrastructure. Other reviews also found that studies measuring outcomes more closely related to the intervention (for example: cycling) were more likely to find intervention effects than studies measuring more general outcomes (for example: physical activity or BMI). Bike count data may support the findings from other evaluations on cycling behavior, but it cannot directly be translated into health gains in the population. Another important design element is whether to include a control population when evaluating built environment changes. The changes in cycling differed for controlled and uncontrolled studies that assessed usage of the infrastructure, but not for cycling behavior. Uncontrolled studies have a stronger basis for causal inference if they can provide evidence that the observed effects do not solely reflect underlying time trends in cycling in the wider area. For example, Cranecounted the number of bikes passing 2 locations along the new infrastructure. They also presented city-wide cycling trends during the same time period. An increase of 3.7% of cyclist was found along the intervention road, whereas a decrease of 2.0% was seen in the city as a whole. This finding suggests that the number of cyclist increased in the area with the new infrastructure, and this increase does not solely reflect underlying time trends in cycling. To strengthen causal inference, we recommend that studies use controlled designs where possible, and present different measures of cycling and physical activity. Evaluating similar interventions across different sites could give further insights in the variation in the change in these sites if controlled designs are not possible. For example, Lanzendorfevaluated improvements made to the cycling infrastructure in 4 German cities. Cycling frequency on average increased by 27%, which differed between cities from 3 to 38%. They also reported an average increase of cycling frequency by 31% in all big German cities. This approach illustrates that the observed changes in cycling in the intervention sites were comparable to the countrywide increase in cycling. The large range in changes in cycling in the 4 intervention sites also gives insight into the potential range of effects which could be expected in other cities.
The duration of time that populations are exposed to the new infrastructure is another important design element, which can be difficult to control in large infrastructural projects. In studies that assessed changes in cycling behavior we found that the changes were larger when exposure time was longer than 1 year. In studies that assessed the usage of cycling infrastructure, those with shorter exposure time reported larger changes than those with longer exposure time. We noted that some count studies did not count on rainy days, or only collected data during peak hours, which may have resulted in larger changes than what could be expected if data was measured throughout by means of automatic counters. Most studies that found changes that were not in favor of the intervention were less than 6 months exposed, suggesting that longer follow-up periods may be needed to allow behavioral changes to be detected. Including questions on infrastructure usage within ongoing surveys, or nested within cohorts, may ensure that if the construction work is delayed, there is data available with sufficient exposure time to measure the impact.
## Data collection methods and implications for causal inference
Studies were categorized according to whether the focus was on usage or cycling behavior, and large differences in results were found between these two types of outcome. Studies presenting count data of infrastructure found larger changes than studies that assessed behavioral change in the population. Studies counting the number of bikes that passed tracking locations are at risk of assessing the displacement of existing riders to the new infrastructure, and seven studies specifically mentioned this phenomena. Some studies had offset some of the so-called funneling biases by selecting strategic counting locations where most cyclist pass, or used multiple counting locations to capture cycling behavior in a wider area. Some studies complemented bike count data with intercept surveys among users of the infrastructure, and asked about their previous travel behaviors. These studies showed that the proportion of users that would not have cycled, had the infrastructural improvement not taken place, was much smaller than the increase in counts of cyclists. Bike count data is useful when aiming to describe at what times of the day, and under which weather conditions, cyclists are using the facility.
Another important consideration is choosing between objective or self-reported measures to collect data on cycling behavior. We found that studies using GPS and other objective measures of cycling reported smaller changes than those using self-reported measures. Using GPS and objective assessments of activity could potentially be used to distinguish cycling on and off the new infrastructure, and yields estimates of total physical activity levels. However, such measures are often applied to a small sample, are limited to a short period of time, and participants who wear such devices might be quite different to the general population. Therefore the findings might be subject to some selection biases. Furthermore, it is possible that the novelty of wearing such devices might lead to changes in physical activity behaviors. Subjective measures of cycling behaviors, such as travel diaries and surveys, provide alternatives when interested in larger groups of people, but many of these have not been validated for cycling specifically.
It is attractive to use already available data when studying so-called "natural experiments" in which researchers lack control over the intervention. Collecting new data to match the timescale of intervention delivery is challenging. A third of the studies evaluating cycling behavior used data that were already collected for a regular monitoring or as part of other studies for the evaluation of other built environment interventions . For example, four US studies used census data to estimate changes in cycling after the introduction of new cycling facilities. Other evaluations of natural experiments were planned, allowing to collect specific data to evaluate the intervention of interest in detail. This resulted in powerful analyses in which the method of data collection was tailored to the research questions, but sometimes resulted in limited time being exposed to the intervention. For example, Dillassessed cycling at baseline and after 2years of follow-up. The construction work was significantly delayed, resulting in a short time period between the opening of the facilities and the second assessment of cycling. Moreover, two of the nine projects were not completed within this period. This may have influenced study outcomes. Using existing data may be useful if researchers were not aware of the new intervention, did not obtain funding in time to design a study around the natural experiment, or if large delays in the construction are expected.
## Analytical approaches and implications for causal inference
Like other reviews, we found that many studies did not perform statistical tests (for cycling behavior: 15% (8/52) and for usage: 67% (14/21)). Smaller changes were found for studies that did not test for statistical significance than those that performed statistical tests. We recommend that studies test for statistical significance which provides more robust evidence that the results are not due to chance, as recommended by guidance for the clear reporting of observational studies. This review included some studies that used more complex analytical methods, such as fixed-effect models, interrupted time series, or estimated the difference in cycling over time by using a regression analyses that included group, period, and an interaction term between group and period. Fixed-effects models allow to account for observed time-varying and unobserved timeinvariant characteristics. Perhaps most prominently, individual attitudes towards physical activity may both determine living at a place with opportunities to be physically active and their physical activity behavior. Fixed-effect models allow to control for such unobserved time-invariant confounding, allowing for better causal inference. One study conducted a time series analyses by using GPS tracking information from a mobile phone application, thereby correcting for time trends prior to the intervention. Studies that specified an interaction term between group and period are able to control for observed differences between groups, thereby reducing the risk of bias. The usage of multiple analytical strategies, and the usage of methods that are able to correct for time trends, and measured or unmeasured confounders at the individual or neighborhood level may strengthening the basis of causal inference.
# Strengths and limitations
In this review, we focused on the methodological aspects in the evaluation of infrastructural interventions to promote cycling and extracted information on the magnitude of the change in cycling. This allowed us to examine differences in change in cycling according to the methods used. This study was comprehensive by searching multiple electronic databases without date or language restrictions, and we included studies published in public health journals and transportation journals. Controlled and uncontrolled studies were considered for inclusion, and the final selection of studies had a large variety in study designs and methods. We added valuable information by calculating the relative and absolute changes in cycling behavior or usage of the infrastructure, which brought together different outcomes in a simple but interpretable way.
Some limitations also have to be noted. We included only studies that reported on measures of cycling and were unable to examine unreported data on cycling that were included in composite measures of active transportation, walking and cycling, or physical activity. The detail of the information provided in the papers differed between studies, which made it difficult to synthesise and interpret study findings. A pragmatic approach was used to calculate relative changes where possible, but for some studies other approaches may have been better. The evidence presented in the review came from studies that were all conducted in high-income countries. Moreover, only a few studies evaluated the impact on physical activity behaviors and studied equity effects. We focused on structural interventions here, but future research should explore the importance of and interactions with other interventions, such as financial incentives, cycle training, or behavioral interventions, together with the introduction and maintenance of high-quality cycling infrastructure.
## Recommendations
Each study design, data collection method and analytical strategy has its advantages and disadvantages. To further strengthen causal inference from observational data, studies are needed that triangulate different methodologies to evaluate the effect of built environment interventions. Studies published in public health journals often report on changes in cycling behavior, while studies published in transportation journals report on usage of cycling infrastructure. Bringing experts from both fields together could result in study designs that better capture the range of impacts of new cycling infrastructure. We are not recommending a specific method or approach, as the research questions of interest should drive the method of data collection. When existing data are used, careful consideration needs to be given to the appropriateness of that data. The reporting of evaluations should adhere to guidelines, such as STROBE which seeks to strengthen the quality of work reported. We suggest, where possible, to combine count data that provides information on how many people are using new infrastructure, with behavioral outcomes of duration and frequency of cycling to ensure estimates of the population health impact. Such estimates could be used in combination with modelling or scenario building tools to estimate the current or future health impacts on outcomes that cannot be observed in studies with limited follow-up. Future studies should focus on the question who are benefiting from the intervention, and identify contexts, barriers and choice constraints to better understanding why cycling changed. This review focused on interventions that changed the cycling infrastructure, but findings and recommendations are likely applicable to other built environment interventions to promote health behaviors.
# Conclusion
Introducing cycling facilities in cities is likely to increase the number of cyclist using the facilities, and may result in increases in cycling. Evidence on total physical activity following cycling facilities was mixed. Equity effects were rarely studied. Research questions interest should drive the method of data collection and reporting of evaluations should adhere to published guidelines. Triangulation of methods is warranted to overcome potential issues that evaluators may encounter when evaluating infrastructural interventions within the built environment, and to strengthen the basis of causal inference.
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Real-Time Measurement of Drilling Fluid Rheological Properties: A Review
# Introduction
In the drilling industry, almost every step requires drilling fluid. The drilling fluid's properties have a significant impact on drilling efficiency and safety. The successful completion and cost of an oil well depend mainly on the drilling fluid's performance. The cost of the drilling fluid itself is relatively small, but the choice of the right drilling fluid program and maintenance of fluid properties while drilling profoundly influence the total well costs. The cost of drilling fluid accounts for 5% to 15% of the entire drilling cost, but it can solve 100% of drilling problems. The physical and chemical properties of drilling fluid, such as its density and rheological properties, have a significant impact on the processing and control of well conditions. A high-viscosity drilling fluid is desirable to transport cuttings from downhole up to the surface and suspend weighting agents (such as barite). However, if the viscosity is too high, the friction is high, which may hinder the circulation of the mud, resulting in excessive pump pressure, reducing the drilling speed and hindering the solids removal equipment.
The drilling fluid properties play an important role in the optimization of the rate of penetration. In the rate of penetration models established by many scholars, such
## Rheological properties of drilling fluid
The drilling fluid's rheological properties refer to the characteristics of flow and deformation under the action of external force. The drilling fluid's rheological properties are essential for the following determinations: estimation of hole cleaning efficiency, calculation of frictional pressure losses in pipes and annuli, determination of equivalent circulating density (ECD) under downhole conditions, determination of prevailing flow regime in pipes and annuli, estimation of swab and surge pressures, and hydraulic optimization for improved drilling efficiency. Proper rheological properties can carry bottom hole cuttings to the ground quickly, increase the rate of penetration, reduce power consumption, ensure drilling safety, and improve economic benefits. In order to estimate the above-mentioned functions of the drilling fluid in time, it is important to measure the drilling fluid rheological properties in real time.depicts how fluids can be classified based on their rheological behavior. There are Newtonian and non-Newtonian fluids. The viscosity of a Newtonian fluid does not change under different shear rates, while the viscosity of a non-Newtonian fluid changes under different shear rates (e.g., water, salt solutions, and light oil). The behavior of non-Newtonian fluid mainly includes three types: dilatant behavior (e.g., water-based polyethylene glycol drilling fluid, pseudoplastic behavior (e.g., drilling fluid with high clay content, high waxy crude oil, and paint), and plastic behavior (e.g., aqueous solutions and emulsions of polymer compounds). The viscosity of the dilatant fluid increases with the increase in the shear rate, while the viscosity of the pseudoplastic fluid and the plastic fluid decreases with the increase in the shear rate. Plastic fluid starts to flow after a given shear stress is applied, while pseudoplastic fluid can flow under any shear force. Drilling fluids rarely exhibit dilatant behavior, as most of the drilling fluids used are plastic and pseudoplastic fluids. Through the rheological curve of the drilling fluid, we can more accurately evaluate the ECD and the ability of carry cuttings, optimize the hydraulic parameters, etc. The most common models used to describe the drilling fluid rheology in the petroleum industry are the Bingham plastic model (Equation (1)), Power law (PL) model (Equation (2)), and Herschel-Bulkley model (Equation (3)):
= YP + PV * (1)
[formula] = (2) = +(3) [/formula]
where YP is yield point, PV is the plastic viscosity, τ0 is the fluid yield stress, K is the consistency factor, n is the flow behavior index, and γ is the shear rate. The total ability for a Bingham plastic fluid to resist flow could be expressed by an apparent viscosity (AV) or effective viscosity for a given shear stress. Generally, the shear rate of apparent viscosity is 1021 s −1 in API.
[formula] AV = = PV + YP(4) [/formula]
The Herschel-Bulkley model is used when the accuracy of the rheological parameter measurement is high or in laboratory research. The API recommends using this model; it consistently provides good simulations of measured rheological data for both waterbased and non-aqueous drilling fluids. For this reason, it has become the de facto rheological model for engineering calculations in the petroleum industry.
## Real-time measurement technologies
The current measurement of rheological properties utilizes a typical manually controlled rotational Couette viscometer. In recent years, many scholars have researched realtime measurement of drilling fluid rheological properties. The real-time measurement technology of drilling fluid rheology mainly includes the following four methods: (1) online rotational Couette viscometer, (2) pipe viscometer, (3) mathematical and physical model or artificial intelligence (AI) model based on a Marsh funnel, and (4) tuning fork technology. The most common models used to describe the drilling fluid rheology in the petroleum industry are the Bingham plastic model (Equation (1)), Power law (PL) model (Equation (2)), and Herschel-Bulkley model (Equation (3)):
[formula] τ = YP + PV * γ (1) τ = Kγ n (2) τ = τ 0 + Kγ n(3) [/formula]
where YP is yield point, PV is the plastic viscosity, τ 0 is the fluid yield stress, K is the consistency factor, n is the flow behavior index, and γ is the shear rate. The total ability for a Bingham plastic fluid to resist flow could be expressed by an apparent viscosity (AV) or effective viscosity for a given shear stress. Generally, the shear rate of apparent viscosity is 1021 s −1 in API.
[formula] AV = τ γ = PV + YP γ(4) [/formula]
The Herschel-Bulkley model is used when the accuracy of the rheological parameter measurement is high or in laboratory research. The API recommends using this model; it consistently provides good simulations of measured rheological data for both water-based and non-aqueous drilling fluids. For this reason, it has become the de facto rheological model for engineering calculations in the petroleum industry.
## Real-time measurement technologies
The current measurement of rheological properties utilizes a typical manually controlled rotational Couette viscometer. In recent years, many scholars have researched real-time measurement of drilling fluid rheological properties. The real-time measurement technology of drilling fluid rheology mainly includes the following four methods: (1) online rotational Couette viscometer, (2) pipe viscometer, (3) mathematical and physical model or artificial intelligence (AI) model based on a Marsh funnel, and (4) tuning fork technology.
## Online rotational couette viscometer
The recommended methods for drilling fluid analysis are presented in API 13B. The drilling fluid's rheological properties are measured using a standard rotational Couette viscometer as shown in. The drilling fluid is placed in the measurement chamber, the motor drives the rotor sleeve to rotate at a constant speed through the transmission device. The viscosity of the measured liquid acts on the bob to generate a certain torque that drives the torsion spring, which is connected to the bob to produce an angle (dial reading). The dial reading is proportional to the viscosity of the fluid; thus, the viscosity of the fluid is calculated by the measured value of the dial reading. The standard rotational
The recommended methods for drilling fluid analysis are presented in API 13B. The drilling fluid's rheological properties are measured using a standard rotational Couette viscometer as shown in. The drilling fluid is placed in the measurement chamber, the motor drives the rotor sleeve to rotate at a constant speed through the transmission device. The viscosity of the measured liquid acts on the bob to generate a certain torque that drives the torsion spring, which is connected to the bob to produce an angle (dial reading). The dial reading is proportional to the viscosity of the fluid; thus, the viscosity of the fluid is calculated by the measured value of the dial reading. The standard rotational Couette viscometer has six rotation speeds: 3, 6, 100, 200, 300, and 600 r/min, related to shear rate:The parameters of the Bingham model (PV, YP, and AV), PL model (n and K), Herschel-Bulkley model (τ0, n, and K), and 10 s and 10 min gel strength can be obtained using a standard rotational Couette viscometer. Many scholars developed an online rotational Couette viscometer based on a standard rotational Couette viscometer to measure the drilling fluid's rheological properties. The main improvement method is to add a control circuit to control the speed of the motor, turn the reading into an electrical signal output, and add the drilling fluid pipeline to automatically fill the measurement chamber with the drilling fluid.shows a typical online rotational Couette viscometer schematic diagram. The parameters of the Bingham model (PV, YP, and AV), PL model (n and K), Herschel-Bulkley model (τ 0 , n, and K), and 10 s and 10 min gel strength can be obtained using a standard rotational Couette viscometer. Many scholars developed an online rotational Couette viscometer based on a standard rotational Couette viscometer to measure the drilling fluid's rheological properties. The main improvement method is to add a control circuit to control the speed of the motor, turn the reading into an electrical signal output, and add the drilling fluid pipeline to automatically fill the measurement chamber with the drilling fluid.shows a typical online rotational Couette viscometer schematic diagram.
The recommended methods for drilling fluid analysis are presented in API 13B. The drilling fluid's rheological properties are measured using a standard rotational Couette viscometer as shown in. The drilling fluid is placed in the measurement chamber, the motor drives the rotor sleeve to rotate at a constant speed through the transmission device. The viscosity of the measured liquid acts on the bob to generate a certain torque that drives the torsion spring, which is connected to the bob to produce an angle (dial reading). The dial reading is proportional to the viscosity of the fluid; thus, the viscosity of the fluid is calculated by the measured value of the dial reading. The standard rotational Couette viscometer has six rotation speeds: 3, 6, 100, 200, 300, and 600 r/min, related to shear rate:The parameters of the Bingham model (PV, YP, and AV), PL model (n and K), Herschel-Bulkley model (τ0, n, and K), and 10 s and 10 min gel strength can be obtained using a standard rotational Couette viscometer. Many scholars developed an online rotational Couette viscometer based on a standard rotational Couette viscometer to measure the drilling fluid's rheological properties. The main improvement method is to add a control circuit to control the speed of the motor, turn the reading into an electrical signal output, and add the drilling fluid pipeline to automatically fill the measurement chamber with the drilling fluid.shows a typical online rotational Couette viscometer schematic diagram. Saasen et al.developed an online rotational Couette viscometer. The control box is used to change the rotational speed; shear stress readings were collected and transmuted to the acquisition device. In order to calibrate the drilling fluid's temperature response over a suitable temperature range, a temperature sensor was attached to the measuring unit. The measurements of the online rotational Couette viscometer were compared to and corresponded well with the standard rotational Couette viscometer. Broussard et al.also developed an online rotational Couette viscometer and conducted experiments in water-based and oil-based mud wells. It demonstrated the possibilities of automated drilling fluid measurements by using an online rotational Couette viscometer. However, there were some shortcomings such as that the viscometer was easily plugged up by gels particles and solids. No data were recorded during the brief testing of the waterbased fluid at the first well due to the revealed heavy buildup of gel particles within the unit. During the first oil-based mud well, the automated sensor, equivalent to 600 rpm, measurements were consistently higher than the standard rotational Couette viscometer measurements. The 6 and 3 rpm equivalent measurements taken by the online viscometer trended very well when compared to the standard viscometer. For the second well, the online viscometer measurements for the water-based mud were significantly lower than the corresponding standard viscometer in the measurements below 300 rpm. Rheological property measurements taken by the online viscometer had an increased amount of noise in the measurements due to the significant accumulation of solids on the equipment. In order to avoid solids accumulation, Stock et al.and Ronaes et al.used a built-in pump and valve system for fluid sampling and cleaning cycles.
Magalhães et al.used a rheology measurement instrument obtained by a modifying a Brookfield process viscometer, TT-100. The operational condition limits of the device were 1 to 15 bar (14.7 to 220.5 psi) of total pressure, temperature up to 160 - C (256 - F), and volumetric flow rate between 1 and 3 m 3 /h. There were four types of drilling fluid tested, namely, Newtonian fluid, pseudoplastic fluid, water-based drilling fluid, and synthetic-based drilling fluid. The measurement results showed that Newtonian fluid, represented by glycerin, and pseudoplastic fluid, represented by CMC solution, were statically consistent in terms of curve fitting. The results of the water-based mud and non-aqueous drilling fluid found some divergences. Compared with the standard viscometer measurement, the TT-100 tended to underestimate the shear stress in waterbased mud, while in the non-aqueous drilling fluid, TT-100 overestimated the shear stress. The limitation of the viscometer was the size of the solids suspended. This article points out that the maximum diameter of the solids in the drilling fluid tested by the instrument must be less than 1 mm. Magalhães et al.installed it on an onshore rig site near the northeast of Brazil, and operated it for several weeks. The proposed device and methodology for measuring online rheology produced similar results to the standard offline technique.
Dotson et al.used a different rotor-bob geometry Couette viscometer from the standard rational Couette viscometer. The relevant non-Newtonian correction factor was applied to agree with the standard rational Couette viscometer reading. Rheological property measurements were performed by batch analysis of the fluid every 10 to 60 min. Once the previous sample was fully displaced, the inlet and outlet valves on the respective flowlines were closed. The sample was then pressurized to 0.55 to 0.69 MPa to collapse large air/bubbles in the fluid and to help ensure the viscometer was filled. Then, the fluid was agitated and heated to a user-defined temperature, typically 120 - F (48.9 - C) or 150 - F (65.5 - C). After each rheology measurement, the measurement chamber was cleaned before the next fluid sample entered the viscometer, and the process was repeated. The online viscometer measurements were in agreement with those from the API viscometers. The rheology data from the two instruments were well within the desired tolerance of ±1.5 dial readings at all rotational speeds investigated. This technology is combined with a density meter to form a density rheological unit (DRU). The DRU helps reduce nonproductive time and manage pressure drilling. However, the conventional mud tests still must be performed and recorded. This can verify the accuracy of the DRU measurement and provide redundant measurements in the event of a DRU failure.
## Pipe viscometer
Because the online rotational Couette viscometer is easily blocked, Vajargah, Magalhães, Sercan Gul, Knut Taugbøl, Hansen, Krogsaeter, and Frøylandused pipe viscometers to test drilling fluid rheological properties. According to Ahmed and Miska, the reliability and accuracy of pipe viscometers often outweigh rotational viscometers.shows a schematic example of the pipe viscometer. The test equipment requires a variable pump, a flow meter, mud with a known density, and a differential pressure sensor to measure the pressure difference in the test section of the straight pipe. Because screw pumps have no pressure pulsation and Coriolis flow meters can measure density and flow rate, pipe viscometers usually use a screw pump and a Coriolis flow meter. Pipe viscometer can measure drilling fluid rheological properties under laminar, transitional, and turbulent flow conditions. The data in the laminar flow state is calculated to characterize the rheological constant of the non-Newtonian fluid. The data obtained in transitional and turbulent conditions can be used to calculate the critical Reynolds number and friction factor in real time.shows the velocity profile in pipe laminar flow. The drilling fluid's rheological properties can be obtained from the follow equations.
## Pipe viscometer
Because the online rotational Couette viscometer is easily blocked, Vajargah, Magalhães, Sercan Gul, Knut Taugbøl, Hansen, Krogsaeter, and Frøylandused pipe viscometers to test drilling fluid rheological properties. According to Ahmed and Miska, the reliability and accuracy of pipe viscometers often outweigh rotational viscometers.shows a schematic example of the pipe viscometer. The test equipment requires a variable pump, a flow meter, mud with a known density, and a differential pressure sensor to measure the pressure difference in the test section of the straight pipe. Because screw pumps have no pressure pulsation and Coriolis flow meters can measure density and flow rate, pipe viscometers usually use a screw pump and a Coriolis flow meter. Pipe viscometer can measure drilling fluid rheological properties under laminar, transitional, and turbulent flow conditions. The data in the laminar flow state is calculated to characterize the rheological constant of the non-Newtonian fluid. The data obtained in transitional and turbulent conditions can be used to calculate the critical Reynolds number and friction factor in real time.shows the velocity profile in pipe laminar flow. The drilling fluid's rheological properties can be obtained from the follow equations. In laminar flow, wall shear stress can be obtained when the differential pressure across the measurement section is known:
[formula] = 4 Δ Δ (5) [/formula]
where τw is the wall shear stress (Pa), D is pipe inner diameter (m), ΔP is the friction pressure loss (Pa), and ΔL is the pipe length of the test section (m). It can be shown that for pipes, the shear rate at the wall can be obtained from:
[formula] = 1 4 3 + ( 8 ) ( ) 8(6) [/formula]
where is the shear rate (1/s) and v is the velocity (m/s). Introducing the generalized flow behavior index, N, as: weigh rotational viscometers.shows a schematic example of the pip The test equipment requires a variable pump, a flow meter, mud with a kn and a differential pressure sensor to measure the pressure difference in the the straight pipe. Because screw pumps have no pressure pulsation and Cor ters can measure density and flow rate, pipe viscometers usually use a screw Coriolis flow meter. Pipe viscometer can measure drilling fluid rheological der laminar, transitional, and turbulent flow conditions. The data in the lam is calculated to characterize the rheological constant of the non-Newtonian f obtained in transitional and turbulent conditions can be used to calculate the olds number and friction factor in real time.shows the velocity p laminar flow. The drilling fluid's rheological properties can be obtained fr equations. In laminar flow, wall shear stress can be obtained when the differe across the measurement section is known:
[formula] = 4 Δ Δ [/formula]
where τw is the wall shear stress (Pa), D is pipe inner diameter (m), ΔP is the sure loss (Pa), and ΔL is the pipe length of the test section (m). It can be shown that for pipes, the shear rate at the wall can be obtaine
[formula] = 1 4 3 + ( 8 ) ( ) 8 [/formula]
where is the shear rate (1/s) and v is the velocity (m/s). Introducing the generalized flow behavior index, N, as: In laminar flow, wall shear stress can be obtained when the differential pressure across the measurement section is known:
[formula] τ w = D 4 ∆P ∆L (5) [/formula]
where τ w is the wall shear stress (Pa), D is pipe inner diameter (m), ∆P is the friction pressure loss (Pa), and ∆L is the pipe length of the test section (m). It can be shown that for pipes, the shear rate at the wall can be obtained from:
[formula] . γ w = 1 4 3 + d ln 8v D d(τ w ) 8v D(6) [/formula]
where . γ w is the shear rate (1/s) and v is the velocity (m/s). Introducing the generalized flow behavior index, N, as: Then Equation (7) is now rewritten as:
[formula] N = d(lnτ w ) d ln 8v D(7) [/formula]
.
[formula] γ w = 3N + 1 4N 8v D (8) [/formula]
According to Equation (7), the slope of the "flow curve" (ln τ w vs. ln(8v/D)) represents the generalized flow behavior index, N. Once N is obtained from the flow curve, the shear rate at the wall can be calculated by using Equation. Subsequently, rheological parameters for any desired rheological model can be obtained by plotting the shear stress vs. shear rate at the wall and applying a proper curve fitting technique. The Herschel-Bulkley model (Equation (3)) exhibits an acceptable accuracy for the majority of drilling, completion, and cementing fluids and is therefore usually used to fit rheological curves.
Once the rheological constants are obtained, the Reynolds number can be calculated by mud velocity, density, and the wall shear stress as:
[formula] Re = 8ρv 2 τ w (9) [/formula]
where ρ is the mud density (kg/m 3 ).
The friction factor f is calculated from the frictional pressure loss measurements using:
[formula] f = D 2ρv 2(10) [/formula]
Vajargah et al.designed and tested a pipe viscometer in 2016. The main measurement section was approximately 5.5 m long and consisted of two pipe sections 1.27 cm and 0.9525 cm in diameter. The pipe viscometer was calibrated with water before testing. Three types of drilling fluid (i.e., bentonite drilling fluid, polymer-based drilling fluid, and synthetic-based drilling fluid) were used to perform the pipeline rheometer. A standard viscometer was also used to obtain rheology data. The rheological diagrams of the two different methods are relatively similar. The test results are shown in.
The pipe viscometer designed by Sercan Gul et al.has the test sections of the flow loop 1.25 m and 3.80 m long with an outside diameter of 2.54 cm. A comprehensive system calibration was achieved by circulating water at different flow rates through the flow loop. Excellent agreement was observed between the measurement results and the theoretical results. The mean absolute percentage error (MAPE) was calculated by taking the mean of the absolute percentage error (APE) for each single data point, as shown in Equations (11) and. The maximum APE was 3.5%, the MAPE was 1.6%, and the coefficient of determination (R 2 ) was 0.99. In experimental verifications, a total of fifteen tests were performed using various water-based mud and oil-based mud formulations at 25 - C, 50 - C, and 65.5 - C. It showed the precision and robustness of the pipe viscometer method and that it could be used to provide a quality and frequent fluid characterization for field muds. In field testing, the pipe viscometer measurements of both PV and YP were a perfect match to the data reported in daily mud reports by the mud engineer.
[formula] APE = ∆p i−experimental − ∆p i−theoretical ∆p i−theoretical * 100%(11)MAPE = 1 n n ∑ i=1 | ∆p i−experimental − ∆p i−theoretical ∆p theoretical | * 100%(12) [/formula]
where ∆p is the pressure loss (Pa), i is each measured data point, and n is the total number of measurements. Taugbøl et al.use the above principles to design pipe automatic drilling fluid measurement. The total length of the equipment was 3.3 m, the width was 0.7 m, and the height was 0.9 m. It converted the measurement results of the pipe viscometer to the standard Couette viscometer's dial readings and achieved good results. It was set to measure the viscosity every 15 min. It was installed and ran on several offshore drilling platforms and provided high-quality real-time drilling fluid data. It highly reduced the sampling time intervals, enabling improved fluid control and improved fluid quality.
Magalhães Filho et al.compared three drilling fluid rheological property real-time testing methods: a standard Couette viscometer FANN35A, online Couette viscometer TT-100, and pipe viscometer. For the Newtonian fluids, the viscosities measured by the three instruments were consistent. For non-Newtonian fluids, the PL parameters provided by each instrument were different, including drilling fluid (with suspended solids) and polymer solutions. These variations can mainly be caused by the consequences of fluid/gap interfaces, homogeneity, and slipperiness. However, these differences were not severe when the pressure drop was estimated using these parameters. The error between the pressure drop calculated by the TT-100 and the experimental value was the smallest. The error of the FANN35A was 17.81%, the error of the TT-100 was 0.41%, and the error of the pipe viscometer was 6.27%.
Some methods have the same theory as the pipe viscometer. Compared with the abovementioned pipe viscometers, these methods are convenient and do not take up much space. A novel downhole sensor was developed by Rondon et al.. It can be inserted into the drill string to measure the rheological properties of fluids in real-time. The mixtures of glycerin and water were used to test and calibrate this sensor. Real crude oil samples were also used to test the performance of this sensor. The error between the designed sensor's measurement value and the standard measurement value was within 2%. However, drilling fluids need further testing to evaluate the performance of the sensor. This sensor needs to further consider the flow rate and viscosity range of the drilling fluid and optimize the dimensions of the sensor. Carlsen et al.measured the pressure at various positions in the drilling fluid's circulation system. Various flow rates and pressures were used to measure the friction coefficient of the drilling fluid. The results show that it can also be used to calculate other drilling fluid rheological properties such as shear stress and viscosity. Vajargah et al.proposed a method to determine rheology in real time from downhole measurements of pressure drop and temperature, considering the well as an annulus pipe viscometer. It can directly obtain the ECD of the well. The results were compared to offline data taken from an offline high-pressure, high-temperature rheometer. It can estimate the gel strength by peak pressure loss. However, the time-dependent behavior of the drilling fluid theory needs to be developed through this method. We think, with the development of measurement while drilling (MWD) technology, it is a good method to obtain the rheological properties of drilling fluid by measuring downhole pressure. This method does not require further surface measurements, which can greatly simplify the rheological measurement methods and equipment and eliminate the labor required for operation and maintenance.
Pipe viscometers usually use a round pipe, the sensor installed in the round pipe may affect the flow rate, resulting in inaccurate pressure measurement. Therefore, Liu et al.developed a rectangle pipe viscometer. During the test, a 5% bentonite slurry with a density of 1.03 g/cm 3 was prepared, and 0.1% polyacrylamide glue solution, 0.1% polyacrylonitrile ammonium, and xanthan gum was added successively. The pipe viscometer continuously recorded the change process of the rheological properties of the drilling fluid, the measurement results were the same as the standard viscometer measurement results, and the error was small. Sun et al.developed a type of altered-diameter rectangle pipe viscometer to realize continuous online monitoring. Through altered-diameter pipes, different flow rates can be generated under constant flow. Fresh water and bentonite drilling fluid are tested in the laboratory. Compared with the standard viscometer, the results show that the error of AV and PV are both within the allowable range. The field test results show that the performance of the tested data was stable and reliable. Compared with the API standard method, the error was within the allowable range. While a pipe viscometer takes up a lot of space, Sercan Guldeveloped a helical pipe viscometer to measure rheological properties. The system included four test parts (two horizontal straight pipes and two vertical spiral pipes), four differential pressure sensors, a 40 L liquid storage tank, a Coriolis flowmeter, and a variable frequency drive screw pump. It tested the rheological properties of 20 polymer-based fluids. These fluids were based on water and added xanthan gum to increase the viscosity. The equivalent straight pipe pressure losses needed to be calculated accurately by using the pressure loss data obtained from a helical pipe viscometer. However, none of the papersreported correlations of PL fluids were valid for Herschel-Bulkley fluids. Thus, a random forest regression model was used to predict the friction coefficient with friction pressure loss, the mean absolute error was 0.803 × 10 −3 , and the mean absolute percentage error was 4.55%. The algorithm was developed using the trained machine learning model and the pipe viscometer equations. The rheogram results matched the standard Couette viscometer.shows a comparative study of the online Couette viscometers and the pipe viscometers conducted by previous researchers, and the results are similar to the standard Couette viscometer.
## The technology based on marsh funnel
Marsh funnel is a commonly used instrument for viscosity analysis, calculating the final fluid release time of almost 1.5 L. Marshinvented the Marsh funnelin 1931 as a quick and easy way to estimate the viscosity of drilling fluids. Pitt et al.analyzed the relationship between rheological properties and the Marsh funnel and established models for estimating drilling fluid rheological properties by using Marsh funnel time such as Equation (13), Equation, and Equation:
[formula] AV = ρ(t − 25)(13) [/formula]
## The technology based on marsh funnel
Marsh funnel is a commonly used instrument for viscosity analysis, final fluid release time of almost 1.5 L. Marshinvented the Marsh funn 1931 as a quick and easy way to estimate the viscosity of drilling fluids. analyzed the relationship between rheological properties and the Mar established models for estimating drilling fluid rheological properties by funnel time such as Equation (13) An artificial neural network (ANN) is an effective technology that im logical neurons of the human brain. The main processing element of the ral network system is the neuron. The ANN model is composed of netwo with at least three layers (i.e., input, hidden, and output layers), a training a a transfer function. Each layer is connected to other layers, and the con layers are called weights. The backpropagation technology is used in the t ficial neural networks. By Comparing the estimated data and actual data layer, it updates the weight between the deviation of each connection and e An artificial neural network (ANN) is an effective technology that imitates the biological neurons of the human brain. The main processing element of the artificial neural network system is the neuron. The ANN model is composed of network architecture with at least three layers (i.e., input, hidden, and output layers), a training algorithm, and a transfer function. Each layer is connected to other layers, and the constants of these layers are called weights. The backpropagation technology is used in the training of artificial neural networks. By Comparing the estimated data and actual data in the output layer, it updates the weight between the deviation of each connection and each layer. This is repeated until the desired improvement is achieved and the error is reduced to a certain threshold.
According to the above theories, artificial intelligence methods are used to estimate rheological properties more accurately in real time based on parameters such as the Marsh funnel viscosity, mud weight, and solid content. Bispo et al.used temperature, xanthan gum, bentonite, and barite to estimate AV. The main model used was the ANN.shows a schematic of the ANN model to estimate rheological properties. The model usually consists of three layers: an input layer-in addition to the Marsh funnel viscosity, the other drilling fluid parameters, such as mud weight and solid content, are also added as input elements; a hidden layer, which contains an optimized number of neurons; an output layer, which contains output parameters (PV, YP, AV, K, n, and τ 0 ). Abdelgawad et al.and Elkatatny et al.used the self-adaptive differential evolution (SaDe) algorithm to optimize the best combination of the ANN's parameters for rheological property estimation. SaDe proposed by Qin et al.is a special differential evolution algorithm with adaptive control parameters and mutation strategies based on learning experience. The trial-and-error procedure required to obtain the optimized solution is avoided in SaDe, which reduces the time required for optimization problems.
As shown in, scholars use the ANN model to predict the drilling fluid rheological properties (PV, AV, n, K, τ0, reading of 300 rpm and 600 rpm) in real time and show good results. The methods to evaluate the performance of the ANN model are average absolute error (AAE) (Equation (16)), average absolute percentage error, AAPE (Equation (17)), R 2 , and mean square error (MSE). In all these papers, the predicted result for R 2 was greater than 0.89, AAE was less than 4.7, and AAPE was less than 8.6%. This inexpensive technique will help drilling engineers to control the drilling operation better and predict drilling problems before they occur. Moreover, it will reduce the total cost of drilling operations. However, different ANN models need to be established by training drilling fluid data in different mud systems. One artificial intelligence model can be used in wells located in the same block or in the same drilling fluid system.
[formula] AAPE = ∑ |( ) * 100|(16)AAE = ∑ | − |(17) [/formula]
where i is each measured data point, yreal is real measured value, and ypredict is predict measured value. Abdelgawad et al.and Elkatatny et al.used the self-adaptive differential evolution (SaDe) algorithm to optimize the best combination of the ANN's parameters for rheological property estimation. SaDe proposed by Qin et al.is a special differential evolution algorithm with adaptive control parameters and mutation strategies based on learning experience. The trial-and-error procedure required to obtain the optimized solution is avoided in SaDe, which reduces the time required for optimization problems.
As shown in, scholars use the ANN model to predict the drilling fluid rheological properties (PV, AV, n, K, τ 0 , reading of 300 rpm and 600 rpm) in real time and show good results. The methods to evaluate the performance of the ANN model are average absolute error (AAE) (Equation (16)), average absolute percentage error, AAPE (Equation (17)), R 2 , and mean square error (MSE). In all these papers, the predicted result for R 2 was greater than 0.89, AAE was less than 4.7, and AAPE was less than 8.6%. This inexpensive technique will help drilling engineers to control the drilling operation better and predict drilling problems before they occur. Moreover, it will reduce the total cost of drilling operations. However, different ANN models need to be established by training drilling fluid data in different mud systems. One artificial intelligence model can be used in wells located in the same block or in the same drilling fluid system.
[formula] AAPE = 1 n ∑ n i=1 | y real − y predict y real * 100|(16)AAE = 1 n ∑ n i=1 |y real − y predict |(17) [/formula]
where i is each measured data point, y real is real measured value, and y predict is predict measured value.
## Acoustic technology
In 2011, Miller et al.introduced a system for continuously measuring and recording mud density and viscosity, using an instrument based on tuning fork technology as shown in. The instrument can measure density and viscosity, and the viscosity is in units of equivalent Marsh funnel seconds. The instrument was mounted in-line with a constant fluid flow rate past it. This method has a wide spectrum of industrial applications; it is robust and reliable and takes into account the separation of the fork teeth, so it is not easy to block. The instrument measures the standing wave generated by the vibrating teeth of the fork and calculates the density and viscosity of the fluid based on the amplitude and frequency measurement results. The density and viscosity values are output with 4-20 mA signals, which can be displayed locally in any required oilfield unit and output to the drilling rig data collection or logging tool computer software package.
## Acoustic technology
In 2011, Miller et al.introduced a system for continuously measur ing mud density and viscosity, using an instrument based on tuning fork shown in. The instrument can measure density and viscosity, and in units of equivalent Marsh funnel seconds. The instrument was mounted constant fluid flow rate past it. This method has a wide spectrum of ind tions; it is robust and reliable and takes into account the separation of the is not easy to block. The instrument measures the standing wave generated ing teeth of the fork and calculates the density and viscosity of the fluid amplitude and frequency measurement results. The density and viscosity put with 4-20 mA signals, which can be displayed locally in any required o output to the drilling rig data collection or logging tool computer software The tuning fork instrument kit operates at a back pressure of 0.7-0 density read is equal to the density of the normal pressurized drilling flui viscosity are calibrated by manual readings per shift. The instrument ki brated based on the new readings of the standard Marsh funnel viscometer sity or viscosity significantly changes. Experience in fields showed that a ca is sufficient at the start of each shift to ensure that readings are compara manual readings. The sensor is small and easy to install but requires manu Ofoche et al.presents a novel approach of continuously measurin rheology and density by use of sound signals.shows the schemati flow of drilling fluid in the pipeline is Poiseuille flow. Sound waves gene zoelectric transducer are passed though the fluid and the resultant dampi signals are used to drive a receiver piezo disc. The data acquisition dev frequency response and voltage by use of a fast Fourier transform routin with a constant rate designed to simultaneously calculate the six API recom rates. Therefore, six pipe sections were designed that have diameters corres six normal shear rates used in the petroleum industry. Since both density will affect the signal response, a multivariate random forest method was e used to predict the dial readings. All dial readings measured by the acousti within ±1 of the dial readings of the rotational viscometer. The tuning fork instrument kit operates at a back pressure of 0.7-0.8 MPa, so the density read is equal to the density of the normal pressurized drilling fluid. Density and viscosity are calibrated by manual readings per shift. The instrument kit can be recalibrated based on the new readings of the standard Marsh funnel viscometer when the density or viscosity significantly changes. Experience in fields showed that a calibration check is sufficient at the start of each shift to ensure that readings are comparable directly to manual readings. The sensor is small and easy to install but requires manual calibration.
Ofoche et al.presents a novel approach of continuously measuring drilling fluid rheology and density by use of sound signals.shows the schematic diagram. The flow of drilling fluid in the pipeline is Poiseuille flow. Sound waves generated by a piezoelectric transducer are passed though the fluid and the resultant damping effect of the signals are used to drive a receiver piezo disc. The data acquisition device records the frequency response and voltage by use of a fast Fourier transform routine. A flow loop with a constant rate designed to simultaneously calculate the six API recommended shear rates. Therefore, six pipe sections were designed that have diameters corresponding to the six normal shear rates used in the petroleum industry. Since both density and viscosity will affect the signal response, a multivariate random forest method was established and used to predict the dial readings. All dial readings measured by the acoustic method were within ±1 of the dial readings of the rotational viscometer. frequency response and voltage by use of a fast Fourier transform routine. A flow loop with a constant rate designed to simultaneously calculate the six API recommended shear rates. Therefore, six pipe sections were designed that have diameters corresponding to the six normal shear rates used in the petroleum industry. Since both density and viscosity will affect the signal response, a multivariate random forest method was established and used to predict the dial readings. All dial readings measured by the acoustic method were within ±1 of the dial readings of the rotational viscometer.briefly compares the real-time measurement techniques, and the detailed analysis are as follows.(1) The online Couette viscometer is the most similar to the API standard measurement method, so it has the highest accuracy and can measure all drilling fluid rheological properties. However, the gap between the rotor and the stator is narrow, and the diameter of solids must less than 1 mm. Solid or gels particles may be sedimented in the viscometer, so the online Couette viscometer is easily plugged. It is inconvenient to use and requires regular cleaning and maintenance. This viscometer is suitable for drilling fluids with low viscosity and low solid content. (2) Compared with the online Couette viscometer, the pipe viscometer provides better automatic measurement technology. The solid and gel particles in the drilling fluid will not settle in the pipe. By adding additional sensors to the pipe, additional variables can be obtained such as fluid density, temperature, critical Reynolds number, and real-time friction coefficient. However, it cannot measure the 10 s and 10 min gel strength. The pipe viscometer is large, and it occupies a large space for installation. Compared with the pipe viscometer, the helical pipe viscometer has obvious advantages, having a compact size and more general friction pressure loss curve. At the same time, the helical pipe increases the friction pressure loss and delayed flow state transition, so the helical pipe viscometer can be used to collect more data in the laminar flow state, thereby improving the accuracy of low shear rheological parameter estimation. However, the theoretical basis for the helical pipe viscometer is still under development. (3) Artificial intelligence technology is the cheapest method, because only the Marsh funnel is needed, and the mud balance and solid content meter may be added optionally. Although the test of the Marsh funnel, density, and sand content was simple and quick, it still required manual testing. The neural network model was different when the drilling system was different. One artificial intelligence method can be used in wells which are in the same block or in the same drilling system. The test results of the tuning fork technology were Marsh funnel viscosity and density, which can be combined with artificial intelligence technology to form an automatic online measurement of drilling fluid rheological properties. (4) The current drilling fluid rheology measurement is offline in API recommended practice for field testing drilling fluids, which can no longer meet the needs of intelligent drilling. In order to control and optimize rheological parameters in real-time, it is necessary to consider developing a criterion that can measure drilling rheological properties in real time. The standard Couette viscometer also has shortcomingsanalyzing the end-effect, correction, and reliability of the Couette viscometer. It is an opportunity to use real-time drilling fluid rheological properties measurement to improve the current criteria. The pipe viscometer is good in automation and will not be plugged by solids and gel. The reliability and accuracy of pipe viscometers often outweigh rotational viscometers. At present, the pipe viscometer used is large, so it is necessary to miniaturize the instrument for convenient use in the field. The helical pipe viscometer requires further mechanism research in theory to promote the real-time measurement of rheology. (5) The practicability of the instrument is relatively not good. We think two convenient methods can be considered in the future. One method is using MWD technology to measure the pressure in the drill string and annulus while drilling, the ECD can be accurately obtained. The drill pipe and annulus are considered as a large pipe viscometer, which can calculate the drilling fluid rheological properties. The other method is acoustic technology, including tuning fork technology, ultrasonic technology, etc. There are many articles using ultrasound to measure fluid parameters, but the composition of the drilling fluid is complex, and the ultrasonic attenuation is related to many factors(temperature, density, viscosity, solid content, etc.). For example, if an increase in ultrasonic attenuation is due to the entrance of solids into the system, the density should also increase, and some increase may be observed in the viscosity.
# Discussion and prospects
On the other hand, if an increase in the attenuation is observed due to the addition of polymers, the density may change slightly or not even change, but viscosity will significantly increase. The sound speed is also important since it helps the system to discern when the density is rising due to the solids suspended or solids dissolved. Therefore, the theory of ultrasound technology needs to be developed by simulation and experiment. Magalhães et al.used density, viscosity, ultrasound attenuation, and sound speed as inputs to establish an ANN model of concentration of the suspended solids. The installation of these two methods is convenient, but further technology and theoretical research are needed. (6) The current drilling fluid performance testing mainly measures the drilling fluid that samples from the mud pit. The drilling fluid returning from the annulus contains a lot of stratum information, so its testing is also very important and can help judge the formation. Testing its performance can also make better decisions for processing to maintain the performance of drilling fluid. However, the mud returning from the annulus contains many solid particles, and some particles have large diameters. Therefore, the allowable particle diameter of the instrument needs to be further considered in the selection of equipment.
# Conclusions
This article analyzes the four real-time measurement technologies of drilling fluids, and the measurement results of each technology are within the tolerance range compared with the standard rotational Couette viscometer in field test. At present, there is no industry standard handbook for real-time drilling fluid measurement. This status hinders the automatic control of drilling fluid. In the future, technical standards for real-time drilling fluid measurement will be established based on standard laboratory testing methods. This article analyzed the four methods in terms of principles, implementation methods, advantages, limitations, etc. Engineers can choose one of the four methods for real-time measurement according to their requirements in the field. The online rotational Couette viscometer is suitable for the low-viscosity and low-solid content drilling fluid. The pipe viscometer is a reliable real-time measurement method. As a result, a standard and small pipe viscometer may be formed to test the drilling fluid rheological properties in the future. The technology of MWD downhole pressure measurement and ultrasound technology can also be considered to measure drilling fluid rheological properties. Through real-time measurement of the rheological properties of drilling fluids, the performance of drilling fluids can be grasped in real-time, the drilling status can be judged in time, and relevant drilling fluid processing decisions can be made to ensure drilling safety. Through big data, artificial intelligence, and other technologies, the drilling fluid performance can be optimized in real time to achieve the optimal rate of penetration, thereby improving economic benefits.
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Nickel‐Catalyzed Inter‐ and Intramolecular Aryl Thioether Metathesis by Reversible Arylation
An ickel-catalyzed aryl thioether metathesis has been developed to access high-value thioethers.1 ,2-Bis(dicyclohexylphosphino)ethane (dcype) is essential to promote this highly functional-group-tolerant reaction. Furthermore,s ynthetically challenging macrocycles could be obtained in good yield in an unusual example of ring-closing metathesis that does not involve alkene bonds.In-depth organometallic studies support ar eversible Ni 0 /Ni II pathway to product formation. Overall, this work not only provides am ore sustainable alternative to previous catalytic systems based on Pd, but also presents new applications and mechanistic information that are highly relevant to the further development and application of unusual single-bond metathesis reactions.Aryl thioethers are often found in natural products,[1]Scheme 1. Context of the work.
pharmaceuticals,materials,photoinitiators,and fragrances.Thus,t he manipulation of C(sp 2 )ÀSb onds has recently become an active research area in transition-metal catalysis. The formation of C À Sb onds has greatly benefited from the advent of modern cross-coupling reactions,a nd aw ide variety of electrophilic precursors can now be readily employed to generate complex aromatic thioethers,u sing ad iverse range of metal catalysts such as Pd, Cu,and Ni. [10a-c] (Scheme 1a). In contrast, the cleavage of CÀSbonds under catalytic conditions has only recently become asubject of intense research efforts in catalysis (Scheme 1b). In these reactions,t he thioether group can effectively act as an alternative to traditional cross-coupling handles such as (pseudo)halogens,t hereby providing new approaches to synthesize and derivatize complex synthetic intermediates. We have recently introduced an ew concept for the manipulation of thioethers that involves both the formation and cleavage of carbon-sulfur bonds through ar eversible single-bond metathesis process(Scheme 1c). However, the previously reported Pd-based catalytic system exhibited several limitations with regards to potential applications: 1) the cost of the Pd precatalyst can be prohibitive for largerscale reactions;the scope with respect to the alkyl thiols was limited to unfunctionalized compounds,t hus precluding many synthetic applications such as ring-closing reactions; 3) the lack of mechanistic information about the reaction limits further catalyst development.
Herein, we report an ickel system for the CÀSb ond metathesis of ab road range of functionalized coupling partners (Scheme 1d). Thei mproved reactivity of the Ni system was key to developing the first example of ar ingclosing metathesis reaction of CÀSb onds that enables rapid access to synthetically challenging macrocycles.T he isolation and characterization of three catalytically competent complexes provide clear support for aNi 0 /Ni II catalytic cycle.
