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Comparison of sequential multiplex PCR, sequetyping and whole genome sequencing for serotyping of Streptococcus pneumoniae
Streptococcus pneumoniae is one of the major causes of pneumonia, meningitis and other pneumococcal infections in young children and elders. Determination of circulating S. pneumoniae serotypes is an essential service by public health laboratories for the monitoring of putative serotype replacement following the introduction of pneumococcal conjugate vaccines (PCVs) and of the efficacy of the immunization program. The Quellung method remains the gold standard for typing S. pneumoniae. Although this method is very effective, it is also costly, time consuming and not totally reliable due to its subjective nature. The objectives of this study were to test and evaluate the efficiency of 3 different molecular methods compared to the Quellung method. Sequential multiplex PCR, sequetyping and whole genome sequencing (WGS) were chosen and tested using a set of diverse S. pneumoniae. One-hundred and eighteen isolates covering 83 serotypes were subjected to multiplex PCR and sequetyping while 88 isolates covering 53 serotypes were subjected to WGS. Sequential multiplex PCR allowed the identification of a significant proportion (49%) of serotypes at the serogroup or subset level but only 27% were identified at the serotype level. Using WGS, 55% to 60% of isolates were identified at the serotype level depending on the analysis strategy used. Finally, sequetyping demonstrated the lowest performance, with 17% of misidentified serotypes. The use of Jin cpsB database instead of the GenBank database slightly improved results but did not significantly impact the efficiency of sequetyping. Although none of these molecular methods may currently replace the Quellung method, WGS remains the most promising molecular pneumococcal serotyping method.
# Introduction
The Gram-positive lancet-shaped cocci bacteria Streptococcus pneumoniae is frequently associated with meningitis, pneumonia and sepsis in humans in addition to be the major cause of PLOS ONE | https://doi.org/10.1371/journal.pone.0189163 December 13, 2017 1 / 16 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mortality in children [bib_ref] Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995-1997, Feikin [/bib_ref]. Pneumococcus infections mainly occur among young children and the elderly, under 5 years old and above 65 years old, respectively. More than 90 S. pneumoniae capsular polysaccharide (CPS) types exist resulting in a large variety of serotypes belonging to 46 different serogroups [bib_ref] Revisiting molecular serotyping of Streptococcus pneumoniae, Camargo [/bib_ref]. In Canada, the introduction of the seven-valent pneumococcal conjugate vaccine (PCV-7) in 2005 targeting the seven predominant serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) led to a significant decrease in invasive pneumococcal diseases (IPD) associated to these serotypes [bib_ref] Streptococcus pneumoniae infection: a Canadian perspective, Deng [/bib_ref]. However, replacement of vaccine serotypes by non-vaccine serotypes (NVT) led to the emergence of serotype 19A as the new predominant multi-drug resistant serotype [bib_ref] Emergence of Invasive Pneumococcal Disease Caused by Nonvaccine Serotypes in the Era..., Munoz-Almagro [/bib_ref]. Following the advent of NVT, two others vaccines were released in 2008 and 2010, PCV-10 and PCV-13, respectively. The monitoring of IPD serotypes became essential as new NVT may have emerged making the introduction of new vaccines necessary. Serotyping methods of S. pneumoniae can be grouped in two different categories: phenotype-based methods and genotype-based methods [bib_ref] Current methods for capsular typing of Streptococcus pneumoniae, Jauneikaite [/bib_ref]. The Quellung method (based on antisera reactions) still remains the Gold Standard method used in most laboratories [bib_ref] Typing of pneumococci by using 12 pooled antisera, Sørensen [/bib_ref]. However this method is expensive, laborious and not fully reliable. Following the sequencing of the cps loci of 90 pneumococcal serotypes, methods based on the detection of serotype-specific genes were developed in order to provide cost-effective and reliable assays for the serotyping of S. pneumoniae [bib_ref] Current methods for capsular typing of Streptococcus pneumoniae, Jauneikaite [/bib_ref] [bib_ref] Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes, Bentley [/bib_ref].