We began our investigation using Ni(COD) 2 as aprecatalyst and thioanisole and cyclohexanethiol as benchmark substrates.I nitial evaluation of different ligands led to the recovery of starting materials (see [fig_ref] Table 1: Substrate scope with respect to thioanisoles [/fig_ref] in the Supporting Information). After detecting the metathesis product in 8% yield after using Xantphos as al igand, we evaluated several other bidentate phosphine ligands.T he combination of a1,2bis(dicyclohexylphosphino)ethane (dcype) ligand and Ni-(COD) 2 (5 %) gave the best result, with the product obtained in 95 %yield. Awide range of thioanisole derivatives worked efficiently with the Ni catalyst [fig_ref] Table 1: Substrate scope with respect to thioanisoles [/fig_ref]. Indeed, electron-neutral (3aa, 3ab and 3ac), electron-rich (3ad, 3ae, 3af, 3ag and 3ah), and electron-poor (3ai, 3aj, 3ak, 3al, 3am and 3an)a renes were tolerated under our reaction conditions,providing the desired products in good to excellent yield. This method proved to be highly efficient in the presence of heterocyclic compounds (3ao, 3ap, 3aq and 3ar). Bicyclic compounds such as naphthalene (3as,9 5%), quinoline (3at,9 5%)a nd benzoxazole (3au,7 1%)w ere also found to be competent. Product (3ax)w as obtained in 86 %y ield, thus demonstrating that alkenyl substrates are suitable substrates for the reaction. The reaction also worked with at hiomethylether in the benzylic position, affording (3ay)i n7 8% yield, which hints at the possibility to reversibly cleave activated C(sp 3 ) À Sb onds under nickel catalysis.N otably,t he same reaction using the previously reported palladium system resulted in poor conversion. Commercially relevant molecules bearing at hioanisole moiety were modified next. Thew idely used photoinitiator MMMPled to product (3az)f ormation in 91 % yield. Aderivative of fexinidazole,adrug for treating sleeping sickness,also resulted in the formation of the desired product (3aaa)i n7 5% yield, thus demonstrating the potential of this reaction for derivatizing ArSR-containing bioactive molecules.N otably,t his method proved to be efficient with arenes bearing an ortho substituent (3aab & 3aac). Remarkably,a sl ittle as 0.5 mol %o ft he catalyst was sufficient to reach 86 %yield on alarger scale (3aa), aresult that clearly shows that the more sustainable nickel system can be as active as palladium.
Next, we evaluated the scope of the reaction with regards to the thiol partner [fig_ref] Table 2: Substrate scope with respect to alkylthiols [/fig_ref] , since only few functionalized thiols were tested previously.W ef irst tried readily available alkyl thiols such as primary (2a-2d), secondary (2e-2g), and tertiary (2h-2j)t hiols.I na ll of these cases,t he metathesis product was obtained in more than 80 %y ield. Thiocitronellol, which bears an alkene functionality that could deactivate the nickel catalyst through chelation,was ac ompetent partner,g iving the expected product (3bk)i n9 3% yield. Moreover,(S)-thioprolinol, which contains abasic amine,also led to the coupling product (3bl)i ng ood yield (80 %). We further explored the scope by using alkyl thiols bearing important functional groups such as ether (3bn,90%), nitrile (3bo,7 5%), thioether (3bp,9 1%), amide (3bq,8 0%), sulfone (3br,4 3%), tetrahydropyran (3bs,4 6%), and azetidine (3bt,4 6%)g roups.T he efficiency of the Ni system encouraged us to develop the first example of ar ing-closing thioether metathesis,w hich led to the formation of the corresponding thiochromane product (3bu,9 3%). At hiospirocycle was also obtained in ah igh yield (3bv,9 3%), thereby paving the way to an ew family of spirocycles.T he utility of the method was finally tested in al ate-stage functionalization of av itamin Ed erivative,d elivering 3bw in good yield (65 %).
Thea pplication of another metathesis reaction, alkene metathesis,t om acrocycle synthesis has had at remendous impact on the synthesis of drug candidates,n atural products and supramolecular assemblies.We thus envisaged that synthetically challenging sulfur macrocyclescould be accessed by taking advantage of the potential self-correcting ability of our reversible C À Sbond metathesis reaction.
Having established an efficient and scalable strategy toward the thiol precursors of these macrocycles,w et hen optimized our reaction conditions for the macrocyclization [fig_ref] Table 3: Substrate scope of the ring-closing metathesis for macrocycle formation [/fig_ref]. Thef irst macrocycle (3ca)w as obtained in 68 % yield using this ring-closing sulfur-bond metathesis strategy. Interestingly,using our previous catalytic system (Pd-SingaCycle A1), even with ahigher catalyst loading of 10 %, the same macrocycle was formed in less than 20 %yield. This result suggests that the Pd catalyst is not active enough to catalyze the metathesis reaction under the highly diluted reaction conditions necessary for the macrocyclization. We then explored the scope with respect to starting materials bearing different chain lengths such as 15, 16, and 18 methylene units between the phenol and the thiol. All substrates readily cyclized under these conditions,a ffording the corresponding macrocycles (3cb, 3cc and 3cd)i n7 5, 48, and 37 %y ield, respectively.S urprisingly,t he dimer of (3cb) was also formed under the reaction conditions in as ynthetically useful 22 %isolated yield, affording arare 40-membered ring macrocycle,a sc onfirmed by X-ray analysis.T he para product 3ce,w hich has ac hain of 16 methylene units,c ould also be obtained in 44 %yield.
While palladium-catalyzed cross-coupling processes have usually been reported to proceed via aP d 0 /Pd II pathway the analogous nickel-catalyzed processes can proceed through different pathways because of the intrinsic higher stability of Ni I and Ni III species,although their role in catalysis has not yet been completely unraveled. While there have been examples of oxidative addition from Ni 0 to Ni II species, with pioneering example from Itamisg roup on Ni-dcype catalytic system,CÀX( N, O, S) reductive elimination can often proceed through aN i III /Ni I pathway, particularly in the case of nitrogen or carbene ligands. Indirect evidence has also been provided through computational analysis and radical trapping experiments. In contrast, the direct observation of C À Xreductive elimination from Ni II is extremely rare. We thus became interested in [a] Yield of isolated product (%). Forc onditions, see the Supporting Information. [a] Yield of isolated product over 2s teps (%). 1) Substrate (0.
## Angewandte chemie
Communications 2112 www.angewandte.org studying the reaction mechanism through stoichiometric experiments and isolation of potential organometallic intermediates.T he ability of nickel complexes to span several oxidation states has been widely observed with some examples of Ni I species reported as active catalysts even when starting from Ni 0 pre-catalysts. To investigate this,w e commenced with the synthesis of aN i(dcype)(COD) complexand tested its stoichiometric reactivity toward the aromatic thioether.W ed ecided to use thioanisole 1n and adamantanethiol 2h as model substrates since their crystalline nature may ease the isolation and generation of single crystals suitable for X-ray diffraction. Treatment of nickel complex 4 with lithium adamantanethiol salt did not result in the formation of any new Ni-ate species in the Supporting Information ).
We next proceeded to evaluate the reaction of complex 4 with the thioanisole derivative 1n (Scheme 2A). After 2hours at 75 8 8C, formation of complex 5 was observed by 31 P{H} NMR with resonances at d = 65 and 61, leading to product isolation (54 %y ield) after 16 hours . Single-crystal X-ray analysis confirmed the identity of the oxidative addition product 5 (Scheme 2A). Thep utative transmetallation step was next studied through astoichiometric experiment between complex 5 and the isolated Li adamanthylthiol salt (Scheme 2B). Ther eaction was very slow at room temperature,h owever,i ncreasing the temperature to 90 8 8Cr esulted in the formation of an ew set of doublets (d = 62), reaching up to 30 %c onversion overnight . Unfortunately,i tw as not possible to isolate as uitable crystal for X-ray analysis.A lternatively,w ew ere able to gather complementary information by synthesizing complex 6 through adifferent pathway.Oxidative addition of adamanthyl S-phenyl to complex 4 gave complex 6 in 50 % yield . Gratifyingly,t he 31 P{H} NMR resonances matched the resonances of the previously observed compound, (Scheme 2B), thus confirming the viability of the transmetallation step.T he identity of complex 6 was further confirmed by X-ray analysis.W hile reductive elimination from Ni III has often been reported,such apathway is not viable under our catalytic conditions because of the microscopic reversibility principle.Reductive elimination from Ni II complex 6 provides am ore reasonable pathway to close the catalytic cycle (Scheme 2C). Interestingly,c omplex 6 could only undergo reductive elimination in the presence of 2equivalents of COD , while the catalytic reaction can, in contrast, proceed with substoichiometric COD.T his result is consistent with ar eversible reductive elimination process with as mall equilibrium constant favoring the oxidative addition complex 6.U nder stoichiometric conditions,the addition of COD is thus necessary to shift the equilibrium toward the product. We next confirmed the catalyticc ompetence of complexes 4 to 6 by using5mol %o f each complex under standard reaction conditions(Scheme 3A).
Based on the aforementioned mechanistic investigation and additional unsuccessful radical-trapping experiments,we propose af ully reversible catalytic cycle based on aN i 0 / Ni II pathway (Scheme 3B). Thereaction is likely to start with oxidative addition of the S À Ph bond, followed by transmetallation between Ni À SMe and alkylS À Li and final reductive elimination to release the product. Although the intermediacyo fas hort-lived Ni I or Ni III species cannot be completely ruled out at this stage,o ur mechanistic experiments strongly support the Ni 0 /Ni II pathway.
[fig] 2: mmol), LiHMDS (1.1 equiv), Ni(COD) 2 (10 mol %), dcype (10 mol %), toluene (0.002 m), 100 8 8C, 9h.2)m-CPBA (4.0 equiv),C H 2 Cl 2 (0.1 m), rt, 12 h. [b] toluene (0.0025 m). [c] 24 h. [/fig]
[table] Table 1: Substrate scope with respect to thioanisoles. [a] [a] Yield of isolated product (%). Forc onditions, see the Supporting Information.[b] NMR yield. [c] Reaction was performed in o-xylene at 140 8 8C. [/table]
[table] Table 3: Substrate scope of the ring-closing metathesis for macrocycle formation.[a] [/table]
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Early metabolic and transcriptional variations in fruit of natural white-fruited Fragaria vesca genotypes
Strawberry fruits (Fragaria vesca) are valued for their sweet fruity flavor, juicy texture, and characteristic red color caused by anthocyanin pigments. To gain a deeper insight into the regulation of anthocyanin biosynthesis, we performed comparative metabolite profiling and transcriptome analyses of one red-fruited and two natural white-fruited strawberry varieties in two tissues and three ripening stages. Developing fruit of the three genotypes showed a distinctive pattern of polyphenol accumulation already in green receptacle and achenes. Global analysis of the transcriptomes revealed that the ripening process in the white-fruited varieties is already affected at an early developmental stage. Key polyphenol genes showed considerably lower transcript levels in the receptacle and achenes of both white genotypes, compared to the red genotype. The expression of the anthocyanidin glucosyltransferase gene and a glutathione S-transferase, putatively involved in the vacuolar transport of the anthocyanins, seemed to be critical for anthocyanin formation. A bHLH transcription factor is among the differentially expressed genes as well. Furthermore, genes associated with flavor formation and fruit softening appear to be coordinately regulated and seem to interact with the polyphenol biosynthesis pathway. This study provides new information about polyphenol biosynthesis regulators in strawberry, and reveals genes unknown to affect anthocyanin formation.
Strawberry (Fragaria × ananassa) is one of the world's most popular fruit crops with an annual production of more than 8.1 million tons in 2014 (http://faostat3.fao.org/browse/Q/QC/E). The strawberry is considered as "accessory fruit" because the fruit is not formed by the enlargement of the ovary but the edible, fruity pulp, also referred to as receptacle, is derived from adjacent tissue exterior to the carpel. The achenes (seeds) are distributed spirally across the epidermis of the pulp [bib_ref] Growth and ripening of strawberry fruit, Perkins-Veazie [/bib_ref]. The development of the strawberry fruit is regarded independent of increased ethylene biosynthesis and respiration, which is why strawberries are considered non-climacteric fruits [bib_ref] Genetic regulation of fruit development and ripening, Giovannoni [/bib_ref] , although recent studies suggest ethylene plays a role in strawberry fruit ripening [bib_ref] Ethylene is involved in strawberry fruit ripening in an organ-specific manner, Merchante [/bib_ref]. The ripening process can be traced by observing the changes in fruit size and color. The stages are usually classified as green, white, turning, and red, and their development is accompanied by changing compositions of plant hormones and metabolites [bib_ref] Metabolic profiling of strawberry (Fragaria × ananassa Duch.) during fruit development and..., Zhang [/bib_ref]. Strawberry fruits ripen quite fast within about 30 days. The plants are also small in size and easy to propagate. These qualities, together with an unusual fruit structure and color formation, have made the strawberry plant an advantageous model system to study fruit development. Woodland strawberry F. vesca has a small, sequenced genome (240 Mb), and is commonly used as a genetic model plant for the Rosaceae family and, in particular, the Fragaria genus.
Besides the appealing flavor, much of the attractiveness of strawberries is based on the bright red fruit-color caused by anthocyanin pigments [bib_ref] Metabolic profiling of strawberry (Fragaria × ananassa Duch.) during fruit development and..., Zhang [/bib_ref] [bib_ref] Flavonoids: a colorful model for the regulation and evolution of biochemical pathways, Koes [/bib_ref]. Pelargonidin 3-O-glucoside, its 6′ -O malonated derivative and cyanidin 3-O-glucoside are the major anthocyanins of strawberry fruit and are biosynthesized from phenylalanine by the phenylpropanoid-flavonoid-anthocyanin pathway, which has been thoroughly investigated by genetic, biochemical and metabolite profiling studies [bib_ref] Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis..., Almeida [/bib_ref]. Anthocyanins are also associated with a large number of health-promoting effects. They possess anti-oxidative properties, have positive impacts on cardiovascular disorders and degenerative diseases, and are able to protect DNA integrity [bib_ref] Berry anthocyanins as novel antioxidants in human health and disease prevention, Zafra-Stone [/bib_ref] [bib_ref] Flavanols and anthocyanins in cardiovascular health: a review of current evidence, Pascual-Teresa [/bib_ref] [bib_ref] Anthocyanins: natural colorants with health-promoting properties, He [/bib_ref]. The basic biosynthetic pathway of anthocyanins is known, Scientific RepoRts | 7:45113 | DOI: [bib_ref] Flavonoids as Flower Pigments. The formation of the natural spectrum and its..., Forkmann [/bib_ref].1038/srep45113 and most plant species share a large number of enzymatic reactions, although there are differences regarding the types of anthocyanins that accumulate [bib_ref] Flavonoids as Flower Pigments. The formation of the natural spectrum and its..., Forkmann [/bib_ref] [bib_ref] Recent advances in the biosynthesis and accumulation of anthocyanins, Springob [/bib_ref].
In contrast to the red-fruited F. vesca genotypes, there are also varieties that produce white fruits, even when they are fully ripened. This is not the result of continuous breeding or genetic modification, but a naturally occurring phenomenon. In the white-fruited varieties the pigment formation seems to be impaired by down-regulation of a single or multiple biosynthetic genes, or because an essential gene is non-functional. Key factors in the regulation of the flavonoid and anthocyanin pathway are MWB (MYB-bHLH-WD40) complex proteins [bib_ref] MYB transcription factors in Arabidopsis, Dubos [/bib_ref]. In order to gain a deeper insight into the regulation of anthocyanin biosynthesis, we performed comparative metabolite profiling and transcriptome analysis of one red-fruited (F. vesca cv. Reine des Vallees (RdV)), and two white-fruited (F. vesca cv. Yellow Wonder (YW) and Hawaii 4 (HW4)) woodland strawberry varieties [fig_ref] Figure 1: Fruits of F [/fig_ref] by liquid-chromatography coupled with mass spectrometry analysis (LC-MS), and RNA-sequencing (RNA-seq), respectively. To survey gene expression during fruit development we performed RNA-Seq on green, intermediate and ripe (white-ripe and red-ripe, respectively) fruits, and separated the achenes (seeds) from the receptacle (pulp). To determine the metabolic differences between the three genotypes, the level of anthocyanins and relevant precursors were analyzed by LC-MS, and the expression pattern of candidate genes was validated by qPCR.
Our analysis completes a recently published F. vesca transcriptome data set, which provides gene expression data of the fruit development stages from fertilized flower to big green fruit [bib_ref] Genome-scale transcriptomic insights into early-stage fruit development in woodland strawberry Fragaria vesca, Kang [/bib_ref] [bib_ref] SGR: an online genomic resource for the woodland strawberry, Darwish [/bib_ref]. Thus, the transcriptome of the complete strawberry fruit ripening process from flower to ripe fruit is now available, with our data covering green to ripe developmental stages (Supplementary data File S1). The analyses of the white-fruited genotypes show that the phenylpropanoid/flavonoid/anthocyanin metabolism and the gene transcript levels are already perturbed at early developmental stages in YW and HW4.
# Results
Metabolite Analysis. Untargeted and targeted metabolite analyses of phenols, phenylpropanoids, flavonoids, proanthocyanidins, and anthocyanins were performed by LC-MS in receptacles and achenes of green, intermediate and ripe fruits of F. vesca RdV, YW and HW4 [fig_ref] Figure 2: Untargeted analysis of secondary metabolites in receptacle and achenes of strawberry varieties... [/fig_ref]. 271 untargeted metabolites showed variance between the three genotypes (p-value ≤ 0.01). Their analysis uncovered much lower variance in the data sets of the metabolites found in the achenes of RdV, YW, and HW4 than in the data of the receptacles [fig_ref] Figure 2: Untargeted analysis of secondary metabolites in receptacle and achenes of strawberry varieties... [/fig_ref]. The receptacles of the three F. vesca varieties clustered according to their developmental stages. Thus, metabolites that confer the red color to ripe RdV fruits did not strongly contribute to the variance in the data. Overall, according to the untargeted analysis, the major metabolites were similar in the three genotypes at the identical ripening stage. However, the targeted metabolite analysis showed, in line with the color change of the receptacle and achenes of RdV [fig_ref] Figure 1: Fruits of F [/fig_ref] , high levels of anthocyanins such as pelargonidin glucoside, pelargonidin glucoside malonate, and cyanidin glucoside in ripe receptacle and achenes of RdV, but not in fruits of YW and HW4 [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref]. Each developmental stage and fruit tissue is dominated by a certain group of phenolic compounds. For instance, phenols such as gallic acid, gallic acid glucose ester and ellagic acid were major metabolites in green achenes of RdV, whereas flavonoids were abundant in intermediate achenes of RdV, and anthocyanins and phenylpropanoids dominated in the late developmental stage. In most cases, the levels of secondary metabolites in achenes and receptacle of the white-fruited genotypes differed considerably from the concentrations determined in the respective tissues of RdV. Although, gallic and ellagic acid accumulated in green achenes of YW to levels found in green achenes of RdV, the concentrations of flavonoids were significantly reduced. In contrast, in green receptacles of YW, the levels of flavonoids even exceeded the concentrations detected in the same tissue of RdV. Thus, the differential metabolite levels suggest that changes in secondary metabolism reflect organ and developmental specificities.
Global Analysis of the Fruit Transcriptome. Global mRNA sequencing of the receptacle and achenes of red-fruited F. vesca RdV variety, and both white-fruited F. vesca YW and HW4 varieties was performed to investigate the differential accumulation of transcripts. RNA was pooled from receptacles and achenes of ten fruits per sample to ensure high reliability regarding the stage of fruit ripening . Sequencing yielded 249,582,360 reads of 100 bp in length [fig_ref] Table 2: Fold change [/fig_ref] , giving ~25 billion nucleotides of total sequence data. After quality clipping, 245,603,827 reads were selected. The mapping of the selected reads to the F. vesca reference genome resulted in a total pool of 204,888,523 transcript counts (greater than 83% overall mapping rate, [fig_ref] Table 3: Flavonoid genes differentially expressed in the red-and white-fruited genotypes and transcription factors... [/fig_ref]. Subsequently, the read counts were normalized by DESeq2 size factors, and scaled to per million range (rpm: reads per million). Genes with fewer than 20 normalized counts summed across samples were considered as not expressed. Out of 33673 annotated F. vesca genes, 19208 were expressed above this threshold.
To investigate global gene expression relationships, we performed a principle component analysis (PCA), and visualized the correlations also by dendrograms of the achenes and receptacle data sets [fig_ref] Figure 4: Global analysis of gene expression among samples and strawberry varieties [/fig_ref]. When the 500 most highly expressed transcripts were employed for PCA analysis, the receptacles and achenes were clearly set apart (left and right), as well as the green and ripe developmental stages (top and bottom, [fig_ref] Figure 4: Global analysis of gene expression among samples and strawberry varieties [/fig_ref]. The receptacles and achenes of the intermediate ripening stage of YW and HW4 grouped with the green tissues, whereas the receptacle and achenes of intermediately ripened RdV clustered with the ripe tissues of all varieties. Thus, gene expression of the white genotypes at the intermediate stage is more closely related to green unripe tissue, and the intermediate stage of the red genotype RdV to the ripe tissues. This observation was also confirmed by hierarchical clustering of the achenes and receptacle data [fig_ref] Figure 4: Global analysis of gene expression among samples and strawberry varieties [/fig_ref] and C).
Analysis of differentially expressed genes between RdV and both white genotypes YW and HW4. To find candidate genes that might explain the loss-of-color phenotype in YW and HW4, differential expression between genotypes was assessed. Thirty-three genes were significantly down-regulated in white genotypes (YW, HW4) compared to the red genotype RdV . Five genes encode enzymes with confirmed biochemical functions in F. vesca or F. × ananassa. Transcript levels of early (chalcone synthase FaCHS2-2: gene26826, chalcone isomerase FaCHI: gene 21346, and flavanone 3-hydroxylase FHT: gene14611), and late (dihydroflavonol reductase DFR: gene15176, anthocyanin synthase ANS: gene32347, and anthocyanin 3-O-glucosyltransferase FaGT1: gene12591) anthocyanin biosynthesis genes were equally reduced. Furthermore, gene31672 a predicted glutathione S-transferase is among the genes showing highest differential expression (logFC = − 7.2), with transcripts accumulating almost exclusively in ripe and intermediate tissues of the red genotype RdV (Supplementary data File S1). On the contrary, 31 genes were significantly up-regulated in both white genotypes [fig_ref] Table 2: Fold change [/fig_ref]. Many candidates currently lack a functional prediction but show remarkable differences between the genotypes. For example candidate gene20847 (logFC = 10.7), almost not expressed in any tissues of RdV exhibits very high expression (up to 1,785 RPM) in tissues of HW4, and lower but considerable expression in tissues of YW. Furthermore, gene27422 a predicted transcription factor ORG2-like of the bHLH class is exclusively expressed in the white genotypes.
Transcript level profiles of known anthocyanin and flavonoid pathway genes during strawberry fruit development. Next, we analyzed the transcript level profiles of anthocyanin and flavonoid pathway genes, whose encoded proteins have already been biochemically characterized. Gene expression levels in achenes and receptacle of RdV, YW, and HW4 of three developmental stages (green, intermediate and ripe) were compared . Four groups of genes could be clearly distinguished by means of their transcript profiles. Early anthocyanin and flavonoid pathway genes such as PAL, CA4H, 4CL, FLS, and a flavonoid glucosyltransferase (FGT) gene show high expression levels in green achenes of RdV, YW, and HW4 as well as in achenes of YW and HW4 of the intermediate ripening stage [fig_ref] Figure 1: Fruits of F [/fig_ref]. CHS, CHI, F3H, DFR, Expression levels of genes involved in fruit softening and flavor formation. Finally, we wanted to know whether the impaired anthocyanin pathway in YW and HW4 affects the expression of genes involved in fruit flavor formation, and fruit softening. Transcript levels of well-characterized genes associated with volatile terpene (pinene synthase and hydroxylase), ester (acyltransferases FcAAT1, and SAAT), furaneol (FaQR), and eugenol (eugenol synthase) formation, as well as genes affecting fruit softening (pectin esterase, pectate lyase, polygalacturonase and beta-galactosidase) were analyzed in the data sets of RdV, YW, and HW4 . The genes showed a similar ripening-related expression profile in the receptacles of the three genotypes, peaking at the ripe stage. It seems that the ripening process is slowed down in the white-fruited genotypes in comparison to RdV, because transcripts of genes involved in fruit flavor production, and degradation of cell wall polysaccharides are already abundant in receptacle of RdV at the intermediate stage, whereas these genes are almost solely expressed in ripe receptacle of YW and HW4.
# Discussion
Considerable information on the polyphenolic composition of commercial strawberry fruit (F. × ananassa) [bib_ref] Characterization of phenolic compounds in strawberry (Fragaria × ananassa) fruits by different..., Aaby [/bib_ref] [bib_ref] Phenolic compounds in strawberry (Fragaria × ananassa Duch.) fruits: Composition in 27..., Aaby [/bib_ref] [bib_ref] HPLC-MS Analysis of Proanthocyanidin Oligomers and Other Phenolics in 15 Strawberry Cultivars, Buendía [/bib_ref] and woodland strawberry F. vesca fruit [bib_ref] Liquid chromatographic/electrospray ionization tandem mass spectrometric study of polyphenolic composition of four..., Bubba [/bib_ref] [bib_ref] Profiling polyphenols of two diploid strawberry (Fragaria vesca) inbred lines using UHPLC-HRMSn, Sun [/bib_ref] [bib_ref] Genetic dissection of the (poly)phenol profile of diploid strawberry (Fragaria vesca) fruits..., Urrutia [/bib_ref] exist. However, data on the levels of phenolics in developing F. vesca fruits is missing, completely. The bright color of red-fruited strawberries is due to four major anthocyanins, pelargonidin 3-glucoside, pelargonidin 3-glucoside 6′ -malonate, pelargonidin 3-rutinoside and cyanidin 3-glucoside [bib_ref] Phenolic composition of strawberry genotypes at different saturation stages, Kosar [/bib_ref] [bib_ref] Comparison of phenolic composition and antioxidant properties of two native Chilean and..., Simirgiotis [/bib_ref] [bib_ref] Antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes, Tulipani [/bib_ref] [bib_ref] Polyphenol composition in the ripe fruits of Fragaria species and transcriptional analyses..., Muñoz [/bib_ref] , which are formed by the phenylpropanoid/flavonoid/anthocyanin pathway during fruit ripening [bib_ref] Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis..., Almeida [/bib_ref] [bib_ref] Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development, Fait [/bib_ref] [bib_ref] Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in..., Griesser [/bib_ref]. In white colored strawberries, these anthocyanins are reduced in the receptacle, and in some cases also in the achenes [bib_ref] E-cinnamic acid derivatives and phenolics from Chilean strawberry fruits, Fragaria chiloensis ssp...., Cheel [/bib_ref] [bib_ref] Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of..., Salvatierra [/bib_ref] [bib_ref] Determination of phenolic composition and antioxidant activity in fruits, rhizomes and leaves..., Simirgiotis [/bib_ref]. Similarly, only trace amounts of pelargonidin 3-glucoside, pelargonidin 3-glucoside 6′ -malonate, and cyanidin 3-glucoside were detected in the ripe receptacle and achenes of YW and HW4; in contrast to their high abundance in fruit of RdV [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref]. Untargeted analysis of secondary metabolites by PCA separated the achenes from the receptacles, whereas the receptacles were further subdivided according to their ripening stage [fig_ref] Figure 2: Untargeted analysis of secondary metabolites in receptacle and achenes of strawberry varieties... [/fig_ref]. Ripe fruit of RdV, YW, and HW4 clustered in the PCA plot, but can be readily differentiated by the different pigmentation [fig_ref] Figure 1: Fruits of F [/fig_ref]. Thus, the anthocyanin level in RdV fruit is not the primary variance in the data. Although, green achenes of RdV, YW, and HW4 accumulated comparable levels of gallic acid, gallic acid glucose ester, and ellagic acid, the immature seeds of the three genotypes can be clearly differentiated by their varying flavonoid concentration [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref]. Achenes of RdV exhibited a tri-phasic polyphenol accumulation profile. The levels of phenols, flavonoids, and anthocyanins/phenylpropanoids peaked in green, intermediate, and ripe seeds of the red-fruited genotype, respectively. During ripening of YW and HW4, flavonoids did not reach the concentrations found in RdV, except for kaempferol glucuronide in intermediate and ripe achenes of both white-fruited genotypes, and epicatechin catechin and epiafzelechin catechin dimers in ripe achenes of YW. In addition, the total amount of polyphenols is reduced in the white-fruited genotypes. Receptacles of RdV showed a bi-phasic formation of polyphenols, as flavonoid levels peak at the intermediate ripening stage, and anthocyanins, quercetin glucuronide, phenylpropanoids, and ellagic acid are abundant in the ripe pulp. In contrast, receptacles of YW contained high levels of ellagic acid and flavonoids with declining concentrations during ripening, except kaempferol glucoside. HW4 displayed a mixed pattern of polyphenol accumulation in the pulp, whereas the lowest levels were found at the ripe developmental stage. Overall, the divergent profiles of secondary metabolites suggest an interference of the pathway in the white-fruited genotypes YW and HW4 at an early developmental stage.
In addition to metabolite profiling, gene transcript abundance was quantified by RNA-seq analysis in achenes and receptacles of the red-and white fruited varieties during fruit ripening. Analysis of global gene expression by PCA separated the achenes from the receptacles, as well as green from ripe tissues [fig_ref] Figure 4: Global analysis of gene expression among samples and strawberry varieties [/fig_ref]. Intermediate receptacle and achenes of YW and HW4 clustered with green fruit samples, and intermediate pulp and seeds of RdV with ripe fruit samples. This indicates that variance in gene expression is highest between samples of the intermediate ripening stage, and confirms the hypothesis that the ripening process in YW and HW4 is already affected at an early stage. In contrast, the untargeted analysis of the metabolites did not show equal variance, as the achene samples of all genotypes grouped together [fig_ref] Figure 2: Untargeted analysis of secondary metabolites in receptacle and achenes of strawberry varieties... [/fig_ref]. On the other hand, ripe receptacle of RdV, YW and HW4 grouped together in the PCA of the transcripts [fig_ref] Figure 4: Global analysis of gene expression among samples and strawberry varieties [/fig_ref] , similar to the PCA of secondary metabolites [fig_ref] Figure 2: Untargeted analysis of secondary metabolites in receptacle and achenes of strawberry varieties... [/fig_ref].
Analysis of differentially expressed genes revealed that expression of major genes in the anthocyanidin/flavonoid biosynthesis pathway was down-regulated in the white varieties . The expression of the branch point gene CHS (gene26826 polyketide synthase 1) was severely reduced. CHS expression is known to be associated with fruit coloring, because artificial down-regulation of CHS function via antisense and RNAi methods leads to pigment loss in flowers or fruits of different plant species [bib_ref] An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus..., Deroles [/bib_ref] [bib_ref] Distinct patterns of pigment suppression are produced by allelic sense and antisense..., Que [/bib_ref] [bib_ref] RNAi-induced silencing of gene expression in strawberry fruit (Fragaria × ananassa) by..., Hoffmann [/bib_ref] [bib_ref] Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria..., Lunkenbein [/bib_ref] [bib_ref] Acylphloroglucinol biosynthesis in strawberry fruit, Song [/bib_ref]. Furthermore, five genes acting downstream of CHS were also clearly down-regulated . The protein encoded by CHI (gene21346 chalcone-flavonone isomerase 3) catalyzes the conversion of naringenin chalcone to the flavanone naringenin, thereby producing the basic skeleton of all flavonoid metabolites [bib_ref] Flavonoids as Flower Pigments. The formation of the natural spectrum and its..., Forkmann [/bib_ref]. The protein encoded by FHT (gene14611 naringenin, 2-oxoglutarate 3-dioxygenase) oxidizes the central B ring of the flavanone naringenin to produce dihydrokaempferol [bib_ref] Flavonoids as Flower Pigments. The formation of the natural spectrum and its..., Forkmann [/bib_ref]. The enzyme encoded by DFR (gene15176 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase) reduces dihydrokaempferol to colorless leucoanthocyanidins [bib_ref] Functional characterization of dihydroflavonol-4-reductase in anthocyanin biosynthesis of purple sweet potato underlies..., Wang [/bib_ref] [bib_ref] Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent..., Miosic [/bib_ref]. The polypeptide encoded by ANS (gene32347 leucoanthocyanidin dioxygenase) generates colored anthocyanidins like pelargonidin [bib_ref] The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for..., Abrahams [/bib_ref] , and the protein encoded by FaGT1 (gene12591 anthocyanidin 3-O-glucosyltransferase 2) stabilizes the anthocyanidins by glucosylation [bib_ref] Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in..., Griesser [/bib_ref]. The resulting anthocyanins accumulate, and are responsible for the coloring of fruits and flowers. Thus, formation of anthocyanin precursors is considerably hampered in the white varieties. Among the significantly down-regulated candidates is also gene31672, a glutathione S-transferase (GST, orthologous to GST Solyc02g081340 from tomato (Solanum lycopersicum). Transgenic tomato fruits exhibiting higher anthocyanin content showed increased expression of Solyc02g081340 40,41 , whereas expression in the anthocyanin absent mutant was barely detectable [bib_ref] Fine mapping and molecular marker development of anthocyanin absent, a seedling morphological..., Zhang [/bib_ref]. It has been proposed that anthocyanins might be transported into vacuoles via the noncovalent activity of GSTs [bib_ref] Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione..., Alfenito [/bib_ref]. Several GST genes with such functions have been characterized in plants including the TT19 gene (encoding a type I GST) of Arabidopsis, Bronze-2 (encoding a type III GST) of maize, AN9 (encoding a type I GST) of petunia [bib_ref] TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and..., Kitamura [/bib_ref] [bib_ref] A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene..., Marrs [/bib_ref] [bib_ref] AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding..., Mueller [/bib_ref] and two GST genes from grape 47 . Therefore, gene31672 might act in anthocyanin transport.
In addition to genes down-regulated in the white genotypes, also candidates significantly up-regulated were found [fig_ref] Table 2: Fold change [/fig_ref] , such as a yet uncharacterized transcription factor (TF) of the bHLH class (gene27422 ORG2-like). It is widely acknowledged that TFs of the MYB and bHLH protein classes regulate the expression of anthocyanin biosynthesis genes [bib_ref] MYB transcription factors that colour our fruit, Allan [/bib_ref] [bib_ref] New insights into the regulation of anthocyanin biosynthesis in fruits, Jaakola [/bib_ref]. MYBs linked to the anthocyanin pathway possess a highly conserved DNA-binding domain, which usually comprises two repeats (R2R3) [bib_ref] New insights into the regulation of anthocyanin biosynthesis in fruits, Jaakola [/bib_ref] , and are suggested to interact with bHLH TFs 48 . Both, activators and repressors are known [bib_ref] MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during..., Medina-Puche [/bib_ref] [bib_ref] The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic..., Aharoni [/bib_ref]. The bHLH proteins have not been extensively studied in plants. Those that have been characterized function in anthocyanin biosynthesis, phytochrome signaling, globulin expression, fruit dehiscence, and carpel and epidermal development [bib_ref] Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry..., Schaart [/bib_ref] [bib_ref] Central role of FaGAMYB in the transition of the strawberry receptacle from..., Vallarino [/bib_ref]. Consequently, gene27422 could encode a bHLH TF regulating pigment formation in strawberry fruit. Recently, MYB10 (encoded by gene31413) was Scientific RepoRts | 7:45113 | DOI: 10.1038/srep45113 characterized as positive regulator of anthocyanin biosynthesis in F. × ananassa 50 , and F. vesca 54 . RNAi-mediated down-regulation of MYB10 resulted in significant reduction of anthocyanin concentration in ripe receptacle of red-fruited F. × ananassa, and F. vesca varieties, while over-expression resulted in dark red fruits [bib_ref] MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during..., Medina-Puche [/bib_ref] [bib_ref] Engineering the anthocyanin regulatory complex of strawberry (Fragaria vesca), Lin-Wang [/bib_ref] [bib_ref] An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic..., Lin-Wang [/bib_ref]. Furthermore, recent transcriptomic and SNP variant analysis revealed a single amino acid change in the MYB10 protein of the white-fruited varieties YW and HW4 that could be responsible for the loss-of-colour phenotypes [bib_ref] Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow..., Hawkins [/bib_ref]. Our data showed that MYB10 transcripts were more abundant in ripe receptacles of the white-fruited varieties YW and HW4 than in red-fruited RdV (Supplemental [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref] contradicting the observation that MYB10 is not differentially expressed in YW in comparison to the red-fruited F. vesca variety Ruegen 57 . Although MYB10 seems to be an important regulator of anthocyanin biosynthesis in receptacle, our data indicated that MYB10 might also have a significant role in ripe achenes due to the high transcript level. However, metabolic and transcriptional variations in fruit of natural white-fruited Fragaria vesca genotypes were already found at early developmental stages where MYB10 is almost not transcribed. Therefore, additional transcriptions factors might account for these differences. MYB1, a transcriptional repressor in regulating the biosynthesis of anthocyanins in strawberry 29,51,58 , was not among the differentially expressed genes (Supplemental File S1). This indicates that MYB1 might not be the anthocyanin biosynthesis repressor responsible for the loss-of-color phenotype in the white-fruited F. vesca genotypes.
Amongst the differentially expressed genes [fig_ref] Table 2: Fold change [/fig_ref] are candidates featuring comparable RPM levels in both white genotypes, such as gene01839 and gene13191. In contrast, other candidates showed diverging levels, such as gene30676 and gene30960. This indicates that in addition to metabolic differences among the two white genotypes [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref] , also transcriptional differences can be found.
The general biosynthesis pathway of anthocyanins has been thoroughly investigated at both, the biochemical and the genetic level, in particular in A. thaliana and Vitis sp. [bib_ref] New insights into the regulation of anthocyanin biosynthesis in fruits, Jaakola [/bib_ref] [bib_ref] Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology, Cheynier [/bib_ref]. Also in the Fragaria genus the key flavonoid pathway genes have been cloned, and their encoded proteins functionally characterized [bib_ref] Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis..., Almeida [/bib_ref]. Enzymatic analysis of [bib_ref] Two-Phase Flavonoid Formation in Developing Strawberry (Fragaria× ananassa) Fruit, Halbwirth [/bib_ref]. The high activity at the immature stage corresponds to the formation of flavanols, while the second peak is clearly related to anthocyanin and flavonol accumulation [bib_ref] Two-Phase Flavonoid Formation in Developing Strawberry (Fragaria× ananassa) Fruit, Halbwirth [/bib_ref]. According to our data, a biphasic transcript expression pattern for the flavonoid pathway genes was not observed . Instead, genes could be grouped into classes according to their expression profiles in receptacle and achenes. Transcripts of key genes of the phenylpropanoid pathway (PAL, CA4H, and 4CL) were highly abundant in immature seeds of F. vesca, and their expression profile suggests a coordinated transcriptional regulation in receptacles and achenes during fruit ripening . At the gateway of primary metabolism PAL, CA4H, and 4CL play a pivotal role as they are producing the precursor molecules of all polyphenols, including lignin. High degree of coordination in the overall expression of these three genes has been shown in parsley leaves, and cell cultures treated with UV light or fungal elicitor [bib_ref] Modes of expression and common structural features of the complete phenylalanine ammonia-lyase..., Logemann [/bib_ref]. The gene expression profile of FLS and a putative flavonoid GT (gene30947 FGT) matched the transcript expression pattern of the phenylpropanoid genes, but act more downstream in the flavonoid pathway . The fruit ripening program in red-fruited RdV is characterized by down-regulation of PAL, CA4H, and 4CL in achenes of the intermediate developmental stage, which did not occur in seeds of YW and HW4. Thus, in immature fruit the early polyphenol biosynthesis pathway is already differently regulated in the red-and white-fruited genotypes. Similarly, flavonoid genes (CHS, CHI, F3H, DFR, and ANS) were coordinately expressed in a spatial and temporal manner . They are involved in the supply of precursor molecules for proanthocyanidin, flavonoid, and anthocyanin production. In apple fruit, the anthocyanin biosynthetic genes, CHS, F3H, DFR, and ANS, are coordinately expressed during red coloration in skin, and their levels of expression are positively related to anthocyanin concentration [bib_ref] Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin, Honda [/bib_ref]. Transcript levels of the flavonoid genes were particularly abundant in green receptacle of YW in comparison to HW4 and might, therefore, contribute to the high levels of flavonoids and proanthocyanidins in green pulp of YW [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref]. The differential expression of the anthocyanidin glucosyltransferase gene FaGT1 27 (gene12591), in the red-fruited and white-fruited F. vesca genotypes is striking . Similarly, white-colored grape cultivars appear to be lacking anthocyanins because of the absence of an anthocyanidin GT [bib_ref] Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera)..., Kobayashi [/bib_ref]. In apple fruits, the transcript expression level of an anthocyanidin GT is positively related to anthocyanin concentration, and the gene is coordinately expressed with CHS, F3H, DFR, and ANS during red coloration in apple skin [bib_ref] Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin, Honda [/bib_ref]. Moreover, late genes in the anthocyanin biosynthetic pathway are coordinately expressed during red coloration of litchi fruits, where low expression of DFR and GT result in absence or extremely low anthocyanin concentrations [bib_ref] Differential Expression of Anthocyanin Biosynthetic Genes in Relation to Anthocyanin Accumulation in..., Wei [/bib_ref]. Interestingly, the transcript expression pattern of the putative GST candidate gene (gene31672) matched exactly the expression of FaGT1 , emphasizing its putative role in anthocyanin transport. The transcript expression profile of FaGT2 (gene26265), a gene encoding a (hydroxyl)cinnamate GT [bib_ref] Characterization of a UDP-glucose:cinnamate glucosyltransferase from strawberry, Lunkenbein [/bib_ref] suggests that GT2 might contribute to the production of gallic acid glucose ester [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref] in early developmental stages and to the production of (hydroxyl) cinnamic acid glucose esters in stages [bib_ref] Formation of β -glucogallin, the precursor of ellagic acid in strawberry and..., Schulenburg [/bib_ref]. The formation of red pigments would require the maintenance of high expression levels of CHS, CHI, F3H, DFR, and ANS in the receptacle and achenes, as well as the down-regulation of PAL, CA4H, and 4CL in achenes of the intermediate ripening stage. In the white-fruited F. vesca genotypes, transcript expression profiles of the proanthocyanidin biosynthesis genes (F3′ H, ANR, and LAR) and the pattern of flavonoid genes (CHS, CHI, F3H, DFR, and ANS) were coordinately regulated, and their ripening program appears to be unable to switch from the biosynthesis of flavonoids and proanthocyanidins occurring at the early stage to the production of anthocyanins in later stages. The polyphenol biosynthesis pathway in fruit of HW4 seems to be disturbed at an even earlier stage, as proanthocyanidin biosynthesis genes are already weakly expressed in green receptacle of HW4.
The interaction of polyphenol metabolism and fruit flavor formation has been frequently demonstrated as phenolic compounds can act as precursors of flavor molecules [bib_ref] Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction..., Koeduka [/bib_ref]. Thus, expression profiles of functionally characterized flavor biosynthesis genes pinene synthase and hydroxylase 68 , AAT 69 , QR 70 , and eugenol synthase 67 were analyzed in the three genotypes . Transcripts of flavor genes were already abundant in red-fruited RdV at the intermediate fruit ripening stage, whereas in YW and HW4 mRNA of genes involved in flavor formation were only detectable at the ripe stage. It appears that the ripening program in the white-fruited genotypes is delayed, which is also supported by the comparison of the transcript profiles of the fruit softening genes pectin esterase [bib_ref] Pectin esterase gene family in strawberry fruit: study of FaPE1, a ripening-specific..., Castillejo [/bib_ref] , pectate lyase 72 , polygalacturonase [bib_ref] A fruit-specific and developmentally regulated endopolygalacturonase gene from strawberry (Fragaria × ananassa..., Redondo-Nevado [/bib_ref] , and beta-galactosidase [bib_ref] Antisense down-regulation of the strawberry β -galactosidase gene Faβ Gal4 increases cell..., Paniagua [/bib_ref] in RdV and YW, as well as in HW4 . Overall, the flavor and softening genes seem to be coordinately regulated. It has been shown, that ripe fruits of red and white F. vesca varieties share most volatile organic compounds. Varying levels among the genotypes occur, but the main compounds such as esters presumably formed by AATs are present 75 .
# Material and methods
Plant Material. F. vesca Reine des Vallees (RdV), Yellow Wonder (YW) and Hawaii 4 (HW4) are three botanical forms of F. vesca, all of which produce small-sized plants and propagate without runners, except HW4. RdV has fruits with red flesh and red skin, whereas YW and HW4 fruits have both yellow flesh and skin [fig_ref] Figure 1: Fruits of F [/fig_ref]. The genetic and growth characteristics of YW and HW4 have been described [bib_ref] Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow..., Hawkins [/bib_ref] [bib_ref] Metabolic profiling of strawberry (Fragaria × ananassa Duch.) during fruit development and..., Zhang [/bib_ref] [bib_ref] Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development, Fait [/bib_ref]. Fruits were sampled between May and August 2014/2015, frozen in liquid nitrogen directly after harvest, and stored at − 80 °C until further usage [fig_ref] Figure 1: Fruits of F [/fig_ref].
## Chemicals.
Except where otherwise stated, chemicals were purchased from Sigma-Aldrich (Steinheim, Germany), Fluka (Steinheim, Germany) or Roth (Karlsruhe, Germany). where RNA sequencing and library preparation was carried out. The 3′ fragment cDNA library was generated through fragmentation of total RNA by ultrasound before poly(A)-tailed 3′ -RNA fragments were isolated using oligo-dT chromatography. Then, an RNA adapter was ligated to the 5′ -ends of the poly(A)-tailed RNA fragments. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and reverse transcriptase. The resulting cDNA was PCR-amplified using a high fidelity DNA polymerase. Each final cDNA library was purified, size selected, quantified and analyzed by capillary electrophoresis before RNA-Seq analysis was performed on an Illumina HiSeq2000 platform (Illumina Inc., USA). A PhiX library (Illumina Inc., USA) was added before sequencing to estimate the sequencing quality. Reads were processed by the CASAVA 1.8 package. Sequencing results are summarized in [fig_ref] Table 2: Fold change [/fig_ref].
## Rna-isolation, -quantity and -quality
Quality Trimming, Mapping and Data Normalization. RNA-seq data processing was performed on Galaxy, a free public server that was installed locally [bib_ref] Galaxy: a platform for interactive large-scale genome analysis, Giardine [/bib_ref] [bib_ref] Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research..., Goecks [/bib_ref] [bib_ref] Galaxy: a web-based genome analysis tool for experimentalists, Blankenberg [/bib_ref]. The Application Programming Interface (API) and the Galaxy Data Manager were used for automation of the pipeline analyses, and handling of built-in reference data [bib_ref] Wrangling Galaxy's reference data, Blankenberg [/bib_ref] , respectively. The bioinformatics tools were installed and organized via the Galaxy ToolShed 81 . http://www. rosaceae.org/Reads were trimmed using the Trimmomatic tool 82 with default settings for single end reads. The TruSeq3 adapters were removed in an initial ILLUMINACLIP step. Quality trimming was performed with a SLIDINGWINDOW step, and finally reads below 20 bp were discarded with a MINLEN step. Before and after trimming, the overall data quality was evaluated with the FastQC software (quality control tool for high throughput sequence data http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmed reads were aligned to the F. vesca reference genome (version 2.0.a1 downloaded from Genome Database for Rosaceae, GDR, www.rosaceae.org 83 ) with TopHat 84 using default settings. The results of the read mapping are summarized in [fig_ref] Table 3: Flavonoid genes differentially expressed in the red-and white-fruited genotypes and transcription factors... [/fig_ref]. Aligned reads were quantified using HTSeq-count 85 in "Union" mode for stranded reads with a minimum alignment quality of 10. The gene prediction input file was downloaded from GDR [bib_ref] Evolutionary origins and dynamics of octoploid strawberry subgenomes revealed by dense targeted..., Tennessen [/bib_ref]. As poly A-tail selection was performed after fragmentation of the RNA, reads were derived from only the 3′ ends of transcripts and normalization by gene length to Reads Per Kilobase of exon per Million mapped reads (RPKM) or Transcripts per Million (TPM) would be inappropriate. Instead, the raw read counts were normalized for library size using the DESeq2 R package [bib_ref] Differential expression analysis for sequence count data, Anders [/bib_ref] , and adjusted to per million scale (divided by total normalized counts for all samples, times 18 for sample number, times 1,000,000), to produce normalized rpm. Differential Expression. Deferentially expressed genes were defined using the general linear models in edgeR [bib_ref] edgeR: a Bioconductor package for differential expression analysis of digital gene expression..., Robinson [/bib_ref]. Specifically, models were fitted with a factor for tissue, stage, and mature color of genotype and likelihood ratio test was performed comparing the white genotypes (YW and HW4) to RdV. The false discovery rate (FDR) was calculated according to [bib_ref] Controlling the false discovery rate: a practical and powerful approach to multiple..., Benjamini [/bib_ref] and genes with FDR < 0.05 were considered significant. Accession numbers of flavonoid genes differentially expressed in the red-and white-fruited genotypes and transcription factors analyzed in this study are summarized in [fig_ref] Table 3: Flavonoid genes differentially expressed in the red-and white-fruited genotypes and transcription factors... [/fig_ref].
Global Sequencing Data Analysis. The data were analyzed in R (R Core Team, 2015), employing appropriate packages mostly accessed via the open source software framework Bioconductor 89 . Before the cluster dendrogram was generated, the dataset was transformed using variance stabilization [bib_ref] Variance stabilization applied to microarray data calibration and to the quantification of..., Huber [/bib_ref]. Subsequently, hierarchical clustering was performed using the complete method and Spearman distance metric. The PCA analysis was per- Metabolite Extraction. 50 mg of lyophilized fruit powder was weighed (n = 3-5). The resulting samples were extracted with 250 μ l of internal standard solution (0.2 mg ml −1 biochanin A and 4-methylumbelliferyl-β-D-glucuronide in methanol) and 250 μ l methanol. After vortexing (1 min), sonication (10 min), and centrifugation (10 min, 16,000 g) the supernatant was collected. The residue was re-extracted with 500 μ l methanol, and the supernatants were combined and dried in a vacuum concentrator. The secondary metabolites were re-dissolved in 50 μ l of water, vortexed, sonicated and centrifuged. The clear supernatant was used for LC-MS analysis.
Liquid chromatography-mass spectrometry (LC-MS). Levels of secondary metabolites were determined on an Agilent 1100 HPLC/UV system (Agilent Technologies, Germany) equipped with a reverse-phase column (Luna 3 u C18(2) 100 A, 150 × 2 mm; Phenomenex, Germany), a quaternary pump, and a variable wavelength detector. Connected to the HPLC was a Bruker esquire3000plus ion-trap mass spectrometer (Bruker Daltonics, Germany). HPLC and mass spectrometry were performed at optimized conditions [bib_ref] RNAi-induced silencing of gene expression in strawberry fruit (Fragaria × ananassa) by..., Hoffmann [/bib_ref] [bib_ref] Metabolic interaction between anthocyanin and lignin biosynthesis is associated with peroxidase FaPRX27..., Ring [/bib_ref]. Resulting data were analyzed with Data Analysis 5.1 software (Bruker Daltonics, Germany), and metabolites were identified using the in-house database. Levels (per mil equivalents of the dry weight, ‰ equ. dw.) of secondary metabolites quantified during targeted analyses are summarized in Untargeted Metabolite Data Analysis. Untargeted metabolite profiling analysis of the LC-MS data set was done according to published reports [bib_ref] XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment,..., Smith [/bib_ref] [bib_ref] Highly sensitive feature detection for high resolution LC/MS, Tautenhahn [/bib_ref] [bib_ref] Correction of mass calibration gaps in liquid chromatography-mass spectrometry metabolomics data, Benton [/bib_ref] in R [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref]. Peaks were grouped together across samples after correction of retention time deviations. After integration of the peak areas, the Wilcoxon Rank-Sum Test was used to determine differences across genotypes (RdV, YW, and HW4). Only metabolites with a p-value ≤ 0.01 were used for computation of subsequent data analysis. The secondary metabolites were quantified according to the internal standard method [bib_ref] Metabolic interaction between anthocyanin and lignin biosynthesis is associated with peroxidase FaPRX27..., Ring [/bib_ref] , and the values are expressed as per mil equivalent of the dry weight (‰ equ. dw). Hierarchical clustering and PCA analysis were generated by the same R packages used for the sequencing data 91 .
Real-time RT-PCR analysis. The same total, DNAse I treated RNA used for RNA-sequencing, was also used to confirm candidate gene expression by RT-PCR analysis. First strand cDNA synthesis was performed in 20 μ l reactions, containing 1 μ g of total RNA template, 10 μ M of Oligo (dT) 15 primers, and 200 U M-MLV reverse transcriptase (both Promega, Mannheim, Germany) according to the manufacturer's instructions. Analyses were carried out on a StepOnePlus TM System (Applied Biosystems TM , ThermoFisher Scientific, Waltham, US-MA) equipped with StepOne TM software v2.1. For each PCR reaction (10 μ l in total), 2 μ l cDNA, 400 nM primers, and 5 μ l 2x master mix (SensiFast TM SYBR Hi-Rox Kit, Bioline,) were used. Prior to gene expression analysis, PCR reactions were optimized in cDNA concentration, primer concentration, and annealing temperature (FvGT1 gene12591: 1x cDNA, 61 °C; FvMYB10 gene31413: 0.1x cDNA, 57 °C; FvUBC9 gene12591: 0.1x cDNA, 55 °C; 400 nM primers for all three genes). The efficiency of each primer pair was determined using the standard curve of a serial cDNA dilution. Several possible reference genes from literature were tested, but in the end only FvUBC9 was suitable, amplified by a primer set according to literature [bib_ref] Engineering the anthocyanin regulatory complex of strawberry (Fragaria vesca), Lin-Wang [/bib_ref]. It was, however, differentially expressed between achenes and receptacle, but showed uniform levels within the respective tissue [fig_ref] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry... [/fig_ref]. Achenes and receptacle samples were, consequently, normalized separately. The cycling program was 2 min at 95 °C, followed by 40 cycles of 5 sec at 95 °C, 10 sec at 55-61 °C, and 20 sec at 72 °C, and ending in a melting curve detection of 15 sec at 95 °C, 1 min at 60 °C, and 15 sec at 95 °C. Analyses were performed in triplicates. Relative quantification was performed according to [bib_ref] A new mathematical model for relative quantification in real-time RT-PCR, Pfaffl [/bib_ref] using UBC9 as reference gene.
[fig] Figure 1: Fruits of F. vesca variety Reine des Vallees (A), Yellow Wonder (B) and Hawaii 4 (C) of the ripening stages ripe (left), intermediate (middle) and green (right). Cross-section of a green fruit of F. vesca Hawaii 4 (D), with arrows indicating the tissues (receptacle, achenes) separated before RNA and metabolite extraction. Scale bars = 5 mm. Scientific RepoRts | 7:45113 | DOI: 10.1038/srep45113 [/fig]
[fig] Figure 2: Untargeted analysis of secondary metabolites in receptacle and achenes of strawberry varieties RdV, YW, and HW4 of three developmental stages (green, intermediate, and ripe). 3-5 biological replicates per data point. The variance in the data is depicted by principal component analysis (PCA). Scientific RepoRts | 7:45113 | DOI: 10.1038/srep45113 [/fig]
[fig] Figure 3: Quantification of secondary metabolites by LC-MS in achenes and receptacle of strawberry fruits. Heatmaps show levels that are expressed as per mil equivalents of the dry weight (relative concentration), with lowest levels shown in blue, and highest levels in red. Individual min. and max. values are given in parentheses. (A) Colour code presentation of metabolite levels in achene (Ac) tissues of different ripening stages (green, intermediate, ripe), and F. vesca varieties (RdV, YW, and HW4). (B) Metabolite levels in receptacle (Rc) tissues. n.d. not detected. Three to five replicates were analyzed. and ANS formed the second group. Transcript abundance of these genes was high in achenes and receptacles of RdV of all developmental stages but low in fruit of YW and HW4, except for green achenes and receptacle, and intermediate receptacle. Gene transcript levels of the first two groups differed considerably between the red-and white-fruited genotypes in achenes and receptacle at the intermediate (and ripe) developmental stage. However, the most significant difference was observed for the mRNA abundance of the anthocyanidin glucosyltransferase gene FaGT1 and an uncharacterized glutathione S-transferase gene. Both were exclusively expressed in fruit of RdV at the intermediate and ripe developmental stage. F3′ H, ANR, and LAR formed the fourth group. They were primarily expressed in the green fruit of the three genotypes, and in intermediate receptacle of HW4. The transcript profiles of F3′ H, ANR, and LAR in fruit of HW4 was clearly different from that of RdV and YW. Thus, gene expression perturbation of flavonoid pathway genes in HW4 occurs already in the green developmental stage. The glucosyltransferase gene FaGT2 was mainly expressed in green achenes of the three genotypes, and ripe receptacles of RdV. [/fig]
[fig] Figure 4: Global analysis of gene expression among samples and strawberry varieties. (A) Principle component analysis (PCA) of transcripts (top 500) in F. vesca varieties RdV, YW, and HW4, in two different tissues (achenes and receptacle) and of three ripening stages green, intermediate and ripe. (B) Cluster dendrogram showing global relationship among achenes samples. (C) Cluster dendrogram showing global relationship among receptacle samples. Scientific RepoRts | 7:45113 | DOI: 10.1038/srep45113 [/fig]
[fig] Figure 5 srep45113, Figure 6: Schematic illustration of the shikimate, ellagic acid, phenylpropanoid, flavonoid and anthocyanin pathway. Red dots indicate biochemically characterized enzymes in strawberry fruit: ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; CA4H, cinnamic acid 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; 4CL, 4-coumaroyl-CoA ligase; DFR, dihydroflavonol reductase; FGT, flavonoid glucosyltransferase; F3H, flavanone 3-hydroxylase; F3′ H, flavonoid 3′ -hydroxylase; FLS, flavonol synthase; GT1, anthocyanidin glucosyltransferase; GT2, (hydroxy)cinnamic acid and (hydroxy)benzoic acid glucosyltransferase; LAR, leucoanthocyanidin reductase; PAL, phenylalanine ammonia lyase. Blue dots indicate putative GST, glutathione S-transferase. Identical genes are connected by a dotted red line when adjacent. Enzymes shown in the same color are co-regulated. Heatmaps show relative transcript levels (% Max) of genes in receptacle (Rc) and achenes (Ac) of F. vesca RdV, YW, and HW4 at the green, intermediate and ripe developmental stage. Scientific RepoRts | 7:45113 | DOI: 10.1038/Transcript levels (normalized RPM) of genes encoding enzymes involved in fruit flavor formation (A) and softening (B) in receptacle (Rc) and achenes (Ac) of F. vesca RdV, YW, and HW4 at the green, intermediate (int) and ripe developmental stage. [/fig]
[table] Table 2: Fold change (logFC) of genes significantly up-regulated in both white genotypes F. vesca YW and HW4 compared to the red genotype RdV. [/table]
[table] Table 3: Flavonoid genes differentially expressed in the red-and white-fruited genotypes and transcription factors analyzed in this study. [/table]
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Development and validation of a tool to assess the physical and social environment associated with physical activity among adults in Sri Lanka
Background: Environmental characteristics are known to be associated with patterns of physical activity (PA). Although several validated tools exist, to measure the environment characteristics, these instruments are not necessarily suitable for application in all settings especially in a developing country. This study was carried out to develop and validate an instrument named the "Physical And Social Environment Scale -PASES" to assess the physical and social environmental factors associated with PA. This will enable identification of various physical and social environmental factors affecting PA in Sri Lanka, which will help in the development of more tailored intervention strategies for promoting higher PA levels in Sri Lanka. Methods: The PASES was developed using a scientific approach of defining the construct, item generation, analysis of content of items and item reduction. Both qualitative and quantitative methods of key informant interviews, in-depth interviews and rating of the items generated by experts were conducted. A cross sectional survey among 180 adults was carried out to assess the factor structure through principal component analysis. Another cross sectional survey among a different group of 180 adults was carried out to assess the construct validity through confirmatory factor analysis. Reliability was assessed with test re-test reliability and internal consistency using Spearman r and Cronbach's alpha respectively. Results: Thirty six items were selected after the expert ratings and were developed into interviewer administered questions. Exploration of factor structure of the 34 items which were factorable through principal component analysis with Quartimax rotation extracted 8 factors. The 34 item instrument was assessed for construct validity with confirmatory factor analysis which confirmed an 8 factor model (x 2 = 339.9, GFI = 0.90). The identified factors were infrastructure for walking, aesthetics and facilities for cycling, vehicular traffic safety, access and connectivity, recreational facilities for PA, safety, social cohesion and social acceptance of PA with the two non-factorable factors, residential density and land use mix. The PASES also showed good test re-test reliability and a moderate level of internal consistency. Conclusions: The PASES is a valid and reliable tool which could be used to assess the physical and social environment associated with PA in Sri Lanka.
# Background
The burden of mortality, morbidity and disability due to non-communicable diseases (NCDs) is high and is increasing in the developing countries. Physical inactivity is identified as the fourth leading risk factor for mortality due to NCDs and contributes to 6% of deaths globally. In 2001, 71% of all deaths in Sri Lanka were due to chronic NCDs and chronic NCD mortality is reported to be 20-30% higher in Sri Lanka than in many developed countries. According to the Annual Health Statistics, coronary heart disease was the leading cause of hospital deaths in Sri Lanka since 1997. The World Health Survey data collected in 2002-2003 revealed that in Sri Lanka 7.3% of the males and 13.8% of the females were physically inactive [bib_ref] Worldwide variability in physical inactivity: a 51-country survey, Guthold [/bib_ref].
Being physically active is influenced by both the physical and social environment [bib_ref] An ecological approach to creating active living communities, Sallis [/bib_ref] [bib_ref] Translating social ecological theory into guidelines for community health promotion, Stokols [/bib_ref] and is best explained through a socio-ecological model of health related behaviours. Many studies have recognized that environmental factors have a significant role in promoting PA among adults [bib_ref] Associations between self-reported and objective physical environmental factors and use of a..., Troped [/bib_ref] [bib_ref] Critical assessment of the literature on the relationships among transportation, land use,..., Handy [/bib_ref] [bib_ref] Understanding environmental influences on walking: review and research agenda, Owen [/bib_ref] [bib_ref] Environmental determinants of physical activity and sedentary behavior, Owen [/bib_ref] [bib_ref] Environmental correlates of walking and cycling: findings from the transportation, urban design..., Saelens [/bib_ref] [bib_ref] Perceived environment and physical activity: a meta-analysis of selected environmental characteristics, Duncan [/bib_ref] and changing behaviours in an entire community. Literature identifies some common physical (built) environment factors associated with PA. They are landuse patterns, transport systems, urban design, green space, availability of pavements, heavy traffic, street lights, unattended dogs, enjoyable scenery, high levels of crime, and easy access to recreation and retail shops [bib_ref] Multiple impacts of the built environment on public health: walkable places and..., Frank [/bib_ref] [bib_ref] Associations of perceived social and physical environmental supports with physical activity and..., Addy [/bib_ref]. Income, equity, culture and social support are identified in literature as elements in the social environment that influences participation in PA [bib_ref] Environmental, psychological, and social influences on physical activity among Japanese adults: structural..., Ishii [/bib_ref] [bib_ref] The importance of the social environment for physically active lifestyle-results from an..., Stahl [/bib_ref].
Considering the apparent importance of the environment for PA, there is limited information in the literature on how best to measure various aspects of the environment. Evidence on the associations between the physical environment and PA behaviour is derived mostly from self-reported data on individuals' perceptions of their environments [bib_ref] Environmental factors associated with adults' participation in physical activity: a review, Humpel [/bib_ref]. Observational methods is another form where individuals using checklists, rates the environment. The introduction of geographic information systems into PA research has revolutionised the measurement of the physical environment, and is still in its early stages [bib_ref] Measuring physical activity environments-a brief history, Sallisj [/bib_ref]. Two major types of PA that have been studied in relation to the environment are the recreational PA and PA through non motorized transportationwalking/cycling. An accepted method of measuring the perceived physical environment is through population based studies and surveillance systems [bib_ref] Measuring the determinants of physical activity in the community: current and future..., Baker [/bib_ref]. Individual responses can then be aggregated to identify patterns in environment characteristics. Thereafter, it is possible to determine the association between the design characteristics of the environment and behaviour [bib_ref] Environmental and policy determinants of physical activity in the United States, Brownson [/bib_ref]. There are different tools developed for assessment of environment characteristics that are related to different types of PA. Abbreviated Neighbourhood Environment Walkability Scale (ANEWS) and the International Physical Activity Questionnaire-environmental (IPAQ-e) are two tools that have been extensively used. ANEWS, a 98 question, selfadministered instrument to determine the perception of neighbourhood design features hypothesised to be related to PA [bib_ref] Measuring the environment for friendliness towards physical activity: a comparison of the..., Brownson [/bib_ref] was developed in San Diego. It consists of six subscales of land use mix-access, street connectivity, infrastructure for walking/cycling, aesthetics, traffic safety and crime safety [bib_ref] Neighbourhood environment walkability scale: validity and development of a short form, Cerin [/bib_ref]. IPAQ-e is a 17 item, 4 factor tool which is considered to be relevant to all countries regardless of the stage of economic development [bib_ref] Test-retest reliability of IPAQ environmental-module in an African population, Adegoke [/bib_ref] , with the factors being the degree of urbanisation, traffic intensity, aesthetics and opportunity and fear of crime [bib_ref] The association between health enhancing physical activity and neighbourhood environment among Swedish..., Bergman [/bib_ref]. There are several other tools which have been developed in America, Europe and Australia to measure the environment associated with PA. All the above mentioned instruments are known to have an average interviewer administration time of 24-30 minutes [bib_ref] Measuring the environment for friendliness towards physical activity: a comparison of the..., Brownson [/bib_ref]. Although tools to assess physical and social environment associated with PA in adults have been developed, validated and utilized extensively in developed countries, South Asia and particularly Sri Lanka lacks a validated tool to assess physical and social environment associated with PA. The purpose of conducting this study was to develop and pilot a tool to assess the physical and social environment associated with PA among adults in the district of Colombo and to assess the construct validity and reliability of it.
# Methods
## Study setting
This study was conducted in the district of Colombo in the western province of Sri Lanka which encompasses the economic capital of Sri Lanka. It extends over an area of 696 square kilometers with a population of 2,390,871 and a population density of 3330 persons per square kilometre.
Arriving at a definition for the physical and social environment associated with PA Physical and social environment associated with PA was defined initially by considering the definitions given by different authors through a literature search carried out on medical sciences, urban development and design, transport studies and social sciences publications. These definitions were then reviewed with several experts in the fields of community medicine, environment studies, urban planning and architecture, engineering including transport engineering, sociology, health promotion, sports medicine and psychology. The most suitable definition was formulated based on the outcome of the above process.
## Item generation
Item generation was initiated with a literature review which was conducted to identify all aspects of the physical and social environment relevant to PA behaviour among adults in urban and rural communities. Experts who had developed similar questionnaires in the developed countries were contacted. After reviewing several different instruments, all the items used were identified, listed and adopted in a culturally acceptable manner.
Thereafter key informant interviews were conducted with the above mentioned experts to generate items. A purposive sampling method was adopted to select the key informants. This was complimented with in-depth interviews with the general public between the ages of 20-59 years living in the Colombo district. Fifteen indepth interviews were carried out using an interviewer guide. Notes were taken after prior permission. Transcripts of the interviews were made and were coded to identify the main items.
## Analysis of the content of items and item reduction
The items were rated by the experts on a five-point scale (1-least important and 5-most important). An item with a mean score of 3 or more was considered for inclusion in the next round. Items were finalized after two iterations of independent ratings by experts.
Formulating draft instrument to measure physical and social environment associated with PA and translation An interviewer administered questionnaire was developed conferring to the measures described by Streiner and Norman. A direct continuous judgment scale with 5 response choices was adopted. The scoring was a simple scoring with scores ranging from 1 to 5. The lowest value 1 indicated the least likelihood of having a conducive environment for PA whereas the highest value 5 indicated the most likelihood of having a conducive environment for PA. The question on residential density and distance to facilities was assessed differently according to the consensus of experts.
The draft instrument developed in English was independently translated to Sinhala by two translators, with a high level of proficiency in English and Sinhala. This was back translated to English and was checked with the original English version and necessary modifications were carried out.
## Finalising the pases with exploratory factor analysis
In order to assess how the selected 36 items were related to each other, and to see if there was a need for further reduction of items, exploratory factor analysis was carried out using the 34 factorable items. Two items, residential density and land use mix were not included, for the reason that the response categories of these two items were not appropriate to be included in exploratory factor analysis.
Adults aged 20-59 years living in the Colombo district for a period of not less than 6 months were invited to assess their environment using the developed instrument. Institutionalised adults, adults with any physical disability preventing engagement in PA, pregnant females up to a postpartum period of 3 months, adults with severe psychiatric illness and adult visitors to the area were excluded from the study. One hundred and eighty people were interviewed which is more than the recommendation (5 participants per item in the tool) for the sample size in multivariate analysis. A trained interviewer visited the households during weekends to collect data. Data collection was done after information on the purpose of the study was given and written consent was obtained from the selected participant.
Principal Component Analysis (PCA) was applied using Statistical Package for Social Sciences version 17 to explore the factor structure. After assessing the sampling adequacy and factorability, those factors with eigen values of more than one were selected. Scree plots were examined and factors were rotated to optimize the interpretability of the scale. A pre-test was carried out prior to finalizing the instrument -PASES. The PASES is shown as Additional file 1.
# Confirmatory factor analysis
Confirmatory factor analysis (CFA) was carried out to assess the extent to which the underlying eight factor model was replicated in a new data set. Although many factors including size of the model, distribution of variables, amount of missing data, reliability of the variables and strength of the relationship among variables affect the sample size, a recommended sample size for CFA is more than 5 times the number of items in the instrument. The instrument was administered to a different group of 180 adults between the ages of 20-59 years living in the Colombo district for a period of not less than 6 months with exclusion criteria similar as above. Data collection too was similar to the data collection procedure for PCA. Data on basic socio demographic and PA were also obtained.
The 34 factorable items were deployed for CFA using LISREL 8.8. after ensuring that the statistical assumptions required for CFA was met. Normality of the data was assessed by inspecting item histograms and calculating the standardized skewness and kurtosis. Multicollinearity was explored through bivariate correlations between the items.
Considering the non-normal distribution of the items of PASES and according to the recommendations offered in LISREL 8.8 [bib_ref] LISREL 8: User' s reference guide, Jöreskogk [/bib_ref] , Robust Maximum Likelihood (RML) estimation method with the Satorra-Bentler scaled chi-square for fit estimation was used for the CFA. The CFA was performed on the covariance matrix of the items of the PASES. Assessment of the appropriateness of the models was based on several fit indices. The absolute fit indices considered were the χ2 with degrees of freedom (df ) and the P value, Goodness of fit index (GFI) and the standardized root mean square residual (SRMR). The Parsimony correlations that were assessed were the root mean square error of approximation (RMSEA). Comparative fit indices that were used were the comparative fit index (CFI) and the Non-normed Fit Index (NNFI). The judgments about how well the model fit data in this study were made on the basis of RMSEA < 0.05, SRMR < 0.08 CFI > 0.90 and NNFI > 0.90 which have been adopted when assessing model fit in similar situations [bib_ref] Scale development: factors affecting diet, exercise, and stress management (FADESM), Chang [/bib_ref]. The reliability of the instrument was assessed by test re-test method, by administering the same questionnaire to 50 randomly selected participants after a two week interval. Internal consistency which is a measure of "item homogeneity" was assessed by calculating Cronbach's Alpha for each sub-division score of the PASES.
The Ethics Review Committee of Faculty of Medicine, University of Colombo approved the study protocol (reference number EC-09-084) and data collection was carried out after obtaining informed written consent from each participant.
# Results
Arriving at a definition for the physical and social environment associated with PA.
For the purpose of this study, physical and social environment associated with PA was defined as the "external context consisting of the characteristics of the natural and built environment and the characteristics of the people in the neighbourhood which influences participation in PA".
## Item generation
The literature review, key informant interviews and indepth interviews generated 80 items which was reduced to 36 after independent rating by the experts.
## Finalising the pases with exploratory factor analysis
The sample for the survey to carry out exploratory factor analysis consisted of, 52% males and 48% females. Twenty eight percent of the participants were educated up to G.C.E. O/L or less, while the majority had education above G.C.E. O/L. Majority (84%) of the participants were employed. The sampling adequacy was assessed through inspection of the inter correlation matrix which showed that there were many correlations that were more than 0.3. In the anti-image correlation matrix the coefficients were well above the accepted level of 0.5. Factorability of the data assessed by Bartlett's test of sphericity, was significant at p < 0.01. The Kaiser-Meyer-Olkin measure was 0.742 which was well above the requirement of 0.6. The items which grouped together were identified as latent factors and were considered relevant only if its eigen value exceeded 1.0. PCA with Quartimax rotation technique gave the best results. [fig_ref] Table 1: Item distribution in PCA [/fig_ref] shows the factor coefficients of individual items after rotation. Eigen values ranged from 7.18 to 1.16. All of the items loaded well to the factors (factor loading >0.4), requiring no further reduction of items. This method initially identified 9 latent factors with one factor retaining only one item. However, as this item also cross loaded with another factor with factor loading of >0.4 and as the cross loading appeared sensible the 8 factor model was selected as the final model after PCA. The factors identified were named as follows:
1 Infrastructure for walking 2 Aesthetics and facilities for cycling 3 Vehicular traffic safety 4 Access and connectivity 5 Recreational facilities for PA 6 Safety 7 Social cohesion 8 Social acceptance of PA The PASES was validated using CFA for Sri Lanka after the pre-test.
# Confirmatory factor analysis
The response rate of the cross sectional survey was 100%. The socio demographic characteristics of the sample of 180 adults are given in. The standardised skewness and kurtosis was calculated by dividing the unstandardised skew or kurtosis by its corresponding standard error, which is interpreted as the z test of skew or kurtosis. The ratios greater than 1.96 and have a p value of 0.05, indicate significant skew or kurtosis. The values show that in this sample 22 items had high standard skewness while 10 items had high standard kurtosis. Items in the model should not be highly correlated or perfectly correlated because multicollinearity hinders the interpretability of the results. When the bivariate correlations between the items were examined, although the highest correlation observed between two items was 0.78, 95% of the correlations were less than 0.06 showing that no two items were highly correlated or perfectly correlated. Therefore several models were evaluated using RML method and were assessed for fit indices.
Initially a 2 factor model was tested where all items in the physical environment were grouped to one and those of the social environment were grouped to another. This model failed to converge and did not show acceptable fit. Thereafter a 6 factor model was tested with 'infrastructure for walking' and 'access and connectivity' combined as one . A seven factor model was also tested with item of 'infrastructure for walking' and 'access and connectivity' combined as one factor. This model showed an acceptable fit with a chi square value of 360 (df = 506). Another 7 factor model with 'social cohesion' and 'social acceptance of PA' combined together as one factor increased the chi square value to 397 (df = 506). A 8 factor model was tested according to factors derived from PCA. This model showed a better fit with a chi square value of 339.94 (df = 499), p value of 1.00, GFI of 0.90, and RMSEA of 0.001. The summary findings of the model fit statistics of different models are shown in the [fig_ref] Table 3: Summary of model fit statistics of the PASES χ2 = Chi-square test [/fig_ref]. Therefore the 8 factor model was accepted as the best fit model.
The results of the test re-test reliability assessed through Spearman's r coefficients are given in [fig_ref] Table 4: Test re-test reliability of the mean scores of 8 factors of the... [/fig_ref]. The correlation scores ranged from 0.628-0.916. The lowest correlation 0.628 was in the domains of aesthetics and recreational facility. The internal consistency measured by Chronbach's Alpha for the physical environment was 0.49, while for the social environment it was 0.82. Both values were significant at p < 0.01 level.
# Discussion
An active lifestyle is a complex behavioural process that is influenced by various factors of which environmental factors are well recognized [bib_ref] Environmental factors associated with adults' participation in physical activity: a review, Humpel [/bib_ref] [bib_ref] Environmental influences on food choice, physical activity and energy balance, Popkin [/bib_ref]. This study was designed to develop a valid and reliable tool to assess the physical and social environment associated with PA in the Colombo district, considering the socio-ecological model for PA behaviour.
The procedure adopted to develop PASES was similar to the procedures used for the development of many other study instruments to assess the physical and social environment for PA in other countries [bib_ref] Constructing indices representing supportiveness of the physical environment for walking using the..., Mccormack [/bib_ref] [bib_ref] Assessment of environmental correlates of physical activity: development of a European questionnaire, Spittaels [/bib_ref]. The steps included: defining the construct, item generation, analysis of the content of items and item reduction, field testing and validation of the developed instrument [bib_ref] The sequential approach to measurement of health behavior constructs: issues in selecting..., Redding [/bib_ref] [bib_ref] Development of the rural active living assessment tools: measuring rural environments, Yousefian [/bib_ref]. Both the quantitative and qualitative research methods provided comprehensive methodologies for exploration of ideas [bib_ref] The sequential approach to measurement of health behavior constructs: issues in selecting..., Redding [/bib_ref] , including key informant interviews and in-depth interviews. Item reduction initially was through a simple and non statistical method where a group of experts rated the importance of each item for the appropriateness of the item to the main construct independently. This method facilitated uninfluenced views of each expert as they did not meet each other [bib_ref] R: Indicators of organizational readiness for clinical information technology/ systems innovation: a..., Snyder-Halpern [/bib_ref]. According to guidelines of developing new instruments [bib_ref] The sequential approach to measurement of health behavior constructs: issues in selecting..., Redding [/bib_ref] , PCA was carried out on a data set that was gathered by administering the translated items to a group of people considered to be similar to the population that the developed instrument was intended to be used [bib_ref] Perceived characteristics of the environment associated with active travel: development and testing..., Ogilvie [/bib_ref]. PCA explored the factor structure of the scale and showed that all the items loaded well (factor loadings >0.4) to 8 factors. Hence, no further reduction of data was required through PCA. CFA on the multi-dimensional construct showed adequate model fit despite emergence of 8 latent factors for the 34 items. Although the PASES was a 8 factor model, the ANEWS had a 6 factor structure after CFA [bib_ref] Residents' perception of walkability attributes in objectively different neighbourhoods: a pilot study, Lesliee [/bib_ref] and the IPAQ-e module had 4 factors after PCA [bib_ref] The association between health enhancing physical activity and neighbourhood environment among Swedish..., Bergman [/bib_ref]. This was expected as some of the items in the three instruments differed. The PASES had two social factors while the ANEWS and IPAQ-e had no social factors.
In the reliability assessment, the Chronbach's Alpha for physical environment sub-division was 0.487 and for the social environment subdivision was 0.823, indicating a moderate reliability. However, the reliability findings of the present study are comparable with most of reliability tests carried out on environment assessment tools [bib_ref] Neighbourhood-based differences in physical activity: an environment scale evaluation, Saelens [/bib_ref] [bib_ref] Prediction of leisuretime walking: an integration of social cognitive, perceived environmental and..., Rhodes [/bib_ref]. The test re-test reliability in the present study was between 0.6-0.9, confirming the ability of the PASES to generate reproducible results.
The factors identified and validated were comparable with other tools developed in the USA and Europe, with some variation. Infrastructure for walking has been a common factor identified in many instruments [bib_ref] Measuring the environment for friendliness towards physical activity: a comparison of the..., Brownson [/bib_ref] and have shown to be associated with the total PA and walking both for leisure and transport [bib_ref] Perceived environment and physical activity: a meta-analysis of selected environmental characteristics, Duncan [/bib_ref] [bib_ref] How the built environment affects physical activity: views from urban planning, Handy [/bib_ref]. Items relating to access and connectivity and the infrastructure for walking were seen to load together as 'degree of urbanisation' [bib_ref] The association between health enhancing physical activity and neighbourhood environment among Swedish..., Bergman [/bib_ref] in the IPAQ-e module but the PASES identified it separately as in the ANEWS [bib_ref] Neighbourhood environment walkability scale: validity and development of a short form, Cerin [/bib_ref]. Aesthetics and facilities for cycling were identified as one factor in the PASES as the perception of the beauty of the environment and facilities for cycling were perceived in a similar manner in the Sri Lankan setting. However aesthetics were a separate factor in both the ANEWS and IPAQ-e tools. Vehicular traffic safety, and safety which are important factors in the environment, were identified in the newly developed and validated tool, with additional factor of 'recreational facilities for PA'. Two social factors, namely social cohesion and social acceptance of PA were identified as important and were incorporated to the PASES. Although not factor analysed 'residential density' and 'land use mix diversity' were components of the newly developed tool PASES. These two components measure proximity, indicating how close different travel destinations are to one another in space. Density indicates the concentration of people, dwelling units or households [bib_ref] How the built environment affects physical activity: views from urban planning, Handy [/bib_ref] and mixture of use of land refers to the spatial placement of different types of land uses (industrial, residential, commercial). The factors identified in the PASES were in some ways similar to other environmental assessment tools with some variation which might reflect the Asian setting.
However, it should be noted that findings of factor analysis assessments are often sample specific [bib_ref] Sample size in factor analysis, Maccallum [/bib_ref] and generalizability of these findings to other populations would depend on their similarity to this study population. The specific "aspects" that could be studied may vary from within and between countries. This study instrument could be used to assess the physical and environmental factors associated with PA in South Asia and other parts of Sri Lanka after testing for reliability and validity in that particular setting.
# Conclusion
A valid and reliable tool to assess the physical and social environment associated with PA was developed in Sri Lanka. This work contributes to a set of tools which can be used by researchers to identify the current perception of the environment for participation in PA by the community and to assess any change in the perception with interventions and with time.
[table] Table 1: Item distribution in PCA (the highest correlation coefficient for the item is in bold) [/table]
[table] Table 3: Summary of model fit statistics of the PASES χ2 = Chi-square test. p = (>0.05 desired). GFI = goodness of fit index (>0.9 desired). SRMR = standardized root mean square residual (<0.08 desired). NNFI = Non-normed fit index (>0.9 desired). CFI = comparative fit index (>0.9 desired). RMSEA = root mean square error of approximation (<0.05 desired). [/table]
[table] Table 4: Test re-test reliability of the mean scores of 8 factors of the PASES [/table]
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Self-Sensing of Damage Progression in Unidirectional Multiscale Hierarchical Composites Subjected to Cyclic Tensile Loading
The electrical sensitivity of glass fiber/multiwall carbon nanotube/vinyl ester hierarchical composites containing a tailored electrically-percolated network to self-sense accumulation of structural damage when subjected to cyclic tensile loading-unloading is investigated. The hierarchical composites were designed to contain two architectures differentiated by the location of the multiwall carbon nanotubes (MWCNTs), viz. MWCNTs deposited on the fibers and MWCNTs dispersed within the matrix. The changes in electrical resistance of the hierarchical composites are associated to their structural damage and correlated to acoustic emissions. The results show that such tailored hierarchical composites are able to self-sense damage onset and accumulation upon tensile loading-unloading cycles by means of their electrical response, and that the electrical response depends on the MWCNT location.
# Introduction
With the increased use of fiber-reinforced polymer composites (FRPCs) for structural applications, such as aerospace, marine, wind turbine, and automotive industries, the interest in developing structural health monitoring (SHM) techniques that ensure a safe structural performance of the composite has also increased [bib_ref] Evaluation (NDE) of Polymer Matrix Composites, Non-Destructive [/bib_ref]. Failure mechanisms of FRPCs are more complex than those of monolithic materials, such as metals or ceramics, given the contrasting mechanical properties between the matrix and fibers, the existence of an interface, the reinforcement orientation, and the manufacturing defects, to name a few. Traditional SHM methods used for periodic inspections of composite structures, such as ultrasonics, X-ray radiography, infrared thermography, holographic interferometry, and eddy currents require extensive human involvement and expensive procedures, becoming more difficult to implement for in situ SHM. A promising SHM approach that can overcome these issues consists in making the composite electroconductive in such a way that, for an applied stress/strain, the composite experiences a change in its electrical conductivity. An advantage of this SHM technique is that the composite itself is capable of tracking its own damage progression (i.e., embedment of external sensors is not required) and hence, does not suffer from problems associated with stress concentrations (as for common embedded sensors). In addition, this method allows the entire structure to be monitored [bib_ref] Sensing of Damage Mechanisms in Fiber-Reinforced Composites under Cyclic Loading using Carbon..., Gao [/bib_ref] [bib_ref] Damage Detection Using Self-Sensing Concepts, Chung [/bib_ref] [bib_ref] Coupled Carbon Nanotube Network and Acoustic Emission Monitoring for Sensing of Damage..., Gao [/bib_ref]. This technique also circumvents the issue of sensitivity to damage accumulation of acoustic emission (AE) by using the residual electrical resistance after unloading the structure. Electrical resistance measurements have been widely used to detect damage in carbon fiber-reinforced composites [bib_ref] Damage Detection Using Self-Sensing Concepts, Chung [/bib_ref] [bib_ref] Load and Failure Analyses of CFRP Laminates by Means of Electrical Resistivity..., Schulte [/bib_ref] [bib_ref] Self-monitoring of Fatigue Damage and Dynamic Strain in Carbon Fiber Polymer-Matrix Composite, Wang [/bib_ref] [bib_ref] Delamination Monitoring of Graphite/Epoxy Laminated Composite Plate of Electric Eesistance Change Method, Todoroki [/bib_ref] [bib_ref] Continuous Carbon Fibre Epoxy-Matrix Composite as a Sensor of its Own Strain, Wang [/bib_ref]. Due to their inherent electrical conductivity, breakage of the load-carrying carbon fibers result in changes in the composite electrical conductivity [bib_ref] Damage Detection Using Self-Sensing Concepts, Chung [/bib_ref] [bib_ref] Load and Failure Analyses of CFRP Laminates by Means of Electrical Resistivity..., Schulte [/bib_ref] [bib_ref] Self-monitoring of Fatigue Damage and Dynamic Strain in Carbon Fiber Polymer-Matrix Composite, Wang [/bib_ref] [bib_ref] Delamination Monitoring of Graphite/Epoxy Laminated Composite Plate of Electric Eesistance Change Method, Todoroki [/bib_ref]. Many works have been conducted to improve the understanding and interpretation of this approach. For instance, Abry et al. [bib_ref] Situ Detection of Damage in CFRP Laminates by Electrical Resistance Measurements, Abry [/bib_ref] measured the longitudinal and transverse changes in electrical resistance of unidirectional carbon fiber/epoxy specimens loaded in tension; given material anisotropy, the electrical resistance response under tension depends also on the fiber orientation. The authors found that when the composite is loaded in tension along the fiber direction, the changes in electrical resistance are dominated by fiber breakage, while when it is loaded transversally, the electrical response is due to the loss of contacts among the adjacent fibers. To extend this concept to different loading scenarios, the group led by Deborah Chung monitored the electrical resistance changes for tensile monotonic and cyclic, flexural, and impact loadings in carbon fiber/epoxy composites [bib_ref] Damage Detection Using Self-Sensing Concepts, Chung [/bib_ref] [bib_ref] Self-monitoring of Fatigue Damage and Dynamic Strain in Carbon Fiber Polymer-Matrix Composite, Wang [/bib_ref] [bib_ref] Real-time Monitoring of Fatigue Damage and Dynamic Strain in Carbon Fiber Polymer-matrix..., Wang [/bib_ref]. Since then, significant research has been devoted to characterize and implement the electrical resistance approach using carbon fibers as reinforcements and self-sensing components [bib_ref] Delamination Monitoring of Graphite/Epoxy Laminated Composite Plate of Electric Eesistance Change Method, Todoroki [/bib_ref]. Given the recent advances in nanotechnology and the commercial availability of nanomaterials with outstanding properties, they have been mixed with thermosetting polymers and infiltrated through fiber preforms to fabricate what has been named "multiscale hierarchical composites". Carbon nanofibers, carbon black nanoparticlesand carbon nanotubes (CNTs) [bib_ref] Sensing of Damage Mechanisms in Fiber-Reinforced Composites under Cyclic Loading using Carbon..., Gao [/bib_ref] [bib_ref] Coupled Carbon Nanotube Network and Acoustic Emission Monitoring for Sensing of Damage..., Gao [/bib_ref] [bib_ref] Carbon Nanotube-Based Health Monitoring of Mechanically Fastened Composite Joints, Thostenson [/bib_ref] [bib_ref] Carbon Nanotube Networks: Sensing of Distributed Strain and Damage for Life Prediction..., Thostenson [/bib_ref] [bib_ref] Load and Health Monitoring in Glass Fibre Reinforced Composites with an Electrically..., Böger [/bib_ref] are some examples of nanomaterials used to build such multiscale hierarchical composites. In the particular case of CNTs, they can form electrically-conductive networks within a vast number of thermosetting polymers at less than 0.5 wt. % [bib_ref] A review and analysis of electrical percolation in carbon nanotube polymer composites, Bauhofer [/bib_ref]. Given the size difference between conventional micrometric fibers and carbon nanotubes (nanometric diameters), it is possible to place carbon nanotubes in matrix-rich areas among fibers as well as between adjacent plies [bib_ref] Coupled Carbon Nanotube Network and Acoustic Emission Monitoring for Sensing of Damage..., Gao [/bib_ref] [bib_ref] Load and Health Monitoring in Glass Fibre Reinforced Composites with an Electrically..., Böger [/bib_ref] [bib_ref] Modeling of Damage Sensing in Fiber Composites Using Carbon Nanotube Networks, Li [/bib_ref]. The CNT network allocated within the matrix allows to track the evolution of damage of the composite by measuring the changes in its electrical resistance during mechanical deformation, showing remarkable sensitivity to matrix-dominated failure mechanisms [bib_ref] Coupled Carbon Nanotube Network and Acoustic Emission Monitoring for Sensing of Damage..., Gao [/bib_ref]. Using this configuration (CNTs dispersed within the matrix) a number of works have been devoted to correlate the electromechanical response to the composite's damage under different loading scenarios [bib_ref] Sensing of Damage Mechanisms in Fiber-Reinforced Composites under Cyclic Loading using Carbon..., Gao [/bib_ref] [bib_ref] Carbon Nanotube-Based Health Monitoring of Mechanically Fastened Composite Joints, Thostenson [/bib_ref] [bib_ref] Carbon Nanotube Networks: Sensing of Distributed Strain and Damage for Life Prediction..., Thostenson [/bib_ref] [bib_ref] Spatial Damage detection in Electrically Anisotropic Fiber-Reinforced Composites Using Carbon Nanotube Networks, Gallo [/bib_ref] [bib_ref] Situ Monitoring of Through-Thickness Strain in Glass Fiber/Epoxy Composite Laminates Using Carbon..., Naghashpour [/bib_ref] [bib_ref] Structural Health Monitoring of Glass Fiber Reinforced Composites Using Embedded Carbon Nanotube..., Alexopoulos [/bib_ref] [bib_ref] Impact Damage Sensing of Multiscale Composites Through Epoxy Matrix Containing Carbon Nanotubes, Arronche [/bib_ref]. A different hierarchical configuration of CNT-based composites for self-sensing applications through electrical resistance consists in placing CNTs directly over the (non-conductive) fibers. Current research has reported the use of this technique and material architecture for single-fiber composites [bib_ref] Functional Interphases with Multi-walled Carbon Nanotubes in Glass Fibre/Epoxy Composites, Zhang [/bib_ref] [bib_ref] Single MWNT-Glass Fiber as Strain Sensor and Switch, Zhang [/bib_ref] [bib_ref] A Single Glass Fiber with Ultrathin Layer of Carbon Nanotube Networks Beneficial..., Zhang [/bib_ref] , but the technique needs to be extended to more realistic configurations as composite laminates. Given this motivation, this work investigates the capability of multiwall carbon nanotube (MWCNT)/glass fiber/vinyl ester composites with a tailored electrical MWCNT network to self-sense composite damage initiation and accumulation when they are subjected to tensile loading-unloading cycles; acoustic emission is used as a benchmark technique to validate the correlation between the electrical resistance variations and composite's damage. In order to tailor the composite's electrical sensitivity, the multiscale hierarchical composites are manufactured into two architectures differentiated by location of the MWCNTs: (i) with MWCNTs randomly dispersed within the matrix, and (ii) with MWCNTs deposited onto the glass fibers.
# Materials and methods
# Materials
Commercial E-glass fibers (Poliformas Plásticas S.A de C.V. Mérida, Mexico) with an average diameter of 15 µm, density of 2.54 g/cm 3 in the form of fiber tows containing~4000 filaments per tow were used as unidirectional fibers. Commercial MWCNTs (Cheap Tubes Inc., Cambridgeport, VT, USA) with purity >95%, 30-50 nm outer diameter, 5-10 nm inner diameter, and 1-6 µm length range were used. All MWCNTs used were chemically oxidized using a solution of H 2 SO 4 /HNO 3 at 3.0 mol/L for 2 h, following the procedure reported in [bib_ref] Evaluation of Mild Acid Oxidation Treatments for MWCNT Functionalization, Avilés [/bib_ref]. An epoxy vinyl ester Hetron 992 FR resin from Ashland composites (Dublin, OH, USA) was used as the thermosetting matrix for composite manufacturing. Cobalt naphthenate (CoNap) in a proportion of 0.2 wt. % and 0.6 wt. % of methyl ethyl ketone peroxide (MEKP) were employed to manufacture the composites.
## Composite manufacturing
To tailor the composite's electrical sensitivity, the multiscale hierarchical composites were manufactured into two architectures depending on the location of the MWCNTs: (i) with MWCNTs randomly dispersed within the matrix, and (ii) with MWCNTs deposited onto the glass fibers. These multiscale composites will be named hereafter as architecture "m" for composites containing MWCNTs randomly dispersed within the matrix [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref] , and architecture "f " for those where the MWCNTs are deposited onto the glass fibers [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref]. The deposition of oxidized MWCNTs onto the glass fibers was conducted following the procedure reported in our previous works [bib_ref] Interactions Between the Glass Fiber Coating and Oxidized Carbon Nanotubes, Ku-Herrera [/bib_ref] [bib_ref] On the Role of Fiber Coating in the Deposition of Multiwall Carbon..., Ku-Herrera [/bib_ref] [bib_ref] An Assessment of the Role of Fiber Coating and Suspending Fluid on..., Ku-Herrera [/bib_ref]. Briefly, oxidized MWCNTs are first ultrasonically dispersed in water, and then glass fiber tows are immersed into the MWCNT/water mixture, assisted by ultrasonic agitation. The glass fiber tows containing MWCNTs are then extracted from the container and dried in an oven.
## Composite manufacturing
To tailor the composite's electrical sensitivity, the multiscale hierarchical composites were manufactured into two architectures depending on the location of the MWCNTs: (i) with MWCNTs randomly dispersed within the matrix, and (ii) with MWCNTs deposited onto the glass fibers. These multiscale composites will be named hereafter as architecture "m" for composites containing MWCNTs randomly dispersed within the matrix [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref] , and architecture "f" for those where the MWCNTs are deposited onto the glass fibers [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref]. The deposition of oxidized MWCNTs onto the glass fibers was conducted following the procedure reported in our previous works [bib_ref] Interactions Between the Glass Fiber Coating and Oxidized Carbon Nanotubes, Ku-Herrera [/bib_ref] [bib_ref] On the Role of Fiber Coating in the Deposition of Multiwall Carbon..., Ku-Herrera [/bib_ref] [bib_ref] An Assessment of the Role of Fiber Coating and Suspending Fluid on..., Ku-Herrera [/bib_ref]. Briefly, oxidized MWCNTs are first ultrasonically dispersed in water, and then glass fiber tows are immersed into the MWCNT/water mixture, assisted by ultrasonic agitation. The glass fiber tows containing MWCNTs are then extracted from the container and dried in an oven. To manufacture the hierarchical composites, a layup consisting of three layers of 14 cm long glass fibers was used. For the composite with architecture m, the preform was made of as-received glass fibers while, for architecture f, the preform was made using glass fibers with deposited MWCNTs. Both composite architectures depicted in [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref] were manufactured by vacuum-assisted resin transfer molding. For composites with architecture m, a modified matrix with dispersed MWCNTs was used to impregnate the glass fiber preform. Such a MWCNT-modified matrix was achieved by mixing 0.5 wt. % of MWCNTs with the vinyl ester resin prior to infusion. The dispersion of MWCNTs within the resin was conducted as indicated in [fig_ref] Figure 2: Procedure used to disperse MWCNTs within the vinyl ester resin prior to... [/fig_ref]. This procedure consisted in adding 0.5 g of oxidized MWCNTs into 100 g of vinyl ester (1) and mixing them by mechanical stirring for 1 h (2) followed by dispersion aided by an ultrasonic bath operated at 42 kHz and 70 W for 3 h (3). The MWCNT-modified vinyl ester (4) was used to impregnate the preform made of as-received glass fibers. For the composite architecture f, a conductive MWCNT-modified vinyl ester with a concentration of 0.5 wt. % MWCNTs was applied only at the ends of the fiber preform, to promote electrical contact among fibers and to consolidate the electrodes. Then the glass fiber preform with the defined electrodes was impregnated with neat vinyl ester resin (without MWCNTs) by resin To manufacture the hierarchical composites, a layup consisting of three layers of 14 cm long glass fibers was used. For the composite with architecture m, the preform was made of as-received glass fibers while, for architecture f, the preform was made using glass fibers with deposited MWCNTs. Both composite architectures depicted in [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref] were manufactured by vacuum-assisted resin transfer molding. For composites with architecture m, a modified matrix with dispersed MWCNTs was used to impregnate the glass fiber preform. Such a MWCNT-modified matrix was achieved by mixing 0.5 wt. % of MWCNTs with the vinyl ester resin prior to infusion. The dispersion of MWCNTs within the resin was conducted as indicated in [fig_ref] Figure 2: Procedure used to disperse MWCNTs within the vinyl ester resin prior to... [/fig_ref]. This procedure consisted in adding 0.5 g of oxidized MWCNTs into 100 g of vinyl ester (1) and mixing them by mechanical stirring for 1 h (2) followed by dispersion aided by an ultrasonic bath operated at 42 kHz and 70 W for 3 h (3). The MWCNT-modified vinyl ester (4) was used to impregnate the preform made of as-received glass fibers.
## Composite manufacturing
To tailor the composite's electrical sensitivity, the multiscale hierarchical composites were manufactured into two architectures depending on the location of the MWCNTs: (i) with MWCNTs randomly dispersed within the matrix, and (ii) with MWCNTs deposited onto the glass fibers. These multiscale composites will be named hereafter as architecture "m" for composites containing MWCNTs randomly dispersed within the matrix [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref] , and architecture "f" for those where the MWCNTs are deposited onto the glass fibers [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref]. The deposition of oxidized MWCNTs onto the glass fibers was conducted following the procedure reported in our previous works [bib_ref] Interactions Between the Glass Fiber Coating and Oxidized Carbon Nanotubes, Ku-Herrera [/bib_ref] [bib_ref] On the Role of Fiber Coating in the Deposition of Multiwall Carbon..., Ku-Herrera [/bib_ref] [bib_ref] An Assessment of the Role of Fiber Coating and Suspending Fluid on..., Ku-Herrera [/bib_ref]. Briefly, oxidized MWCNTs are first ultrasonically dispersed in water, and then glass fiber tows are immersed into the MWCNT/water mixture, assisted by ultrasonic agitation. The glass fiber tows containing MWCNTs are then extracted from the container and dried in an oven. To manufacture the hierarchical composites, a layup consisting of three layers of 14 cm long glass fibers was used. For the composite with architecture m, the preform was made of as-received glass fibers while, for architecture f, the preform was made using glass fibers with deposited MWCNTs. Both composite architectures depicted in [fig_ref] Figure 1: Hierarchical composite architectures [/fig_ref] were manufactured by vacuum-assisted resin transfer molding. For composites with architecture m, a modified matrix with dispersed MWCNTs was used to impregnate the glass fiber preform. Such a MWCNT-modified matrix was achieved by mixing 0.5 wt. % of MWCNTs with the vinyl ester resin prior to infusion. The dispersion of MWCNTs within the resin was conducted as indicated in [fig_ref] Figure 2: Procedure used to disperse MWCNTs within the vinyl ester resin prior to... [/fig_ref]. This procedure consisted in adding 0.5 g of oxidized MWCNTs into 100 g of vinyl ester (1) and mixing them by mechanical stirring for 1 h (2) followed by dispersion aided by an ultrasonic bath operated at 42 kHz and 70 W for 3 h (3). The MWCNT-modified vinyl ester (4) was used to impregnate the preform made of as-received glass fibers. For the composite architecture f, a conductive MWCNT-modified vinyl ester with a concentration of 0.5 wt. % MWCNTs was applied only at the ends of the fiber preform, to promote electrical contact among fibers and to consolidate the electrodes. Then the glass fiber preform with the defined electrodes was impregnated with neat vinyl ester resin (without MWCNTs) by resin For the composite architecture f, a conductive MWCNT-modified vinyl ester with a concentration of 0.5 wt. % MWCNTs was applied only at the ends of the fiber preform, to promote electrical contact among fibers and to consolidate the electrodes. Then the glass fiber preform with the defined electrodes was impregnated with neat vinyl ester resin (without MWCNTs) by resin infusion. For both composite architectures, f and m, the vinyl ester resin (neat or MWCNT-modified) was infused into the fiber preform assisted by vacuum at a rate of~10 mL/min. The laminate was left for curing at room temperature for 2 h and then taken out of the mold for post-curing at 82˝C for 4 h in a convection oven.