Among these methods, three were chosen for comparison in this study: sequential multiplex PCR, sequetyping and whole genome sequencing (WGS). The sequential multiplex PCR protocol developed by the Centers for Disease Control and Prevention (CDC) relies on the use of primers targeting serotype-or serogroup-specific regions in the cps loci [bib_ref] Revisiting Pneumococcal Carriage by Use of Broth Enrichment and PCR Techniques for..., Carvalho M Da [/bib_ref]. PCR has been extensively used for the serotyping of S. pneumoniae and had the advantage of being easy to use and can be performed on a large quantity of samples [bib_ref] Evaluation of PCR-based approach for serotype determination of Streptococcus pneumoniae, Shakrin [/bib_ref] [bib_ref] From Quellung to multiplex PCR, and back when needed, in pneumococcal serotyping, Siira [/bib_ref] [bib_ref] Direct Streptococcus pneumoniae real-time PCR serotyping from pediatric parapneumonic effusions, Slinger [/bib_ref] [bib_ref] Development of an automated and multiplexed serotyping assay for Streptococcus pneumoniae, Yu [/bib_ref]. The sequetyping method was developed by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] and is based on the cpsB gene sequence which appears to be specific to serotypes [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref]. WGS became a suitable method for serotyping with the improvement in accuracy and a decrease in cost which has allowed the identification of serotype by comparing cps loci sequences [bib_ref] Genetic Characterisation of Malawian Pneumococci Prior to the Roll-Out of the PCV13..., Everett [/bib_ref] [bib_ref] Five winters of pneumococcal serotype replacement in UK carriage following PCV introduction, Gladstone [/bib_ref] [bib_ref] Strain features and distributions in pneumococci from children with invasive disease before..., Metcalf [/bib_ref].
The replacement of the Gold Standard Quellung method in routine laboratories by a genotype-based method is a current issue for many laboratories, requiring preliminary estimations of the efficiency and adaptability of different methods. Such comparisons and evaluations for some methods have already been conducted [bib_ref] Distribution of Serotypes and Antibiotic Susceptibility Patterns Among Invasive Pneumococcal Diseases in..., Al-Sheikh [/bib_ref] [bib_ref] Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping, Dube [/bib_ref] [bib_ref] A comparison of conventional and molecular microbiology in detecting differences in pneumococcal..., Ogami [/bib_ref] [bib_ref] Evaluation of Pneumococcal Serotyping by Multiplex PCR and Quellung Reactions, Richter [/bib_ref] [bib_ref] Comparison of serotyping, pulsed field gel electrophoresis and amplified fragment length polymorphism..., Shaaly [/bib_ref]. Unfortunately, inter-strain genome variations led to an increase in cps loci rearrangement and diversity. Thus the efficiency of molecular serotyping methods may vary between strains and/or between different regions [bib_ref] Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes, Bentley [/bib_ref].
In this study, a large number of serotypes were included, but a focus on the most prevalent serotypes in Québec/Canada and serotypes targeted by PCV-13 were chosen. The evaluation of a potential molecular replacement for the Quellung identification method was considered.
# Material and methods
Isolates, culture conditions and DNA extraction One hundred eighteen invasive S. pneumoniae representing 83 serotypes previously identified by the Quellung reaction were selected from the Laboratoire de santé publique du Québec (LSPQ) provincial surveillance program (see S1 . All the isolates were subjected to sequential multiplex PCR and sequetyping methods. Six serotype 35A isolates and six serotype 34 isolates were added to the pool tested with the sequential multiplex method as well as six serotype 29 isolates were added to the sequetyping pool. A subset of 53 isolates were tested with WGS and represented 32 different serotypes. The selection of the serotypes was performed on the basis of the most prevalent serotypes in the province of Québec in 2012-2016 [fig_ref] Fig 1: S [/fig_ref]. Rare serotypes were also included in order to test the robustness of the method. WGS data for 35 S. pneumoniae was also provided by the National Microbiology Laboratory (NML, Winnipeg, Canada), totaling 88 isolates representing 53 serotypes subjected to serotyping using WGS approach. Finally, three Streptococcus pseudopneumoniae and three Streptococcus mitis were used as specificity controls for sequential multiplex PCR and sequetyping.