## Specimens preparation and test setup
The changes in electrical resistance of the hierarchical composites were measured using unidirectional (0˝) laminates with the fibers aligned with the loading direction. The specimens' instrumentation for the electromechanical characterization involved tabbing the laminates, bonding strain gages, and electrode instrumentation, as schematized in [fig_ref] Figure 3: Tensile test specimen dimensions and instrumentation for in situ electrical monitoring coupled... [/fig_ref]. In this figure the conventional (1,2,3) material coordinate system is used to indicate the fiber (1), in-plane transverse (2), and through-thickness (3) directions. Tensile specimens were obtained from the unidirectional laminates with dimensions scaled down (ratio of 1:2) from the dimensions recommended by the ASTM standard D3039 for 0˝specimens. The specimens were 120 mm long and 7 mm wide, with a thickness of~1.0 mm defined by the three plies employed. The 25 mm long tabs were made of plain wave glass fiber/vinyl ester laminates, and were adhesively bonded to the specimen ends. The electrical resistance of the specimens was measured from a pair of copper wires running around the periphery of the tabs bonded to the specimen with silver paint. In order to calculate the volume electrical conductivity (σ e ) of the hierarchical composites, their electrical resistances without loading (R 0 ) was measured before tensile testing. infusion. For both composite architectures, f and m, the vinyl ester resin (neat or MWCNT-modified) was infused into the fiber preform assisted by vacuum at a rate of ~10 mL/min. The laminate was left for curing at room temperature for 2 h and then taken out of the mold for post-curing at 82 °C for 4 h in a convection oven.
## Specimens preparation and test setup
The changes in electrical resistance of the hierarchical composites were measured using unidirectional (0°) laminates with the fibers aligned with the loading direction. The specimens' instrumentation for the electromechanical characterization involved tabbing the laminates, bonding strain gages, and electrode instrumentation, as schematized in [fig_ref] Figure 3: Tensile test specimen dimensions and instrumentation for in situ electrical monitoring coupled... [/fig_ref]. In this figure the conventional (1,2,3) material coordinate system is used to indicate the fiber (1), in-plane transverse, and through-thickness (3) directions. Tensile specimens were obtained from the unidirectional laminates with dimensions scaled down (ratio of 1:2) from the dimensions recommended by the ASTM standard D3039 for 0° specimens. The specimens were 120 mm long and 7 mm wide, with a thickness of ~1.0 mm defined by the three plies employed. The 25 mm long tabs were made of plain wave glass fiber/vinyl ester laminates, and were adhesively bonded to the specimen ends. The electrical resistance of the specimens was measured from a pair of copper wires running around the periphery of the tabs bonded to the specimen with silver paint. In order to calculate the volume electrical conductivity (σe) of the hierarchical composites, their electrical resistances without loading (R0) was measured before tensile testing. A Shimadzu AG-I (Shimadzu, Kyoto, Japan) universal testing machine equipped with a 20 kN load cell was employed for all tests. The crosshead displacement rate of the universal testing machine was 1 mm/min. The specimens' strain was recorded by means of unidirectional strain gages (350 Ω, gage factor of 2.125) using a Vishay P3 strain indicator. The electrical resistance (R) of the specimen was measured in real-time during the tests using an Agilent DMM 3441A digital multimeter, synchronizing all instruments via an in-house data acquisition software. For the AE analysis, two PICO-type piezoelectric transducers were attached onto the specimen´s surface leaving a measurement span of 40 mm, as depicted in [fig_ref] Figure 3: Tensile test specimen dimensions and instrumentation for in situ electrical monitoring coupled... [/fig_ref]. The two PICO-type sensors were connected to a PCI-2-based AE system (Physical acoustic, Princeton Junction, NJ, USA) to acquire the acoustic events during the tensile test. A threshold of 40 dB was used to filter-out the noise not related to the acoustic events within the specimen. Additionally, all acoustic events not coming from the gap between sensors were discarded by data post-processing. Five replicates for each composite architecture were tested. The axial stress (σ), strain (ε), the electrical resistance, and the acoustic events occurring within the specimens were acquired simultaneously. Cyclic loading-unloading tests were conducted in order to investigate damage accumulation. A total of six incremental loading-unloading tension cycles were applied to the specimens controlled by the maximum applied load. These load levels sequentially reached maximum forces of Fmax = 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 kN, A Shimadzu AG-I (Shimadzu, Kyoto, Japan) universal testing machine equipped with a 20 kN load cell was employed for all tests. The crosshead displacement rate of the universal testing machine was 1 mm/min. The specimens' strain was recorded by means of unidirectional strain gages (350 Ω, gage factor of 2.125) using a Vishay P3 strain indicator. The electrical resistance (R) of the specimen was measured in real-time during the tests using an Agilent DMM 3441A digital multimeter, synchronizing all instruments via an in-house data acquisition software. For the AE analysis, two PICO-type piezoelectric transducers were attached onto the specimen´s surface leaving a measurement span of 40 mm, as depicted in [fig_ref] Figure 3: Tensile test specimen dimensions and instrumentation for in situ electrical monitoring coupled... [/fig_ref]. The two PICO-type sensors were connected to a PCI-2-based AE system (Physical acoustic, Princeton Junction, NJ, USA) to acquire the acoustic events during the tensile test. A threshold of 40 dB was used to filter-out the noise not related to the acoustic events within the specimen. Additionally, all acoustic events not coming from the gap between sensors were discarded by data post-processing. Five replicates for each composite architecture were tested. The axial stress (σ), strain (ε), the electrical resistance, and the acoustic events occurring within the specimens were acquired simultaneously. Cyclic loading-unloading tests were conducted in order to investigate damage accumulation. A total of six incremental loading-unloading tension cycles were applied to the specimens controlled by the maximum applied load. These load levels sequentially reached maximum forces of F max = 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 kN, and were carefully chosen to cover the mechanical response of the composite from the elastic region until evident damage is detected by AE. The change in electrical resistance (∆R = R´R 0 ) normalized by the electrical resistance of the load-free state (R 0 ), the stress (σ), and the acoustic events were plotted as function of the strain (ε) for each loading-unloading cycle. Additionally, for two selected specimens, 15 additional loading cycles up to a maximum force of 3.0 kN were applied to the same specimen that was previously loaded under the six incremental cycles discussed earlier. These experiments allowed to investigate damage accumulation due to cyclic loading-unloading conditions.
# Results and discussions
## Electrical conductivity of the hierarchical composites
The composites' electrical conductivity was characterized along the fiber direction. The electrical conductivity of the composites was obtained by measuring the electrical resistance of the specimens used for the tensile test free of any mechanical load (R 0 ) and using the specimen's gage length (~60 mm) and cross-sectional area (~7 mm 2 ). The composites with architecture m exhibited a mean volumetric electrical conductivity of 3.43ˆ10´3 S/m (with a standard deviation of˘1.03ˆ10´3 S/m), while for the composites with architecture f the electrical conductivity was 1.70ˆ10´1˘4.00ˆ10´3 S/m. The higher electrical conductivity of the composite with architecture f results from the contribution of electrically-conductive pathways created along the fibers upon MWCNT deposition and due to additional lateral contacts among adjacent fibers of the laminate.plots the results of the mechanical, electromechanical and AE characterization of a representative composite with architecture m subjected to monotonic tensile loading up to failure. A representative σ-ε curve is shown inwhile the electrical (∆R/R 0 ) and AE signals (amplitude and cumulative counts, circles, and diamonds, respectively) as a function of elapsed time (t) are shown in. As seen from, the composites with architecture m exhibit a linear trend for almost the whole stress-strain curve, and a drop of stress is observed close to failure, portraying the loss of load-bearing capacity. As seen in, during the firsts 20 s of the test (corresponding to ε < 0.2% inno acoustic events are detected, suggesting that the ∆R/R 0 response measured is due to elastic deformation of the composite, i.e., piezoresistivity (see inset in. In this region the tensile strain applied to the composite equally stretches the matrix and fibers, modifying the separation among MWCNTs and resulting in an increase in the electrical resistance of the composite. At t~20 s the first acoustic events are detected, indicating the onset of composite damage. Thereafter, the number of acoustic events progressively increase as the specimen is loaded up to failure. Analysis of the amplitude of the acoustic events [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref] [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref] suggests that the composite´s damage is generated by a combination of three sequential damage mechanisms, viz. matrix microcracking, fiber/matrix debonding, and fiber breakage. For low levels of strain (ε < 1.0%) the damage is mainly attributed to matrix microcraking as indicated by the amplitudes of the AE events lower than 60 dB (t < 170 s in [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref] [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref]. In addition to the acoustic events associated to matrix cracking (60-40 dB) for 20 < t < 170 s some acoustic events with amplitudes of 60-70 dB are detected, which are associated to fiber/matrix debonding [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref] [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref]. In this region, the initiation and propagation of matrix cracks destroy the conductive pathways of the MWCNT network, which is reflected in the ∆R/R 0 response exhibiting some oscillations with an overall increase in resistance. The oscillations observed in ∆R/R 0 may arise from occurrence of unstable matrix cracks. The acoustic events associated to fiber/matrix debonding increase for t > 170 s (ε > 1.0%) until the specimen's collapse. The onset of fiber breakage is expected around t~170 s (ε~1.0%), as indicated by a few acoustic events with amplitudes >70 dB [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref] [bib_ref] Clustering of Acoustic Emission Signals Collected During Tensile Tests on Unidirectional Glass/Polyester..., Godin [/bib_ref] [bib_ref] Identification of Fiber Breakage in Fiber Reinforced Plastic by Low-Amplitude Filtering of..., Ativitavas [/bib_ref]. For larger strains the occurrence of fiber breakage increase until specimen's collapse. However, for the composite with architecture m, ∆R/R 0 does not clearly reflect fiber breakage until imminent collapse. Since for this configuration, the MWCNTs are dispersed within the matrix, the sensitivity of the composite with architecture m to fiber breakage is relatively poor. For this composite adrchitecture, such a signal may stem from cracking of matrix layers surrounding the fibers, which is triggered by fiber breakage. Results of the mechanical and electromechanical behaviors of the composite with architecture f are shown in [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref]. Similar to the composite with architecture m under monotonic tensile loading, the composite with architecture f shows a linear stress-strain response for almost the whole curve, and a drop in stress is observed close to failure. For low levels of strain (ε < 0.2%, t < 25 s), no acoustic events are detected, suggesting that the structural integrity of the composite is still intact and hence, the ∆R/R0 signal arises purely from piezoresistivity (see inset in. The piezoresistive response of the composite with architecture f is originated from the deformation of the MWCNT network located at the fiber/matrix interface as a consequence of the load transfer from the matrix to the fibers. At ε ~0.2% the onset of composite damage occurs as suggested by the few acoustic emissions (40-60 dB) detected at t ~25 s. The main damage mechanism for low strain levels (ε < 1.0%) is attributed to matrix microcracking, as indicated by the acoustic events with amplitudes lower than 60 dB (t < 170 s in [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref] [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref]. Different to composites with architecture m, composites with architecture f show a smoother trend in ∆R/R0 with minimal oscillations. This is associated to a lower damage sensitivity of such composites to matrix cracking, given that MWCNTs are located onto the fibers in such a particular material architecture. Furthermore, the overall change in electrical resistance of composites with architecture f [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref] is smaller than that of composites with architecture m; this is also likely because of increased damage sensitivity to matrix cracking for architecture m. Damage associated to fiber/matrix debonding is also detected for ε < 1.0% (t < 170 s) as indicated by acoustic emissions with amplitudes of 60-70 dB [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref] [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref]. In [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref] the first acoustic events with amplitudes >70 dB (related to fiber breakage [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref] [bib_ref] Clustering of Acoustic Emission Signals Collected During Tensile Tests on Unidirectional Glass/Polyester..., Godin [/bib_ref] [bib_ref] Identification of Fiber Breakage in Fiber Reinforced Plastic by Low-Amplitude Filtering of..., Ativitavas [/bib_ref] are detected at t ~ 160 s (ε ~ 1%). Thereafter, the ∆R/R0 response exhibits few sudden peaks, which coincides with important changes in the cumulative counts of the AE signals. In fact, the shape of the ∆R/R0 curve outstandingly follows that of the AE cumulative counts, pinpointing the high sensitivity of the electrical signal to detect composite damage. It is assumed that when the fibers break at those strain levels, fiber breakage is accompanied by more damage within the composite in the form of matrix cracking and fiber/matrix debonding (see amplitudes of the AE in [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref]. Above t > 160 s, ∆R/R0 increases sharply indicating continued fiber breakage. At t ~330 s (ε ~ 2.0%) an abrupt change in ∆R/R0 is observed, suggesting that the composite experiences severe damage associated to fiber breakage. However, at such a strain level the composite still maintains limited load bearing capacity since the load is redistributed as the fibers continue breaking; final collapse of the specimen occurs at t ~360 s. Results of the mechanical and electromechanical behaviors of the composite with architecture f are shown in [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref]. Similar to the composite with architecture m under monotonic tensile loading, the composite with architecture f shows a linear stress-strain response for almost the whole curve, and a drop in stress is observed close to failure. For low levels of strain (ε < 0.2%, t < 25 s), no acoustic events are detected, suggesting that the structural integrity of the composite is still intact and hence, the ∆R/R 0 signal arises purely from piezoresistivity (see inset in. The piezoresistive response of the composite with architecture f is originated from the deformation of the MWCNT network located at the fiber/matrix interface as a consequence of the load transfer from the matrix to the fibers. At ε~0.2% the onset of composite damage occurs as suggested by the few acoustic emissions (40-60 dB) detected at t~25 s. The main damage mechanism for low strain levels (ε < 1.0%) is attributed to matrix microcracking, as indicated by the acoustic events with amplitudes lower than 60 dB (t < 170 s in [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref] [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref]. Different to composites with architecture m, composites with architecture f show a smoother trend in ∆R/R 0 with minimal oscillations. This is associated to a lower damage sensitivity of such composites to matrix cracking, given that MWCNTs are located onto the fibers in such a particular material architecture. Furthermore, the overall change in electrical resistance of composites with architecture f [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref] is smaller than that of composites with architecture m; this is also likely because of increased damage sensitivity to matrix cracking for architecture m. Damage associated to fiber/matrix debonding is also detected for ε < 1.0% (t < 170 s) as indicated by acoustic emissions with amplitudes of 60-70 dB [bib_ref] Mechanical Behavior and Health Monitoring by Acoustic Emission of Unidirectional and Cross-Ply..., Masmoudi [/bib_ref] [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref]. In [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref] the first acoustic events with amplitudes >70 dB (related to fiber breakage [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref] [bib_ref] Clustering of Acoustic Emission Signals Collected During Tensile Tests on Unidirectional Glass/Polyester..., Godin [/bib_ref] [bib_ref] Identification of Fiber Breakage in Fiber Reinforced Plastic by Low-Amplitude Filtering of..., Ativitavas [/bib_ref] are detected at t~160 s (ε~1%). Thereafter, the ∆R/R 0 response exhibits few sudden peaks, which coincides with important changes in the cumulative counts of the AE signals. In fact, the shape of the ∆R/R 0 curve outstandingly follows that of the AE cumulative counts, pinpointing the high sensitivity of the electrical signal to detect composite damage. It is assumed that when the fibers break at those strain levels, fiber breakage is accompanied by more damage within the composite in the form of matrix cracking and fiber/matrix debonding (see amplitudes of the AE in [fig_ref] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f [/fig_ref]. Above t > 160 s, ∆R/R 0 increases sharply indicating continued fiber breakage. At t~330 s (ε~2.0%) an abrupt change in ∆R/R 0 is observed, suggesting that the composite experiences severe damage associated to fiber breakage. However, at such a strain level the composite still maintains limited load bearing capacity since the load is redistributed as the fibers continue breaking; final collapse of the specimen occurs at t~360 s.
## Damage progression under monotonic tensile loading
## Damage accumulation
The unidirectional hierarchical composites were subjected to incrementally increasing cyclic tensile loading in order to investigate the electrical sensitivity of such composites to damage accumulation. The applied load was gradually increased in each cycle reaching a maximum strain of ε ≈ 1.2%, which is about half the failure strain seen in. [fig_ref] Figure 6: Coupled mechanical [/fig_ref] shows the mechanical (top), AE cumulative counts (middle), and the electromechanical (bottom) responses of a representative composite with architecture m subjected to incrementally increasing cyclic tensile loading. In [fig_ref] Figure 6: Coupled mechanical [/fig_ref] the load level in cycle I (Fmax = 0.5 kN) was chosen to ensure that the composite's integrity is intact. As seen from this figure the mechanical behavior is linearly elastic during the first loading-unloading cycle and no evidence of damage is detected by AE during this first cycle [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , middle). The electromechanical response [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , bottom) for this first cycle is also linear with the applied strain during the whole cycle and does not show residual (permanent) changes of electrical resistance after unloading, i.e., after the first cycle ∆R = R − R0 = 0 as seen in the inset of [fig_ref] Figure 6: Coupled mechanical [/fig_ref]. A very similar scenario regarding σ − ε, AE, and ∆R/R0 occurred for cycle II (Fmax = 1.0 kN), indicating that the composite was yet in the linear elastic regime. For cycle III (Fmax = 1.5 kN), the level of applied load induced limited damage to the composite, which is evidenced by few acoustic events. At this cycle, although the permanent change in electrical resistance is yet small (∆R ≈ 0), the ∆R/R0 vs ε curve loses linearity, which is associated to damage initiation, likely by matrix cracking and/or matrix viscoelastic phenomena. By applying a higher load level in cycle IV (Fmax = 2.0 kN), the cumulative AE counts evidently increase up to ~4000 acoustic events. In this cycle, composite damage increased given the higher load level, and the ∆R/R0 response followed a nonlinear trend; after unloading ∆R/R0 shows only a modest permanent change (∆R/R0 ≈ 0.2%). For cycle V (Fmax = 2.5 kN), the cumulative acoustic events markedly increase since at this load level matrix cracks are expected to propagate, inducing fiber/matrix debonding. For this higher load level the permanent ∆R/R0 after unloading is still small, but the unloading path of ∆R/R0 is slightly different to that of the loading one, indicating certain hysteresis. This hysteresis is attributed to the presence of irreversible phenomena (damage) in the matrix and probably at the fiber/matrix interface. Such a hysteresis may be driven by the release of residual stresses from the curing process [bib_ref] Strain Sensing Using Carbon Fiber, Wang [/bib_ref] and/or matrix viscoelastic phenomena [bib_ref] Cyclic Tension and Compression Piezoresistivity of Carbon Nanotube/Vinyl ester Composites in the..., Ku-Herrera [/bib_ref]. For the last cycle (cycle VI, Fmax = 3.0 kN), the AE cumulative counts indicate significant matrix and fiber/matrix damage which is also reflected in the ∆R/R0 response by not returning to zero upon unloading. It is important to notice that the permanent value attained by ∆R/R0 after the VI th cycle is negative. This irreversible (negative) value of ∆R/R0 upon unloading was
## Damage accumulation
The unidirectional hierarchical composites were subjected to incrementally increasing cyclic tensile loading in order to investigate the electrical sensitivity of such composites to damage accumulation. The applied load was gradually increased in each cycle reaching a maximum strain of ε « 1.2%, which is about half the failure strain seen in. [fig_ref] Figure 6: Coupled mechanical [/fig_ref] shows the mechanical (top), AE cumulative counts (middle), and the electromechanical (bottom) responses of a representative composite with architecture m subjected to incrementally increasing cyclic tensile loading. In [fig_ref] Figure 6: Coupled mechanical [/fig_ref] the load level in cycle I (F max = 0.5 kN) was chosen to ensure that the composite's integrity is intact. As seen from this figure the mechanical behavior is linearly elastic during the first loading-unloading cycle and no evidence of damage is detected by AE during this first cycle [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , middle). The electromechanical response [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , bottom) for this first cycle is also linear with the applied strain during the whole cycle and does not show residual (permanent) changes of electrical resistance after unloading, i.e., after the first cycle ∆R = R´R 0 = 0 as seen in the inset of [fig_ref] Figure 6: Coupled mechanical [/fig_ref]. A very similar scenario regarding σ´ε, AE, and ∆R/R 0 occurred for cycle II (F max = 1.0 kN), indicating that the composite was yet in the linear elastic regime. For cycle III (F max = 1.5 kN), the level of applied load induced limited damage to the composite, which is evidenced by few acoustic events. At this cycle, although the permanent change in electrical resistance is yet small (∆R « 0), the ∆R/R 0 vs ε curve loses linearity, which is associated to damage initiation, likely by matrix cracking and/or matrix viscoelastic phenomena. By applying a higher load level in cycle IV (F max = 2.0 kN), the cumulative AE counts evidently increase up to~4000 acoustic events. In this cycle, composite damage increased given the higher load level, and the ∆R/R 0 response followed a nonlinear trend; after unloading ∆R/R 0 shows only a modest permanent change (∆R/R 0 « 0.2%). For cycle V (F max = 2.5 kN), the cumulative acoustic events markedly increase since at this load level matrix cracks are expected to propagate, inducing fiber/matrix debonding. For this higher load level the permanent ∆R/R 0 after unloading is still small, but the unloading path of ∆R/R 0 is slightly different to that of the loading one, indicating certain hysteresis. This hysteresis is attributed to the presence of irreversible phenomena (damage) in the matrix and probably at the fiber/matrix interface. Such a hysteresis may be driven by the release of residual stresses from the curing process [bib_ref] Strain Sensing Using Carbon Fiber, Wang [/bib_ref] and/or matrix viscoelastic phenomena [bib_ref] Cyclic Tension and Compression Piezoresistivity of Carbon Nanotube/Vinyl ester Composites in the..., Ku-Herrera [/bib_ref]. For the last cycle (cycle VI, F max = 3.0 kN), the AE cumulative counts indicate significant matrix and fiber/matrix damage which is also reflected in the ∆R/R 0 response by not returning to zero upon unloading. It is important to notice that the permanent value attained by ∆R/R 0 after the VI th cycle is negative. This irreversible (negative) value of ∆R/R 0 upon unloading was observed for the three tested specimens and is associated to matrix dominated processes, such as matrix yielding and viscoelasticity [bib_ref] Cyclic Tension and Compression Piezoresistivity of Carbon Nanotube/Vinyl ester Composites in the..., Ku-Herrera [/bib_ref]. Releasing of curing stresses at the fiber/matrix interface could also be a contributing factor for this irreversible ∆R/R 0 [bib_ref] Strain Sensing Using Carbon Fiber, Wang [/bib_ref]. Since, in this composite the MWCNTs are dispersed within the matrix, the ∆R/R 0 response is very sensitive to events occurring in such a polymer matrix. Limited fiber breakage is also expected during cycle VI and is correlated to a few acoustic events with amplitudes >70 dB; however, for this composite architecture (m) ∆R/R 0 does not conspicuously reflect the onset of fiber breakage, since the MWCNTs in this composite are randomly located within the matrix, rather than onto the fibers. During the loading segment of the first cycle, no acoustic events are detected until σ ≈ 400 MPa, which corresponds to the maximum stress in cycle V, see [fig_ref] Figure 7: Evolution of stress [/fig_ref]. Then the acoustic events accumulate ~25 × 10 3 counts at the maximum stress level (500 MPa). For the loading segment of the first cycle, ∆R/R0 increases up to 6.2% at the maximum stress level and decreases to ~−0.3% upon unloading. For the second cycle in [fig_ref] Figure 7: Evolution of stress [/fig_ref] , a lower number of acoustic events are detected compared to the first one, given that a higher load level is necessary to generate more damage in the composite. For this second cycle, ∆R/R0 increases during loading and its maximum value was ~5.9%, a bit lower than that of the first cycle. For the unloading segment, again ∆R/R0 decreases as the load was released but in this case it reaches ~−0.43% upon full unloading. After each loading-unloading The corresponding results of the composites with architecture f are shown by a representative plot in [fig_ref] Figure 6: Coupled mechanical [/fig_ref]. As for the mechanical response of the composites with architecture m, the composites with architecture f behaves linearly elastic during cycles I and II (F max = 0.5 and 1.0 kN). The lack of detected acoustic events during cycles I and II indicates that the structural integrity of the composite remains intact during those cycles. The electromechanical responses of the two first cycles [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , bottom) is also linear with the applied strain during the loading-unloading cycles, without any permanent change in electrical resistance upon unloading (see inset in [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , bottom). For cycle III (F max = 1.5 kN), the applied load induces marginal matrix cracking to the composite, as confirmed by a few acoustic events with amplitudes of 40-60 dB [fig_ref] Figure 6: Coupled mechanical [/fig_ref]. Despite that according to the acoustic events matrix cracking occurs during cycle III, ∆R/R 0 returns to zero upon unloading. This is because at this stress level, damage occurs at the matrix level and the MWCNTs in this configuration are located onto the fibers. Notice that for cycle III ∆R/R 0 loses its linear trend for the last part of the curve, which can be associated to the onset of matrix damage. By applying a higher load level (F max = 2.0 kN) in cycle IV, the accumulated acoustic events increase up to~2500 events. In this cycle, composite damage increases with increased load and ∆R/R 0 follows a nonlinear trend. After unloading, ∆R/R 0 shows only a small permanent change (~0.2%) and an evident hysteresis in the curve, suggesting damage accumulation. At this loading level, it is expected that matrix cracking reaches the fiber/matrix interface (AE events of 60-70 dB [bib_ref] Damage Characterization of Polymer-based Composite Materials: Multivariable analysis and Wavelet Transform for..., Marec [/bib_ref] which modifies the tailored MWCNT network in such a region. In cycle V, the cumulative AE signal doubles reaching~5000 events at the maximum load. At this load level matrix cracks are expected to propagate inducing significant damage associated to fiber/matrix debonding, as previously discussed. In this cycle the electromechanical response shows an evident permanent ∆R/R 0 of~0.35%, in addition to the more pronounced hysteresis seen from the unloading curve of ∆R/R 0 [fig_ref] Figure 6: Coupled mechanical [/fig_ref]. The destruction of the MWCNT conductive pathways at the fiber/matrix interface region is probably induced by the propagation of fiber/matrix debonding. In cycle VI, a pronounced increase of the accumulated acoustic events evidence the onset of fiber breakage (70-100 dB, amplitudes not shown in the plot). For this composite architecture (f ) a positive permanent ∆R/R 0 of~0.65% is attained upon unloading cycle VI as showed in [fig_ref] Figure 6: Coupled mechanical [/fig_ref] (bottom). This behavior contrasts that of the composite with architecture m, where the permanent change in electrical resistance upon unloading is negative. These contrasting electromechanical behaviors between the composites with architectures f and m are due to the deliberately different location of the MWCNT network in such composites, which is differently affected depending on the composite's damage mechanism.
After the sixth cycle shown in [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , the same specimen was additionally subjected to 15 identical loading-unloading cycles. [fig_ref] Figure 7: Evolution of stress [/fig_ref] shows the evolution of stress (σ, diamonds), ∆R/R 0 (circles) and the cumulative acoustic events (continuous red line) for both composite architectures, m [fig_ref] Figure 7: Evolution of stress [/fig_ref] and f [fig_ref] Figure 7: Evolution of stress [/fig_ref]. To better interpret this analysis, the cumulative acoustic events and ∆R/R 0 were reset to zero for the first cycle presented in [fig_ref] Figure 7: Evolution of stress [/fig_ref] (which correspond to the last cycle (VI) in [fig_ref] Figure 6: Coupled mechanical [/fig_ref] , and the new cumulative acoustic events were associated to accumulation of damage in the composite due to these new repetitive loading-unloading cycles at a constant stress level. For all cycles, the specimens reached a maximum stress of 500 MPa and returns to σ = 0 upon unloading. is required to generate more damage in the specimen. For the second cycle, again ∆R/R0 increases during loading, but in this case ∆R/R0 only reached ~4.8% at the maximum stress level, and ~0.6% upon unloading. In contrast with the composite with architecture m, the ∆R/R0 response of the composite with architecture f does not show negative values upon unloading and there are no increments in the permanent change in electrical resistance upon unloading. The reduction in amplitude of the ∆R/R0 response in this composite is associated to fiber/matrix debonding, since during the loading-unloading cycles the applied load propagates the existing cracks through the fiber/matrix interface region, decreasing the matrix-to-fiber load transfer efficiency. It is also likely that after each cycle, a few fibers break which can also contribute to the decrease in amplitude of the ∆R/R0 signal. The fact that the maximum (peak at σmax = 500 MPa) and minimum (valley at σ = 0) values of ∆R/R0 may be used as a metric of damage accumulation can be better assessed with the aid of [fig_ref] Figure 8: Cumulative ∆R/R0 response [/fig_ref]. Such a figure plots the accumulated difference (absolute value) between subsequent peak (labeled "Max") values of ∆R/R0 as a function of the cycle number (k) which is quantified by: During the loading segment of the first cycle, no acoustic events are detected until σ « 400 MPa, which corresponds to the maximum stress in cycle V, see [fig_ref] Figure 7: Evolution of stress [/fig_ref]. Then the acoustic events accumulatẽ 25ˆ10 3 counts at the maximum stress level (500 MPa). For the loading segment of the first cycle, ∆R/R 0 increases up to 6.2% at the maximum stress level and decreases to~´0.3% upon unloading. For the second cycle in [fig_ref] Figure 7: Evolution of stress [/fig_ref] , a lower number of acoustic events are detected compared to the first one, given that a higher load level is necessary to generate more damage in the composite. For this second cycle, ∆R/R 0 increases during loading and its maximum value was~5.9%, a bit lower than that of the first cycle. For the unloading segment, again ∆R/R 0 decreases as the load was released but in this case it reaches~´0.43% upon full unloading. After each loading-unloading cycle, the amplitude of the ∆R/R 0 response reduces progressively, exhibiting negative permanent values upon unloading. This behavior is associated to matrix yielding and viscoelastic matrix-dominated phenomena [bib_ref] Cyclic Tension and Compression Piezoresistivity of Carbon Nanotube/Vinyl ester Composites in the..., Ku-Herrera [/bib_ref] and is consistent to that observed infor the composite architecture m.
The corresponding loading excursions for architecture f are shown in [fig_ref] Figure 7: Evolution of stress [/fig_ref] , where the additional 15 identical loading-unloading cycles of constant applied stress of 500 MPa are indicated by the curve representing σ. The evolution of σ (diamonds), ∆R/R 0 (circles), and the cumulative AE (continuous red line) over time are shown in [fig_ref] Figure 7: Evolution of stress [/fig_ref]. During the loading segment of the first cycle, no acoustic events are detected until σ « 400 MPa; then the acoustic events accumulate~15ˆ10 3 counts for σ = 500 MPa. After the loading segment of the first cycle, ∆R/R 0 increases up to~5.1% at the maximum stress level, and decreases to~0.6% when the specimen is completely unloaded. For the second cycle, fewer acoustic events are detected compared to the first cycle, since a higher stress level is required to generate more damage in the specimen. For the second cycle, again ∆R/R 0 increases during loading, but in this case ∆R/R 0 only reached~4.8% at the maximum stress level, and~0.6% upon unloading. In contrast with the composite with architecture m, the ∆R/R 0 response of the composite with architecture f does not show negative values upon unloading and there are no increments in the permanent change in electrical resistance upon unloading. The reduction in amplitude of the ∆R/R 0 response in this composite is associated to fiber/matrix debonding, since during the loading-unloading cycles the applied load propagates the existing cracks through the fiber/matrix interface region, decreasing the matrix-to-fiber load transfer efficiency. It is also likely that after each cycle, a few fibers break which can also contribute to the decrease in amplitude of the ∆R/R 0 signal.
The fact that the maximum (peak at σ max = 500 MPa) and minimum (valley at σ = 0) values of ∆R/R 0 may be used as a metric of damage accumulation can be better assessed with the aid of [fig_ref] Figure 8: Cumulative ∆R/R0 response [/fig_ref]. Such a figure plots the accumulated difference (absolute value) between subsequent peak (labeled "Max") values of ∆R/R 0 as a function of the cycle number (k) which is quantified by:
# Conclusions
The electrical capability of glass fiber/carbon nanotube/vinyl ester unidirectional hierarchical composites containing a tailored electrical network of multiwall carbon nanotubes (MWCNTs) to self-sense their damage progression under cyclic loading was investigated. Tailored MWCNT networks were achieved by deliberately placing the MWCNTs either randomly dispersed within the Equation (1) is stated in terms of the maximum values of ∆R/R 0 but is also valid for the minimum ones (valleys, labeled "Min"). Both values, "Max" and "Min" are plotted in [fig_ref] Figure 8: Cumulative ∆R/R0 response [/fig_ref]. As seen from this figure, for composite architecture m [fig_ref] Figure 8: Cumulative ∆R/R0 response [/fig_ref] both accumulated electrical curves ("Max" and "Min") keep a close correlation with the cumulative AE, being the accumulated values of ∆R/R 0 corresponding to the peak values more sensitive. The "Max" curves of both composite architectures correlates better with the damage accumulation detected by AE probably because at the peak stress the microcracks open, while they tend to close when the stress returns to zero. For composite architecture f, a close correlation between the electrical and AE cumulative counts is also observed but only for the maximum values of ∆R/R 0 . As inferred from the numerical values of the vertical axis in [fig_ref] Figure 8: Cumulative ∆R/R0 response [/fig_ref] ,b the composites with architecture m are more sensitive to this kind of damage accumulation than those with architecture f. This again, reveals that the tailored architecture of the MWCNT network within the composite has a paramount effect on its sensitivity to damage accumulation. At the repeated load levels experienced in [fig_ref] Figure 8: Cumulative ∆R/R0 response [/fig_ref] (500 MPa, see [fig_ref] Figure 7: Evolution of stress [/fig_ref] matrix damage (rather than fiber damage) is expected, which is consistent with the location of the MWCNTs in architecture m.
# Conclusions
The electrical capability of glass fiber/carbon nanotube/vinyl ester unidirectional hierarchical composites containing a tailored electrical network of multiwall carbon nanotubes (MWCNTs) to self-sense their damage progression under cyclic loading was investigated. Tailored MWCNT networks were achieved by deliberately placing the MWCNTs either randomly dispersed within the polymer matrix (architecture m) or deposited onto the glass fibers (architecture f ). By using incrementally-increasing cyclic tensile loading tests, damage initiation and progression were identified by acoustic emission (AE) and correlated to in situ measurements of the composite´s electrical resistance. Cyclic loading-unloading tests where the maximum applied stress was gradually increased and cyclic tests where the maximum stress was kept constant for 15 more cycles were conducted. For both composite architectures, the onset of matrix cracking was identified by a deviation of linearity in the ∆R/R 0 vs ε curve. For the composites with MWCNTs dispersed within the matrix, ∆R/R 0 returns to zero upon unloading unless a critical stress is reached where composite damage initiates, which correlates well with the acoustic events. A negative permanent change of electrical resistance upon unloading characterized damage accumulation of the composite with MWCNTs dispersed within the matrix; this was associated to matrix-dominated irreversible phenomena such as yielding and viscoelasticity. On the other hand, the composites with MWCNTs deposited onto the fibers showed a positive permanent change in electrical resistance upon unloading, which was associated to fiber/matrix debonding and fiber breakage. The multiscale composites with MWCNTs on the fibers were able to detect the onset of composite damage by a deviation from linearity and hysteresis in the electromechanical curve, as well as to detect damage progression, being very sensitive to fiber-dominated damage mechanisms. The accumulation and progression of matrix damage due to repetitive loading-unloading cycles at a peak stresses of 500 MPa were tracked by both composite architectures, being the composite with MWCNTs dispersed in the matrix more sensitive to this kind of damage accumulation. The different electrical responses of the composites with architectures m and f when they are subjected to cyclic tensile loading highlights the tailored sensitivity to damage in such multiscale hierarchical composites. Therefore, the multiscale hierarchical composites developed in this study are excellent candidates for health monitoring of structures subjected to cyclic loading, and their sensitivity can be tailored for specificity from their hierarchical structure regarding the MWCNT location within the composite.
[fig] Figure 1: Hierarchical composite architectures. (a) Composite architecture m, with MWCNTs dispersed within the matrix, and (b) composite architecture f, with MWCNTs deposited onto the fibers. [/fig]
[fig] Figure 2: Procedure used to disperse MWCNTs within the vinyl ester resin prior to resin infusion. [/fig]
[fig] Figure 3: Tensile test specimen dimensions and instrumentation for in situ electrical monitoring coupled with acoustic emission. [/fig]
[fig] Figure 4: For this composite architecture, such a signal may stem from cracking of matrix layers surrounding the fibers, which is triggered by fiber breakage. Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture m. (a) Stress-strain curve, (b) evolution of ∆R/R0 (continuous line), AE amplitude (circles), and AE cumulative counts (diamonds) during the tensile test. [/fig]
[fig] Figure 5: Mechanical, electromechanical, and acoustic emission characterization of the composite with architecture f. (a) Stress-strain curve, (b) evolution of ∆R/R0 (continuous line), AE amplitude (circles), and AE cumulative counts (diamonds) during the tensile test. [/fig]
[fig] Figure 6: Coupled mechanical (Top), AE (Middle), and electromechanical (Bottom) responses of hierarchical composites under incrementally-increasing cyclic tensile loading. (a) Composite architecture m, (b) composite architecture f. [/fig]
[fig] Figure 7: Evolution of stress (diamonds), ∆R/R0 (circles) and cumulative AE (red line) signals for a selected specimen subjected to cyclic loading. (a) Composite with architecture m, and (b) composite with architecture f. [/fig]
[fig] Figure 8: Cumulative ∆R/R0 response (Equation (1)) for assessment of damage accumulation under identical loading cycles. (a) Composite with architecture m, and (b) composite with architecture f. [/fig]
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Postoperative radiotherapy in resected non-small cell lung cancer: The never-ending story
# Introduction
## Historical evolution of postoperative radiotherapy
One of the great historical controversies in the field of thoracic oncology is the use of Postoperative radiotherapy (PORT) in patients with non-small cell lung cancer (NSCLC). The rationale for this therapeutic strategy is the high risk of locoregional recurrence (LRR) after radical surgery, especially in patients with pN2 disease, who account for up to 30% of patients. The development of LRR in patients with NSCLC has important clinical implications and is associated with worse survival outcomes . Several different pathological variables have been associated with a higher risk of developing LRR, including tumour size > 3 cm, lymphovascular invasion, visceral pleural invasion, and involvement of multiple lymph nodes .
The role of PORT in NSCLC remains controversial, mainly because studies carried out over the last few decades have reported conflicting safety and efficacy results. Although multiple retrospective and prospective studies have been performed, we still lack high-quality evidence to confirm or definitively rule out PORT in these patients. A meta-analysis published in 1998 found that PORT was associated with lower overall survival (OS) rates in patients with stage I-II disease, with 2-year OS rates of 43% in the non-PORT group vs 30% in the patients that received PORT, although there was no clear evidence that PORT negatively influenced outcomes in patients with stage III pN2 disease . In older studies, the poor outcomes of PORT could be due to the high levels of morbidity and mortality associated with obsolete radiotherapy techniques or inappropriate doses, fractionations, and/or irradiation volumes. In fact, a more recent meta-analysis demonstrated that PORT improves OS outcomes when modern technology (linear accelerators vs cobalt therapy units) is used to deliver the radiation dose .
Despite the contradictory findings described above, several studies have reported a clear benefit for PORT in patients with involved lymph nodes (pN2) in terms of improved local control and even OS . Among those studies with positive findings, the most important is the study carried out by Mikell et al , who evaluated 2115 patients with pN2 NSCLC based on data retrieved from the National Cancer Database (NCBD). In that study, PORT was associated with a significant increase in OS (42 mo vs 38 mo, P = 0.048) in patients treated according to the therapeutic standards of the modern era [three-dimensional conformal radiotherapy (3D-CRT), adjuvant chemotherapy (ChT), etc.] .
The long-awaited preliminary results of the Lung ART trial (NCT00410683)[8], which included patients with NSCLC who underwent complete resection with adjuvant ChT, were recently presented at the ESMO 2020 meeting. Lung ART is a multi-institutional randomized phase III trial which included stage III N2 NSCLC cases comparing mediastinal PORT (54 Gy/27-30 fractions) to no PORT in very selected patients: PS 0-2, complete resection with optimal nodal exploration and proven N2 disease. The main endpoint was disease-free survival (DFS). Between August 2007 and July 2018, 501 patients were randomized after surgery or after ChT: 252 patients allocated to PORT, and 249 to no PORT. With a median FU of 4.8 years DFS HR was 0.85 (95%CI [0.67-1.07]); median DFS was 30.5 mo with PORT and 22.8 without PORT; 3-year DFS was 47.1% with PORT vs 43.8% without PORT (P = ns), and finally, 3-year OS was 66.5% with PORT vs 68.5% without PORT (P = ns). Early and late Gr 3-5 cardio-pulmonary toxicity was respectively 7% and 20% in PORT arm vs 3.2% and 7.7% in control arm. Nonetheless, PORT significantly decreased LRR in the mediastinum (46.1% vs 25% with and without PORT, respectively), a finding that suggests that PORT could offer a clinical benefit in a well-selected subgroup of patients.
However, these preliminary results raised further doubts about the role of PORT in NSCLC. The findings of this landmark trial are extremely important and may come to redefine the role of radiotherapy in NSCLC.
According to these data, PORT should not be routinely recommended to all resected stage III N2 NSCLC patients. The decision to prescribe o not PORT must be individualised according to the patient's specific characteristics. In general, PORT should be indicated only in highly selected patients with good performance status (PS 0-1), significant mediastinal lymph node involvement (pN2, extracapsular extension), and/or residual disease (R1-R2) after surgery. In addition, PORT must be only performed in cases with a favourable dose distribution that fulfils the dose restriction criteria for the organs of risk (OARs), especially cardiopulmonary restrictions.
## Current evidence and recommendations for port
The role of PORT in the treatment of NSCLC remains controversial. Although this therapeutic strategy has been evaluated in numerous retrospective and prospective studies, robust evidence to definitively support the value of PORT is still lacking, as can be seen in the lack of consensus among the clinical guidelines published by the main international scientific societies[9-13].
Currently, the most widely accepted indication for PORT, with the most evidence, is for the treatment of residual disease (including extracapsular extension) after radical surgery. Most international guidelines recommend PORT in patients with involved surgical margins (R1-R2) at the surgical bed due to the high risk of recurrence in this region, with a recommended dose ranging from 54-60 Gy (1.8-2 Gy/fraction) .
By contrast, in patients with stage pN2 disease, the current evidence suggests that the treatment decision should be assessed on a case-by-case basis by a multidisciplinary team to determine if the patient would be likely to benefit from PORT. The treatment decision should consider several key clinical characteristics, including the number of mediastinal nodal stations involved (≥ 1), the patient's general physical condition (PS 0-1), and cardiopulmonary function.summarizes the recommendations proposed by the main international guidelines.
## Management of cases with involved surgical margins
The rate of incomplete resections (microscopic or macroscopic; R1-R2) after radical surgery for lung cancer ranges from 1%-17% . In these cases, the aim of PORT is to reduce the risk of local recurrence and improve OS. Although various clinical guidelines recommend salvage surgery in patients with positive surgical margins, this approach is not supported by robust data. Ghiribelli et al evaluated OS in a series of patients with incomplete resections (R1), finding that survival was not correlated with the type of infiltration, nodal involvement, or histological type. As a result, in patients with microscopic residual tumours, the authors recommended salvage surgery only in patients with early stage (I-II) disease; by contrast, the recommended treatment in stage III pN2 disease is adjuvant radiotherapy.
A study published in 2012 evaluated the efficacy and toxicity of PORT according to histological subtype in patients (n = 41) with incompletely resected NSCLC . Of the 41 patients, 23 had microscopic (R1) and 18 macroscopic (R2) residual disease. The histologic distribution was as follows: squamous cell carcinoma (SCC) (n = 23), adenocarcinoma (14), and other histologies (4). The predominant progression pattern , with significantly lower 5year OS rates in the R1 group for all stages: stage I, 37% vs 62% (P <0.0001); stage II, 29% vs 41% (P < 0.0001); and stage III, 19% vs 33% (P < 0.0001). Administration of adjuvant ChT with PORT in the R1 group was associated with better OS than surgery alone, regardless of stage (stage I, 44% vs 35%, P = 0.05; stage II, 33% vs 21%, P = 0.0013; stage III, 30% vs 12%, P < 0.0001).
In a study published in 2015, Wang et al evaluated 3395 patients with incompletely resected stage II-III NSCLC to determine the influence of PORT on survival outcomes, finding that PORT was associated with significantly better 5-year OS (32.4% vs 23.7%). Radiation doses between 50-70 Gy improved survival rates in the PORT group vs the non-PORT group. However, when higher doses (> 70 Gy) were administered, there were no between-group differences in OS. The authors of that study concluded that PORT improves OS in patients with incompletely resected stage II-III NSCLC and should therefore be considered as an adjuvant treatment. They also suggested that the radiation dose in patients with macroscopic residual disease (R2) should be the same as those used for radical radiotherapy (60-66 Gy).
## Mediastinal staging
## Preoperative mediastinal staging
The appropriate management of NSCLC depends on accurate mediastinal staging. Contrast-enhanced chest computed tomography (CT) is currently the diagnostic test of choice for preoperative mediastinal staging. On CT imaging, nodes with a short-axis diameter ≥ 1 cm are considered pathological . In recent years, 18F-fluorodeoxyglucose (FDG) positron-emission tomography (PET)-CT has transformed lung cancer staging due to its greater sensitivity. However, PET-CT has some limitations in cases October 24, 2021
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Issue 10 with small nodes (< 1 cm) and in certain histologies in which FDG uptake is limited. PET-CT also has a high false positive rate (20%-25%) in the presence of intercurrent infections and inflammatory processes. Consequently, histopathologic confirmation of mediastinal node involvement is usually required, especially when the therapeutic approach depends directly on the results of this assessment[21-23]. Histological confirmation can be omitted in certain patients with small (≤ 3 cm) peripheral tumours without radiological evidence of suspected mediastinal involvement. Mediastinal nodes can be obtained endoscopically through endobronchial ultrasound (EBUS) and/or endoscopic ultrasound (EUS) guided puncture, or surgically, through mediastinoscopy or video-assisted thoracoscopy (VATS). Endobronchial ultrasound (EBUS/EUS) is usually the first step in evaluating suspected mediastinal node involvement . These minimally invasive endoscopic techniques are usually preferred to surgical approaches due to their good sensitivity and specificity profile and relatively low risk of morbidity. If the sample is negative, not assessable, or insufficient (despite radiological suspicion), staging should be completed with invasive techniques, which have a higher negative predictive value (NPV). For many years, conventional mediastinoscopy was the main surgical staging technique, despite the technical limitations of this procedure for the study of the posterior and inferior mediastinum, in which either extended cervical mediastinoscopy or VATS is necessary[26].
## Mediastinal restaging after neoadjuvant therapy
Mediastinal restaging after neoadjuvant therapy (ChT or ChT+RT) is controversial. Some patients with stage IIIA, low volume N2 disease are classified as potentially resectable and may benefit from neoadjuvant therapy, which could increase the likelihood of achieving a complete response (CR) in the mediastinum, thus permitting surgical resection of the tumour . In this clinical scenario, however, the value of CT for mediastinal restaging is questionable since CT-based assessment, although highly predictive of pathologic CR, tends to underestimate the true CR rate.
PET-CT is an excellent tool to assess the response of both the primary tumour and metastatic lesions, but it is less reliable in evaluating mediastinal involvement due to high rates of false negative and false positives (20% and 25%, respectively)[28,29]. Therefore, histopathologic confirmation is necessary in cases with radiological response if surgical resection is being considered.
EBUS/EUS restaging after neoadjuvant therapy has a low sensitivity and a low NPV. If the test is negative, the surgical technique should be escalated to reduce the false negative rate . Restaging via mediastinoscopy has a high sensitivity (> 60%), specificity (≈ 100%), positive predictive value (PPV; 100%) and NPV (> 73%); however, this procedure is not routinely performed due to its technical complexity in this clinical context. Rather, the recommended strategy is initial confirmation of stage N2 disease by EBUS or EUS-guided transbronchial aspiration during the initial workup, thus reserving mediastinoscopy for restaging .
## Selection of candidates for port
Numerous studies have explored a wide range of prognostic factors potentially associated with an increased risk of LRR in order to identify high-risk patients suitable for adjuvant radiotherapy. In patients with NSCLC, the histological type is not currently considered a prognostic factor for adjuvant treatment due to the poor quality of the available data and contradictory findings in the literature. While some studies have found that SCC histology is associated with worse OS rates than adenocarcinoma [32,33], findings from other studies point in the opposite direction .
The findings of a recent meta-analysis involving 25780 patients from 13 studies (most retrospective) underscored the prognostic value of multiple mediastinal node involvement. That study showed that, in patients with pN2 disease with ≥ one positive node and/or multiple N2 station involvement, PORT significantly improved both DFS (HR 0.The studies that have generated the most interest are those that have sought to stratify risk groups according to multiple clinical, pathologic, and molecular parameters. In this regard, the study by Deng and colleaguesis worth highlighting. Those authors evaluated numerous characteristics -age, sex, surgical technique, histological type, degree of differentiation, tumour size, number of nodes evaluated (LNR index) -in a large sample (n = 2329) of patients included in the SEER database. Based on that analysis, the authors proposed a prognostic scoring model that classified patients into two risk categories (high and low), which was a significant predictor of survival outcomes (OS and CSS).
Jiang et alrecently developed a model that incorporated several molecular biomarkers, together with other well-known clinical variables, to predict clinical outcomes in patients with stage IIIA pN2 NSCLC. In that study, the following variables were significantly associated with the risk of LRR: epidermal growth factor receptor (EGFR) status: wild-type vs native (HR 3.666, 95%CI: 1.724-7.797); lymphocyte to monocyte ratio (LMR) < 4.69 (HR 2.364, 95%CI: 1.221-4.574); surgical procedure (VATS vs thoracotomy) (HR 0.348, 95%CI: 0.175 -0.693); and pN2 LNR ≥ 38.9% (HR 3.597, 95%CI: 1.832-7.062). The authors then used those data to develop a predictive modelbased on the four independent risk factors to determine the individual risk of LRR in each patient. This score, in turn, could be used to recommend or not adjuvant radiotherapy.
## Technical recommendations for the treatment of port
## Simulation
The generally accepted recommendations provided by clinical guidelines for the management of NSCLC should be followed for positioning, immobilization, and treatment simulation. Systems designed to improve immobilization and control respiratory motion (4D-CT) should be used, preferably with image-guided radiotherapy (IGRT), to obtain smaller treatment volumes and more precise radiotherapy to achieve a better dosimetric distribution.
In general, CT imaging (slice thickness, 2-3 mm) should be performed with intravenous contrast to improve contouring of the nodal areas. The use of 5FDG-PET-CT for postoperative simulation is not recommended due to the lack of robust data; moreover, interpretation of these images in the immediate postoperative period can be challenging due to the inflammation, which can lead to false positives. Image interpretation after ChT is also difficult and it is easy to underestimate the residual disease (false negatives).
## Target volumes
The most important data for target volume definition were described in the Lung-ART clinical trial and based on contouring performed by 17 experienced thoracic radiation oncologists in two representative cases. The clinical target volume (CTV) should include the bronchial stump, ipsilateral hilum, adjacent mediastinal pleura, and involved nodes (according to the pathology report). The involved nodal station and those immediately superior and inferior to that region should also be contoured, being careful to avoid oversizing the CTV. To generate the PTV (planning target volume), a margin of at least 0.5 cm in the mediolateral and dorsoventral directions (1 cm in the craniocaudal direction) should be applied to the CTV to minimize uncertainties related to tumour motion and patient positioning. The definition of critical organs (OARs)and dose restrictions are the same as in NSCLC, although with more restrictive lung criteria. In post-lobectomy patients, Boonyawan et al, proposed limiting the lung volume that receives 10 and 20 Gy (V10 and V20) to < 30% and < 20%, respectively. In patients older than age 65, the lung V5 should be reduced to ≤ 36%; if IMRT is performed, the recommended V5 is < 64.9%, with mean lung dose (MLD) < 10.8 Gy. In patients undergoing pneumonectomy, to ensure safety, these limitations should be even more restrictive, as follows: V5 < 30%, V20 < 13%, and MLD < 7.5 Gy[51]. If 3D-CRT is used, the V20 should be < 10%.
## Dose and fractionation
In completed-resected (R0) surgeries, the recommended dose is 50-54 Gy using a conventional fractionation scheme (1.8-2 Gy/d). However, in high risk patients with R1 or R2 margins, the total dose may be increased up to 54-60 Gy, or even up to radical doses of 60-66 Gy if there is evidence of macroscopic residue in the surgical bed or mediastinal region.
The use of hypofractionated regimens is not advised due to the risk of increased toxicity. Currently, accelerated fractionation radiotherapy schemes (2 Gy/d, 7 d/wk) are being explored (NCT02189967) .
In terms of treatment sequencing, PORT should be administered after completing ChT if the surgical resection is complete (R0); however, in patients with postoperative R1-R2 margins, there is some controversy surrounding the use of concomitant or sequential RT and ChT. As a result, the treatment sequence should be individualized based on the expected tolerance.
Although several radiotherapy techniques -3D-CRT, intensity modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT), and tomotherapy -all provide optimal dosimetric results in the postoperative context, data from prospective studies support the routine use of the IMRT in NSCLC due to lower cardiac doses and a lower risk of severe pneumonitis.
## Future lines of research in port
At present, there is broad consensus among radiation oncologists that the current level of evidence is insufficient to recommend PORT for all patients with stage III pN2 NSCLC, which is mainly attributable to the heterogeneous characteristics of patients with pN2 disease and treatment-related cardiopulmonary toxicity, which remains high despite efforts to reduce it.
In terms of the lack of homogeneity, it is evident that TNM staging in patients with pN2 NSCLC does not provide sufficient information to indicate or not adjuvant therapy. Consequently, it is essential to explore and evaluate new clinical, pathological, and molecular factors to better differentiate between different risk subpopulations, which would then allow us to tailor the treatment indication based on It is important to note that most of the prognostic factors identified to date have been derived from data obtained in large retrospective series or epidemiological records. Clearly, due to the important methodological limitations of those studies, it is difficult to extrapolate the findings of those studies into routine clinical practice without stronger supporting data. In this regard, new studies with more robust methodological designs are needed to obtain a higher level of evidence.lists the main trials currently underway to evaluate PORT in NSCLC.
The studies performed to date have consistently found an association between PORT and a higher risk of cardiopulmonary morbidity and mortality, a finding that undermines the clinical benefits of this treatment. However, some studies have shown that IMRT is superior to 3D-CRT in NSCLC in terms of dosimetry and survival outcomes. Heavy particle therapy seems to show certain dosimetric advantages vs IMRT in terms of protection of OARs, and could significantly reduce cardiopulmonary toxicity, although prospective studies confirming this clinical benefit are not yet available . For all the reasons described above, it is evident that only advanced radiotherapy techniques, such as VMAT or IMRT, which allow for better dose conformity, should be used for the treatment of NSCLC. In addition, these techniques should be used in all future clinical trials of PORT to better determine the true value of PORT in patients with NSCLC.
# Conclusion
In patients with stage pN2 disease, current evidence suggests that the treatment decision should be evaluated on a case-by-case basis by a multidisciplinary team to determine whether the patient is likely to benefit from PORT. The treatment decision should consider several key clinical features, such as the volume of nodal mediastinal tumor burden, physical condition (performance status) and individual cardiopulmonary risk, but another technological issues, like availability to modern functional imaging devices or high dosimetric conformation radiotherapy (IGRT or VMAT), may be critical for a correct indication.
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Behavior in the use of face masks in the context of COVID‐19
[bib_ref] To mask or not to mask: Modeling the potential for face mask..., Eikenberry [/bib_ref] [bib_ref] Mask or no mask for COVID-19: A public health and market study, Li [/bib_ref] [bib_ref] Efficacy of face mask in preventing respiratory virus transmission: A systematic review..., Liang [/bib_ref] [bib_ref] A rapid systematic review of the efficacy of face masks and respirators..., Macintyre [/bib_ref] [bib_ref] Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1, Schaeffer [/bib_ref]
## | me thods
## | research questions
a. Which behaviors, non-compliant with the WHO guidance on the use of masks, can be observed in the general population? b. How often can behaviors, non-compliant with the WHO guidance on the use of masks, be found in the general population?
## | study design
We conducted a naturalistic observational study.
## | objects and sample
A non-probabilistic convenience sample was observed. We defined to include any adolescent and adult person who would enter the observation corridor after any one-minute time interval to ensure the exclusion of arbitrary selection.
## | observation sites
The observations were conducted in front of 24 grocery stores of different companies in two provinces of Western Austria. We defined an observation corridor of approximately 20-by-20 meters around the entrance areas.
## | observation times
The observations were conducted over a period of 6 weeks from April to June 2020. For each observation unit, a duration of 25 to 40 s was pre-estimated based on the findings from the pre-test.
## | observation procedure
## | observers
The standardized observations were carried out by a total of 13 persons. All observers were nursing science students. The students were instructed face to face as well as via specially designed videos regarding the specific observational conditions. provides information about the structure of the standardized observation process.
## | observation units and standardized assessment
Based on a thematically specific literature research conducted by two independent researchers, various criteria were identified which seemed to be scientifically valid in the correct usage of face masks [bib_ref] Guidance on the use of respiratory and facial protection equipment, Coia [/bib_ref] [bib_ref] Use of face masks in a primary care outpatient setting in Hong..., Ho [/bib_ref]. This pool of identified criteria was topically structured, assessed for relevance and synthesized in a consensus procedure by two researchers.
Subsequently, 12 items were formulated that relate to three main behavioral aspects Transport, Place/Adjust, and Wear. The resulting observation form was tested by the students in a pre-test observation. Based on written and verbal feedbacks of the students on practicability and comprehensibility of the observation form following the pre-test, the observation form was reduced by two items.
Another three items were revised and reformulated due to a reported lack of comprehensibility and clarity.
The final version of the observation form enables a standardized recording of a complete observation unit. It consists of a total of 10 items, which are assigned to three observable main behavioral aspects and are responded by marking the observed behavior corresponding to a dichotomous rating .
# | data analysis
Descriptive data analysis was performed using SPSS Statistics for Windows, version 24.0 (SPSS Inc., Chicago, Ill., USA). Data visualization was performed using SankeyMATIC open source software (www.sanke ymatic.com).
# | ethical considerations
The study protocol was submitted for consideration to the university's competent ethics committee. This ethical committee confirmed in a written statement that there was no need for an ethical committee approval nor an institutional submission requirement.
## F i g u r e 1 structure of the standardized observation process
## | re sults
## | observation characteristics
# | observation results
In total, the carrying, adjusting and wearing behavior of 2,080 per-
## | d iscuss i on
This study aimed to observe behaviors that do not comply with the , [bib_ref] The transtheoretical model of health behavior change, Prochaska [/bib_ref] , [bib_ref] A protection motivation theory of fear appeals and attitude change1, Rogers [/bib_ref]. Due to the acute need for action, however, the implementation of the mask obligation could not be carried out under the mentioned strategically essential aspects. In combination with the negative effects of limited health literacy in the context of COVID-19 [bib_ref] COVID-19: Health literacy is an underestimated problem, Paakkari [/bib_ref] , this could provide an explanation for the insufficient handling of face masks in the sample observed.
## | strengths and limitations
The study design was appropriate for an explorative baseline assessment of the behavior in handling face masks, as no data were available for Austria so far. A strength of the study was the high standardization of the observations by using a structured observation form and professionally trained observers. Nevertheless, it must be critically noted that possible bias cannot be ruled out due to the lack of testing of the observation form for inter-rater reliability. In addition to the standardized observation corridor, observation sites were strictly determined, which minimized bias caused by different conditions within the observations. In order to prevent selection bias, we tried to include people from all income and consumer groups by including grocery stores of different companies (from low budget to exclusive stores). Furthermore, the observed persons were selected by following a strictly regulated procedure with constant time criteria. However, due to the chosen sampling method (convenience sample), selection bias is possible. Due to pandemicrelated travel restrictions at the time of the study, only two of a total of nine provinces in Austria could be included in the data collection.
The results of this observational study can therefore not be generalized to the Austrian population.
We did not choose observation dates according to any defined pattern. This is due to the fact that no theory could be found in the literature which associates preferred shopping times with characteristics of a certain kind of population. Furthermore, at the time of the study, social life and daily routines in Austria were highly influenced by the restrictions and effects of the lockdown. Moreover, the obligation to wear masks in Austria applies without exception to all persons from the age of 6 years onwards and irrespective of individual personal characteristics. Therefore, all observations were carried out on different days of the week at different times of the day in order to reduce bias in this context as far as possible.
The sample size (n = 2,080) would be suitable to generalize the results to the whole Austrian population. Based on a sample calculation assuming an estimated total population of 970,000 persons in the Austrian provinces of Tyrol and Vorarlberg, the minimum sample size would be 384 persons (95% CI). However, the representativeness of the sample cannot be assumed due to the lack of sociodemographic characteristics. Nevertheless, it should be considered that the wearing of face masks is a measure that is equally give any information about the long-term wearing behavior. We decided to conduct the study under the described conditions, as this was the only way to ensure the safety of the observers. A further strength is the comprehensible visualization of the structured observation process and the presentation of the results using a Sankey diagram, which clearly illustrates the error frequencies in relation to the observed carrying and wearing behavior in an innovative form.
## | con clus ions
The usage of face masks in the general public is highly error-prone concerning the behavioral aspects of transporting, adjusting, and wearing. Even if the study is not able to respond to the question of the reasons for the erroneous usage, there is an obvious need for action for the decision makers. Especially because of the potential risk of increase of COVID-19 infections and the associated maintenance of the mask obligation, further research should explore potential associations between erroneous behaviors in the use of masks and various population groups.
## Co n fli c t o f i nte r e s t
## None.
## O rci d
Matthias Rohringer https://orcid.org/0000-0002-7038-0032
## R e fe r e n c e s
[fig] F: WHO guidance for the use of face masks in the general population and to record frequencies in erroneous behavior regarding transport, positioning, and wearing of the mask. Considering the results, erroneous behavior in the use of face masks is highly prevalent in the general population.At least one erroneous behavior could be observed in almost half of all observed persons. With regard to the erroneous carriage of masks, there are potential risks of virus transmission in the sense of self and external hazard. Primarily hands are potentially contaminated with pathogens. This could mean that any contact of the hands with the inside of the mask contributes to the transmission of viruses into the respiratory tract (Chowell et al., 2015). Conversely, especially in the case of multiple use of disposable masks as well as uncleaned cloth masks by infected persons, it can be expected that viruses will be further spread by the contaminated mask as a result of further contact with surfaces (van Doremalen et al., 2020). It could also be observed that a part of all persons wore the mask only under the nose or mouth. In addition to the inadequate protection of the immediate environment caused by this behavior, there is also the possibility that viruses are exhaled directly onto the outside of the mask. As the observation showed, a large proportion of the people who wore the mask below the nose or mouth touched the potentially contaminated mask surface when the mask was completely positioned or before entering the grocery store. Based on the premise that the described cascade of infections can be triggered by touching the mask and that almost half of all persons observed made this mistake in their wearing behavior at least once, the intended the measure may be limited. Taking into account the average observation time (<1 min), this result is concerning inasmuch as these behavioral errors could occur repeatedly with increasing wearing time. urthermore, people who had already worn face masks prior to entering the observation corridor may have gone through adjustments with errors. The time until mouth and nose was completely covered by the mask varied within the time of entering the observation corridor and entering the store. In combination with not reaching the minimum distance, this could have significantly increased transmission risk (Jefferson et al., 2011). The WHO guidance on the use of masks in the context of COVID-19 points out the importance of accompanying information and education measures when the governmental health authority introduces the obligation of wearing masks (World Health Organization, 2020b). Generally speaking, established theories on behavioral change in health-related contexts unanimously point out that the success of systemic public health interventions not only depends on the provision of measure-related information, but that the factors time and the possibility of motivational adaptation are decisive for implementation [/fig]
[table] Table 1: provides information on the observation time per site and the number of persons observed both per site and in total in the respective federal provinces. [/table]
|
Characterizing the genetic diversity of the Andean blueberry (Vaccinium floribundum Kunth.) across the Ecuadorian Highlands
4 blueberry (Vaccinium floribundum Kunth.) across the 5 Ecuadorian Highlands 6 7 8 1 8 4 2 geological history of the Andean region. Our results suggest that elevation could also be 4 3 a key factor in the differentiation of mortiño populations. This study provides an 4 4 extensive overview of the species across its distribution range in Ecuador, contributing 4 5 to a better understanding of its conservation status. These results can assist the 4 6 development of conservation programs for this valuable biological and cultural resource 4 7and for the páramo ecosystems as a whole. 4 8
total of 179 alleles were identified (N A ), with an average number of 11.2 alleles per 2 2 9 locus (ranging from 6 alleles for Mo002 to 20 alleles for Mo020 and Mo025) [fig_ref] Table 1: Genetic diversity indices of the 100 V [/fig_ref].
[formula] 2 3 0 [/formula]
Null alleles presented low to mid frequencies across all the SSR loci, ranging from 2 3 1 0.034 for Mo002 to 0.225 for Mo025 (S3 [fig_ref] Table: The regression equation [/fig_ref]. Expected heterozygosity (H E ) 2 3 2 estimates ranged from 0.26 for Mo001 to 0.91 for Mo025 [fig_ref] Table 1: Genetic diversity indices of the 100 V [/fig_ref]. and PIC (polymorphic information content). Optimum annealing temperature (in °C) for also presented. Primers sequences and SSR motifs are presented in the S2 diversity indicators (S4 [fig_ref] Table: The regression equation [/fig_ref]. Similar results were found when analyzing the mean 2 5 0 allelic richness (A R ) per collection site, an estimate that corrects for the sampling 2 5 1 imbalance between collection sites (S4 [fig_ref] Table: The regression equation [/fig_ref]. The global expected heterozygosity variance explained by the second component (PC2) [fig_ref] Fig 2: Principal coordinate analysis [/fig_ref]. Individuals from the central 2 6 3 region were distributed along the PC2. Furthermore, a clearly differentiated group was in the southern region. Individuals from this group were mainly separated from the 2 6 7 others through the variance explained by the first component (PC1) [fig_ref] Fig 2: Principal coordinate analysis [/fig_ref]. markers. The first three components represent 42.83% of the total variation. Individuals were assigned to their defined latitudinal region (northern, central and A Bayesian analysis was performed to better evaluate the genetic structure of V. [fig_ref] Table: The regression equation [/fig_ref]. Individuals were assigned to four clusters based on their 2 7 9
predominant inferred ancestry . Cluster 1 contained most of the individuals and CS20 (Surimpalti) from the central region (in blue) . Notably, cluster 4 was 2 8 4
highly consistent with the group identified in the PCoA [fig_ref] Fig 2: Principal coordinate analysis [/fig_ref] , comprising the CS27 (Podocarpus in Loja) (S1 [fig_ref] Table: The regression equation [/fig_ref]. occurs within the genetic clusters, and only 21.36% between these clusters (p=0.001) 3 0 2 (S6 [fig_ref] Table: The regression equation [/fig_ref]. Corrected F ST genetic distances show the greater differentiation occurs higher genetic similarities between these clusters [fig_ref] Table 2: Pairwise F ST values between the four genetic clusters identified for V [/fig_ref]. genetic differentiation between the analyzed clusters. Finally, genetic diversity indices for each cluster were calculated ( differences in H E were found between cluster 1 and both clusters 2 (p=0.259) and 3 3 2 1 (p=0.777), nor between cluster 2 and cluster 3 (p=0.331). Among all individual 3 2 2 collection sites, locations assigned to cluster 4 (CS12, CS22-CS24) presented the lowest 3 2 3 diversity indices (S4 [fig_ref] Table: The regression equation [/fig_ref]. H E (expected heterozygosity) are given for all 100 individuals genotyped at 16 SSR loci 3 3 0 distributed in four genetic clusters. A directional relative migration network was inferred for the four genetic clusters, given flow and possible migration rates between the four genetic clusters [fig_ref] Fig 4: Directional relative migration network among the four genetic clusters 3 4 8... [/fig_ref]. As observed 3 3 7 from genetic distances, clusters 1 and 2 presented the greatest genetic similarity (represented by the shorted distances between nodes). In contrast, cluster 4 presented 3 3 9
the greatest differentiation in relation to the others, again in agreement with the results 3 4 0 obtained by the genetic distances' analyses [fig_ref] Table 2: Pairwise F ST values between the four genetic clusters identified for V [/fig_ref]. A relatively high rate of potential 3 4 1 bidirectional asymmetric gene flow was found between clusters 1 and 2, being higher 3 4 2 from cluster 2 to cluster 1 than in the opposite direction. In turn, both clusters present minimal unidirectional gene flow with the other clusters [fig_ref] Fig 4: Directional relative migration network among the four genetic clusters 3 4 8... [/fig_ref]. proportional to the genetic similarity (Nei's Gst) between populations, while the 3 5 0 connecting lines shading reflect the relative migration rate between the populations.
## 5 1
Numbers indicate the gene flow rate (scaled from 0 to 1). Mantel test results revealed a significant correlation between genetic and geographic populations. Furthermore, we observed a significant negative correlation between increment in elevation along our altitude sampling range. The plot shows the correlation between elevation (x-axis) and the genetic diversity (y- individuals were sampled in this study (total of 27 collection sites). The mean number of alleles (N A =11.2 alleles per locus) and the global expected presence of null alleles that tend to bias the estimation of heterozygosity and inflate F ST The total number of alleles obtained for V. floribundum in this study is similar to those The Bayesian approach in STRUCTURE showed that V. floribundum individuals were 4 0 5 grouped into four genetic clusters . From these, only individuals from cluster 4 4 0 6 also grouped separately in the PCoA [fig_ref] Fig 2: Principal coordinate analysis [/fig_ref] , and greatly differentiated from the other this study (3929-4160 masl) (S1 [fig_ref] Table: The regression equation [/fig_ref]. This higher strata of the páramo altitudinal 4 1 2 range has been described as a distinct ecosystem known as superpáramo , and its isolation, such as reproductive barriers due to phenological shifts that results in 4 2 9 differences in flowering time . It is also possible that altitude clines affect and unidirectional from cluster 4 to the other clusters (0.21-0.39) [fig_ref] Fig 4: Directional relative migration network among the four genetic clusters 3 4 8... [/fig_ref]. As a phenomenon that has been proposed as a factor that directly affected the diversity and have come into contact again, a phenomenon known as secondary contact . isolation-by-distance and isolation-by-elevation scenarios is probably more nuanced, as The genetic data presented in this study demonstrates that V. floribundum displays a noteworthy that that elevation also has an effect on the genetic diversity of the was driven by an interplay between isolation-by-distance and isolation-by-elevation processes.
## 0 8
Furthermore, the high values of genetic diversity that were found are encouraging for 5 0 9
the conservation of this species. Since this study assessed the genetic diversity and Ecuadorian Highlands, the results presented here could serve as a basis for the 5 1 2 development of conservation programs for this species and its habitat at the páramo, a 5 1 3 relevant and unique ecosystem. This research was funded with a Chancellor's Grant from Universidad San Francisco de 5 1 7
Quito USFQ (Quito-Ecuador). Genetic data for specimens were obtained under the Ecuadorian law. We would like to thank Diego Urquía for his contribution in data 5 2 1 processing, as well as Dario Ramirez and Andrea Pinos for their assistance in the 5 2 2 collection of the samples. We also thank all the members of the Plant Biotechnology Laboratory (Universidad San Francisco de Quito) who contributed to this research. Conceptualization: María de Lourdes Torres.
## 2 7
Data curation: Pamela Vega-Polo, María Mercedes Cobo.
## 2 8
Formal analysis: Pamela Vega-Polo, María Mercedes Cobo, Bernardo Gutierrez.
## 2 9
Funding acquisition: María de Lourdes Torres. Rowntree, María de Lourdes Torres. Resources: Jennifer Rowntree, María de Lourdes Torres.
## 3 5
Software: Pamela Vega-Polo, Bernardo Gutierrez.
## 3 6
Supervision: María Mercedes Cobo, María de Lourdes Torres.
## 3 7
Validation: Bernardo Gutierrez.
## 3 8
Visualization: Pamela Vega-Polo, María Mercedes Cobo, Bernardo Gutierrez.
## 3 9
Writing -original draft: Pamela Vega-Polo, Andrea Argudo, Jennifer Rowntree. Berlin
[fig] Fig 2: Principal coordinate analysis (PCoA) of V. floribundum using 16 SSR [/fig]
[fig] Fig 4: Directional relative migration network among the four genetic clusters 3 4 8 found in the population structure analysis. The distances between nodes are [/fig]
[fig] Fig 5: Linear regression between V. floribundum genetic diversity and elevation. [/fig]
[fig] V: separated by lower elevation valleys can behave like islands where gene 4 3 5 flow between different locations is decreased. This kind of "sky island" dynamics can and geographic distances (r=0.29; p=0.0001), . floribundum in Ecuador. Under this model, physical barriers could play an additionalTable 2). The moderate degree of differentiation (F ST =0.078) between clusters 1 [/fig]
[table] Table 1: Genetic diversity indices of the 100 V. floribundum individuals analyzed [/table]
[table] Table 2: Pairwise F ST values between the four genetic clusters identified for V. [/table]
[table] Table 3: Genetic diversity indices of the four V. floribundum identified genetic [/table]
[table] Table: The regression equation (H E =0.968-0.00015*Elevation) can be [/table]
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Anorexia nervosa and cancer: a protocol for a systematic review and meta-analysis of observational studies
Background: Anorexia nervosa is characterized by a severe restriction of caloric intake, low body weight, fear of gaining weight or of becoming fat, and disturbance of body image. Pathogenesis of the disorder may include genetic predisposition, hormonal changes and a combination of environmental, psychosocial, and cultural factors. Cancer is the second leading cause of death worldwide. At present, no systematic reviews and meta-analyses have evaluated the risk of cancer in people with anorexia nervosa. The objective of this study will be to evaluate the association between anorexia nervosa and the risk of developing or dying from cancer. Methods/design: This study protocol is part of a systematic collection and assessment of multiple systematic reviews and meta-analyses (umbrella review) evaluating the association of cancer and multiple central nervous system disorders. We designed a specific protocol for a new systematic review and meta-analysis of observational studies of anorexia nervosa with risk of developing or dying from any cancer. Data sources will be PubMed, Embase, Scopus, Web of Science, and manual screening of references. Observational studies (case-control and cohort) in humans that examined the association between anorexia nervosa and risk of developing or dying from cancer will be sought. The primary outcomes will be cancer incidence and cancer mortality in association with anorexia nervosa. Secondary outcomes will be sitespecific cancer incidence and mortality, respectively. Screening of abstracts and full texts, and data abstraction will be performed by two team members independently. Conflicts at all levels of screening and abstraction will be resolved through discussion. The quality of studies will be assessed by using the Ottawa-Newcastle scale by two team members independently. Random effects models will be conducted where appropriate. Subgroup and additional analyses will be conducted to explore the potential sources of heterogeneity. The World Cancer Research Fund (WCRF)/American Institute for Cancer Research (AICR) criteria and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach will be used for determining the quality of evidence for cancer outcomes. Discussion: Findings from this systematic review will inform an ongoing umbrella review on cancer and central nervous system disorders. Our systematic review and meta-analysis of observational studies will establish the extent of the epidemiological evidence underlying the association between anorexia nervosa and cancer. Systematic review registration: PROSPERO CRD42017067462.
# Background
Anorexia nervosa is a mental disorder characterized by a severe restriction of caloric intake, a significantly low body weight for the developmental stage, an intense fear of gaining weight or of becoming fat, and a severe disturbance of body image. Pathogenesis of the disorder may include genetic predisposition, hormonal changes, and a combination of environmental, psychosocial, and cultural factors [bib_ref] Anorexia nervosa: aetiology, assessment, and treatment, Zipfel [/bib_ref] [bib_ref] Eating disorders, Treasure [/bib_ref]. Anorexia nervosa can affect people of all ages and genders, and have been reported worldwide both in high income and low-middle income regions [bib_ref] The global burden of eating disorders, Erskine [/bib_ref] [bib_ref] Global, regional, and national disability-adjusted life-years (DALYs) for 315 diseases and injuries..., Dalys [/bib_ref]. Most recent burden of disease estimates revealed 2.9 million people with anorexia nervosa (representing 653,019 disability-adjusted life years lost) around the world [bib_ref] Global, regional, and national disability-adjusted life-years (DALYs) for 315 diseases and injuries..., Dalys [/bib_ref]. The disorder is more prevalent among adolescent and young women; however, young men may also be affected. Although most patients eventually recover, anorexia nervosa can blight young lives and distort development [bib_ref] Death in anorexia nervosa, Palmer [/bib_ref] [bib_ref] Mortality rates in patients with anorexia nervosa and other eating disorders. A..., Arcelus [/bib_ref] [bib_ref] Re-examining premature mortality in anorexia nervosa: a meta-analysis redux, Keshaviah [/bib_ref] [bib_ref] Quality of life in eating disorders: a meta-analysis, Winkler [/bib_ref].
Cancer is the second leading cause of death worldwide [bib_ref] Global Burden of Disease Cancer Collaboration, Fitzmaurice [/bib_ref] [bib_ref] Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years..., Fitzmaurice [/bib_ref] , with over 8.8 million deaths in 2015 [bib_ref] Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years..., Fitzmaurice [/bib_ref]. There is evidence suggesting that excess body weight is a risk factor for several cancers. For example, a recent umbrella review of 204 meta-analyses [bib_ref] Adiposity and cancer at major anatomical sites: umbrella review of the literature, Kyrgiou [/bib_ref] found strong evidence for the association between body mass index and cancers of digestive organs (esophageal adenocarcinoma and cancers of the colorectum, biliary tract system, and pancreas), hormone-related cancers (such as breast cancer in women), endometrial cancer, kidney cancer, and multiple myeloma; but also between adiposity and the risk of colorectal cancer, gallbladder cancer, gastric cardia cancer, ovarian cancer, and multiple myeloma [bib_ref] Adiposity and cancer at major anatomical sites: umbrella review of the literature, Kyrgiou [/bib_ref]. The underlying mechanisms between excess body weight and cancer are complex and are not yet fully understood. Excess body weight and adiposity might affect immune system function and inflammatory processes, levels of certain hormones (such as insulin and estrogen), factors that regulate cell growth (such as insulin-like growth factor-1 [IGF-1]), and proteins that influence how the body uses certain hormones (such as sex hormone-binding globulin), among others [bib_ref] Overweight, obesity, and mortality from cancer in a prospectively studied cohort of..., Calle [/bib_ref].
Research on how losing body weight might lower the risk of developing cancer is limited. Energy restriction (or calorie restriction) has been found to be protective against the development of cancer in experimental animal studies [bib_ref] Lasting influence of early caloric restriction on prevalence of neoplasms in the..., Ross [/bib_ref] [bib_ref] Energy restriction and the risk of spontaneous mammary tumors in mice: a..., Dirx [/bib_ref] [bib_ref] Calorie restriction and prevention of age-associated chronic disease, Omodei [/bib_ref] [bib_ref] Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms, Calle [/bib_ref]. Energy restriction is difficult to study in human populations. Anorexia nervosa, an excessive form of calorie restriction associated with pathological weight loss, has been proposed as a biomarker of energy restriction [bib_ref] Calorie restriction and cancer prevention: a mechanistic perspective, Hursting [/bib_ref] [bib_ref] Anorexia nervosa and cancer risk, Mellemkjaer [/bib_ref]. Several epidemiological studies [bib_ref] Anorexia nervosa and cancer risk, Mellemkjaer [/bib_ref] [bib_ref] Caloric restriction and incidence of breast cancer, Michels [/bib_ref] [bib_ref] Age at onset of anorexia nervosa and breast cancer risk, Papadopoulos [/bib_ref] [bib_ref] No paradox, no progress: inverse cancer comorbidity in people with other complex..., Tabarés-Seisdedos [/bib_ref] have evaluated whether there exists a general reduction in cancer development among patients with anorexia nervosa (the so-called, "energy-restriction hypothesis". For example, a retrospective cohort study by Mellemkjaer et al. [bib_ref] Anorexia nervosa and cancer risk, Mellemkjaer [/bib_ref] suggested a potential reduction, but not statistically significant, in cancer incidence among women with anorexia nervosa compared with the general population (standardized incidence ratio [SIR] = 0.80; 95% confidence interval [CI] 0. . In another retrospective cohort study, Michels and Ekbom [bib_ref] Caloric restriction and incidence of breast cancer, Michels [/bib_ref] reported that women hospitalized for anorexia nervosa were associated with a lower incidence of breast cancer compared to the general population (SIR = 0.47; 95% CI 0.19-0.97), with a larger effect among parous women (SIR = 0.24; 95% CI 0.03-0.87). However, a recent observational study assessing the risk of cancer among people with anorexia nervosa has suggested that the potential associations in humans remain controversial [bib_ref] Cancer incidence among patients with anorexia nervosa from Sweden, Denmark and Finland, Mellemkjaer [/bib_ref].
At present, no systematic reviews and meta-analyses have evaluated the epidemiological evidence examining the association between anorexia nervosa and cancer. To better understand the body of the evidence, we will conduct a systematic review and meta-analysis of observational studies in order to synthesize and evaluate the validity of the association between anorexia nervosa and the risk of developing or dying from cancer.
# Methods
## Protocol
This study protocol is part of an ambitious ongoing umbrella review (a systematic collection and assessment of multiple systematic reviews and metaanalyses) into the association of cancer and multiple central nervous system disorders [bib_ref] Cancer and central nervous system disorders: protocol for an umbrella review of..., Catalá-López [/bib_ref]. To our knowledge, no previous systematic reviews and metaanalyses have evaluated the risk of developing or dying from cancer in anorexia nervosa. For this reason, we have designed a specific protocol for a new systematic review and meta-analysis. The present protocol has been registered within the PROSPERO database (registration number: CRD42017067462) and is being reported in accordance with the reporting guidance provided in the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) reporting guideline [bib_ref] Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of..., Stroup [/bib_ref] and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) statement [bib_ref] Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P), Moher [/bib_ref] [bib_ref] Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration..., Shamseer [/bib_ref] (see PRISMA-P checklist in Additional file 1).
## Ethics
No ethical approval is required for the performance of this work.
# Search methods
We will systematically search, from inception, PubMed/ MEDLINE, EMBASE, SCOPUS, and Web of Science to identify observational studies examining association between cancer and anorexia nervosa. A date restriction will not be imposed. The final electronic search strategies will be defined by a senior information specialist (AA-A) and by a clinical epidemiologist (FC-L). Keywords related to anorexia nervosa, cancer, and epidemiological studies will be used. A draft search strategy for PubMed/MEDLINE database has been included in Additional file 2. The reference lists of examined full-text papers will be scrutinized for additional relevant publications. We will also contact authors of primary publications and/or collaborators to check if they are aware of any studies we may have missed. There will be no restriction by language of publication, and we will arrange for translation where necessary.
## Eligibility criteria
Studies will be selected according to the following criteria: study design, participants (exposure), comparator(s) or control group, and outcome(s) of interest.
Study design: eligible studies will be observational studies reporting study specific data for cancer outcomes in people with anorexia nervosa. Prospective cohort studies, retrospective cohort studies (also known as historical cohort studies), and case-control studies will be included. Randomized controlled trials will be unavailable for our research question. We will exclude studies in which anorexia nervosa is not the exposure of interest, and cancer is not reported as the outcome of interest. Observational studies not presenting study specific data (e.g., relative risks, 95% confidence intervals, numbers of cases/population, observed and expected cases) or sufficient data for an outcome measure to be calculated will be also excluded. There will be no restriction by study setting.
Participants (exposure): index subjects will be patients with anorexia nervosa (regardless of age or sex). We will use investigator-reported definitions (according to accepted diagnostic criteria such as the International Comparator(s) or control group: the comparator group will be based on subjects with no history of anorexia nervosa (e.g., the general population, the community, unexposed outpatient, or hospital-based controls).
Outcome(s): the primary outcomes will be cancer incidence and cancer mortality (all malignant neoplasms; ICD-9: 140-209; ICD-10: C00-C97) in association with anorexia nervosa. Given the varied biology of cancers, the risk of incident site-specific cancers, and the risk of fatal site-specific cancers will be explored as secondary outcomes. Site-specific cancers will be defined in groups that include ICD codes pertaining to neoplasms [bib_ref] Global Burden of Disease Cancer Collaboration, Fitzmaurice [/bib_ref] (see Additional file 3).
## Screening and selection procedure
Two reviewers will screen all articles identified from the search independently. First, titles and abstracts of articles returned from initial searches will be screened based on the eligibility criteria outlined above. Second, full texts will be examined in detail and screened for eligibility. Third, references of all considered articles will be hand-searched to identify any relevant report missed in the search strategy by two reviewers independently. Any disagreement between reviewers will be resolved by discussion to meet a consensus.
## Data collection process
From each eligible observational study, two reviewers will independently extract information on first author, year of publication, epidemiological design (cohort or case-control, prospective, or retrospective), country of study, follow-up period, setting (mixed, inpatient, outpatient, or community), coverage (multi-center or single center study), general characteristics of participants (age, sex, ethnicity, and parity status), sample size, the outcomes of interest (including definitions and confounding factors that were taken into consideration), the number of cases and controls (in case-control studies) or the number of cases and population participants (in cohort studies) and/ or the maximally adjusted relative risk (reported as odds ratio for case-control studies and hazard ratio or standardized incidence/mortality ratio for cohort studies), and 95% confidence intervals. We will use pre-designed forms that will be piloted initially on a small number of included reviews and observational studies. We will also contact authors of primary publications and/or collaborators for missing outcome data or unclear information.
## Quality and risk of bias assessment
The methodological quality and bias of primary epidemiological studies will be evaluated using the Newcastle-Ottawa scale (NOS) for observational studies. Using the NOS tool, each study is judged on eight items, categorized into three groups: the selection of the study groups, the comparability of the groups, and the ascertainment of either the exposure or outcome of interest for case-control or cohort studies, respectively. Stars are awarded for each item, and the highest quality (low risk of bias) studies are awarded up to nine stars. We will consider studies with 0-3, 4-6, and 7-9 stars to represent low, moderate, and high quality, respectively. The quality (risk of bias) for each observational study will be independently assessed by two reviewers. Discrepant scores will be resolved by discussion and consensus.
## Methods for evidence synthesis
The data from each paper (e.g., population, study characteristics, outcomes, and findings) will be used to build evidence tables. Data from primary observational studies will be used to perform random-effects metaanalyses. We will estimate the summary effect size and its 95% confidence interval using the inverse variance method based on the DerSimonian and Laird random effects model [bib_ref] Meta-analysis in clinical trials, Dersimonian [/bib_ref]. The random-effects model is selected a priori to synthesize the epidemiological data, as it considers both within-study and between-study variation by incorporating the heterogeneity of effects into the overall analyses. We will evaluate heterogeneity by estimating the variance between studies using Cochran's Q test [bib_ref] The combination of estimates from different experiments, Cochran [/bib_ref] and I 2 statistic [bib_ref] Measuring inconsistency in meta analyses, Higgins [/bib_ref]. The Cochran's Q test is obtained by the weighted sum of the squared differences of the observed effect in each study minus the fixed summary effect. The I 2 statistic is the ratio of variance between studies over the sum of the variances within and between studies, and ranges between 0 and 100% (with values of 0-25% and 75-100% taken to indicate low and considerable heterogeneity, respectively). In addition, we will calculate the 95% prediction interval [bib_ref] A re-evaluation of randomeffects meta-analysis, Higgins [/bib_ref] [bib_ref] Interpretation of random effects metaanalyses, Riley [/bib_ref] , which further accounts for between-study heterogeneity and evaluates the uncertainty for the effect that would be expected in a new observational study.
We will apply a set of criteria to conclude whether the evidence for a cancer outcome may be considered convincing, probable, limited-suggestive, limited-not conclusive, or unlikely. As described elsewhere [bib_ref] Cancer and central nervous system disorders: protocol for an umbrella review of..., Catalá-López [/bib_ref] , we will follow the Global Burden of Disease Study approach based on World Cancer Research Fund (WCRF)/American Institute for Cancer Research (AICR) criteria for grading the quality of evidence [bib_ref] Global, regional, and national comparative risk assessment of 79 behavioural, environmental and..., Forouzanfar [/bib_ref]. "Convincing evidence" consists of biologically plausible associations between exposure and outcome based on multiple epidemiological studies in different populations. Evidentiary studies must be substantial, include prospective observational studies, and where relevant, epidemiological studies of sufficient size, duration, and quality and showing consistent effects. A convincing relationship should be robust enough to be highly unlikely to be modified in the foreseeable future as new evidence accumulates. "Probable evidence" is similarly based on epidemiological studies with consistent associations between exposure and outcome but with existing shortcomings, such as insufficient prospective observational studies available. "Limited-suggestive evidence" represents too limited evidence to conclude on a probable or convincing causal association, but where there is evidence suggestive of a direction of effect. "Limited-not conclusive evidence" consists of information that is so limited that no firm conclusion can be made for several reasons (e.g., the evidence might be limited by the amount of evidence in terms of the number of studies available, by inconsistency of direction of effect, by poor quality of studies, or by any combination of these factors). "Substantial effect on risk unlikely" consists of evidence strong enough to support a judgment that a particular exposure is unlikely to have a substantial causal relation to a cancer outcome. The evidence should be robust enough to be unlikely to be modified in the foreseeable future as new evidence accumulates. We will also use the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology for evaluating the quality of evidence for each outcome [bib_ref] GRADE: an emerging consensus on rating quality of evidence and strength of..., Guyatt [/bib_ref] [bib_ref] The GRADE approach is reproducible in assessing the quality of evidence of..., Mustafa [/bib_ref] [bib_ref] GRADE guidelines: 4. Rating the quality of evidence-study limitations (risk of bias), Guyatt [/bib_ref]. For purposes of systematic reviews, the GRADE approach defines the quality of a body of evidence as the extent to which one can be confident that an estimate of effect or association is close to the quantity of specific interest. Using GRADE, the quality of a body of evidence involves consideration of withinstudy risk of bias (methodological quality), directness of evidence, heterogeneity, precision of effect estimates, and risk of small study effects (sometimes called "publication bias") [bib_ref] GRADE: an emerging consensus on rating quality of evidence and strength of..., Guyatt [/bib_ref] [bib_ref] The GRADE approach is reproducible in assessing the quality of evidence of..., Mustafa [/bib_ref] [bib_ref] GRADE guidelines: 4. Rating the quality of evidence-study limitations (risk of bias), Guyatt [/bib_ref]. GRADE rating will be adjudicated as high (further research is very unlikely to change our confidence in the estimate of effect), moderate (further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate), low (further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate), or very low (very uncertain about the estimate of effect) [bib_ref] GRADE: an emerging consensus on rating quality of evidence and strength of..., Guyatt [/bib_ref].
## Additional analyses
If sufficient studies are identified, potential sources of heterogeneity will be investigated further by subgroup or meta-regression analyses according to baseline characteristics and methodological factors [bib_ref] Cancer and central nervous system disorders: protocol for an umbrella review of..., Catalá-López [/bib_ref]. We plan to conduct subgroup analyses by sex (men or women), age (e.g., at first diagnosis of anorexia nervosa), study design (cohort or case-control; prospective, or retrospective), follow-up (0-1, >1-5, or >5 years), setting (mixed, inpatient, outpatient, or community), ethnicity (e.g., Asian or non-Asian), population-based (yes or no), country economic status (developed or developing countries according to International Monetary Fund), year of publication (before 2000 or in 2000 and after), study quality (high or low-moderate risk of bias), adjustment for confounding variables (age, sex or other), and sample size (<500, 500-1000 or >1000 participants). If sufficient information is identified, we will conduct subgroup analyses or meta-regression analyses for cancer types according to relationship with smoking (smoking-related cancer sites or other cancer sites) or sex hormones (cancers occurring in hormone-sensitive tissues such as breast, ovary, uterine endometrium, prostate and colorectal, where sex hormones exert an important role in cancer etiopathogenesis and progression) [bib_ref] Caloric restriction and incidence of breast cancer, Michels [/bib_ref] [bib_ref] Hormones, vitamins, and growth factors in cancer treatment and prevention. A critical..., Lupulescu [/bib_ref] [bib_ref] Influence of sex hormones on cancer progression, Folkerd [/bib_ref] (see Additional file 2). We will conduct a specific subgroup analysis among women based on parity status (parous or nulliparous women) [bib_ref] Caloric restriction and incidence of breast cancer, Michels [/bib_ref] [bib_ref] Age at onset of anorexia nervosa and breast cancer risk, Papadopoulos [/bib_ref]. If sufficient studies are identified, we will perform cumulative metaanalyses in the order of publication year showing the consistency of evidence over time [bib_ref] Cumulative meta-analysis of therapeutic trials for myocardial infarction, Lau [/bib_ref] [bib_ref] Evolution of treatment effects over time: empirical insight from recursive cumulative metaanalyses, Ioannidis [/bib_ref]. Small study effects will be assessed by inspection of the funnel plots for asymmetry and with Egger's test [bib_ref] Bias in meta-analysis detected by a simple, graphical test, Egger [/bib_ref] and Begg's test [bib_ref] Operating characteristics of a rank correlation test for publication bias, Begg [/bib_ref] , with the results considered to indicate potential small study effects when P < 0.10.
## Software considerations
All analyses will be conducted in Stata version 13 or higher (StataCorp LP, College Station, Texas, USA) using the metan (for fixed and random effects meta-analysis), metareg (for meta-regression analysis), metacum (for cumulative meta-analysis), and metabias and metafunnel (for small study effects analysis).
# Discussion
The systematic review presented in this protocol will inform an ongoing umbrella review and meta-analysis of observational studies on cancer and central nervous system disorders [bib_ref] Cancer and central nervous system disorders: protocol for an umbrella review of..., Catalá-López [/bib_ref]. This systematic review will establish the extent of the epidemiological evidence underlying the association between anorexia nervosa and the risk of developing or dying from cancer, in a reproducible and rigorous way. The systematic review and meta-analysis presented in this protocol will be reported in accordance with the reporting guidance provided in the PRISMA statement [bib_ref] Preferred reporting ítems for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref] and the MOOSE reporting guideline [bib_ref] Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of..., Stroup [/bib_ref]. Any amendments or modifications made in the protocol will be outlined and reported in the final paper.
Direct and inverse cancer comorbidity could be a relevant model to investigate common or related pathways or processes and test new therapies and prevention programs, but, most importantly, to understand why certain people might potentially be protected from the malignancy [bib_ref] No paradox, no progress: inverse cancer comorbidity in people with other complex..., Tabarés-Seisdedos [/bib_ref] [bib_ref] Inverse cancer comorbidity: a serendipitous opportunity to gain insight into CNS disorders, Tabarés-Seisdedos [/bib_ref] [bib_ref] Inverse and direct cancer comorbidity in people with central nervous system disorders:..., Catalá-López [/bib_ref]. In this context, understanding the complex connections between anorexia nervosa and cancer might be important for clinical research and practice.
There are several strengths and limitations of our planned methods. We will comprehensively evaluate epidemiological data characterizing the associations between anorexia nervosa and cancer, exploring the extent of heterogeneity and bias in observational studies. We have planned assessments of meta-bias and strength of evidence statements. We anticipate that we will identify knowledge gaps to be filled by new research considering that some outcomes will be poorly covered in the biomedical literature. A key challenge is that based on knowledge from previous reviews on cancer and central nervous system disorders [bib_ref] Inverse and direct cancer comorbidity in people with central nervous system disorders:..., Catalá-López [/bib_ref] [bib_ref] A systematic review of the incidence and prevalence of cancer in multiple..., Marrie [/bib_ref] [bib_ref] Parkinson's disease and cancer risk: a systematic review and meta-analysis, Bajaj [/bib_ref] [bib_ref] Cancer incidence in patients with schizophrenia and their first-degree relatives-a meta-analysis, Catts [/bib_ref] , we anticipate identifying studies using different study designs, populations, durations, and with a variable quality of reporting methods and results.
## Additional files
[fig] Additional file 1: PRISMA-P Checklist. (DOCX 27 kb) Additional file 2: Key terms for PubMed/MEDLINE search. (DOCX 22 kb) Additional file 3: Definitions of specific cancer-site outcomes. (DOCX 27 kb) Abbreviations AICR: American Institute for Cancer Research; AMSTAR: Assessment of Multiple Systematic Reviews; DSM: Diagnostic and Statistical Manual of Mental Disorders; GRADE: Grading of Recommendations Assessment, Development, and Evaluation; ICD: International Classification of Diseases; MOOSE: Meta-analysis Of Observational Studies in Epidemiology; NOS: Newcastle-Ottawa scale; PRISMA-P: Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Protocols; WCRF: World Cancer Research Fund [/fig]
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Whole genome sequence of Corynebacterium kroppenstedtii isolated from a case of recurrent granulomatous mastitis
A B S T R A C TWe describe the case of a 33-year-old woman with recurrent granulomatous mastitis associated with Corynebacterium kroppenstedtii. This organism has been increasingly associated with granulomatous mastitis, specifically the cystic neutrophilic histopathologic variant, although currently there is a paucity both of reported cases and genomic sequence data. We highlight the challenges in the diagnosis and treatment of this entity, in particular focusing on the various methods of microbiologic identification, including MALDI-TOF, 16 s rRNA PCR and whole-genome sequencing.
A 33-year-old woman was referred to the Infectious Diseases clinic with a mass in her left breast. She was pregnant at the time of assessment at 21 weeks and 6 days gestational age (gravida 3, para 1). She had a 2-year-old son and stopped breastfeeding 11 months prior to conception of her current pregnancy. Her past medical history was otherwise significant for a previous cholecystectomy. The patient developed progressive pain and edema along the medial aspect of her left breast 13 days prior to assessment. There were no skin changes or nipple discharge. She presented to the emergency department of our hospital 2 days later and was prescribed cephalexin for presumed bacterial mastitis. She was referred for breast imaging and underwent an ultrasound of her left breast 2 days later. This demonstrated a lobulated, hypoechoic lesion with peripheral vascularity, measuring 7.7 Â 3.4 cm in the largest dimension. There was overlying skin thickening and subcutaneous edema, with no abnormal axillary lymphadenopathy. The radiographic appearance was concerning for a breast abscess. Ultrasound-guided biopsy and aspirate of this lesion was performed, with 6 specimens obtained for evaluation.
Histopathologic examination demonstrated extensive mixed chronic inflammatory infiltrates, including lymphocytes, plasma cells and neutrophils, centred around breast lobules [fig_ref] Figure 1: Hematoxylin and esosin stain of cystic neutrophilic granulomatous mastitis [/fig_ref]. The neutrophils were arranged around microcystic lipid spaces, some of which contained gram positive bacterial organisms. The overall appearance was felt to be in keeping with cystic neutrophilic granulomatous mastitis. Gram stain demonstrated few polymorphonuclear cells with no bacteria. Specimens were planted on Columbia agar with 5% sheep blood, chocolate agar and MacConkey agar and incubated at 37 C, with no growth at 48 h. 16S rRNA PCR was subsequently performed on a tissue specimen, and Sanger sequencing identified Corynebacterium kroppenstedtii.
The patient was seen in the Infectious Diseases clinic after these investigations. She had significant clinical improvement with 2 weeks of cephalexin. The patient had minimal pain at her left breast and was systemically well. Physical examination demonstrated a residual nodule in her left breast, with no overlying inflammatory features. She completed a 4-week course of cephalexin, and her disease remained quiescent throughout her pregnancy. Treatment with doxycycline or trimethoprim-sulfamethoxazole, which have more typically been used to treat breast infections associated with Corynebacterium, was considered, but their use was precluded by teratogenicity.
Unfortunately, the patient was seen 3 months after delivery with worsening pain and drainage from her left breast. Ultrasoundguided aspiration was repeated, with demonstration of a complex fluid collection measuring 4.0 Â 2.3 cm in the largest dimension. Gram stain demonstrated few polymorphonuclear cells with no bacteria. Given the prior identification of C. kroppenstedtii by 16S rRNA PCR, the aspirate was planted on sheep blood agar enriched with 0.1 % Tween 80, in addition to standard media, and incubated at 37 C. Small colonies were seen on the Tween 80-enriched sheep blood agar at 24 h and appeared as non-branching gram-positive bacilli on gram stain [fig_ref] Figure 2: Gram stain of C [/fig_ref]. The colonies were identified as C. kroppenstedtii by matrix-associated laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry. Antibiotic sensitivity testing demonstrated susceptibility to cefotaxime, ciprofloxacin, doxycycline, erythromycin, gentamicin, linezolid, tetracycline and vancomycin based on Clinical and Laboratory Standards Institute breakpoints. The patient completed another 4 weeks of cephalexin, with good response. Repeat ultrasound at end-oftherapy demonstrated interval reduction in the size of the collection, and she has remained well at her most recent assessment, with no significant pain or drainage from her left breast.
Additionally, we characterized the isolate using whole-genome sequencing. To facilitate this, DNA was extracted from a 48 -h sheep blood agar culture using MasterPure Complete DNA and RNA Purification Kit (Lucigen, Middleton, USA) as per the manufacturer's protocol. Extracted DNA was quantified using Qubit 1X dsDNA HS Kit (Thermo Fisher Scientific, Waltham, USA) and used as input for library preparation with Nextera DNA Flex Library Prep Kit (Illumina, San Diego, USA) according to the manufacturer's instructions. Normalized libraries were sequenced on a MiniSeq (Illumina, San Diego, USA) using a mid-output 2 Â 150 bp pairedend run that resulted in 633 642 raw reads, respectively. Genome assembly and analysis was performed using the Shovill pipeline on the Galaxy platform [bib_ref] The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2020 update, Jalili [/bib_ref]. Globally, the draft genome was 2,511,697 bp in length with GC content 56.8 %.
# Discussion
Granulomatous mastitis is an uncommon inflammatory disease of the breast occurring in women of reproductive age, within a few years of giving birth. Patients typically present with a unilateral, firm and mildly tender breast mass, often resembling a malignant lesion [bib_ref] A clinicopathological review of 34 cases of inflammatory breast disease showing an..., Taylor [/bib_ref]. There may be features of local inflammation, including erythema and sinus tract formation [bib_ref] A 9-Point risk assessment for patients who inject drugs and require intravenous..., Eaton [/bib_ref].
Granulomatous mastitis is largely considered an idiopathic condition, with various possible inflammatory and infectious disease associations including amyloidosis, sarcoidosis, hyperprolactinemia, granulomatosis with polyangiitis, tuberculosis and fungal infection [bib_ref] A clinicopathological review of 34 cases of inflammatory breast disease showing an..., Taylor [/bib_ref]. However, cystic neutrophilic granulomatous mastitis, a specific subtype of granulomatous mastitis, has been increasingly associated with C. kroppenstedtii. Histopathologically, cystic neutrophilic granulomatous mastitis is characterized by lipogranulomas, comprised of lipid vacuoles bordered by neutrophils and an outer layer of macrophages, with surrounding mixed inflammatory infiltrate of lymphocytes, neutrophils and giant cells [bib_ref] Cystic neutrophilic granulomatous mastitis: an update, Wu [/bib_ref]. The vacuoles may contain gram positive bacilli, as seen in our case [bib_ref] Cystic neutrophilic granulomatous mastitis: an update, Wu [/bib_ref].
Corynebacterium are catalase-positive, non-sporulating, gram positive bacilli, of which more than 90 species have been identified. Non-diphtheroid species are ubiquitous; they colonize mucocutaneous surfaces and frequently contaminate clinical specimens. However, they are capable of invasive infection in appropriate hosts, such as patients who are immunocompromised or have prosthetic devices.
C. kroppenstedtii was first isolated in 1998 from the sputum sample of a woman with pneumonia [bib_ref] Corynebacterium kroppenstedtii sp. nov., a novel corynebacterium that does not contain mycolic..., Collins [/bib_ref]. The relationship with granulomatous mastitis was described in 2002 in a retrospective study of 24 women with histopathologically-confirmed disease and isolation of Corynebacterium species, of which the majority were C. kroppenstedtii [bib_ref] Corynebacterium species isolated from patients with mastitis, Paviour [/bib_ref]. Since then, the association has been documented in multiple case reports and case series [bib_ref] A clinicopathological review of 34 cases of inflammatory breast disease showing an..., Taylor [/bib_ref] [bib_ref] The brief case: recurrent granulomatous mastitis due to Corynebacterium kroppenstedtii, Johnson [/bib_ref] [bib_ref] Corynebacterium kroppenstedtii: a challenging culprit in breast abscesses and granulomatous mastitis, Saraiya [/bib_ref] [bib_ref] Cystic neutrophilic granulomatous mastitis: the Cleveland Clinic experience with diagnosis and management, Gautham [/bib_ref] [bib_ref] Cystic neutrophilic granulomatous mastitis associated with Corynebacterium including Corynebacterium kroppenstedtii, Johnstone [/bib_ref] [bib_ref] Idiopathic granulomatous mastitis: a 10-year study from a multicentre clinical database, Co [/bib_ref] [bib_ref] Two cases of granulomatous mastitis caused by Corynebacterium kroppenstedtii infection in Nulliparous..., Kutsuna [/bib_ref] [bib_ref] Recurrent breast abscesses due to Corynebacterium kroppenstedtii, a human pathogen uncommon in..., Fleche-Mateos [/bib_ref]. C. kroppenstedtii is a lipophilic organism lacking mycolic acids in its cell membrane, conferring the propensity to infect lipid-rich breast tissue [bib_ref] The brief case: recurrent granulomatous mastitis due to Corynebacterium kroppenstedtii, Johnson [/bib_ref]. This property also explains the organism's fastidious nature, and growth on regular media can be challenging, as seen in our case [bib_ref] A microbiological and clinical review on Corynebacterium kroppenstedtii, Tauch [/bib_ref]. The addition of a lipid component, such as Tween 80, to sheep blood agar may enhance growth [bib_ref] A microbiological and clinical review on Corynebacterium kroppenstedtii, Tauch [/bib_ref]. Microbiologic identification can be performed with MALDI-TOF mass spectrometry and 16S rRNA PCR [bib_ref] A microbiological and clinical review on Corynebacterium kroppenstedtii, Tauch [/bib_ref].
The management of cystic neutrophilic granulomatous mastitis associated with C. kroppenstedtii can be challenging. The diagnosis is often delayed given the non-specific symptomatology and imaging findings, need for biopsy for histopathologic evaluation, and difficulty of isolating C. kroppenstedtii by routine culture methods. Lipid components enhance the usually fastidious growth of C. kroppenstedtii, but are generally not added without strong clinical or microbiologic suspicion. No guidelines exist for the treatment of granulomatous mastitis by C. kroppenstedtii, though typically involves prolonged courses of antibiotics for several weeks. The antibiotic susceptibilities of C. kroppenstedtii have been rigorously investigated. A study of 11 breast tissue and aspirate specimens that grew C. kroppenstedtii in culture found susceptibility to rifampin, tetracycline, trimethoprim-sulfamethoxazole, linezolid and vancomycin; resistance to beta-lactams was observed, though this may have been biased by treatment of betalactam-sensitive strains [bib_ref] Antimicrobial treatment options for granulomatous mastitis caused by Corynebacterium species, Dobinson [/bib_ref]. Beta-lactams, fluoroquinolones, clindamycin, aminoglycosides and macrolides have also been used successfully [bib_ref] Corynebacterium kroppenstedtii: a challenging culprit in breast abscesses and granulomatous mastitis, Saraiya [/bib_ref]. Given the lipophilic nature of C. kroppenstedtii and its tropism for breast tissue, lipophilic antibiotics, such as doxycycline and trimethoprim-sulfamethoxazole, may be more effective due to greater penetration into lipid-rich environments [bib_ref] Antimicrobial treatment options for granulomatous mastitis caused by Corynebacterium species, Dobinson [/bib_ref]. Surgery should be considered and was previously thought to be integral to management, but recent evidence suggests procedures outside of drainage of fluid collections are not indicated, and may be associated with increased recurrence and scarring [bib_ref] Re-evaluating if observation continues to be the best management of idiopathic granulomatous..., Davis [/bib_ref] [bib_ref] Is surgical excision necessary for the treatment of Granulomatous lobular mastitis?, Shin [/bib_ref]. Interestingly, cases of idiopathic granulomatous mastitis, where C. kroppenstedtii has not been identified though is not necessarily absent, are widely treated with corticosteroids [bib_ref] Idiopathic granulomatous mastitis-a prospective study of 49 women and treatment outcomes with..., Pandey [/bib_ref] , and often resolve after diagnosis without medical or surgical intervention. Whether cystic neutrophilic granulomatous mastitis associated with C. kroppenstedtii is phenotypically unique from classic idiopathic granulomatous mastitis, apart from its known histopathologic distinctions, and responds differently to standard treatment requires further elucidation. The natural history of granulomatous mastitis is characterized by long duration to resolution, despite adequate antibiotic and surgical management. A study of 145 episodes found an average time to resolution of 5 months, with a range of up to 20 months [bib_ref] Re-evaluating if observation continues to be the best management of idiopathic granulomatous..., Davis [/bib_ref]. As in our patient, recurrent disease is also common, and several previous published cases have involved multiple antibiotic courses and surgical procedures [bib_ref] The brief case: recurrent granulomatous mastitis due to Corynebacterium kroppenstedtii, Johnson [/bib_ref] [bib_ref] Corynebacterium kroppenstedtii: a challenging culprit in breast abscesses and granulomatous mastitis, Saraiya [/bib_ref] [bib_ref] Recurrent breast abscesses due to Corynebacterium kroppenstedtii, a human pathogen uncommon in..., Fleche-Mateos [/bib_ref].
# Conclusion
Our case highlights the importance of considering C. kroppenstedtii in patients with granulomatous mastitis. Growth on conventional media is challenging due to the organism's fastidious nature; recognition of this disease-bacteria association allows for adjustments to culture methods that will enhance growth. Our case also demonstrates the utility of whole genome sequencing as an adjunctive tool for microbiologic identification.
## Declaration of competing interest
## No coi
## Sources of funding for your research
None Consent AC; Informed consent obtained in writing SM, FL, PA; NA CT, RK; Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request
[fig] Figure 1: Hematoxylin and esosin stain of cystic neutrophilic granulomatous mastitis. Breast parenchyma with granulomatous inflammation and cystic spaces rimmed by neutrophils. Gram-positive bacteria were identified in the cystic spaces. [/fig]
[fig] Figure 2: Gram stain of C. kroppenstedtii. A single colony was selected from blood agar media and bacteria were Gram positive and displayed rod like morphology suggestive of Corynebacterium. [/fig]
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The Role of Facebook® in Promoting a Physically Active Lifestyle: A Systematic Review and Meta-Analysis
Citation: Duregon, F.; Bullo, V.; Di Blasio, A.; Cugusi, L.; Pizzichemi, M.; Sciusco, S.; Viscioni, G.; Cruz-Diaz, D.; Bocalini, D.S.; Bortoletto, A.; et al.
# Introduction
Physical inactivity could be defined as "the 20th-century pandemic" with serious health implications. The most influential aspect is the sedentary lifestyle, which increases the risk of mortality for all causes, doubles the risk of developing cardiovascular disease, diabetes, and obesity, and increases the risk of colon and breast cancer, hypertension, osteoporosis, dyslipidemia, and mood disorders . Moreover, the risk of mortality in people who sat more than 8 h per day or were less active than 2.5 MET-hours per week is similar to the smoker and obese individuals [bib_ref] Does physical activity attenuate, or even eliminate, the detrimental association of sitting..., Ekelund [/bib_ref]. In 2013, the worldwide economic impact of sedentary was about $67.5 billion through healthcare expenditure and productivity losses [bib_ref] The economic burden of physical inactivity: A global analysis of major non-communicable..., Ding [/bib_ref]. These thoughts highlight the need for effective interventions to promote a physically active lifestyle.
## Study quality assessment
The quality of the studies was assessed applying an adapted nine criteria checklist provided by the Cochrane Collaboration Back Review Group [bib_ref] Method Guidelines for Systematic Reviews in the Cochrane Collaboration Back Review Group..., Van Tulder [/bib_ref]. As in previous systematic reviews , the checklist had to be marginally adapted to rate the strength of the evidence.
Each study in the review was scored based on the following nine criteria: (1) "Was the method of randomization adequate?"; (2) "Were the groups similar at baseline regarding the outcome measures?"; (3) "Were inclusion and exclusion criteria adequately specified?"; (4) "Was the drop-out rata described adequately?"; "Were all randomized participants analyzed in the group to which they were allocated?"; [bib_ref] How can interventions increase motivation for physical activity? A systematic review and..., Knittle [/bib_ref] "Was compliance reported for all groups?"; [bib_ref] An ecological approach to creating active living communities, Sallis [/bib_ref] "Was intention-to-treat analysis performed?"; "Was the timing of outcomes assessment similar in all groups?"; (9) "Was a followed-up performed?".
Whether the paper provided a satisfactory description, a positive value was assigned (+). If the criterion description was considered absent, unclear, or lacked the specified content, a negative value was assigned (−). A study was qualitatively judged as high quality if it showed a positive score on 5 out of 9 criteria; otherwise, it was considered a low-quality study.
## Data extraction and synthesis
Three researchers (F.D., V.B., S.G.) independently examined all abstracts of the sourced studies. Several studies were analyzed in more detail to be included in the review. During the reference screening of the included items, additional articles were potentially sourced. Independent searches were then combined, compared, and reviewed for applicability, whereas a consensus was made regarding study inclusion. The review process was repeated in case of discrepancies, and a fourth researcher (S.G.) was consulted. Quality assessment using the modified Cochrane methodological quality criteria was then inde-pendently applied and discussed before final quality scores were assigned . The same researchers who screened titles, abstracts, full texts, and references performed the quality assessment. Several domains were identified for the categorization of the study results. In particular, studies were analyzed in regard to "PA parameters", "anthropometric parameters", "cardiovascular parameters", "diet parameters", "body composition", "lipid profile and glucose tolerance", and "psychosocial parameters". . Quality assessment of the included studies.
## Citation
Randomization Procedure
## Similarity of study groups inclusion or exclusion criteria
Dropouts Blinding Compliance Intention-to-Treat Analysis
## Timing of outcomes assessment
# Follow-up results
Todorovic J et al. (2019)− [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] + + + + − + − + − [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] − − + + − + + + + 6/9 [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] + − + + − + + + + 7/9 [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] − [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] + + + + − + − + − [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] − [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] − + + + − − − − − 3/9 [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] + − + + + + + − − [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] + [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] + − + + + + + + + 8/9 [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] − [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref]
[formula] + − + − − − + − 3/9− + + − − − + − 3/9− + + − + − − − 3/9+ − + + − − − + − 4/9+ + + − + + − − 6/9+ + + + − + − + − 6/9+ + + − − + − + − 5/9− − − − − − − − 0/9+ − + + − + − + + 6/9+ − + + − + + − − 5/9+ − + + − − + + − 5/9 [/formula]
# Data analysis
Meta-analysis was performed for the overall PA parameter, using random-effects models with confidence intervals set at 95%; effect size (ES) was calculated through Review Manager 5.4 software (Copenhagen, Denmark, The Nordic Cochrane Centre, The Cochrane Collaboration, 2020). The ES was calculated as standardized mean difference ∆Mean/SDpooled, where ∆Mean is the difference between the post-intervention mean of the Facebook (FB) Group and Control Group, and SDpooled is the mean of the postintervention standard deviation, summarizing the different tests evaluating the same parameter. Overall, ES consisted of FB group compared with all the different control groups, including established Control Group (CG), Paper Group (PG), Phone Conference Group (PhC-G), Standard Walking Intervention Group (SWI-G), and Exercise Group (EG). The ES is a measure of the effectiveness of a treatment, and it helps determine whether a statistically significant difference is a difference of practical concern. The interpretation was performed according to guidelines by Cohenand Sawilowsky [bib_ref] New effect size rules of thumb, Sawilowsky [/bib_ref] , where an ES value of 0.20 indicates a small effect, ES of 0.50 indicates a medium effect, ES higher than 0.80 indicates a large effect, ES value of 1.20 indicates a very large effect, and an ES value of 2.0 indicates a huge effect.
# Results
A total of 4419 studies resulted from the literature search. Firstly, duplicates were removed, then 3795 records were screened. Based on inclusion and exclusion criteria, 18 articles were considered eligible for this systematic review [fig_ref] Figure 1: Flow chart of the literature research [/fig_ref] ; one study was excluded due to unavailable data [bib_ref] Using facebook and text messaging to deliver a weight loss program to..., Napolitano [/bib_ref]. performed according to guidelines by Cohenand Sawilowsky [bib_ref] New effect size rules of thumb, Sawilowsky [/bib_ref] , where an ES value of 0.20 indicates a small effect, ES of 0.50 indicates a medium effect, ES higher than 0.80 indicates a large effect, ES value of 1.20 indicates a very large effect, and an ES value of 2.0 indicates a huge effect.
# Results
A total of 4419 studies resulted from the literature search. Firstly, duplicates were removed, then 3795 records were screened. Based on inclusion and exclusion criteria, 18 articles were considered eligible for this systematic review [fig_ref] Figure 1: Flow chart of the literature research [/fig_ref] ; one study was excluded due to unavailable data [bib_ref] Using facebook and text messaging to deliver a weight loss program to..., Napolitano [/bib_ref]. Quality appraisal classified twelve studies as high quality [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] , while six as low quality [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref]. From a methodological perspective, randomization was applied in twelve studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] , and seven [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] showed similarity of the groups' participants at baseline. All studies adequately reported inclusion and exclusion criteria, except for two [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref]. Moreover, Quality appraisal classified twelve studies as high quality [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] , while six as low quality [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref]. From a methodological perspective, randomization was applied in twelve studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] , and seven [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] showed similarity of the groups' participants at baseline. All studies adequately reported inclusion and exclusion criteria, except for two [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref]. Moreover, twelve studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] reported the timing of outcomes assessments and the com-pliance [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] of the intervention. All studies reported drop-out ratio, except for while seven studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] applied the intention-to-treat analyses. Finally, four studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] performed follow-up evaluations, and only two studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] performed a single blinding evaluator procedure .
Sample sizes ranged from 8 to 375 subjects, with an average of 33.4 years old. In six studies, participants had chronic diseases, in particular, in three studies [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] , there were cancer survivors, in two studies [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] individuals with overweight or obesity, and one study [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] include individuals with obesity and depressive disorders. Interventions lasted from 4 to 24 weeks, and the majority of the studies proposed a structured intervention of physical activity, predominantly aerobic training. Only one study [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] guaranteed a supervised physical exercise session (1 h per week) with a trainer. [fig_ref] Table 3: Cont. [/fig_ref] summarizes the characteristics of the different protocols. Finally, [fig_ref] Table 4: Cont. [/fig_ref] reported all results of the included studies. [fig_ref] Table 3: Cont. [/fig_ref]. Characteristics of the inclusion study.
## Study subjects and grouping (n) protocol duration and frequency; training modality, program and intensity
Todorovic J et al.375 medical students FB-G (311) CG (64) Duration: 4 weeks Facebook group Students were free to practice any kind of physical activity if and when they wanted to. Facebook group was managed by the research team, that used a participatory approach. All members were allowed to post motivational messages or questions for their peers; the reports and photos from organized events were also posted regularly, so all participants could follow the level of participation of other students. Control group In this group, students were free to practice any kind of physical activity if and when they wanted to, but they did not join the facebook group.
Pope ZC et al. The program also included a secret Facebook group to allow participants to connect and support each other towards achieving their healthy eating and exercise goals. week 1 (overview of the national PA recommendations, health benefits of PA, and PA statistics among African American women); week 2 (developing a PA plan that works for you); week 3 (barriers to PA among African American women and strategies to overcoming barriers); week 4 (developing a social support network to promote PA); week 5 (strategies for incorporating short bouts of PA into your daily routine to achieve national PA recommendations); week 6 (testimonials from African American Women on how they successfully incorporate PA into their daily lives); week 7 (National PA recommendations, barriers to PA among African American Women and strategies to overcoming barriers and incorporate more PA into your life); week 8 (strategies for maintaining a physical active lifestyle after the intervention); 3. Motivational text messages promoting PA: 3 text messages every each week (a. tips on strategies to increase PA throughout the day; b. information on how to overcome barriers to PA; c. reminders of the health benefits of PA; d. motivational/inspirational tips and quotes to participants); 4. Adaptive pedometer-based self-monitoring and goal-setting program: weekly individualized step goals and social reinforcement via email. Printed group 1. Mailed print-based component: booklets mailed every two weeks at home with general information on risk factors for cardiovascular disease, the benefits of PA, tips and strategies to increase daily PA, and encouraged participants to perform a minimum of 150 min of MVPA per week; 2. Static pedometer-based self-monitoring program: participants were instructed to achieve a static goal of 8000-10,000 steps each day.
Rote AE et al. (14) FB1h-G (24) EG (17) CG (7) Duration :
## Physical activity parameters
All included studies analyzed physical activity parameters, adopting mainly pedometers and accelerometers, or administering surveys [fig_ref] Figure 2: Forest plot representing effect size of FB intervention and control, paper and... [/fig_ref] ; only Naslund et al. used a field test. Two investigations showed an increase in the amount of physical activity, within facebook group (FB-G), by 20.6% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] and 45.5% [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] after 12 weeks of a structured program; also, two investigations presented an amelioration by 89.2% [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] and 39.4% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] in FB-G compared to CG (ES = 0.22) after 7 and 12 weeks, respectively. Valle et al. reported increased physical activity in all analyzed parameters, particularly in the light physical activity (LPA), where the FB-G-fitnet ameliorated by 197.1%, likened to FB-G-comparison. ES of 0.41 confirmed this result showing a small to medium effect in favor of the FB-G for which there was a more complete intervention. Moreover, an ES of 0.73 indicates a medium to large effect of FB-G in comparison with PG. As a matter of fact, eight weeks of intervention with Facebook support demonstrated an improvement by 7.8% [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] in light activity, by 6.1% [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] in moderate-lifestyle, and by 52.2% [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] in moderate-to-vigorous activity (MVPA). Also, Chee et al., after 16 weeks of a training program, showed a significant increase in the number of steps per day by 84.5% in FB-G compared to PG; this outcome was confirmed after a 2-month follow-up by 58.1% [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. In 8 weeks of intervention for university students, comparing FB-G to a standard exercise group, it was found an increase of PA by 33.5% [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] and 135.5% [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] , respectively, within and between conditions. In addition, showed amelioration in met calorie goal by 44% in favor of the Phone Conference Group (ES = −0.21) [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref]. Finally, only two studies reported a worsening within FB-G by 4.2% [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] in the number of steps, by 16.7% [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] in MVPA, and by 58.4% [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] in Leisure Vigorous Activity (VA). Phone Conference Group (ES = −0.21) [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref]. Finally, only two studies reported a worsening within FB-G by 4.2% [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] in the number of steps, by 16.7% [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] in MVPA, and by 58.4% [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] in Leisure Vigorous Activity (VA).
## Anthropometric parameters
Twelve studies analyzed the effect of physical activity, supported by Facebook usage, on anthropometric parameters. In particular, interventions lasted from 12 to 24 weeks, reported a significant reduction in body mass index (BMI) (0.8% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] , 4.6% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] , weight (0.7% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] ; 4.8% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] , and waist circumference (WC) (1.3% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] ; 4.7% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] , compared with CG. Valle et al. reported a significant reduction in weight (7.8%) and BMI (6.3%) within FB-G-fitnet [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] , where an administrator encouraged the interaction via social network. Also, Chee et al. found a significant decrease within the group in all the evaluations, with the FB group that achieved a reduction in body weight (7.3%), BMI (7.4%), WC (4.5%), hip circumference (HC) (2.4%), and waist-hip ratio (WHR) (2.3%) [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. Moreover, in the latter study, there was a significant additional reduction in these evaluations (weight: 4.4%, WC: 2.7%, HC: 1.4%, WHR: 1.1%) after a 2-month follow-up. Contrarily, one study [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] showed a significant reduction in BMI (1.6%) within the control group.
## Anthropometric parameters
Twelve studies analyzed the effect of physical activity, supported by Facebook usage, on anthropometric parameters. In particular, interventions lasted from 12 to 24 weeks, reported a significant reduction in body mass index (BMI) (0.8% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] , 4.6% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] , weight (0.7% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] ; 4.8% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] , and waist circumference (WC) (1.3% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] ; 4.7% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] , compared with CG. Valle et al. reported a significant reduction in weight (7.8%) and BMI (6.3%) within FB-G-fitnet [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] , where an administrator encouraged the interaction via social network. Also, Chee et al. found a significant decrease within the group in all the evaluations, with the FB group that achieved a reduction in body weight (7.3%), BMI (7.4%), WC (4.5%), hip circumference (HC) (2.4%), and waist-hip ratio (WHR) (2.3%) [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. Moreover, in the latter study, there was a significant additional reduction in these evaluations (weight: 4.4%, WC: 2.7%, HC: 1.4%, WHR: 1.1%) after a 2-month follow-up. Contrarily, one study [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] showed a significant reduction in BMI (1.6%) within the control group.
## Cardiovascular parameters
Cardiovascular parameters were investigated in eight out of eighteen studies with different methods. Four studies [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] used the YMCA step test to determine the cardiorespiratory fitness (CRF), three studies [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] evaluated diastolic and systolic pressure, while one study [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] also measured the resting heart rate and the augmentation index (AI). Only Chee et al. found a significant improvement within-group comparison, with FB-G that decreased both systolic (5.1%) and diastolic (6.2%) pressure; these data were confirmed after 2-month follow-up with a significant additional reduction of 3.1% and 3.8%, respectively [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. Mixed results were found by Looyestyn et al., where a higher reduction of one-minute post-test (YMCA test) heart rate, not statistically significant, was reported in PG compared with FB-G. On the other hand, the same Paper Group worsened after a 5-month follow-up, while the FB-G confirmed the heart rate reduction [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref]. Conversely, another study [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] , analyzing the heart rate, showed a decrement in both Facebook Groups after ten weeks of intervention, while FB-G compared with CG in other two studies [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] demonstrated an improvement ranged from 0.1% to 11.9% in all cardiovascular evaluations.
## Diet parameters
The influence of physical activity, integrated with a social network, in eating habits was compared in six investigations [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref]. After 12 weeks of intervention, Torquati et al. reported a significant increase in fruit and vegetable intake (26.5%) within the healthy nurses' Facebook Group; however, this data was not confirmed after a 6-month followup [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref]. Another two studies found statistically significant changes in diet parameters. In particular, the first [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] that considered the junk food intake, both days per week and times per day, detected a reduction of 39.4% and 33.3%, respectively, within FB-G. The latter [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] showed an increase of 19.2% within FB-G in diet quality score and 28.6% in vegetable servings per day between groups comparison. Finally, the other two studies [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] , whose sample comprised individuals with overweight or obesity, did not report significant ameliorations in all parameters.
## Body composition
Six [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] out of eighteen studies examined body composition, using the bioelectrical impedance analysis. A statistically significant amelioration in body fat mass (BFM) was detected in three studies after 12 weeks (2.5% [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] , 16 weeks (17.1% [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] , and 24 weeks (2.6% [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] of physical activity intervention in FB-G compared with CG [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] or PG [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. Chee et al. also found a significant improvement within FB-G in BFM expressed in kilograms or percentual changes (22.7%). The latter data were confirmed after a 2-month follow-up, with a further BFM reduction of 10.4% in FB-G, both within and between groups comparison [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. Moreover, a significant increase (1.1%) in lean mass was assessed after 24 weeks of a combined program consisting of physical activity practice and Total Wellbeing Diet for participants with overweight or obesity [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref].
## Lipid profile and glucose tolerance
Three studies [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] investigated the impact of physical activity on lipid profile and glucose parameters, such as insulin and fasting glucose. Comparing FB-G with CG, Ashton et al. showed a statistically significant improvement in total cholesterol (10.3%), lowdensity lipoprotein (LDL) (18.2%), and total cholesterol/high-density lipoprotein (HDL) ratio (9.1%) after 12 weeks of supervised physical activity, combined with a home-based resistance training [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref]. Another study [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] compared FB-G with CG but did not find meaningful differences in all parameters, despite the long duration of the intervention. Only triglycerides improved in FB-G (15.4%), contrarily for CG that worsened (8.3%), even though non-significantly. The same study [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] that also considered glucose parameters found a significant decrease in fasting glucose of 7.3% in favor of FB-G compared with PG. Finally, Chee et al., that focused the content of the FB posts on walking activity, detected significant enhancements within FB-G in all evaluations; specifically, in total cholesterol (14.4%), LDL (21.4%), HDL (15.8%), Triglycerides (34.9%), and Fasting glucose (18.1%) [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. These improvements were confirmed after a 2-month follow-up where FB-G maintained significant changes respect to baseline. Nevertheless, comparison between post-intervention and follow-up evaluations showed significant worsening in above mentioned parameters.
## Psychosocial parameters
Psychosocial parameters were investigated by six studies [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] , using psychometrically validated questionnaires, such as The Self-Efficacy Barriers to Exercise Measure, The Exercise Attitude Questionnaire-18, The Intrinsic Motivation Inventory, The Social Support for Exercise Survey. After eight weeks of intervention, social support significantly improved within FB-G (18.6% [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] and compared with PG (30.8% [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] ; nevertheless, these data were not confirmed after a 5-month follow-up [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref]. Conversely, Pope et al. [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] found a significant worsening in social support (10%) in Facebook Group1, in which there was a more detailed physical activity monitoring with smartwatch compared to Facebook Group2. This data countertrend was also confirmed in another study [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] , which showed a significant improvement in the competence (20.8%) in Facebook2 Group with 1 h of exercise program, compared with Facebook1 Group that performed 3 h per week of fitness class. In the same study [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] , also enjoyment significantly increased in the Facebook2 Group, in within condition. Finally, mixed results were found by specifically, it was detected a significant increase in the outcome expectation (6.6%) in PG compared with FB-G, while self-regulation ameliorated considerably in FB-G (89.9%), both within and between groups comparison [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref]. [fig_ref] Table 4: Cont. [/fig_ref]. Results.
## Study groups (n) results
Todorovic J et al.
## Study groups (n) results
Looyestyn J et al.
(2018) [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] FB-G (41) vs. PG (48); FB-G 5-Mo F-Up (29) [fig_ref] Table 4: Cont. [/fig_ref]. Cont.
## Study groups (n) results
Naslund JA et al.
## Study groups (n) results
Jane M et al. [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] FB-G [bib_ref] The PRISMA Statement for Reporting Systematic Reviews and Meta-Analyses of Studies That..., Liberati [/bib_ref] vs. CG (17) PA parameters
Steps [fig_ref] Table 4: Cont. [/fig_ref]. Cont. [fig_ref] Table 4: Cont. [/fig_ref]. Cont. [fig_ref] Table 4: Cont. [/fig_ref]. Cont.
## Study groups (n) results
## Study groups (n) results
## Study groups (n) results
## Study groups (n) results
## Fb-g 2-month f-up
# Discussion
This systematic review focused on the effectiveness of intervention delivered via Facebook in improving physical activity behavior and health outcomes. Particularly, we investigated the role of Facebook usage in PA promotion on healthy parameters and psychosocial variables. The results showed that the Facebook interaction could improve the amount of physical activity, social support, self-regulation, competence and enjoyment, anthropometric, lipid, and glucose parameters compared to a control group or a paper group. Conversely, the data did not confirm this effect when they compared FB-G to another group or with a different exercise treatment. Our results must be read in light of the primary studies' quality, which showed that most of the studies are of high quality, therefore it can suppose that they used a methodologically correct approach, with a randomization procedure and specific criteria. Conversely, among the low-quality studies, there are pilot studies that need to be implemented in the future to confirm what has already been reported.
In this research analysis, 18 investigations were included; different study designs and different samples, such as healthy adults, cancer survivors, obese or overweight people, underlined the heterogeneity of the studies. All studies reported at least one PA evaluation, and participants involved only used the Facebook social network. Ten studies [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] compared FB-G to other types of physical activity management, eight studies [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] to CG with no exercise intervention, while two studies [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] had not the comparison group.
In addition, we investigated the efficacy of Facebook exercise interventions in improving the total amount of physical activity with a meta-analytic approach; eleven [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] [bib_ref] A Social Media-Based Physical Activity Intervention, Cavallo [/bib_ref] out of eighteen studies were included in the meta-analysis.
## Physical activity parameters
Sedentary habit is a detrimental phenomenon with a major health effect worldwide [bib_ref] Effect of physical inactivity on major non-communicable diseases worldwide: An analysis of..., Lee [/bib_ref]. In contrast, it is well known that regular moderate-intensity physical activity seems to attenuate the increased risk of death associated with high sitting time [bib_ref] Does physical activity attenuate, or even eliminate, the detrimental association of sitting..., Ekelund [/bib_ref]. Nowadays, many different interventions address this issue, including social network involvement that can engage a large number of users at a low cost [bib_ref] Active Team" a social and gamified app-based physical activity intervention: Randomised controlled..., Edney [/bib_ref]. Our results revealed that Facebook usage could potentially involve people to be more physically active, not only in comparison to a sedentary group (CG) but also to other groups with less interactive physical activity management. Interventions consisted of promoting, via Facebook, strategies to increase the number of steps per day [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] , or to maintain a physically active lifestyle [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref] , even with team challenge [bib_ref] A Web-Based, Social Networking Physical Activity Intervention for Insufficiently Active Adults Delivered..., Maher [/bib_ref] or photo posts and opinion polls [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref]. ES analysis confirmed this trend, particularly likening FB-G with PG, for which there were only written form physical activity instructions to follow individually. We can speculate that 8 to 12 weeks of structured program could be sufficient to gain positive effects, even more with an extended period such as Chee et al. that maintained improvements after 2-month follow up [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref]. Nevertheless, according to ES analysis, EG intervention seemed to be more effective than FB-G. A physical fitness class with the presence and the supervision of a trainer [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref] , following a standard intervention [bib_ref] The Efficacy of a Walking Intervention Using Social Media to Increase Physical..., Rote [/bib_ref] , could be more effective than web-based involvement [bib_ref] Effectiveness of physical exercise for people with chronic diseases: The EFIKRONIK study..., Arietaleanizbeaskoa [/bib_ref].
## Anthropometric parameters
Anthropometric parameters were analyzed in many included studies, comparing FB-G with CG, PG, PhC-G, or other FB-G. Nevertheless, few investigations [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] A randomized trial of a Facebook-based physical activity intervention for young adult..., Valle [/bib_ref] found statistically significant improvements. The twelve studies that carried out anthropometric evaluations had 12.5 weeks of average duration of protocol; hence it could be more advisable for a longer period to obtain a considerable change in weight [bib_ref] Long-term weight loss after diet and exercise: A systematic review, Curioni [/bib_ref] or waist and hip circumferences. Two studies [bib_ref] Facebook for Supporting a Lifestyle Intervention for People with Major Depressive Disorder,..., Naslund [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] managed 24 weeks of intervention; however, they did not find significant changes in the mentioned parameters. This is probably because they had a sample composed of individuals with obesity, and the program consisted of only one required weight management session or health lesson per week. Also, Jane et al. observed the 24 weeks of intervention and included participants with overweight and obesity, and it was shown significant improvements in weight, BMI, and WC in favor of FB-G compared with CG [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref]. In this specific case, participants were instructed to follow the "Total Wellbeing Diet" and issued a pedometer. Therefore, we can speculate that a web-based interaction could not be efficient without a multilevel approach [bib_ref] Effectiveness of Approaches to Increase Physical Activity Behavior to Prevent Chronic Disease..., Schwartz [/bib_ref] , especially with people with obesity. In addition, the specificity of a supervised and tailored program should never be overlooked, not even with social network usages, such as Ashton et al. that found significant changes in all anthropometric parameters proposing a supervised PA and an individualized session with targeted goals [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref].
## Cardiovascular parameters
There is consensus that physical activity protects against the development of cardiovascular diseases, and several meta-analyses have concluded that physical exercise has a positive effect on blood pressure levels in both normotensive and hypertensive cases [bib_ref] Exercise as medicine-evidence for prescribing exercise as therapy in 26 different chronic..., Pedersen [/bib_ref]. In this review, eight studies [bib_ref] Use of Wearable Technology and Social Media to Improve Physical Activity and..., Pope [/bib_ref] [bib_ref] Changing Diet and Physical Activity in Nurses: A Pilot Study and Process..., Torquati [/bib_ref] [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] [bib_ref] Feasibility of smartphone application and social media intervention on breast cancer survivors'..., Pope [/bib_ref] analyzed blood pressure and CRF values in an average 13 weeks of intervention, but only Chee et al. showed significant improvements in systolic and diastolic blood pressure within FB-G. Probably, this result could be due to the major attention on the content of the FB posts, focused on the walking activity, the amount of recommended steps per day, and the benefits of walking. It is well known that the beneficial outcomes of aerobic training include improvements in CRF, endothelial function, oxidative stress, and myocardial function [bib_ref] The Effects of Chronic Aerobic Exercise on Cardiovascular Risk Factors in Persons..., Miele [/bib_ref]. Future research could investigate the longer-term effects of a physical activity intervention on cardiovascular parameters, managed with a social network.
## Diet parameters
Lifestyle behaviors, including diet, physical activity, and sedentary behavior, play an important role in maintaining well-being, and improving these behaviors is considered essential also to reduce the health burden of non-communicable diseases [bib_ref] Efficacy of interventions that use apps to improve diet, physical activity and..., Schoeppe [/bib_ref]. A recent study [bib_ref] Attitudes and Barriers to Healthy Diet and Physical Activity: A Latent Profile..., Vaughan [/bib_ref] conducted a profile analysis of attitudes and barriers that influence these habits to tailor an individual-level intervention that implies attention to diet and PA. In this review, six included studies matched physical activity with diet intervention, and three out of six found significant improvements in diet parameters. Particularly, they used a "dinner disc" to guide main meal portion size [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] , and the content of education messages, published in the FB group, was decided by a team of a nutritionist, physical education trainer, and public health specialist [bib_ref] Efficacy of Health Education using Facebook to Promote Healthy Lifestyle among Medical..., Krishnamohan [/bib_ref]. As mentioned above, the multidisciplinary and the specificity of intervention could be more appropriate to change these habits. However, after 24 weeks of participation, individuals with overweight and obesity [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] Distance learning strategies for weight management utilizing online social networks versus group..., Willis [/bib_ref] did not obtain improvements, probably due to the different situation compared to a sample of healthy adults and the lack of more incisive appropriate interventions.
## Body composition
Six out of eighteen included studies evaluated body composition, and three of them [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] showed significant results, particularly in body fat mass. Simultaneously, they significantly improved the total amount of physical activity in favor of FB-G, proposing a prevalent aerobic training supported by social networking. A recent review [bib_ref] Bautmans, I. The effects of exercise on muscle strength, body composition, physical..., Liberman [/bib_ref] underlined that exercise has moderate-to-large effects on body composition and physical functioning. In addition, the duration of interventions from 12 to 24 weeks seems to be in line with the literature. that the practice of aerobic training in older adults could significantly modify BFM after 4-5 months [bib_ref] Effects of aerobic exercise on C-reactive protein, body composition, and maximum oxygen..., Kelley [/bib_ref]. The same authors suggested that resistance training increases muscle mass already after 2-3 months. It is also well known that strength training is a sound method to improve body composition [bib_ref] Strength training and body composition in middle-age women, Burrup [/bib_ref]. The articles included in this review did not introduce a structured resistance training intervention, perhaps due to the major management effort via social networks. However, future research could focus on strength training supported by Facebook usage, test the feasibility of this type of intervention, and obtain an overview of the exercise methods finalized on body composition parameters.
## Lipid profile and glucose tolerance
In this article, limited findings were reported for lipid and glucose profiles. Indeed, only three studies [bib_ref] Feasibility and preliminary efficacy of the 'HEYMAN' healthy lifestyle program for young..., Ashton [/bib_ref] [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref] [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] focused on these parameters, comparing FB-G with CG or PG. Ashton et al. found significant results in all evaluations after 12 weeks of a structured intervention. We can speculate that supervised aerobic exercise training can increase fitness and improvements in some metabolic outcomes, as well as Finucane et al. explained [bib_ref] The effects of aerobic exercise on metabolic risk, insulin sensitivity and intrahepatic..., Finucane [/bib_ref] , even though Jane et al. showed a statistically significant result only for fasting glucose outcome, compared with PG after 24 weeks of intervention [bib_ref] Effects of a weight management program delivered by social media on weight..., Jane [/bib_ref]. Furthermore, there was a positive trend also in other parameters, but not significant enough. Finally, in the mentioned study, participants were obese or overweight, and it is well known that obesity is strongly associated with the development of insulin resistance and, consequently, with metabolic syndrome components [bib_ref] Insulin resistance in obesity: An overview of fundamental alterations, Barazzoni [/bib_ref]. Perhaps, in this case, it could be more efficient a constant supervision, both for PA and diet, not only with social network interaction. Finally, Chee et al. found an improvement in all lipid and glucose parameters within FB-G [bib_ref] A randomised controlled trial of a Facebook-based physical activity intervention for government..., Chee [/bib_ref] , focusing on walking activity as suggested in another study [bib_ref] Daily Walking Combined with Diet Therapy Is a Useful Means for Obese..., Amanouchi [/bib_ref].
## Psychosocial parameters
Psychosocial parameters were largely analyzed through psychometrically questionnaires, investigating social support, self-efficacy, self-regulation, enjoyment, competence, outcome expectations, and barriers. Particularly, social support showed significant results. As a matter of fact, both this outcome in favor of FB-G, encouraging social interaction and facing discussion topics, such as the development of social support networks to promote PA [bib_ref] A Web-Based, Social Networking Beginners' Running Intervention for Adults Aged 18 to..., Looyestyn [/bib_ref] [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref]. Conversely, another study [bib_ref] Effectiveness of Combined Smartwatch and Social Media Intervention on Breast Cancer Survivor..., Pope [/bib_ref] found an amelioration on social support in favor of the Facebook Group2, in which participants not used smartwatch as support to PA. We can suppose that intervention less focused on PA (no smartwatch for exercise monitoring) may have influenced the result, making social support more perceived.
Moreover, Joseph et al. showed a significant improvement in self-regulation, for which FB-G ameliorated by 89.2% [bib_ref] Print versus a culturally-relevant Facebook and text message delivered intervention to promote..., Joseph [/bib_ref]. We can speculate that proposing an intervention with an extensive range of FB discussion topics, such as developing a PA plan, strategies for maintaining an active lifestyle, and overcoming barriers to physical exercise, could be effective for the self-regulation for PA. Finally, Wang et al., comparing FB-G with CG, EG, and another FB-G, found an interesting result in the competence [bib_ref] Use of Facebook in physical activity intervention programme: Test of self-determination theory, Wang [/bib_ref]. In fact, it seems that the FB-G group perceived more competence than the other groups, probably due to the physical fitness class taught by a university lecturer, supported by social networking itself, compared to an ordinary fitness class.
# Limitations
In this study, some limitations should be considered. Firstly, in this review, some investigations were pilot studies and, due to their nature, they did not execute inferential statistics, so we cannot compare all evaluations of each other. Secondly, the meta-analysis was performed only on PA parameters because the other outcome evaluations were considered only in a small part of the studies. Thirdly, included study presented high heterogeneity of the methologies to evaluate pA (e.g., pedometers, smartwatch, questionnaire). Finally, the study analysis focused solely on Facebook, as mentioned above, by methodological choice. Therefore, it could be interesting to consider a larger spectrum of parameters that can influence future research outcomes.
# Conclusions
The Facebook networking can be considered feasible and easily accessible to promote the practice of exercise and to increase the total amount of physical activity. Furthermore, considering Facebook ® as a large social media platform, one of the biggest worldwide and used in different range age, there could be a global influence on awareness of being physically active. Various strategies, for instance counseling with tips, social networking, challenges, can be employ as good practice to obtain an amelioration on healthy parameters, such as cardiovascular and body composition, and social support, with a great impact on public health. However, it is important to maintain a supervision during the exercise practice, and a tailoring to obtain more positive results. Future research on physical activity and social network management could be focus also on strength training, to verify if a more structured intervention, consisted of aerobic and strength training, will be able to show an extensive effect.
[fig] Figure 1: Flow chart of the literature research. [/fig]
[fig] Figure 2: Forest plot representing effect size of FB intervention and control, paper and other groups in total amount of physical activity. FB: Facebook group; CG: control group; PG: paper group; FB+: Facebook with other supports; FB−: Facebook without support. [/fig]
[fig] 2019: [22] FB-G (311) vs. CG(64)PA parametersÎ PAQ(met/min/week) ↑: FB-G (+14.4); CG (−22.4) Pope ZC et al. (2019) [41] FBS-G (19) vs. FB-G (19) PA parametersŜ B (min/day) ↓: FBS-G (+2.9); FB-G (−2.1) LPA (min/day) ↑: FBS-G (−6.9); FB-G (+16.3) MVPA (min/day) ↑: FBS-G (+110.7); FB-G (+44.6) Diet parametersD aily Kcaloric consumption (cals) ↓: FBS-G (−0.9); FB-G (−4.6) Daily Fruit intake (cups) ↑: FBS-G (+36.7); FB-G (−7.0) Daily Vegetable intake ↑: FBS-G (+14.5); FB-G (−0.7) Daily Whole Grain intake (oz. equivalents) ↑: FBS-G (+9.8); FB-G (+93.0) Daily SSB Consumption calories ↓: FBS-G (+47.1); FB-G (−7.5) Psychosocial parametersŜ elf-efficacy (sc) ↑: FBS-G (+33.7); FB-G (+28.7) Social-support (sc) ↑: FBS-G (+36.5); FB-G (+19.0) Enjoyment (sc) ↑: FBS-G (+9.3); FB-G (+5.7) Barriers (sc) ↓: FBS-G (−1.7); FB-G (−4.5) Outcome expectancy (sc) ↑: FBS-G (+8.3); FB-G (+7.7) Intrinsic motivation (sc) ↑: FBS-G (+13.7); FB-G (+12.3) Anthropometric parametersŴ eight (kg) ↓: FBS-G (−0.2); FB-G (−0.5) Body compositionB FM (%) ↓: FBS-G (+11.1); FB-G (+1.7) Cardiovascular parametersĈ ardiorespiratory fitness (heart rate) ↑: FBS-G (+2.6); FB-G (Steps (n/d) ↑: FB-G (−4.2) *, 6-Mo F-up (−6.2) SB (%) ↓: FB-G (−0.9), 6-Mo F-up (+2.1) SB (min) ↓: FB-G (−4.6), 6-Mo F-up (0.0) LPA (%) ↑: FB-G (+2.1), 6-Mo F-up (−1.5) LPA (min) ↑: FB-G (−2.4), 6-Mo F-up (−5.0) MVPA (%) ↑: FB-G (−16.7) *, 6-Mo F-up (0.0) Anthropometric parameters Weight (kg) ↓: FB-G (−0.1), 6-Mo F-up (−7.6) BMI (kg/m2) ↓: FB-G (−0.4), 6-Mo F-up (−7.4) WC (cm) ↓: FB-G (0.0), 6-Mo F-up (−6.6) Cardiovascular parameters SBP (mmHg) ↓: FB-G (−0.4), 6-Mo F-up (−1.6) DBP (mmHg) ↓: FB-G (−1.5), 6-Mo F-up (−3.5) Diet parameters Energy intake (kJ/d) ↓: FB-G (+2.3), 6-Mo F-up (−8.6) ARF score (diet quality) ↑: FB-G (+0.6), 6-Mo F-up (−1.2) Fruits and vegetables (%) ↑: FB-G (+26.5) *, 6-Mo F-up (−9.7) Discretionary food (% energy) ↓: FB-G (−2.9), 6-Mo F-up (−13.7) [/fig]
[fig] Author: Contributions: Conceptualization, M.B. and F.D.; methodology, S.G. and V.B.; software, A.B.; formal analysis, F.D., V.B. and S.G.; data curation, A.D.B. and L.C.; writing-original draft preparation, F.D. and S.S.; writing-review and editing, C.L.A.; visualization, G.V. and D.S.B.; supervision, D.C.-D. and M.P.; project administration, F.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. [/fig]
[table] Table 1: Participants, interventions, comparators, outcomes, study (PICOS) model. Primary outcomes: evaluation of PA before and after the intervention Secondary outcomes: changes on weight, BMI, body circumferences, body composition, diet parameters, cardiovascular parameters Study designs Pilot study, RCT, no-RCT, exploratory study, Randomized pilot trial PA: physical activity; BMI: Body Mass Index; RCT: randomized controlled trial. [/table]
[table] Table 3: Cont. [/table]
[table] Table 4: Cont. [/table]
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Genetic, transcriptional and post-translational regulation of the programmed death protein ligand 1 in cancer: biology and clinical correlations
The programmed death protein 1 (PD-1) and its ligand (PD-L1) represent a well-characterized immune checkpoint in cancer, effectively targeted by monoclonal antibodies that are approved for routine clinical use. The regulation of PD-L1 expression is complex, varies between different tumor types and occurs at the genetic, transcriptional and post-transcriptional levels. Copy number alterations of PD-L1 locus have been reported with varying frequency in several tumor types. At the transcriptional level, a number of transcriptional factors seem to regulate PD-L1 expression including HIF-1, STAT3, NF-κΒ, and AP-1. Activation of common oncogenic pathways such as JAK/STAT, RAS/ERK, or PI3K/AKT/MTOR, as well as treatment with cytotoxic agents have also been shown to affect tumoral PD-L1 expression. Correlative studies of clinical trials with PD-1/PD-L1 inhibitors have so far shown markedly discordant results regarding the value of PD-L1 expression as a marker of response to treatment. As the indications for immune checkpoint inhibition broaden, understanding the regulation of PD-L1 in cancer will be of utmost importance for defining its role as predictive marker but also for optimizing strategies for cancer immunotherapy. Here, we review the current knowledge of PD-L1 regulation, and its use as biomarker and as therapeutic target in cancer.
# Introduction
Cancer development and progression raises a strong antitumor immune response through which the immune system can eliminate cancer cells. This immunosurveillance theory describes the complex interactions between immune and cancer cells, divided in three distinct but often overlapping stages: elimination, equilibrium, and evasion. Thus, tumors can suppress immunity and escape eradication; evading immune destruction has been characterized as a hallmark of cancer [bib_ref] Cancer immunology, Finn [/bib_ref] [bib_ref] Hallmarks of cancer: the next generation, Hanahan [/bib_ref].
Programmed death protein 1 (PD-1) and its ligand (PD-L1) have been recognized as inhibitory molecules that cause impaired immune response against cancer cells. Therapeutic antibodies targeting PD-1/PD-L1 have been introduced into clinical practice, leading to better patient outcomes [bib_ref] Immune checkpoint inhibitors: new insights and current place in cancer therapy, La-Beck [/bib_ref]. Immune checkpoint regulation has been under intense investigation over the last decades, however, the underlying mechanisms regulating the PD1 and PD-L1 expression are not fully understood; several oncogenic signaling pathways, epigenetic modifications, and genetic variations have been suggested. The aim of this review is to summarize the current knowledge on PD-L1 regulation and its emerging role as a target in cancer immunotherapy.
Immune surveillance: the role of PD-1/PD-L1 axis as immune checkpoint PD-1 (CD279) is a transmembrane protein, member of the CD28 family. It is mainly expressed on activated T cells but it can also be detected in other cells such as B-and natural killer (NK) cells upon induction [bib_ref] PD-1 and its ligands in tolerance and immunity, Keir [/bib_ref]. PD-1 has two ligands, PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC), which belong to the B7-CD28 protein family [bib_ref] Signaling pathway and dysregulation of PD1 and its ligands in lymphoid malignancies, Xia [/bib_ref]. PD-L1 is expressed on tumor cells but it can also be present on the surface of other cell types including T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, epithelial, endothelial cells, and as recently shown, brown adipocytes [bib_ref] PD-L1 is an activation-independent marker of brown adipocytes, Ingram [/bib_ref]. PD-L2 is typically expressed in antigenpresenting cells (APCs). PD-L1 is expressed upon stimulation of cytokine interferon-γ (IFNg), secreted by activated T cells [bib_ref] The future of immune checkpoint therapy, Sharma [/bib_ref] [bib_ref] Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion, Dong [/bib_ref].
PD-L1 and PD-L2 are encoded by the CD274 and PDCD1LG2 genes, respectively, located on chromosome 9p.24.1, whereas PD-1 is encoded by the PDCD1 gene located on chromosome 2q37.3 [bib_ref] PD-1 and its ligands in tolerance and immunity, Keir [/bib_ref].
PD-1/PD-L1 axis plays an important role in the regulation of T-cell immunity and has been also implicated in autoimmunity and infection [bib_ref] Basics of PD-1 in self-tolerance, infection, and cancer immunity, Chikuma [/bib_ref]. The PD-1/PD-L1 interaction has been characterized as an "immune checkpoint" due to its impact on the orchestration of immune response against tumor antigens. Along with cytotoxic Tlymphocyte-associated protein 4 (CTLA-4, CD152), they represent immunological "brakes" that modulate T-cell activation leading to an impaired immunosurveillance.
T-cell activation involves a two signal-model; APCs require a first signal from T-cell receptor (TCR), which recognizes the antigen along with the major histocompatibility complex (MHC) presented on the surface of APC. The second signal includes the co-stimulatory interaction between CD28 on the surface of T cells and CD80 (B7.1) or CD86 (B7.2) on the surface of APC [bib_ref] Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity, Chen [/bib_ref] [bib_ref] Regulation of PD-L1: a novel role of pro-survival signalling in cancer, Chen [/bib_ref].
The engagement of PD-1 with its ligands leads to the inhibition of T-cell activation and response, via mechanisms that include blocking of proliferation, induction of apoptosis, and regulatory T-cell differentiation and therefore immune inhibition [bib_ref] Regulation of PD-L1: a novel role of pro-survival signalling in cancer, Chen [/bib_ref]. Blocking the PD-1/PD-L1 axis with potent monoclonal antibodies may reverse the impaired anticancer immunity and thus represents an appealing target of cancer immunotherapy [bib_ref] Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential, Sharma [/bib_ref].
## The genetic basis of pd-l1 expression in cancer
The genetic aberrations of the PD-L1/PD-L2 gene loci represent a key mechanism of PD-L1 expression both in solid and hematologic tumors. Studies of copy number alterations (CNAs) have been reported in several tumor types [fig_ref] Table 1: Copy number alterations [/fig_ref]. The highest frequencies of CNAs have been seen in squamous cell carcinomas of vulva and cervix and triple-negative breast cancer (TNBC), as well as in classical Hodgkin lymphoma (cHL) and primary mediastinal B-cell lymphoma (PMBCL). Contrary, low or absent CNAs have been reported in small and non-small cell lung cancer (NSCLC) and in diffuse large B-cell lymphomas (DLBCL). In general, copy number gains and especially amplifications are well correlated with the protein levels of PD-L1. Given the challenges in determining the protein levels of PD-L1 as detailed below, detection of CNAs is an attractive alternative for identifying patients who could benefit from treatment with checkpoint inhibitors. [fig_ref] Table 1: Copy number alterations [/fig_ref] summarizes the current literature of the genetic regulation of PD-L1 [bib_ref] PD-L1 copy number gain in nonsmall-cell lung cancer defines a new subset..., Goldmann [/bib_ref] [bib_ref] Genomic amplification of CD274 (PD-L1) in small-cell lung cancer, George [/bib_ref] [bib_ref] Genetic basis for PD-L1 expression in squamous cell carcinomas of the cervix..., Howitt [/bib_ref] [bib_ref] PD-L1 expression and CD274 gene alteration in triple-negative breast cancer: implication for..., Guo [/bib_ref] [bib_ref] Genomic amplification of 9p24.1 targeting JAK2, PD-L1, and PD-L2 is enriched in..., Barrett [/bib_ref] [bib_ref] CD274/PD-L1 gene amplification and PD-L1 protein expression are common events in squamous..., Straub [/bib_ref] [bib_ref] CD274 (PDL1) and JAK2 genomic amplifications in pulmonary squamous-cell and adenocarcinoma patients, Clave [/bib_ref] [bib_ref] Genetic basis of PD-L1 overexpression in diffuse large B-cell lymphomas, Georgiou [/bib_ref] [bib_ref] PD-L1 and PD-L2 genetic alterations define classical hodgkin lymphoma and predict outcome, Roemer [/bib_ref] [bib_ref] PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma, Ansell [/bib_ref] [bib_ref] Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 ligand expression, and further..., Green [/bib_ref] [bib_ref] Comprehensive characterization of programmed death ligand structural rearrangements in B-cell non-Hodgkin lymphomas, Chong [/bib_ref] [bib_ref] Genomic rearrangements involving programmed death ligands are recurrent in primary mediastinal large..., Twa [/bib_ref] [bib_ref] Targetable genetic features of primary testicular and primary central nervous system lymphomas, Chapuy [/bib_ref] [bib_ref] PD-L1 protein expression in breast cancer is rare, enriched in basal-like tumours..., Ali [/bib_ref] [bib_ref] Prognostic and predictive value of PDL1 expression in breast cancer, Sabatier [/bib_ref]. In addition to these individual studies, a large in silico analysis of CNAs in PD-L1 has been conducted using the Cancer Genome Atlas datasets (22 cancer types, 9771 tumors). Interestingly, deletions of 9p24.1 were more common than gains in this analysis and were found mostly in melanoma and NSCLC, with gains occurring frequently in ovarian, head and neck, bladder, and cervical carcinomas [bib_ref] Pan-cancer analysis of copy number changes in programmed death-ligand 1 (PD-L1, CD274)-associations..., Budczies [/bib_ref].
Furthermore, a novel genetic regulatory mechanism of PD-L1 gene expression involving the disruption of its 3′ untranslated region (3′-UTR) has been shown in multiple tumor types including T-cell leukemia/lymphoma, DLBCL, and gastric adenocarcinoma. Through interruption of PD-L1 3′-UTR by structural variation, a deviant increase in PD-L1 transcripts occurs leading to immune escape in murine EG7-OVA cancer cells, which in turn can be reversed by PD-L1/ PD-1 inhibition [bib_ref] Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers, Kataoka [/bib_ref].
## Pd-l1 regulation via oncogenic signaling pathways
## Ras/raf/mek/mapk-erk pathway
The mitogen-activated protein kinase (MAPK) pathway is crucial for various functions in normal cells, including growth and differentiation. Its role is also important in carcinogenesis because its activation leads to cancer development [bib_ref] MAP kinase signalling pathways in cancer, Dhillon [/bib_ref]. The ERK-MAPK pathway has been shown to regulate PD-L1 expression in different cancer types. Both pharmacologic inhibition of mitogen-activated protein kinase (MEK) and small interfering RNA (siRNA) knockdown of ERK1/2 resulted in decreased levels of PD-L1 in melanoma cells resistant to BRAF inhibition [bib_ref] The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes..., Jiang [/bib_ref]. Interestingly, in TNBC cells, MEK inhibition resulted in upregulation of MHC II and PD-L1 expression both in vitro and in vivo, whereas combined MEK/PD-1 inhibition increased the effectiveness of antitumor immunity [bib_ref] RAS/MAPK activation is associated with reduced tumorinfiltrating lymphocytes in triple-negative breast cancer:..., Loi [/bib_ref]. MAPK signaling pathway was also responsible for the ectopic expression of PD-L1 in v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant NSCLC cell lines, as revealed by the decrease in PD-L1 levels after both MEK and extracellular signal-regulated MAP kinase (ERK) abrogation [bib_ref] RAS-mitogenactivated protein kinase signal is required for enhanced PD-L1 expression in human..., Sumimoto [/bib_ref]. In another study, Toll-like receptor 4 activation resulted in upregulation of PD-L1 in bladder cancer cells. The use of both ERK and JNK inhibitors abrogated PD-L1 expression, further supporting the contribution of MAPK signaling in PD-L1 regulation [bib_ref] TLR4 signaling induces B7-H1 expression through MAPK pathways in bladder cancer cells, Qian [/bib_ref]. Moreover, the interaction of tyrosine kinase receptor c-Met with its ligand hepatocyte growth factor (HGF) induced Ras activation. Ablation of Ras effect led to downregulation of c-Met-mediated expression of PD-L1 in renal cancer cells [bib_ref] Novel roles of c-Met in the survival of renal cancer cells through..., Balan [/bib_ref].
KRAS activation may also induce PD-L1 expression, as it resulted in stabilization of PD-L1 mRNA transcript assessed through Adenylate-uridylate-rich elements identification in its 3′-UTR in lung cancer cell lines. Additionally, MEK and Phosphoinositide 3-kinase (PI3K) inhibition led to decreased PD-L1 levels and enhanced effectiveness of antitumor immunity in vivo [bib_ref] Cell intrinsic upregulation of PD-L1 through oncogenic KRAS signalling, Coelho [/bib_ref].
## Pi3k/pten/akt/mtor pathway
The PI3K/Akt/mTOR signaling represents another pathway that affects immune surveillance through the regulation of PD-L1. Its activation by either oncogenic PIK3CA mutations (catalytic subunit alpha of PI3K) or by loss-offunction mutations of its negative regulator, phosphatase and tensin homolog (PTEN) modulates immune responses contributing to a survival benefit of cancer cells [bib_ref] The phosphatidylinositol 3-kinase AKT pathway in human cancer, Vivanco [/bib_ref]. In human gliomas, loss of PTEN and activation of PI3K pathway enhanced PD-L1 expression [bib_ref] Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in..., Parsa [/bib_ref]. In TNBC, knockdown of PTEN by short hairpin RNA resulted in elevated levels of both PD-L1 protein expression and mRNA transcripts, whereas inhibition of Akt and mechanistic target of rapamycin (mTOR) decreased PD-L1 expression [bib_ref] PD-L1 expression in triple-negative breast cancer, Mittendorf [/bib_ref]. In a murine model of lung SCC, concurrent inactivation of PTEN and Lbk1 resulted in increased levels of PD-L1 [bib_ref] Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with..., Xu [/bib_ref]. PI3K inhibition, resulted in PD-L1 downregulation in different cancer types including renal cell carcinoma through HGF/c-Met [bib_ref] Novel roles of c-Met in the survival of renal cancer cells through..., Balan [/bib_ref] , KRAS-or EGFRmutated NSCLC [bib_ref] Control of PD-L1 expression by oncogenic activation of the AKT-mTOR pathway in..., Lastwika [/bib_ref] and melanoma [bib_ref] The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes..., Jiang [/bib_ref]. Conversely, LY294002 did not abrogate PD-L1 expression in bladder cancer cells [bib_ref] TLR4 signaling induces B7-H1 expression through MAPK pathways in bladder cancer cells, Qian [/bib_ref]. Moreover, mTOR inhibition with rapamycin reduced levels of PD-L1, both in human cell lines and in murine models of NSCLC and combined treatment with rapamycin and anti-PD-1 antibody inhibited tumor growth in mice [bib_ref] Control of PD-L1 expression by oncogenic activation of the AKT-mTOR pathway in..., Lastwika [/bib_ref].
## Epidermal growth factor receptor (egfr)
EGFR is commonly mutated in NSCLC and has been associated with PD-L1 upregulation in these tumors [bib_ref] Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell..., Azuma [/bib_ref]. PD-L1 was overexpressed in EGFR-mutant murine lung cancer, whereas treatment with an anti-PD-1 antibody restrained tumor growth. Forced ectopic expression of mutant EGFR on bronchial epithelial cells resulted in PD-L1 upregulation, whereas this effect was abolished upon treatment with EGFR tyrosine kinase inhibitors [bib_ref] Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung..., Akbay [/bib_ref] [bib_ref] Dynamic interplay of oncogenes and T cells induces PD-L1 in the tumor..., Rech [/bib_ref]. The EGFR-mediated regulation of PD-L1 in EGFR mutant NSCLC was dependent on MAPK pathway activation. Inhibition of ERK1/2/c-Jun resulted in reduced PD-L1 levels in PD-L1 overexpressing lung cancer cells [bib_ref] Upregulation of PD-L1 by EGFR activation mediates the immune escape in EGFR-driven..., Chen [/bib_ref]. In another more recent study, EGFR was shown to regulate the Transcriptional and posttranscriptional control of PD-L1 in cancer. Regulation of PD-L1 is complex and occurs at different levels. Several signaling pathways are involved including RAS/RAF/MEK/ MAPK-ERK and PI3K/PTEN/ Akt/mTOR. Their activation by oncogenic and/or loss-offunction mutations can lead either to direct action on target genes or to the activation of transcription factors. Such molecules as STAT3, STAT1, c-Jun, HIFs, or NF-κB can shuttle into the nucleus, bind to specific sites on PD-L1 gene promoter and induce its expression. PD-L1 is also regulated posttranscriptionally by microRNAs, which bind to mRNA and lead to its translational repression or enhancement expression of PD-L1 through the activation of Interleukin-6 (IL-6)/Janus Kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in EGFR-driven NSCLC [bib_ref] The EGFR pathway is involved in the regulation of PD-L1 expression via..., Zhang [/bib_ref].
## Eml4-alk
PD-L1 upregulation has been observed in patients with NSCLC harboring the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein like-4 (EML4) chromosomal rearrangement. Activation of EML4-ALK was associated with increased PD-L1 expression; furthermore, treatment with either the ALK inhibitor alectinib or ALK gene silencing with siRNA abrogated this effect. Notably, PD-L1 upregulation was dependent on MAPK/ERK/MEK and PI3K/Akt signaling pathways [bib_ref] Induction of PD-L1 expression by the EML4-ALK oncoprotein and downstream signaling pathways..., Ota [/bib_ref]. In another study using pulmonary adenocarcinoma cell lines, EML4-ALK transcriptionally regulated PD-L1 via STAT3 and HIF-1a [bib_ref] EML4-ALK enhances programmed cell death-ligand 1 expression in pulmonary adenocarcinoma via hypoxia-inducible..., Koh [/bib_ref]. These studies indicate the different ways in which this chimeric protein can regulate the expression of PD-L1 and thus reveal the complexity of signaling pathways and their downstream targets. The various crosstalks in the cellular level can influence anticancer immunity and at the same time offer possible appealing therapeutic targets.
## Transcriptional control of pd-l1
The transcriptional regulation of PD-L1 is summarized in .
The JAK/STAT pathway STAT3 plays a key role in promoting cancer cell survival and proliferation, as well as creating immunosuppressive and thus pro-carcinogenic conditions in the tumor microenvironment (TME) [bib_ref] STATs in cancer inflammation and immunity: a leading role for STAT3, Yu [/bib_ref]. Furthermore, STAT3 is involved in PD-L1 regulation in various cancer types. In nucleophosmin-anaplastic large-cell lymphoma kinase (NPM-ALK) positive anaplastic large-cell lymphoma (ALCL), STAT3 is activated by NPM-ALK oncoprotein through JAK3 activation, binds physically to the PD-L1 gene promoter, and induces its expression in vitro and in vivo [bib_ref] Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1,..., Marzec [/bib_ref]. This STAT3-mediated transcriptional regulation of PD-L1 has been recently shown in another T-cell lymphoma, namely the ALK-negative ALCL. STAT3 gene silencing led to decreased PD-L1 levels in ALK-ALCL [bib_ref] PD-L1 is commonly expressed and transcriptionally regulated by STAT3 and MYC in..., Atsaves [/bib_ref] and also in KRAS-mutant NSCLC cell lines [bib_ref] RAS-mitogenactivated protein kinase signal is required for enhanced PD-L1 expression in human..., Sumimoto [/bib_ref]. By contrast, chromatin immunoprecipitation analysis did not show active binding of STAT3 directly on the promoter of PD-L1 in melanoma cells, despite the presence of putative binding sites of STAT3 on the promoter identified in silico.
Abrogation of STAT3 resulted in enhancement of PD-L1 construct activity mediated by IFNg [bib_ref] Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression, Garcia-Diaz [/bib_ref]. PD-L1 was also induced by latent membrane protein-1 in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) through JAK3/STAT3 activation [bib_ref] EBVdriven LMP1 and IFN-gamma up-regulate PD-L1 in nasopharyngeal carcinoma: implications for oncotargeted..., Fang [/bib_ref].
Another STAT family member, STAT1, is considered to be a tumor suppressor that reduces proliferation, induces apoptosis, and enhances cancer immunosurveillance [bib_ref] The tumor suppressor function of STAT1 in breast cancer, Koromilas [/bib_ref]. Accumulating evidence indicates the emerging role of STAT1 in tumor growth, immune suppression, and therapeutic resistance [bib_ref] The good and the bad faces of STAT1 in solid tumours, Meissl [/bib_ref]. Upon stimulation with IFNg, STAT1 activation resulted in PD-L1 upregulation and in reduction of NK-cell activity against tumor cells in multiple myeloma, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL) [bib_ref] Interferon-gamma-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK..., Bellucci [/bib_ref]. Similarly, STAT1 inhibition led to decreased PD-L1 levels in myeloma cells and thus suppressed the antitumor function of cytotoxic T cells [bib_ref] Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression..., Liu [/bib_ref]. PD-L1 upregulation was JAK2/STAT1-dependent in head and neck cancer with wild-type EGFR, whereas JAK2 inhibition resulted in both basal and EGF-mediated downregulation of PD-L1. Moreover, knockdown of STAT1 gene abolished both IFNg-and EGF-mediated upregulation of PD-L1. Of note, EGFR activation promotes phosphorylation of STAT1, which in turn binds to the promoter of PD-L1 and controls its expression [bib_ref] Identification of the cell-intrinsic and -extrinsic pathways downstream of EGFR and IFN..., Concha-Benavente [/bib_ref]. Although putative binding sites for STAT1 on PD-L1 promoter have been postulated, active binding of STAT1 on PD-L1 gene promoter could not be demonstrated in melanoma cells [bib_ref] Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression, Garcia-Diaz [/bib_ref].
Interferon regulatory factor 1 (IRF1) is a downstream effector of STAT1 upon IFNg stimulation. Its role is crucial in both constitutive and IFNg-mediated upregulation of PD-L1. Inhibition of IRF1 activity or expression resulted in decreased PD-L1 levels in human lung cancer cells [bib_ref] Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation..., Lee [/bib_ref].
The key role of IRF1 and interferon receptor pathway in the regulation of PD-L1 has also been implied in melanoma, where putative binding sites for IRF1 have been identified in the PD-L1 promoter and abrogation of IRF1 site resulted in reduced PD-L1 levels [bib_ref] Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression, Garcia-Diaz [/bib_ref] [bib_ref] A novel link between inflammation and cancer, Grinberg-Bleyer [/bib_ref]. Recently, another novel mechanism of PD-L1 regulation by DNA double-strand breaks (DSBs) was unveiled. This DSB-dependent PD-L1 upregulation was mediated by the activation of STAT1/ STAT3 phosphorylation and IRF1 [bib_ref] DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells, Sato [/bib_ref].
## Hypoxia-inducible factors (hifs)
Hypoxia signaling represents an important pathway in oncogenesis. HIF-1a and HIF-2a are the major components of a transcriptional complex, through which tumor cells adapt to hypoxic conditions. HIF stabilization leads to its binding to specific regions called hypoxia response elements (HRE) on certain gene promoters [bib_ref] Immune response regulation in the tumor microenvironment by hypoxia, Labiano [/bib_ref]. High levels of HIF-1 have been correlated with both worse outcomes and resistance to cytotoxic therapy [bib_ref] Targeting hypoxia in cancer therapy, Wilson [/bib_ref]. Intriguingly, HIF-1 expression by different cellular sub-populations of the innate and adaptive immunity can modify antitumor activity by repressing the effective T-cell response and alter TME to promote tumor cell survival [bib_ref] Immune response regulation in the tumor microenvironment by hypoxia, Labiano [/bib_ref]. A recent study revealed that HIF-1α guided CD8 + T-cell migration and function, whereas its depletion on T cells resulted in increased tumor growth and impaired antitumor control [bib_ref] An HIF-1alpha/VEGF-A axis in cytotoxic T cells regulates tumor progression, Palazon [/bib_ref]. One of the mechanisms by which hypoxia signaling impairs T-cell functionality is the induction of PD-L1 on myeloid-derived suppressor cells under hypoxic conditions. Indeed, HIF-1a transcriptionally regulates PD-L1 expression by binding on HRE of its promoter [bib_ref] PD-L1 is a novel direct target of HIF-1alpha, and its blockade under..., Noman [/bib_ref]. Furthermore, PD-L1 may be a target of HIF2a in clear cell renal cell carcinoma (ccRCC) cells in which the tumor-suppressor pVHL was abrogated. Upon deficiency of pVHL increased PD-L1 levels, associated with HIF-2a activation, were observed in vitro [bib_ref] PD-L1 expression is regulated by hypoxia inducible factor in clear cell renal..., Ruf [/bib_ref]. Similar results were obtained from ccRCC patient samples with VHL loss-of-function mutations, where a positive correlation was seen between PD-L1 expression, HIF-2a expression and VHL mutations. Of note, HIF-2a transcriptionally regulates PD-L1 by binding to the active HRE of its promoter [bib_ref] Renal cell carcinoma programmed death-ligand 1, a new direct target of hypoxia-inducible..., Messai [/bib_ref]. Moreover, STAT3 can cooperate with HIF-1, but not HIF-2, in the regulation of HIF target genes in response to hypoxia. Inhibition of STAT3 expression or activity in breast and RCC cell lines reduced the expression of genes targeted by HIF-1 [bib_ref] STAT3 and HIF1alpha cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4..., Pawlus [/bib_ref]. These findings support the idea of combining HIF-targeting therapies and immunotherapy.
The role of nuclear factor kappa B (NF-κB) NF-κB is a master transcription factor activated in several cancer types, promoting inflammation, inhibiting apoptosis, and impairing effective antitumor immunity [bib_ref] NF-kappaB and the link between inflammation and cancer, Didonato [/bib_ref]. The NF-κB family contains seven members, with the most representative being the p65 RelA/p50. This cytoplasmic heterodimer translocates to the nucleus and acts as a transcription factor of κB upon degradation of the IκΒ-α inhibitor [bib_ref] The diverse and complex roles of NF-kappaB subunits in cancer, Perkins [/bib_ref] [bib_ref] Inducible but not constitutive expression of PD-L1 in human melanoma cells is..., Gowrishankar [/bib_ref]. In melanoma cells, NF-κB mediated PD-L1 overexpression induced by IFN-γ. PD-L1 upregulation by NF-κΒ was independent of STAT3 and c-Jun, whereas targeting of MAPK and PI3K signaling pathways had a minor impact on PD-L1 expression [bib_ref] Inducible but not constitutive expression of PD-L1 in human melanoma cells is..., Gowrishankar [/bib_ref]. Notably, STAT3 regulates and cooperates with NF-κΒ in additional cancer types [bib_ref] NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer, Fan [/bib_ref]. For example, PD-L1 regulation may be dependent on p65/NF-κB and mediated by LMP1 in EBVpositive NPC, as inhibition of NF-κB activity resulted in decreased PD-L1 levels [bib_ref] EBVdriven LMP1 and IFN-gamma up-regulate PD-L1 in nasopharyngeal carcinoma: implications for oncotargeted..., Fang [/bib_ref].
## The myc oncogene
Myc plays a pivotal role in carcinogenesis by controlling cell proliferation and survival in various cell systems.
Tumor regression after Myc inactivation is associated with a not fully understood immune response, as reflected by the accumulation of CD4 + T cells [bib_ref] Sustained loss of a neoplastic phenotype by brief inactivation of MYC, Jain [/bib_ref] [bib_ref] MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer, Shachaf [/bib_ref] [bib_ref] CD4(+) T cells contribute to the remodeling of the microenvironment required for..., Rakhra [/bib_ref]. Furthermore, a novel role of Myc was recently revealed in the context of avoiding effective cancer immunosurveillance. Using a Tetoff MYC-dependent mouse model of T-ALL (MYC T-ALL), Casey et al. showed that Myc transcriptionally regulates PD-L1 and CD47, an inhibitory regulator of the innate immune system [bib_ref] MYC regulates the antitumor immune response through CD47 and PD-L1, Casey [/bib_ref]. Moreover, forced expression of PD-L1 and CD47 upon Myc inactivation was correlated with worse antitumor immune response as indicated by the reduction of macrophages and CD4 + T cells in TME, tumor progression, and maintenance of angiogenesis and senescence [bib_ref] MYC-a thorn in the side of cancer immunity, Spranger [/bib_ref]. Elucidating the role of Myc in the regulation of immune-mediated antitumor response, the potential crosstalks with other oncogenic pathways and the immune infiltrate in TME may pave the way for the use of immune checkpoint inhibitors in patients with Mycoverexpressing tumors [bib_ref] MYC regulates the antitumor immune response through CD47 and PD-L1, Casey [/bib_ref]. A recent work on ALKnegative ALCL also supports a Myc-mediated regulation of PD-L1, as forced expression of Myc led to PD-L1 upregulation in cell lines showing low baseline levels of PD-L1. Similarly, both inhibition and silencing of Myc resulted in PD-L1 downregulation in lymphoma cells [bib_ref] PD-L1 is commonly expressed and transcriptionally regulated by STAT3 and MYC in..., Atsaves [/bib_ref].
## The bromodomain and extraterminal (bet) protein brd4
BET proteins modulate gene expression through enzymes that regulate chromatin and histone modification [bib_ref] BET domain co-regulators in obesity, inflammation and cancer, Belkina [/bib_ref]. Specifically, the BET protein BRD4 acts through RNA polymerase II by binding to the acetyl-lysine region of histones [bib_ref] Selective inhibition of BET bromodomains, Filippakopoulos [/bib_ref]. Inhibition of BRD4 by the JQ1 inhibitor decreased PD-L1 expression and tumor growth. BRD4 gene silencing also resulted in decreased PD-L1 levels in mouse models and in ovarian cancer cell lines. Notably, BRD4 transcriptionally regulated PD-L1 by binding on its promoter [bib_ref] BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression, Zhu [/bib_ref]. Similarly, in a recent study on B-cell lymphoma, BET inhibitors enhanced effective antitumor immunity through regulation of PD-L1, whereas inhibition and genetic ablation of BRD4 resulted in suppression of PD-L1 expression in a transcriptional, Myc-independent, manner. Moreover, BRD4 synergized with IRF1 to regulate PD-L1 expression induced by IFN-γ [bib_ref] BET-bromodomain inhibitors engage the host immune system and regulate expression of the..., Hogg [/bib_ref]. Also, another BET inhibitor (I-BET151) was shown to abrogate NF-Κβ activity in melanoma, both in vitro and in vivo, thus indirectly affecting PD-L1 expression [bib_ref] Control of NF-kB activity in human melanoma by bromodomain and extra-terminal protein..., Gallagher [/bib_ref].
## Histone deacetylases (hdacs)
The role of the epigenetic modifiers HDACs in the modification of non-histone targets, including those participating in tumor-host interactions, has recently been investigated [bib_ref] Targeting histone deacetylase 6 mediates a dual antimelanoma effect: enhanced antitumor immunity..., Woan [/bib_ref] [bib_ref] Modulation of antigen-presenting cells by HDAC inhibitors: implications in autoimmunity and cancer, Woan [/bib_ref]. In a study in melanoma, both inhibition and depletion of HDAC6 resulted in reduced PD-L1 levels in vitro and in vivo. PD-L1 regulation by HDAC6 was mediated by STAT3 and both HDAC6 and STAT3 were recruited to the PD-L1 gene promoter [bib_ref] Essential role of HDAC6 in the regulation of PD-L1 in melanoma, Lienlaf [/bib_ref]. It should be noted that HDAC have pleiotropic effects within both the innate and adaptive immune response, and may thus affect PD-L1 levels via interferons [bib_ref] Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders, Falkenberg [/bib_ref].
## The role of cell cycle
Cyclin-dependent kinases (CDKs) have a key role in cell cycle [bib_ref] Cell cycle, CDKs and cancer: a changing paradigm, Malumbres [/bib_ref]. Cyclin-dependent kinase 5 (Cdk5) is a serinethreonine kinase important in central nervous system function [bib_ref] A decade of CDK5, Dhavan [/bib_ref] and other cellular functions [bib_ref] Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis,..., Ohshima [/bib_ref] [bib_ref] Molecular roles of Cdk5 in pain signaling, Utreras [/bib_ref]. In a study of medulloblastoma, depletion of Cdk5 led to the upregulation of interferon regulatory factor 2 and interferon regulatory factor binding protein 2, which in turn, suppressed the expression of PD-L1. Cdk5 was thus necessary for PD-L1 upregulation after IFN-γ stimulation through STA1/IRF1 axis and its disruption led to tumor rejection in a CD4 + T-cell-dependent manner in medulloblastoma mouse models [bib_ref] Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity, Dorand [/bib_ref]. These data highlight Cdk5 as a novel target for interventions in combination with immune checkpoint blockade. Additionally, CDK4/6 inhibition has been recently shown to enhance antitumor immunity through increased T-cell cytotoxicity and Treg suppression [bib_ref] CDK4/6 inhibition triggers anti-tumour immunity, Goel [/bib_ref]. This is discussed in detail in the post-translational regulation of PD-L1 hereunder.
The AP-1 transcription factors c-Jun, the best known member of the AP-1 family, represents another transcription factor that is implicated in PD-L1 gene regulation. Knockdown of c-Jun resulted in decreased levels of PD-L1 in melanoma cells resistant to BRAF inhibitors [bib_ref] The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes..., Jiang [/bib_ref] , and co-activation of STAT3 and the subsequent formation of a transcriptional complex further enhanced these effects [bib_ref] Cooperative transcriptional activity of Jun and Stat3 beta, a short form of..., Schaefer [/bib_ref]. Similarly, combined knockdown of c-Jun and STAT3 genes in the same melanoma model showed a synergistic effect on PD-L1 downregulation [bib_ref] The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes..., Jiang [/bib_ref]. Additionally, c-Jun and JUNB have been shown to bind AP-1 sites in the PD-L1 promoter in HL cells [bib_ref] Constitutive AP-1 activity and EBV infection induce PD-L1 in Hodgkin lymphomas and..., Green [/bib_ref] and in KRAS-mutant NSCLC. In lung adenocarcinoma cell lines, the transcriptional activity was subjected to MAPK signaling pathway [bib_ref] RAS-mitogenactivated protein kinase signal is required for enhanced PD-L1 expression in human..., Sumimoto [/bib_ref]. MAPK/AP-1 was also shown to contribute to LMP1-mediated upregulation of PD-L1 in EBV-associated NPC [bib_ref] EBVdriven LMP1 and IFN-gamma up-regulate PD-L1 in nasopharyngeal carcinoma: implications for oncotargeted..., Fang [/bib_ref].
## The ambivalent role of p53
The tumor-suppressor gene p53 has been implicated in antitumor immunity by regulating several genes involved in the immune system. Indeed, immune checkpoint regulation has been shown to represent a major target of p53 [bib_ref] Emerging roles of p53 and other tumour-suppressor genes in immune regulation, Munoz-Fontela [/bib_ref]. Paradoxically, activation of wild-type p53 using the small molecule Nutlin-3a resulted in increased expression of PD-L1 in human breast cancer [bib_ref] Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53, Yoon [/bib_ref] and in ALK-negative ALCL cells [bib_ref] PD-L1 is commonly expressed and transcriptionally regulated by STAT3 and MYC in..., Atsaves [/bib_ref]. In p53-mutated NSCLC, downregulation of miR-34 resulted in increased PD-L1 levels [bib_ref] PDL1 regulation by p53 via miR-34, Cortez [/bib_ref] , whereas an inverse correlation between miR-34a and PD-L1 was also confirmed in AML [bib_ref] Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target..., Wang [/bib_ref].
## Micrornas
MicroRNAs can bind to 3′-UTR of mRNAs and lead to their degradation or translational repression [bib_ref] MicroRNAs: target recognition and regulatory functions, Bartel [/bib_ref]. MiR-513 was shown to increase PD-L1 expression in cholangiocytes [bib_ref] MicroRNA-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in..., Gong [/bib_ref] , whereas mutation in the 3′-UTR of PD-L1 mRNA led to overexpression of the protein by preventing miR-570 binding in gastric cancer [bib_ref] A frequent somatic mutation in CD274 3'-UTR leads to protein overexpression in..., Wang [/bib_ref] [bib_ref] A miR-570 binding site polymorphism in the B7-H1 gene is associated with..., Wang [/bib_ref]. On the contrary, miR-197 downregulated PD-L1 by affecting STAT3 in platinumresistant NSCLC [bib_ref] The clinical relevance of the miR-197/CKS1B/ STAT3-mediated PD-L1 network in chemoresistant non-smallcell..., Fujita [/bib_ref] , whereas miR-138-5p was associated with decreased levels of PD-L1 in colorectal cancer (CRC) [bib_ref] The tumor suppressor miR-138-5p targets PD-L1 in colorectal cancer, Zhao [/bib_ref]. Also in CRC, miR-20b, miR-21, and miR-130b caused PD-L1 upregulation through attenuation of PTEN [bib_ref] MiR-20b, -21, and -130b inhibit PTEN expression resulting in B7-H1 overexpression in..., Zhu [/bib_ref].
## Post-translational regulation of pd-l1
The role of ubiquitination In a recent study by Lim et al., a novel regulatory mechanism involving the fifth protein element of COP9 signalosome complex (CSN5), also known as Jab1, was revealed in breast cancer. CSN5 has been associated with increased proliferation, decreased apoptotic rates, and survival of cancer cells [bib_ref] JAB1/CSN5: a new player in cell cycle control and cancer, Shackleford [/bib_ref]. Under chronic inflammatory conditions, tumor necrosis factor alpha (TNF-α), secreted mostly by macrophages, led to PD-L1 stabilization and therefore to an immunosuppressive profile of the tumor environment [bib_ref] A novel link between inflammation and cancer, Grinberg-Bleyer [/bib_ref]. The stabilization of PD-L1 by TNF-a was shown to be mediated by NF-κΒ subunit RelA/p65, which binds on the promoter of CSN5 gene and has a direct effect on its regulation. CSN5 in turn, prevents the ubiquitination of PD-L1, hinders its degradation and as a result enhances tumor escape from immunosurveillance. Indeed, CSN5 inhibition or gene silencing abolished PD-L1 expression and tumor proliferation in vivo. Curcumin, a CSN5 inhibitor, induced better responses to anti-CTLA-4 treatment in vitro, indicating the potential of combinational administration of immune checkpoint with CSN5 inhibitors [bib_ref] A novel link between inflammation and cancer, Grinberg-Bleyer [/bib_ref] [bib_ref] Deubiquitination and stabilization of PD-L1 by CSN5, Lim [/bib_ref] [bib_ref] Targeted inhibition of the COP9 signalosome for treatment of cancer, Schlierf [/bib_ref]. In another in vitro study, induction of both PD-L1 ubiquitination and PD-L1 protein levels was noted upon treatment with epidermal growth factor. An increase of The results presented here concern the primary endpoint of the study in the IC2/3 group of PD-L1 expression The results presented here concern the primary endpoint of the study in the intermediate and poor risk group mono-and multiubiquitination of PD-L1 was seen, an effect that was abrogated upon inhibition of the EGFR pathway and/or ubiquitin E1 activating enzyme [bib_ref] Identifying regulatory posttranslational modifications of PD-L1: a focus on monoubiquitinaton, Horita [/bib_ref]. Furthermore, a recent study demonstrated a novel role of cyclin D-CDK4 and cullin 3-speckle-type POZ protein (SPOP) E3 ligase in regulating the expression of PD-L1. Cyclin D1-CDK4 was shown to phosphorylate SPOP and lead to ubiquitinationmediated PD-L1 destabilization. Thus, either inhibition of CDK4/6 or loss-of-function mutations of SPOP led to increased levels of PD-L1 and reduced tumor-infiltrating lymphocytes. Additionally, treatment with a CDK4/6 inhibitor and an anti-PD-1 antibody resulted in tumor regression and improved survival in vivo [bib_ref] Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune..., Zhang [/bib_ref].
## Lysosomal-mediated degradation
CKLF-like MARVEL transmembrane domain containing protein 6 (CMTM6) was recently identified as a novel regulator of PD-L1 [bib_ref] Identification of eight genes encoding chemokine-like factor superfamily members 1-8 (CKLFSF1-8) by..., Han [/bib_ref] [bib_ref] MARVEL: a conserved domain involved in membrane apposition events, Sanchez-Pulido [/bib_ref]. CMTM6-a tetraspanin protein-interacted with PD-L1 through its transmembrane domain and regulated PD-L1 expression in cancer and myeloid cells, both in vitro and in vivo [bib_ref] CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity, Burr [/bib_ref]. Depletion of CMTM6 did not influence the CD274 transcript, but led to reduction of PD-L1 protein expression and augmentation of antitumor immunity. The mechanism of action of CMTM6 involves the avoidance of PD-L1 lysosome-mediated degradation, probably through prevention of its ubiquitination, as these two proteins are co-localized in the plasma membrane [bib_ref] CMTM6 controls PD-L1, Visan [/bib_ref].
## The role of glycosylation
N-glycosylation represents a crucial post-translational modification determining protein formation, functionality, and interaction with other proteins [bib_ref] Scanning N-glycosylation mutagenesis of membrane proteins, Cheung [/bib_ref]. A novel association between procedure-glycosylation and ubiquitination in the regulation of PD-L1 has recently been unveiled. In basal-like breast cancer cells, N-glycosylation of PD-L1 (highly at sites N35, N192, N200, and N219) led to protein stabilization and avoidance of its degradation by 26S proteasome. In contrast, non-glycosylated forms interrelated with Glycogen synthase kinase 3 beta (GSK3β), which in turn phosphorylated PD-L1 resulting in its degradation. Inhibition of GSK3β activity augmented immune suppression by tumor cells both in vitro and in vivo. Furthermore, EGFR promoted inactivation of GSK3β, and EGFR signaling blockade reversed stabilization of PD-L1 and led to enhanced antitumor responses [bib_ref] Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity, Li [/bib_ref]. In another study, N-linked glycosylation of PD-L1 (gPD-L1) was shown to increase PD-L1/PD-1 interaction, and consequently immunosuppression in TNBC. Its targeting with monoclonal antibodies or drug-conjugated gPD-L1 was thus proposed as a promising target of post-translational modifications of immune checkpoints [bib_ref] Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1, Li [/bib_ref].
## Effect of chemotherapy in pd-l1 expression
Chemotherapeutic agents, apart from their direct cytotoxic effects on cancer cells, can also modulate immune responses against tumors [bib_ref] The secret ally: immunostimulation by anticancer drugs, Galluzzi [/bib_ref] [bib_ref] The effect of chemotherapy on programmed cell death 1/programmed cell death 1..., Luo [/bib_ref]. Treatment with paclitaxel, etoposide and 5-fluorouracil induced PD-L1 expression in breast cancer cell lines in a dose-dependent manner [bib_ref] Chemopreventive agents induce programmed death-1-ligand 1 (PD-L1) surface expression in breast cancer..., Zhang [/bib_ref]. Paclitaxel was also associated with elevated levels of PD-L1 in human CRC and hepatocellular carcinoma cell lines. This regulation was dependent on MAPK activation [bib_ref] Paclitaxel induced B7-H1 expression in cancer cells via the MAPK pathway, Gong [/bib_ref]. Likewise, cisplatin induced PD-L1 expression in hepatoma cells in ERK1/2 phosphorylation-dependent manner [bib_ref] Cisplatin induces programmed death-1-ligand 1(PD-L1) over-expression in hepatoma H22 cells via Erk/MAPK..., Qin [/bib_ref].
In another study, doxorubicin led to PD-L1 downregulation on cell surface and a simultaneous PD-L1 upregulation in the nucleus of breast cancer cells. Nuclear PD-L1 expression was accompanied by nuclear AKT phosphorylation and proved to be dependent on PI3K/AKT pathway, whereas knockdown of PD-L1 was associated with enhanced doxorubicin-mediated apoptosis [bib_ref] Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in..., Ghebeh [/bib_ref].
## Targeting immune checkpoint regulators: the era of immunotherapy in cancer
The introduction of systemic cancer immunotherapy in clinical practice significantly predates the first randomized trials of immune checkpoint inhibitors. Despite the occurrence of rare, prolonged complete remissions in patients with metastatic melanoma and ccRCC [bib_ref] High-dose recombinant interleukin-2 therapy in patients with metastatic melanoma: long-term survival update, Atkins [/bib_ref] [bib_ref] Cancer-specific survival outcomes among patients treated during the cytokine era of kidney..., Belldegrun [/bib_ref] , the use of high-dose IL-2 was restricted by the significant, often fatal adverse events and the need for intensive monitoring and experience in its administration, whereas the use of IFNg in ccRCC was characterized by its perceived low efficacy. The clinical application of cancer immunotherapy had remained stagnant until the first checkpoint inhibitor received regulatory approval for use in metastatic melanoma, the CTLA-4 inhibitor ipilimumab. Ipilimumab exhibits several recurring characteristics of immunotherapy: slow induction of response, a striking disassociation between imaging-assessed objective responses and survival, which led to the introduction of immune-related response criteria [bib_ref] iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics, Seymour [/bib_ref] , unique patterns of toxicity termed "immunerelated adverse events" [bib_ref] Immune-related adverse events, need for systemic immunosuppression, and effects on survival and..., Horvat [/bib_ref] and robust, durable improvements in terms of patient survival [bib_ref] Pooled analysis of long-term survival data from phase II and phase III..., Schadendorf [/bib_ref]. Shortly after the approval of ipilimumab the first trials of PD-1 and later PD-L1 inhibitors were published. Their results have vastly changed the treatment landscape in multiple human malignancies, adding a new category of effective and, compared with cytotoxic chemotherapy, less toxic agents to the therapeutic armamentarium. The results of the published phase 3 trials are presented in [fig_ref] Table 2: Randomized phase 3 trials of PD-1 and PD-L1 inhibitors Trial [Ref] [/fig_ref] [bib_ref] Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer, Reck [/bib_ref] [bib_ref] First-line nivolumab in stage IV or recurrent non-small-cell lung cancer, Carbone [/bib_ref] [bib_ref] Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010):..., Herbst [/bib_ref] [bib_ref] Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer, Brahmer [/bib_ref] [bib_ref] Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer, Borghaei [/bib_ref] [bib_ref] Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a..., Schachter [/bib_ref] [bib_ref] Nivolumab in previously untreated melanoma without BRAF mutation, Robert [/bib_ref] [bib_ref] Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4..., Weber [/bib_ref] [bib_ref] Combined nivolumab and ipilimumab or monotherapy in untreated melanoma, Larkin [/bib_ref] [bib_ref] Pembrolizumab as second-line therapy for advanced urothelial carcinoma, Bellmunt [/bib_ref] [bib_ref] Nivolumab versus everolimus in advanced renal-cell carcinoma, Motzer [/bib_ref] [bib_ref] Nivolumab for recurrent squamous-cell carcinoma of the head and neck, Ferris [/bib_ref] [bib_ref] Durvalumab after chemoradiotherapy in stage III nonsmall-cell lung cancer, Antonia [/bib_ref] [bib_ref] Adjuvant nivolumab versus ipilimumab in resected stage III or IV melanoma, Weber [/bib_ref] [bib_ref] Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial..., Powles [/bib_ref] [bib_ref] LBA5CheckMate 214: efficacy and safety of nivolumab+ipilimumab (N+I) v sunitinib (S) for..., Escudier [/bib_ref] , whereas a selection of ongoing randomized trials in an ever-expanding list of indications, both at refractory disease, as well as in earlier lines of therapy or at the adjuvant setting is presented in [fig_ref] Table 3: Selected ongoing phase 3 trials of PD-1 and PD-L1 inhibitors [/fig_ref]. The results of these trials are eagerly awaited, because there are high unmet needs in many of the indications that these agents are being tested. Of interest are also hematologic malignancies; preliminary trials report impressive response rates in otherwise refractory disease [bib_ref] Nivolumab in patients with relapsed or refractory hematologic malignancy: preliminary results of..., Lesokhin [/bib_ref] , believed to be driven by both the inherent role of the PD-1/PD-L1 axis in the evasion of immunosurveillance in lymphoid tumors, particularly in those with a viral etiology [bib_ref] PD-1-PD-L1 immunecheckpoint blockade in B-cell lymphomas, Goodman [/bib_ref] , and by the presumed significance of PDL1 and PDL2 amplification in the biology of certain neoplasms such as Hodgkin lymphoma [bib_ref] PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma, Ansell [/bib_ref]. In contrast, the recent discontinuation of the ongoing phase 3 trials in multiple myeloma due to an increased risk of death underscores the fact that better understanding of the underlying immune mechanisms is still needed. Importantly, a new generation of clinical trials has been initiated and initial results are already available regarding a multi-faceted attempt to improve upon the efficacy of PD-1/ PD-L1 inhibitors as monotherapy: their combination with CTLA-4 inhibitors, already shown to improve outcomes in metastatic melanoma [bib_ref] Combined nivolumab and ipilimumab or monotherapy in untreated melanoma, Larkin [/bib_ref] and pursued in other malignancies including NSCLC and SCLC; their combination with cytotoxic chemotherapy, based upon the premise of the prevention of early disease progression due to the simultaneous administration of chemotherapy and the release of neoantigens due to the cytotoxic effects of the combinatory treatment, which may potentiate the activity of PD-1 inhibitors, an approach that has shown promising results in advanced NSCLC and at the neoadjuvant setting of TNBC [bib_ref] Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-smallcell lung..., Langer [/bib_ref] [bib_ref] Pembrolizumab plus standard neoadjuvant therapy for high-risk breast cancer (BC): results from..., Nanda [/bib_ref] ; the combination of targeted agents and PD-1 axis blockade [bib_ref] Prospects for combining targeted and conventional cancer therapy with immunotherapy, Gotwals [/bib_ref] , with preliminary results showing that combining immunotherapy with inhibitors of known effectors of the axis, such as CDK4/6, results in promising activity [bib_ref] A phase 1b study of abemaciclib plus pembrolizumab for patients with hormone..., Rugo [/bib_ref] ; and finally, the combination with inhibitors or stimulators of modulatory molecules such as indoleamine 2,3-dioxygenase (IDO) inhibitors, because IDO is a major negative feedback pathway regulated by IFNg. Preliminary results of the IDO inhibitor epacadostat with nivolumab in a variety of tumors and with pembrolizumab in melanoma are promising and phase 3 results are eagerly awaited [bib_ref] Epacadostat plus nivolumab in patients with advanced solid tumors: preliminary phase I/II..., Perez [/bib_ref] [bib_ref] Epacadostat plus pembrolizumab in patients with advanced melanoma and select solid tumors:..., Gangadhar [/bib_ref].
In short, the current era of cancer immunotherapy could be characterized as the "end of the beginning". A variety of agents is available for use in multiple indications and clinical experience is accumulating. The next phase, namely the optimization of the use of the available agents and the exploration for novel combinations, has already begun.
## Immune checkpoint regulators as novel biomarkers: prognostic and predictive value
Taking into account the significant clinical efficacy of PD-1/ PD-L1 blockade in a small subset of patients, the considerable costs and potential for devastating immune-related adverse events associated with the use of these inhibitors and the robust theoretical background explaining the biology of their mechanism of action, considerable efforts have been undertaken in order to identify putative predictive biomarkers. The best characterized biomarker is the immunohistochemistry (IHC)-assessed PD-L1 expression. The conflicting results of individual trials have been summarized in meta-analyses, which indicate that increased levels of PD-L1 expression are associated with an improved probability for objective response [bib_ref] PD-L1 expression in cancer patients receiving anti PD-1/PD-L1 antibodies: a systematic review..., Gandini [/bib_ref] [bib_ref] Programmed cell death 1 (PD-1) ligand (PD-L1) expression in solid tumors as..., Khunger [/bib_ref]. Supporting these results are two recently published clinical trials in the first line of advanced NSCLC, KEYNOTE-024, and CheckMate 026. In the former, overall survival (OS) in patients selected for PD-L1 positivity ≥50% was improved with pembrolizumab compared with platinum-based chemotherapy [bib_ref] Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer, Reck [/bib_ref]. Contrary, in the latter trial there were no OS gains in PD-L1 ≥5% patients treated with nivolumab versus chemotherapy [bib_ref] First-line nivolumab in stage IV or recurrent non-small-cell lung cancer, Carbone [/bib_ref]. As there are no perceived differences in the potency of these antibodies, the obvious discrepancy in the patient population could account for the different outcome. However, several observations hinder the routine selection of appropriate candidates according to PD-L1 expression. First, in addition to the modest concordance rates between the various antibodies used to assess PD-L1 expression reported in the literature, questions still remain regarding the uncontrolled pre-analytical conditions and the assay and inter-pathologist discrepancies [bib_ref] PD-L1 immunohistochemistry in clinical diagnostics: inter-pathologist variability is as high as assay..., Micke [/bib_ref] , which can lead to PD-L1 status misclassifications despite the similar analytical performance of the available assays [bib_ref] PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the..., Hirsch [/bib_ref]. Second, PD-L1 expression exhibits significant intratumoral, intertumoral and temporal heterogeneity [bib_ref] Quantitative assessment of the heterogeneity of PD-L1 expression in non-small-cell lung cancer, Mclaughlin [/bib_ref] [bib_ref] Heterogeneity of tumor and immune cell PD-L1 expression and lymphocyte counts in..., Casadevall [/bib_ref] , putting into question the widespread practice of assessing PD-L1 IHC expression on archival tissue. Third, as clearly shown in individual randomized trials such as the CheckMate 017 trial at the second line of lung SCC [bib_ref] Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer, Brahmer [/bib_ref] , characterizing patients as appropriate for anti-PD-1 therapy according to PD-L1 expression both includes patients who do not respond to treatment and also excludes potential responders. Fourth, in the aforementioned CheckMate 026 trial, nivolumab was not more effective than chemotherapy even in the subgroup of 50% or higher PD-L1 expression. As this was not a stratification factor, imbalances such as the sex of the patients could have confounded the results, implying that PD-L1 positivity by itself is not a strong predictive biomarker [bib_ref] First-line nivolumab in stage IV or recurrent non-small-cell lung cancer, Carbone [/bib_ref]. Finally, the association of objective response rates and PD-L1 expression in the triallevel meta-analyses is of unsure clinical importance, since checkpoint inhibitors can confer prolonged, clinically meaningful periods of disease stabilization and because their use beyond progression in patients deemed to derive clinical benefit has been found to improve outcomes in a diverse selection of solid malignancies [bib_ref] Impact of atezolizumab (atezo) treatment beyond disease progression (TBP) in advanced NSCLC:..., Gandara [/bib_ref] [bib_ref] Treatment beyond progression in patients with advanced renal cell carcinoma treated with..., Escudier [/bib_ref] [bib_ref] Nivolumab for patients with advanced melanoma treated beyond progression: analysis of 2..., Long [/bib_ref].
Keeping in mind the shortcomings of PD-L1 expression, other biomarkers have been explored. Following the observation that smokers with NSCLC seem to derive improved benefit from anti-PD-1 agents [bib_ref] Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients, Herbst [/bib_ref] , it was postulated that this effect may be a surrogate marker for an increased mutational load and subsequent increased neoantigen production and exposure and more effective immune response in patients chronically exposed to a strong mutagenic factor such as smoking. Indeed, mutational load has been found to be a predictive factor in NSCLC [bib_ref] Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell..., Rizvi [/bib_ref]. TNBC PD-L1 amplification Unfavorable [bib_ref] PD-L1 expression and CD274 gene alteration in triple-negative breast cancer: implication for..., Guo [/bib_ref] Residual after neoadjuvant ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 expression of the residual tumor serves as a prognostic marker in..., Chen [/bib_ref] Gastrointestinal cancer
All digestive tumors ↑ PD-L1 expression Unfavorable [bib_ref] Prognostic and predictive values of PD-L1 expression in patients with digestive system..., Dai [/bib_ref] Hepatocellular cancer ↑ PD-L1/2 expression Unfavorable [bib_ref] Overexpression of PD-L1 and PD-L2 is associated with poor prognosis in patients..., Jung [/bib_ref] [bib_ref] Increased programmed death ligand-1 expression predicts poor prognosis in hepatocellular carcinoma patients, Gu [/bib_ref] Colorectal cancer ↑ PD-L1 expression Favorable [bib_ref] Prognostic implication of CD274 (PD-L1) protein expression in tumorinfiltrating immune cells for..., Lee [/bib_ref] [bib_ref] Prognostic impact of programed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression..., Li [/bib_ref] Colorectal cancer ↑ PD-L2 expression Unfavorable [bib_ref] PD-L2 expression in colorectal cancer: independent prognostic effect and targetability by deglycosylation, Wang [/bib_ref] Gastric cancer ↑ PD-L1 expression Unfavorable [bib_ref] Prognostic significance of PD-L1 expression in patients with gastric cancer in East..., Liu [/bib_ref] [bib_ref] PD-L1 and gastric cancer prognosis: a systematic review and meta-analysis, Gu [/bib_ref] Cholangiocarcinoma ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 and PD-1 expression correlate with prognosis in extrahepatic cholangiocarcinoma, Ma [/bib_ref] Esophageal cancer ↑ PD-L1 expression Favorable [bib_ref] Increased intraepithelial CD3.T-lymphocytes and high PD-L1 expression on tumor cells are associated..., Jesinghaus [/bib_ref] Pancreatic cancer ↑ PD-1 expression Favorable [bib_ref] Prognostic value, localization and correlation of PD-1/PD-L1, CD8 and FOXP3 with the..., Diana [/bib_ref] Genitourinary cancer
Clear cell renal ↑ PD-L1/2 expression Unfavorable [bib_ref] Intratumoral expression of programmed death ligand 1 (PD-L1) in patients with clear..., Abbas [/bib_ref] [bib_ref] PD-L1 expression in renal cell carcinoma clear cell type is related to..., Leite [/bib_ref] [bib_ref] Clinicopathologic analysis of PD-L1 and PD-L2 expression in renal cell carcinoma: association..., Shin [/bib_ref] Non-clear cell renal ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 expression in nonclear-cell renal cell carcinoma, Choueiri [/bib_ref] Papillary renal ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 expression in papillary renal cell carcinoma, Motoshima [/bib_ref] Chromophobe renal ↑ PD-L2 expression Unfavorable [bib_ref] PD-L2: a prognostic marker in chromophobe renal cell carcinoma?, Erlmeier [/bib_ref] Bladder cancer ↑ PD-L1 expression Unfavorable [bib_ref] Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis..., Nakanishi [/bib_ref] [bib_ref] The prognostic significance of PD-L1 in bladder cancer, Huang [/bib_ref] Prostate cancer ↑ PD-1 expression Unfavorable [bib_ref] The prognostic role of immune checkpoint markers programmed cell death protein 1..., Ness [/bib_ref] Prostate cancer ↑ PD-L1 expression Unfavorable [bib_ref] The immune checkpoint regulator PD-L1 is highly expressed in aggressive primary prostate..., Gevensleben [/bib_ref] Ovarian cancer ↑ PD-L1 expression Favorable [bib_ref] PD-L1 expression is associated with tumor-infiltrating T cells and favorable prognosis in..., Webb [/bib_ref] [bib_ref] Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression..., Darb-Esfahani [/bib_ref] Lung and head and neck cancer NSCLC ↑ PD-L1 expression Favorable [bib_ref] PD-1 and PD-L1 expression in NSCLC indicate a favorable prognosis in defined..., Schmidt [/bib_ref] [bib_ref] Programmed death ligand-1 expression in non-small cell lung cancer, Velcheti [/bib_ref] NSCLC ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 protein expression assessed by immunohistochemistry is neither prognostic nor predictive of..., Tsao [/bib_ref] [bib_ref] PD-L1 overexpression is associated with a poor prognosis in Asian nonsmall cell..., Xia [/bib_ref] [bib_ref] The expression of PD-L1 protein as a prognostic factor in lung squamous..., Takada [/bib_ref] [bib_ref] Impact of PD-L1 expression in patients with surgically resected non-small-cell lung cancer, Igawa [/bib_ref] [bib_ref] PD-L1 overexpression is partially regulated by EGFR/ HER2 signaling and associated with..., Okita [/bib_ref] NSCLC ↑ PD-L1 expression Not predictive [bib_ref] PD-L1 protein expression assessed by immunohistochemistry is neither prognostic nor predictive of..., Tsao [/bib_ref] NSCLC PD-L1 amplification Unfavorable [bib_ref] PD-L1 is upregulated by simultaneous amplification of the PD-L1 and JAK2 genes..., Ikeda [/bib_ref] SCLC ↑ PD-L1 expression Unfavorable [bib_ref] High PD-L1 expression is associated with stage IV disease and poorer overall..., Chang [/bib_ref] Pulmonary neuroendocrine ↑ PD-L1 expression Unfavorable [bib_ref] Prognostic value of PD-L1 and PD-1 expression in pulmonary neuroendocrine tumors, Fan [/bib_ref] Head and neck cancer ↑ PD-L1 expression Favorable [bib_ref] PD-L1 expression confers better prognosis in locally advanced oral squamous cell carcinoma, Kogashiwa [/bib_ref] [bib_ref] PD-L1 expression on immune cells, but not on tumor cells, is a..., Kim [/bib_ref] Head and neck cancer ↑ PD-L1 expression Unfavorable [bib_ref] High PD-L1 expression correlates with metastasis and poor prognosis in oral squamous..., Lin [/bib_ref] Melanoma and sarcoma Melanoma ↑ PD-L1 expression Favorable [bib_ref] Association of PD-1/PD-L axis expression with cytolytic activity, mutational load, and prognosis..., Danilova [/bib_ref] Melanoma ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1, PD-L2 and PD-1 expression in metastatic melanoma: correlation with tumor-infiltrating immune..., Obeid [/bib_ref] Soft tissue sarcoma ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 expression is associated with FOXP3+regulatory T-cell infiltration of soft tissue sarcoma..., Que [/bib_ref] Hematologic malignancies
Hodgkin's lymphoma ↑ PD-1 expression Unfavorable [bib_ref] PD-L1 expression correlates with VEGF and microvessel density in patients with uniformly..., Koh [/bib_ref] Hodgkin's lymphoma PD-1/L-1 co-expression Unfavorable [bib_ref] Programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and EBV-encoded RNA (EBER) expression..., Paydas [/bib_ref] Hodgkin's lymphoma PD-L1 amplification Unfavorable [bib_ref] The effect of chemotherapy on programmed cell death 1/programmed cell death 1..., Luo [/bib_ref] DLBCL ↑ PD-L1 expression Unfavorable [bib_ref] Expression of programmed cell death ligand 1 is associated with poor overall..., Kiyasu [/bib_ref] [bib_ref] The expression and clinical relevance of PD-1, PD-L1, and TP63 in patients..., Fang [/bib_ref] NK/T-cell lymphoma ↑ PD-L1 expression Unfavorable [bib_ref] PD-L1 is upregulated by EBV-driven LMP1 through NF-kappaB pathway and correlates with..., Bi [/bib_ref] Multiple myeloma ↑ Soluble PD-L1 Unfavorable [bib_ref] Serum levels of soluble programmed death ligand 1 predict treatment response and..., Wang [/bib_ref] [bib_ref] Soluble PD-L1: a biomarker to predict progression of autologous transplantation in patients..., Huang [/bib_ref] All tumor types Meta-analyses ↑ PD-L1 expression Unfavorable [bib_ref] Prognostic significance of programmed cell death 1 (PD-1) or PD-1 ligand 1..., Zhang [/bib_ref] [bib_ref] Prognostic role of PD-L1 in malignant solid tumors: a meta-analysis, Pyo [/bib_ref] [bib_ref] Prognostic significance of PD-L1 in solid tumor: an updated meta-analysis, Wang [/bib_ref] HER2 human epidermal growth factor receptor, TNBC triple-negative breast cancer, NSCLC non-small cell lung cancer, SCLC small cell lung cancer, DLBCL diffuse large B-cell lymphoma, NK natural killer cells Supporting this association is the observation that mismatch repair defective, and thus hypermutated tumors, are exquisitely sensitive to PD-1 blockade [bib_ref] PD-1 Blockade in tumors with mismatch-repair deficiency, Le [/bib_ref] [bib_ref] Mismatch-repair deficiency predicts response of solid tumors to PD-1 blockade, Le [/bib_ref]. In addition, NSCLC harboring driver molecular aberrations such as EGFR mutations, which exhibit lesser mutational loads have been shown to be relatively resistant to immune checkpoint inhibition [bib_ref] EGFR mutations and ALK rearrangements are associated with low response rates to..., Gainor [/bib_ref] , a finding supported by a recently published meta-analysis on the prediction of response in NSCLC patients. EGFR mutant and KRAS wildtype status were associated with a lack of sensitivity to PD-1/PD-L1 inhibition, whereas clinical factors such as smoking status, histology, sex, performance status, and age did not affect the magnitude of benefit [bib_ref] Clinical and molecular characteristics associated with survival among patients treated with checkpoint..., Lee [/bib_ref]. The quantitative and qualitative assessment of the host immune response has also been explored as a predictor in checkpoint inhibition. Factors such as the abundance of preexisting CD8 (+) T cells, a restricted (clonal) TCR repertoire, a TH1-type response, increased levels of IFN-γ and IL-18 and decreased levels of IL-6, among others, have been correlated with improved responses [bib_ref] Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients, Herbst [/bib_ref] [bib_ref] PD-1 blockade induces responses by inhibiting adaptive immune resistance, Tumeh [/bib_ref] , but these results need to be evaluated prospectively in randomized trials. The implementation of multiparametric, highthroughput flow cytometry, and multiplex immunohistochemical staining techniques that vastly improve the T-cell population analysis [bib_ref] New flow cytometric assays for monitoring cell-mediated cytotoxicity, Zaritskaya [/bib_ref] and of whole-exome sequencing for the evaluation of the mutational load and the presence of specific, predictive molecular alterations will aid in this respect.
On the other hand, PD-1 and PD-L1 expression both at the tissue level and on circulating tumor cells have been evaluated in a wide variety of malignancies for their prognostic impact [fig_ref] Table 4: Examples of studies reporting a correlation of PD-1/L1 status and prognosis [/fig_ref] [bib_ref] Genomic amplification of 9p24.1 targeting JAK2, PD-L1, and PD-L2 is enriched in..., Barrett [/bib_ref] [bib_ref] PD-L1 and PD-L2 genetic alterations define classical hodgkin lymphoma and predict outcome, Roemer [/bib_ref]. The results have been thus far inconsistent among tumor types and somewhat confusing, with reports supporting both an improved and a decreased OS conferred by high expression, a phenomenon that resonates the previously mentioned shortcomings of the assessment of PD-L1. The biologic background of these observations is as of yet uncertain. Moreover, as the expansion of the indications of PD-1/PD-L1 blockade continues with the conduct and report of clinical trials, these associations could be affected due to the increasing use of these agents, making their clinical utility questionable at the moment.
## Open questions for future research
Despite the progress in genetic and epigenetic regulation of PD-L1 expression, several gaps in the literature should be covered by intensive laboratory-based research. For instance, the signaling transduction pathways involved in PD-L1 regulation are only partially understood. Better understanding of the signaling mechanisms could provide the biologic rationale for combined targeted therapy with immunotherapy strategies in cancer. Furthermore, little is known about the post-translational modifications of PD-L1 protein including tyrosine or serine/threonine phosphorylation, acetylation, ubiquitination, and SUMOylation. It is also largely unknown how possible post-translational modifications not only regulate PD-L1 levels in the tumor cells, but also how they might affect its physiologic function or its interaction with the PD-1 receptor. In addition to PD-L1, the non-genetic mechanisms underlying PD-L2 expression and function in solid tumors and hematologic malignancies should be investigated, as both ligands compete for the same receptor, PD1, and therefore the relative levels of both proteins may impact certain immunotherapy approaches.
Regarding clinical practice, regulatory authorities both in Europe (European Medicine Agency), and the United States (Food and Drug Administration) have approved the use of PD-1/PD-L1 inhibitors for a variety of malignancies regardless of the presence or absence of predictive biomarkers. Exceptions include the use of pembrolizumab at the first and second line of NSCLC, which requires PD-L1 expression levels of ≥50% and ≥1% respectively, as well as the site agnostic indication for mismatch repair deficient tumors. In addition, the financial burden of the generalized use of these agents is considerable even in high-resource settings [bib_ref] Economic sustainability of anti-PD-1 agents nivolumab and pembrolizumab in cancer patients: recent..., Tartari [/bib_ref]. Overcoming this obstacle and achieving the personalized use of these agents requires a stepwise approach: first, taking into account the previously mentioned shortcomings of PD-L1 as a potential biomarker, it is important to retrospectively identify, in the large amount of collected tumor material from prospective studies, novel predictive biomarkers. These would ideally be prospectively validated, although the logistics of repeating single agent trials might be prohibitive. Instead, these biomarkers could form the basis of the next-generation combinatorial trials, of trials addressing the as yet unanswered question of the optimal duration of treatment or of trials in earlier disease settings where the overtreatment of already cured individuals in a massive scale could pose a significant public health burden.
## Summary
Despite the clinical success of immune checkpoint inhibition in many tumors through PD-L1/PD-1 blockade, relatively little is known regarding the biology of these regulators of cancer immune surveillance. Many mechanisms have been demonstrated to regulate the expression of PD-L1 including signaling pathways, transcriptional factors, and post-transcriptional modulators. The oncogenic signaling pathways such as JAK/STAT, RAS/ERK, or PI3K/AKT/MTOR are activated by gene mutations and growth factors. At the transcriptional level, a number of transcriptional factors seem to regulate PD-L1 expression including HIF-1, STAT3, NF-κΒ, and AP-1. PD-L1 is subject to post-transcriptional regulation by several miR-NAs, CSN5, CMTM6, CDK4 and possibly other, still unknown mechanisms. Better understanding of PD-L1 regulation may pave the way for combinational treatments with both immune checkpoint inhibitors and targeted therapies against kinases or transcription factors many of which are already available for clinical use. adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.
[table] Table 1: Copy number alterations (CNAs) of CD274 gene in cancer NSCLC non-small cell lung carcinoma, SCLC small-cell lung carcinoma, SCC squamous cell carcinoma, BC breast cancer, TNBC triple-negative breast carcinoma, PDA pancreatic ductal adenocarcinomas, PDJ amplicon the loci for PD-L1, PD-L2, and JAK2, [/table]
[table] Table 2: Randomized phase 3 trials of PD-1 and PD-L1 inhibitors Trial [Ref] [/table]
[table] Table 3: Selected ongoing phase 3 trials of PD-1 and PD-L1 inhibitors [/table]
[table] Table 4: Examples of studies reporting a correlation of PD-1/L1 status and prognosis [/table]
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Epigenetic Regulatory Dynamics in Models of Methamphetamine-Use Disorder
# General introduction
Methamphetamine (METH) is a powerful psychostimulant that belongs to the class of amphetamine-type stimulants (ATS) and has a high potential for abuse. The other ATS include amphetamine and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). At low therapeutic doses, METH can cause elevated mood and increased alertness, improved concentration, and increased energy in fatigued individuals. In the past few years, the prevalence of METH abuse has exceeded that of cocaine use worldwide because it produces euphoria that lasts longer due to its 12-h half-life compared to the 90 min half-life of cocaine. METH is also popular because of cost differencesand the ease of its synthesis from precursors such as ephedrine (derived from the plant Ephedra sinica), pseudoephedrine, or 1-phenyl-2-propanone. Recently, new alternative "designer precursors", including methyl α-phenylacetoacetate (also known as MAPA)and its optical isomers, have been used by clandestine laboratory operators to circumvent federal controls placed on other known precursors. With the development of various synthetic production methods, METH continues to dominate global market scenes. According to reported seizure data for 2015-2019, METH accounted for 72% of all global ATS-related seizures. The three countries with most ATS-related seizures are the United States (US), Thailand, and Mexico.
## Epidemiology of meth use
Prevalence of METH use is often used to gauge the significance of its abuse. According to the United Nations Office on Drug and Crime (UNODC, 2021), 27 million people worldwide used amphetamines, suggesting a significant increase in the geographical spread of METH trafficking at the global level. METH use in the USA displays a stable trend, with the highest incidence of METH use among the general population
## Clinical presentations of meth-use disorder
The clinical presentations of MUD depend on the route of administration, (smoked, snorted, swallowed, or injected), patterns of drug use, and amounts of drug taken. When smoked, snorted, or injected, the user experiences, within minutes, an intense "rush" or "flash" that is described as extremely pleasurable. With oral intake, these effects are less prominent and occur after 2-4 h. Administration of METH can also result in cardiovascular, cerebrovascular, and other autonomic dysfunctions.
METH use usually starts as a recreational activity wherein users take relatively low doses at relatively long intervals between drug-taking behaviors. Users progress over time to taking the drug in binges followed by intervals of abstinence after they run out of drug. Binges can also be accompanied by crashes during which individual users can feel various adverse consequences that include mood disturbances and intense craving. These withdrawal symptoms often serve to accelerate the escalation of METH taking that is accompanied by significantly shortened intervals between successive METH injections. METH doses self-administered during recreational use in humans are estimated at 20-40 mg, which, for a 60-80 kg person, are equivalent to 0.25-0.67 mg/kg doses.have reported that METH can cause positive drug effects in subjects even at a very low dose equivalent to 0.06-0.08 mg/kg. A binge dose can be as high as 0.5-1 g/day and METH self-administration might occur 4-6 times per day over a 24-h period.
During withdrawal, most people experience depression, anxiety, tiredness, and intense craving for the drug. The clinical scenario can also include complex cardiovascular and cerebrovascular symptomsin addition to the psychostimulatory effects of the drug. Pre-existing psychiatric comorbidities are also known phenomena of the MUD diathesis.
As mentioned earlier, only some people who use METH develop MUD. The development of MUD is thought to be related to genetic and environmental factors that result in either resilient or susceptible phenotypes during the chronic use of rewarding drugs. The switch from recreational use to meeting clinical criteria for MUD is likely related to epigenetically driven transcriptional and translational changes that can impact synaptic functions and responses to METH over decades. Recently, we published a brief review on the epigenetic landscape of MUDwhere we mainly focused on animal models of METH administration. In this review, we focus on both pre-clinical and clinical aspects of METH-induced chromatin modifications. Herein, we have provided individual tables that summarize the experimental design, assays, and results of all the epigenetic alterations (histone, DNA and non-coding RNA modifications) in MUD. Besides, this review addresses the gender differences and the effects of pre-natal exposure of METH in relation to chromatin modification. Overall, we discuss potential METH-induced epigenetic alterations that might serve as substrates to promote a permissive state that can lead to the behavioral manifestations called addiction.
The term "epigenetics" was coined by Conrad Waddington in 1942 to explain "the causal interactions between genes and their products", which he defined as changes in phenotype without changes in genotype. A reprint of this original article was published in 2012. Epigenetic regulation refers to stable and heritable transcriptional changes in gene expression that are not secondary to alterations in DNA sequences. Epigenetic changes enable the same DNA sequences to function differently under different circumstances and promote changes in behaviors, learning and memory formation, and in cognitive functions.
The chromatin can undergo a complex network of events that are responsible for registering and maintaining gene regulation via "marks". As illustrated in, the specific editing process is carried out by "writers" and "erasers" that modify the histone proteins that are tightly bound to the DNA. Writers include histone acetyl transferase (HATs), histone methyl transferase (KMTs/HMTs), and SET family of methyl transferase (SUV). These enzymes target promoters and enhancers to facilitate transcriptional activation. Erasers include histone deacetylases (HDACs) and demethylases (KDM), which govern transcriptional repression. In addition, a set of proteins, chromo and bromodomains, are called "readers" and are located within the chromatin. They function as chromatin remodelers and adaptor proteins by interacting with transcriptional machinery (reviewed by Zhu et al. 2020. Illustration of aberrant alterations in METH-induced epigenetic modifications and effectors represented by "writers", "readers", and "erasers". Epigenetic writers include histone acetyl-transferases (HATs: p300/CBP; PCAF; GCN5; TAF/Kat4; Tip60/Kat5), histone methyl-transferases (HMTs: PRMT5; MLL1/KMT2a; SUV39H1), DNA methyltransferases (DNMT: DNMT1; DNMT3b), ten-eleven translocase (TET: TET1, 2 and 3), readers include, corepressor for the RE1-silencing transcription factor (CoREST), methyl-binding protein (MBP: MeCP2) and erasers include, histone demethylase (HDM: Kdm5c), histone deacetylases (HDACs). The green arrow represents transcriptional activation and red block-head arrow represents transcriptional repression. The red circle on DNA strand (DNA methylation) indicate methyl-cytosine; yellow pentagon on the DNA strand (DNA hydroxymethylation) indicates hydroxymethyl-cytosine. ac: acetylation; me: methylation.
In the following pages, we will discuss the identified core molecular actors that appear to significantly contribute to the development of MUD (see. These. Illustration of aberrant alterations in METH-induced epigenetic modifications and effectors represented by "writers", "readers", and "erasers". Epigenetic writers include histone acetyl-transferases (HATs: p300/CBP; PCAF; GCN5; TAF/Kat4; Tip60/Kat5), histone methyl-transferases (HMTs: PRMT5; MLL1/KMT2a; SUV39H1), DNA methyltransferases (DNMT: DNMT1; DNMT3b), ten-eleven translocase (TET: TET1, 2 and 3), readers include, corepressor for the RE1silencing transcription factor (CoREST), methyl-binding protein (MBP: MeCP2) and erasers include, histone demethylase (HDM: Kdm5c), histone deacetylases (HDACs). The green arrow represents transcriptional activation and red block-head arrow represents transcriptional repression. The red circle on DNA strand (DNA methylation) indicate methyl-cytosine; yellow pentagon on the DNA strand (DNA hydroxymethylation) indicates hydroxymethyl-cytosine. ac: acetylation; me: methylation.
In the following pages, we will discuss the identified core molecular actors that appear to significantly contribute to the development of MUD (see. These include, modifications to the histone proteins along with changes in DNA (methylation and hydroxymethylation). These alterations influence the overall chromatin dynamics to make DNA more (euchromatin) or less (heterochromatin) accessible to the transcriptional machinery, with consequent activation or suppression of transcription. These changes can significantly impact the development and/or progression of diseased states.
## Histone modifications in meth-use disorder
Histone modifications are covalent modifications where the N-terminal tail of histone proteins undergoes post-translational changes by addition of acetyl, methyl, ubiquitin, phosphoryl, sumoyl, ADP ribosyl moieties along with deamination and proline isomerization.
## Histone acetylation
Histone modifications are poised to play a role in setting up the dynamic responsivity and range of brain regulation of physiology and behavior. The first histone modification was identified in the mid-1960s bywho mulled over the role of acetylated histones in transcription regulation. Acetylation of histone proteins requires a balance between the activities of both HATs and HDACs. Histone acetylation is generally associated with increased gene expression while a hypo-acetylated state results in decreased gene expression. Under normal conditions, HAT and HDAC are maintained in a balanced homeostatic state in the neuron. However, in the neurodegenerative state the acetylation homeostasis is disturbed. HDACs are critical regulators of gene expression and play important roles in the pathophysiology of several neurodegenerative diseases. These include Parkinson's, Alzheimer's, dementia, schizophrenia, bipolar disorder, and depression. Similarly, the most investigated chromatin modification in drugs of abuse is histone acetylation. Histone acetylation states have been investigated after various METH dosing regimens and in the presence of HDAC inhibitors (HDACi).
## Acute effects of meth on histone acetylation
Scientists in the Cadet laboratory have published several papers in which they interrogated the status of histone modifications using experimenter-administered METH in rodent models. Most of these reports pertain to deacetylated histones, which lead to a reduction in transcription activity.
In 2019, González and colleagues reported that an acute injection of METH (1 mg/kg) in mice increased HDAC1, HDAC2, and pan-acetylated histone H3 protein levels in the prefrontal cortex (pCTX) whereas pan-acetylated histone H4 protein showed a decrease(see. However, in the nucleus accumbens (NAc), a large single dose of METH (20 mg/kg) in rats decreased HDAC1 but increased HDAC2 protein levels. These results were associated with decreased abundance of histone H3 acetylated at lysine 9 (H4K5ac) and at lysine 18 (H3K18ac). In contrast, there was increased abundance of histone H4 acetylated at lysine 5 (H4K5ac) and at lysine 8 (H4K8ac). These observations suggest that METH can cause differential changes in different histone acetylation on histone proteins and support the idea that changes in chromatin dynamics can occur in independent histone-specific fashion. These findings also suggest that METH can impact gene expression according to its impact on various histone markers at specific gene promoters. This suggestion is supported by chromatin immunoprecipitation (ChIP) studies after METH administration. Specifically, pull-down assays using a pan-H3ac antibody revealed a significant enrichment of this marker at the promoters of Drd2, Adra1a, Hcrtr1, and Hrh1 genes but decreased abundance at the Hrh3 promoter. Moreover, ChIP using a pan-H4ac antibody documented increased enrichment at Drd1 and Hcrtr1 promoters but decreased abundance at Hcrtr2, Grin1, and Hrh3 promoters. These results underlie the potential complex epigenetic responses when METH is administered.
It is also interesting to compare the epigenetic effects of METH to those of modafinil, a drug used clinically to treat ADHD, narcolepsy. Modafinil has also been used in clinical trials for treatment of cocaineand METH dependence. Modafinil treatment did not alter the protein levels of pan-H3Ac, H4ac, or of HDACs-1 and 2. ChIP data from modafinil-treated mice using pan-H3ac and pan-H4ac identified similar results to those reported for METH-treated animals, with additional findings of increased enrichment of H3ac at Drd1 and Adra1b promoters and of H4ac at the Drd2 promoter. The authors also reported decreased H4ac at the Hrh1 promoter after modafinil treatment. Drug-induced comparable and differential epigenetic changes might be responsible, in part, for some of the behavioral similarities and differences reported after administration of various stimulants.
In vitro studies have also been used to study the epigenetic effects of METH. Exposure to METH (10uM) upregulated HDAC1, HDAC4, and HDAC6 expression, but decreased HDAC5 expression levels in astrocytes. The paper also reported increased p300, a histone acetyltransferase, but decreased expression of the HATs, PCAF, and GCN5. Taken together, these studies indicate that METH can have differential effects on epigenetic markers both in vivo and in vitro, supporting the idea that epigenetic mechanisms are very important events to understand when planning future therapeutic interventions against MUD.
## Chronic effects of meth on histone acetylation
In addition to investigating the effects of the administration of single doses of METH,also studied alterations in histone acetylation after repeated injections of METH over several days. They focused their analysis on various receptors involved in cognitive control such as the dopamine (Drd1 and Drd2), adrenaline (Adra1a and Adra1b), orexin (Hcrtr1 and Hcrtr2), histamine (Hrh1 and Hrh3), and glutamate (Gria1 and Grin1) receptors in the medial pCTX. They found that repeated injections of METH (1 mg/kg, daily for 7 d) followed by withdrawal for 4 days significantly impacted recognition memory. However, modafinil, an FDA-approved drug used for treatment of narcolepsy did not elicit similar behavioral changes. They showed further that METH caused increased H4ac enrichment at Drd1, Hcrtr1, and Grin1 promoters but less enrichment of H3ac on the promoters of Drd2, Hcrtr1, Hcrtr2, Hrh1, Hrh3, and Grin1 in the medial pCTX. However, they found no differences in the effects of the two drugs on the protein expression of pan-H3ac and H4ac. These results support the notion that enrichment at gene promoters is not directly related to the levels of the histone protein under investigation. A recent ChIP-chip analysis using a rat model of METH-induced behavioral sensitization discovered increased H3 acetylation in a large number of gene promoters (241 genes) and increased H4 acetylation in a fewer number of gene promoters (10 genes) in the pCTX. Interestingly, H4 hyperacetylation was found to contribute to increased expression of Angp32a, a gene is involved in the regulation of synaptic plasticity and memory.
Additional studies to test the effects of METH on specific acetyl marks on lysine residues located on histone tails have revealed that acute and repeated METH exposure can cause enrichment of H4K5ac on the promoter regions of several immediate early genes (IEGs), with some differences in the observed patterns for the two conditions.
Interestingly, repeated METH injections caused decreased H4K5ac protein levels and of its binding to the promoter regions of AMPA and NMDA glutamate receptor subunits.
Chronic administration of METH mimicking the "binge" model showed reduction in the mRNA levels of Hdac8 (Class I), Hdac9 (Class IIa), Hdac6, and Hdac10 (Class IIb), Sirt2, Sirt5 and Sirt6 (Class III), and Hdac11 (Class IV) in the dorsal striatum (DStr), a region for habit formation.
The self-administration (SA) animal model has been used to model drug addiction in humans because animals escalate their drug intake, spend considerable time to obtain the drug, and will continue to take drugs despite the adverse consequences. Using the SA approach, Cadet and his group have shown that METH can cause changes in histone deacetylation in brain regions that participate in the reward circuitry. This approach involved allowing rats to self-administer and escalate METH intake followed by contingent footshocks as representative of adverse consequences and a subsequent period of METH-seeking behavior after 30 days of forced withdrawal from METH SA. The investigators reported that there were increased levels of HDACs (Hdac2, Hdac8, and Hdac9) in the dorsal striatum but decreased levels of HDACs (Hdac1, Hdac5, Hdac7, Hdac10, and Hdac11) in the nucleus accumbens of compulsive METH takers. These observations support the notion that HDACs are important participants in mediating certain aspects of METH-induced behavioral consequences associated with METH-taking behaviors.
## Role of hdac inhibitors in meth exposure
Given the critical role of HDACs in SUDs, it will be important to investigate how manipulations of HDAC activities might influence the drug-taking behaviors in animal models of addiction. If successful, these studies can be followed in experiments using clinical settings. In fact, since there are several HDAC inhibitors (HDACis) that are in clinical use against cancer, it should be possible to use the drugs with the least side-effects in present clinical practice.
METH use is known to be accompanied with behavioral sensitization that can last for several weeks to months in rodents. Interestingly, the class I HDACi, valproic acid (VPA), can attenuate behavioral sensitization responses. In another study, it was reported that microinjections of NaB and VPA into the prefrontal cortex, ventricle, amygdala, and dorsal striatum but not into the hippocampus could attenuate METHinduced hyperactivity. Together, these results further support a role for HDAC in the acute and chronic behavioral effects of the drug.
As previously reported, chronic METH injection resulted in decreased H4K16ac binding and expression of GluA1 and GluA2 and using VPA-normalized binding and expression. A specific HDAC6i that is thiazolidinedione-based has been shown to normalize the abundance of acetylated α-tubulin and to reverse METH-induced morphological changes in a neuroblastoma cell line. Moreover,used another HDAC6i, MeBib derived from a benzimidazole scaffold, and were able to reduce the METH SA by rats.
Many investigators have sought to determine the role of HDACs in the effects of METH by using HDAC knockout mice. For example, using a HDAC2 knockout mouse model,investigated the effects of METH on HDACs and found that METH decreased the expression of Hdac3 and Hdac8 (class I), Hdac4 and Hdac7 (class IIa), as well as and Hdac11 (class IV) in wild-type mice. In contrast, those changes were normalized in HDAC2 KO mice.
Other investigators have used METH SA in rats to assess the role of HDACs in drugtaking behaviors. They found that Hdac5 overexpression in the dorsal striatum using viral vectors increased whereas Hdac5 knockdown decreased METH-seeking behavior. In addition, HDAC5 knockdown rats exhibited increased HDAC1, HDAC4, and HDAC5 target genes, namely Gnb4 and Suv39h1.
## Role of hats in meth exposure
Many investigators have examined the potential involvement of HATs in METHmediated gene transcription. For example,reported that METH increased histone H3 acetylation and upregulated the expression of several synaptic plasticity genes known to be regulated after acute or repeated injections of METH. Cadet and co-workers have also provided evidence that METH SA is associated with up-regulation of the HATs, Kat4, and Kat5 in the DStr. Recently, an in vitro study byusing human primary astrocyte reported that METH significantly upregulated p300/CBP and down-regulated GCN5 and PCAF levels. The authors also reported that METH exposure upregulated global acetylation levels of histone H3 lysine, H3K27ac, H3K56ac and down-regulated H3K14ac. These observations support the notion that METH can cause a combination of histone modifications that can control chromatin dynamics during acute or repeated injections of METH.
One of the most extensively studied signaling pathway after METH administration involves cAMP activation, CREB (cAMP response element binding protein), and CBP (CREB binding protein). p300/CBP serves as an adaptor between CREB and the transcription initiation complex. METH administration was found to increase CREB phosphorylation in both ventral and dorsal striata. In addition, these investigators observed increased recruitment of pCREB onto the promoter regions of c-fos, fosB, Bdnf and Syp, and Cartpt.
## Histone methylation
Cross-talks between different types of histone modifications are often observed in various brain regions that control the sensitivity to the psychostimulant exposure, (reviewed by .found that intermittent subcutaneous injections of METH-induced behavioral sensitization in mice and significantly increased the mRNA expression of the chemokine receptor CCR2, implicated in drugrewarding properties. Activation of CCR2 was due to increased trimethylation of histone H3 at lysine 4 (H3K4me3) at the promoter site of CCR2. Consistent with this result, METH-associated memory was accompanied by increased H3K4me2/3 in the NAc. These changes were secondary to augmented expression of the "writer" enzyme, histone methyltransferase myeloid/lymphoid, or mixed-lineage leukemia 1 (MLL1), but decreased expression of "eraser" enzyme, histone demethylase KDM5C. These two enzymes are known to regulate H3K4 methylation. To validate the role of H3K4 methylation modifiers, the investigators used siRNA-mediated focal, intra NAc knockdown of Mll1 that led to reduced H3K4me3 and reduced cfos and Oxtr mRNA levels; however knockdown of Kdm5C resulted in hypermethylation of H3K4. Together, these findings identified histone methylation as a novel molecular mechanism that can also influence METH-induced behavioral sensitivity.
It should be pointed out that, in the DStr, METH SA caused increased H3K4me3 protein but no changes in H3K4me3 binding on the promoter of cfos. These discrepant findings suggest that there are regional effects of METH on the epigenetic regulation of gene expression in the brain. The data suggest that experimenter-injected drug and drug self-administration might result in different epigenetic consequences and associated changes in gene expression.
## Dna methylation
In 1948, Rollin Hotchkiss first detected chemical modifications on the fifth position of cytosine DNA base, where hydrogen group was replaced by methyl group, and suggested that this modified methyl cytosine existed naturally in DNA. Involvement of DNA methylation in gene regulation was not explored until the 1980s when it came into the limelight with studies that demonstrated its role in gene regulation and cell differentiation. This reversible process of DNA methylation is catalyzed by DNA methyltransferase (DNMT). The added methyl group does not affect the base pairing itself, but the protrusion of methyl groups into the DNA major groove can affect DNA-protein interactions.
## Pre-clinical studies
Animal models studying the effects of METH on DNA methylation (Table 2) revealed that acute and chronic METH injections increased striatal Dnmt1 mRNA expression in a strain-specific manner (increased in the Fisher 344 and not in the Lewis strain). Similarly, Jayanthi et al. (2018)validate this finding of increased striatal Dnmt1 mRNA in their study using single dose of METH in Sprague Dawley rats.
Enzyme-linked immunosorbent assay (ELISA-based DNA methylation determination found increased 5-mC levels in the pCTX after chronic METH exposure. A recent study using a pyrosequencing method to measure DNA methylation marks in chronic METH in pCTX and hippocampus found that several CpG sites in the IEGs including Arc, Fos, Klf10, and Nr4a1 had significant changes in their DNA methylation levels.found the extended role of hippocampal DNA methylation by studying behavioral response after in-utero METH exposure and identified several candidate genes involved in cognition and memory alteration in DNA methylome.
Using methylated DNA immunoprecipitation (MeDIP), Jayanthi et al. (2014; 2018 and 2020)had shown changes in DNA methylation after different paradigms of METH. Acute injection of METH followed by a 30-day withdrawal period led to increased mRNA expression of the stress-related genes Crh and Avp due to DNA hypomethylation at CpG sites near the promoter region for Crh and at intragenic region for Avp. In association with DNA methylation, chronic METH increased methylated CpG binding protein 2 (MeCP2) protein levels and caused MeCP2 co-precipitation with HDAC2. The MeCP2-HDAC2 protein led to transcription repression of AMPA glutamate receptors (GluA1 and GluA2). This observation was supported by ChIP assay that showed METH increased MECP2 enrichment at the promoter regions of GluA1 and GluA2. Moreover, MeDIP PCR revealed decreased cytosine methylation at CpG sites located near the promoter site of GluA2 and at CpG-rich site located at −23 kb from promoter region for GluA1.
To model the escalation of drug-use criterion of MUD, Jayanthi et al. (2020)used noncontingent injections of METH before SA training and found that METH pretreatment caused enhancement of escalated METH SA and down-regulated mRNA and protein expression of voltage-gated K+ channels (Kcna1, Kcna3, and Kcnn1). The epigenetic mechanisms underlying the transcriptional alterations observed may be due to increased DNA methylation at the CpG-rich sites on their promoter sequences. In addition to changes in the expression of stress-related genes-Crh, Avp, AMPA glutamate receptors and voltage-gated K+ −channels recently, the hallmark protein in Parkinson's disease SCNA was significantly increased after exposure to high, prolonged dose of METH. The elevation in gene expression was associated with hypomethylation within the SCNA promoter sequences.
Beyond changes in CpG sites, the long-interspersed element-1 (LINE-1) in the DNA known to cause genome instability when altered was found to be regulated by DNA methylation and histone modifications. According to the report by, METH injections exhibit increased activity of LINEs in the dentate gyrus of hippocampus and DStr regions that contribute to METH-induced impairment in cognition and memory.
In addition, reports from Mong's lab demonstrated that repeated exposure to METH enhances sexual motivation in hormonally primed female rats on an epigenetic level. They observed a significant increase in DNMT3b protein level in female rats treated with METH and exogenous steroids in comparison to METH-alone treated rats. This suggests that increased sex drive is not a consequence of METH use but could be the reason behind drug use.
## Clinical studies
Detection of DNA methylation levels by methylight qPCR in male METH addicts revealed significant correlation between the methylation levels of chimerin 2 (CHN2) and METH dependence. CHN2 is a protein involved in remodeling of the actin cytoskeleton and hippocampal axonal pruning. This suggests that prolonged abuse of METH induces abnormal methylation of CHN2 gene that interferes in actin skeleton remodeling leading to irregular formation of neurites and growth cones that is considered crucial to maintaining long-lasting addictive behavior.
Another study that explored the genome-wide methylation analysis of METH users differentiated by their clinical diagnostic criteria qualification for MUD. Subjects that do meet the criteria belong to low METH addictive quality group (LMAQ) and the ones who qualified for METH dependence as high METH addictive quality group (HMAQ). The two addiction phenotypes (LMAQ and HMAQ) was distinctly segregated by the percentage of methylated Caveolin-2 (CAV2) that was significantly increased only in the low addictive group.
Pyrosequencing studies showed a significant increase in parvalbumin (PVALB) methylation levels in the psychotic subgroup of METH-dependent patients and no change was seen in the non-psychotic METH group. The change in PVALB level might contribute to the GABAergic deficits associated with METH dependence. In addition,found promoter DNA hypomethylation of candidate genes involved in DA regulation (DRD3, DRD4, MB-COMT, and AKT1) associated with increased expression of the corresponding genes in psychotic METH-dependent patients. Another molecular mediator of memory consolidation process linked to psychostimulants that was found hypomethylated in METH addicts was the brain-derived neurotrophic factor (BDNF).
Among the five CpGs (CpG1-5) measured on the BDNF promoter using pyrosequencing analysis only CpG5 methylation was significantly reduced in METH abusers. Moreover, the report also revealed a functional connection between the methylation of the CpG5 fragment and Bdnf gene expression level using cell-based luciferase assay. Interestingly, a research group from Japanhas recently identified the gene Shati/Nat8L as a medical marker for MUD diagnosis after gaining support from several pre-clinical research data. Research byreported that the ratio of DNA methylation in SHATI/NAT8L was significantly higher at six CpG sites in METH users when compared to healthy subjects.
One of the significant METH-related public health consequences concerns the longterm effects of METH on brain development and associated behaviors in children born to addicted mothers. This issue was addressed by a longitudinal study in children with in-utero exposure to METH. The authors used sodium bisulfite pyrosequencing and found increased DNA methylation at the CpG2 site of HSD11B2, a stress-related gene.
Another study has reported that METH-induced changes in LINE-1 methylation patterns were associated with METH-induced paranoia. Thus, it is possible that METH-induced neuro-oxidative pathways may have altered LINE-1 partial methylation patterns, which in turn may increase risk to develop METH-induced paranoia(seefor details).
## Dna hydroxymethylation
DNA hydroxymethylation (5-hmC), was discovered in T-even bacteriophageand later in rats in 1972. Only recently 5-hmC has been implicated in gene regulation and has been identified as a novel epigenetic mark. In 5-hmC the methyl group of cytosine is replaced by a hydroxy-methyl group catalyzed by the ten-eleven-translocation (TET) family of proteins. Significant levels of 5-hmC, a ten-fold enrichment was observed in the brainespecially within gene bodies that are strongly transcribed.
Several studies have focused on the role of 5-hmC in learning and memory using conditional knockout of the Tet genes. Though understudied, Cadet and his team have published data on METH-mediated effects on 5-hmC. Using a rat model of METH SA and footshocks as adverse consequences, they found that METH-addicted rats showed differential DNA hydroxymethylation in the NAc in comparison to both control and METH-abstinent rats (rats that suppressed METH intake after footshock). These changes occurred mostly at intergenic sites located on long interspersed elements (LINEs). Moreover, they also observed differential DNA hydroxymethylation and increased expression of specific members of potassium channels in the NAc that appear to be promoters of the abstinence from METH-taking behaviors.
Using experimenter-administered doses of METH, the same group documented increased DNA hydroxymethylation at the promoter region of the stress gene, corticotrophinreleasing hormone (Crh), and at the intragenic DNA sequence of vasopressin (Avp). Interestingly, there were also METH-induced increased TET1 and TET3 levels in the NAc. Importantly, METH increased TET1 binding at the Crh promoter and increased TET3 binding at Avp intragenic regions suggesting that TET-induced DNA hydroxymethylation is an important driver of the effects of METH in the NAc. The use of the TET inhibitor, 1,5-isoquinolinediol (IQD) provided conclusive evidence that the TETs were involved in the regulation of Crh and Avp mRNA expression levels. In contrast, increasing doses of METH over a period of two weeks led to decreased enrichment of 5-hmC at the promoter regions of striatal AMPA glutamate receptors in rats.
## Wb chip-pcr medip and hmedip-pcr rt-pcr
## Rat male nac
Single dose METH (10 mg/kg) for 1, 2, 3, and 4 wk, co-treatment with 1,5-isoquinolinediol (IQD, 3 mg/kg) on days 1, 2, 4, and 6 per wk, i.p.
[formula] ↑ METH [/formula]
## Non-coding rna
Potential involvement of non-coding RNA (ncRNA) have not yet been fully examined in MUD. Elucidation of their potential roles may also impact therapeutic options for MUD.
Non-coding RNAs refer to RNAs that do not translate into proteins but still perform crucial roles in transcription and post-transcriptional events. These include microRNAs (miRNAs), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), nucleolar RNAs (snoRNAs), and long non-coding RNA (lncRNA). The accumulated evidence supports the role of miRNAs (15-25-nucleotides in length) as regulators of genes involved in METH-mediated changes in dendritic spines, synaptic transmission, and unfolded protein response(seefor details).
## Pre-clinical studies
A preclinical study using conditioned place preference (CPP) has reported that METH CPP is accompanied by the upregulation of 276 and downregulation of 25 miRNAs in serum exosomes. Using the KEGG pathway analysis, the authors found that these miRNAs-regulated genes are involved in vesicular transport, amphetamine addiction, cGMP-PKG signaling pathway, dopaminergic synapse, and GABAergic synapse.
Another study by found that chronic administration of METH to mice induced miR-31-3p in the dorsal HIP. This miRNA targets RhoA, an enzyme that mediates actomyosin signaling, vesicular trafficking, and dendritic spine morphology. Interestingly, overexpression of miR-31-3p increased METH-induced CPP score whereas miR-31-3p knockdown attenuated CPP in mice. Using the CPP model in rats,also measured the expression of a large number of miRNAs in the NAc and showed changes in miRNAs that target genes involved in Wnt signaling, tuberculosis, toxoplasmosis, spliceosome, and axon guidance. Moreover,showed that METH-CPP was associated with the downregulation of miRNA, miR-181a-5p, in the DStr. They also identified 36 target genes from online bio-informatic databases and were able to validate up-regulation of 11 of these genes that are members of the ER chaperone complex. These results support other experiments that have demonstrated that METH can impact the function of the ER.
It is of interest that miRNA expression profiling in the DStr of METH-injected rats using high-throughput sequencing analysis identified 167 miRNAs that were dysregulated (113 up-regulated and 54 down-regulated). Using network enrichment analysis, the authors reported changes in the expression of genes that regulate PI3K-Akt and FoxO signaling. In addition to changes in gene expression in the NAc and DStr, miRNA profiling is also altered in the midbrain that mainly contributes to dopaminergic signaling in MUD. Using short access of drug self-administration over a period of 40 days,reported that METH SA was accompanied by 7 up-regulated and 71 down-regulated miRNAs in the VTA. Most of these genes participated in dopamine metabolic process, biological quality, and plasma membrane integrity.
METH intake also dysregulates miRNA biogenesis. Specifically, METH-induced behavioral sensitization in mice is associated with decreased Argonaute2 (Ago2) mRNA expression. These changes were negatively correlated with the development phase of behavioral sensitization. Measurements of Ago2-dependent miRNAs in NAc neurons found that miR-3068-5p could disrupt METH-induced locomotor sensitization. These effects of Ago2/miR-3068-5p occurred through interactions with the glutamate receptor, GluN1/Grin1.also found increased expression of miR-128 during METH-induced locomotor sensitization. Moreover, AAV-mediated overexpression of miRNA-128 had significant additional effects on the locomotor activity. In contrast, AAV-mediated inhibition of miRNA-128 attenuated METH-induced locomotion. Interestingly, the researchers identified three differentially expressed proteins (Arf6, Cpeb3, and Nlgn1) in miR-128-dependent METH-induced sensitization. Importantly, these proteins are involved in controlling dendritic morphology and synaptic transmissionthat are known to be impacted by METH administration.
It is important to note that a study has suggested that METH might impact the expression of miRNAs in extracellular vesicles (EV). METH-dependent CPP in mice caused an increased expression of EV-containing miRNAs (miR-183-5p, miR-9a-5p, and miR-369-3p) in the hippocampus. These miRNAs are known to play a crucial role in cell communication in the CNS and peripheral system. These investigators also showed decreased hippocampal protein levels of ErbB4 and NRG1, that might serve as markers of METH-induced psychosis.
## Clinical study
Studies in humans with MUD have documented changes in plasma EVs. Specifically, , using a miRNA array platform, reported differential expression (19 up-regulated and 69 down-regulated) of miRNAs in the peripheral blood of female patients. The authors found that age of first use was correlated positively with changes in miR-628-5p expression but negatively correlated with miR-301a-3p and miR-382-5p. In addition, lifetime of METH use correlated positively with miR-301a-3p and miR-382-5p expression but negatively with miR-628-5p expression. Finally, the frequency of METH use was negatively correlated with miR-382-5p. This study illustrates the need to collect all essential data regarding the clinical history of patients who meet the criteria for a substance-use disorder diagnosis.
A recent study byusing plasma EVs of METH patients reported potential relationships between symptoms of anxiety and depression and changes in miRNAs in EVs. Specifically, changes in anxiety and depression scores were negatively correlated with the expression of miR-363-3p, miR-16-5p, miR-129-5p, and miR-92a-3p. These studies support, in part, the idea of using changes in EVs as potential biomarkers of psychiatric disorders, mainly anxiety and depression, in patients exposed to METH. Before such conclusions can be reached, however, there is a need to carry out large-scale clinical studies that compare METH-associated anxiety and depression to other clinical populations that have not been exposed to any psychostimulant. Another study conducted bycompared serum miRNA expression profiles in male and female METH-dependent patients to those of normal controls. They reported 5 up-regulated and 9 down-regulated miRNAs. More clinical work is necessary to identify specific mRNAs and non-coding RNAs whose expression might be altered in plasma EVs. These studies promise to provide potential windows to occurrences in the CNS. Such panoramic views may help to decipher the molecular substrates of MUD and help to better plan therapeutic interventions against this malady that affects so many patients. Abbreviation: METH, methamphetamine; DStr, dorsal STR; HIP, hippocampus; dHIP, dorsal HIP; VTA, ventral tegmental area; NAc, nucleus accumbens; i.p., intraperitoneal; s.c., subcutaneous; ↑, significantly increased; ↓, significantly decreased; CT, control; KI CT, knock-in with control vector; KD CT, knock-down with control vector; AcMETH, acute METH; ChMETH, chronic METH; KI METH, knock-in with METH treatment; KD METH, knock-down with METH treatment; EV, extracellular vesicles; AAV, Adeno-associated virus; AAV-SYN, AAV using synapsin-1 promoter for expression; AAV-SYN-spmIR-30a-5p, AAV-SYN miRNA sponge to inhibit miR-30a-5p; Rhy, rhynchophylline extract from Uncaria rhyncophylla (Miq.) Miq. ex Havil.; HAM-A, scale that grades the severity of anxiety; HAM-D, scale that grades the severity of depression; SA, self-administration; CPP, conditioned place preference; METHiHS, METH-induced hyperlocomotor sensitization; h, hour(s); d, day(s); wk, week(s); miR/miRNA, microRNA; rno, Rattus norvegicus; hsa, Homo sapiens; WB, Western Blot/immunoblot; RNA -Seq, RNA sequencing; miRNA -Seq, miRNA sequencing; RT-PCR, reverse transcriptase-polymerase chain reaction; miRNA-PCR, miRNA-polymerase chain reaction.
# Conclusions
Research studies investigating the molecular effects of METH have used high-throughput sequencing to measure global epigenetic changes and altered gene expression. These studies have identified important correlations between epigenetic modifications and transcriptional changes. In most cases, combinatorial epigenetic events will be responsible for METH-induced changes in transcription in brain regions implicated in reward circuitries. Nevertheless, much more work is necessary in order to identify general and specific targets that would provide panoramic details for the various molecular switches that could trigger acceleration from recreational intake to compulsive and pathological drug use. Here, our review has summarized the accumulated evidence that histone and DNA modifications as well as changes in ncRNAs are involved in the acute and chronic effects of METH on the brain and periphery. These observations form the initial steps toward elucidat-
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