Isolates were cultured on TSA II (Trypticase Soy Agar with 5% sheep blood) agar plate and incubated overnight at 35˚C in a 5% CO 2 atmosphere. Bacteria were collected with a loop and suspended in G2 buffer solution with RNase A (QIAGEN inc, Toronto, ON, Canada). Samples were then frozen at -20˚C until extraction. DNA extraction was performed with the MagAttract DNA Mini M48 Kit (QIAGEN inc, Toronto, ON, Canada) and the QIAGEN TM BioRobot M48 workstation according to manufacturer's instructions.
## Sequential multiplex pcr
The CDC sequential multiplex PCR protocol was used as described by [bib_ref] Revisiting Pneumococcal Carriage by Use of Broth Enrichment and PCR Techniques for..., Carvalho M Da [/bib_ref]. Briefly, primers pairs were designed to target serotype-or serogroup-specific regions in the wzy or wzx genes. The choice of primers was modeled on those included in the CDC protocol as they were adapted to the 22 most prevalent serotypes in Quebec (2012-2016). These serotypes represent 90% of IPD in Quebec. All serotypes included in the PCV-13 (4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F, 3, 6A and 19A) were also covered by this protocol. Positive and negative controls were used in each reaction. Positive controls consisted of a mix of S. pneumoniae DNA extract of serotypes present in each multiplex. S. pseudopneumoniae and S. mitis DNA extracts were tested in each multiplex as a control of specificity.
## Sequetyping
Sequetyping procedures were conducted as described by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] with some modifications. Briefly, master mix was composed of 0.3 μl of Amplitaq DNA polymerase (5 U/μl), 38.85 μl of DNA-free water, 5 μl of 10x PCR buffer (ThermoFisher Scientific, Whitby, Canada), 1.5 μl of MgCl 2 (50 mM), 0.75 μl of dNTPs (10 mM), 0.8 μl of cps1 and cps2 primers (25 μM) and 2 μl of DNA extract for a final volume of 50 μl. Cycling conditions was performed as described by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref]. Sequencing was performed using the BigDye1 Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Whitby, Canada) in a 3130xl Genetic Analyzer (ThermoFisher Scientific, Whitby, Canada).
Assembled cpsB sequences were blasted (default parameters) against a local and comprehensive cpsB database developed by [bib_ref] Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae..., Jin [/bib_ref] [bib_ref] Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae..., Jin [/bib_ref]. This database extended the previous database created by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] by covering 95 serotypes instead of 93 and including a total of 390 sequences. Then, cpsB sequences were used to interrogate the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). In-house Python scripts allowed the automation of these processes. Hits with the highest identity value and High Scoring Pair (HSP) length were retained for serotype attribution. cpsB sequences were deposited in the GenBank database and are accessible under accession numbers MF693230-MF693347. Scripts have been deposited in the GitHub database and are available (https://github.com/Mauffrey/serotyping).
## Whole genome sequencing
Libraries for whole genome sequencing were prepared with the Nextera XT DNA library preparation kit and sequenced using an Illumina MiSeq reagent kit v3 (600 cycles, paired ends) following the manufacturer's instructions. Reads quality was evaluated with FastQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc/). De novo genome assemblies were performed using SPAdes version 3.9.0 [bib_ref] SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing, Bankevich [/bib_ref] assembler on Calcul Quebec public resources (http://www. calculquebec.ca/en/) with standard parameters and the option flag '-careful' activated. Assemblies' quality was assessed with Quast. Following assembling, in-house Python scripts allowed to remove contigs with length below 500 bp and coverage below 5x and to compute assembly's statistics. Concerning the identification of the different cps loci, a local cps database was created with 107 cps sequences representing 92 different serotypes [bib_ref] Revisiting molecular serotyping of Streptococcus pneumoniae, Camargo [/bib_ref] retrieved from the NCBI GenBank database. Assembled contigs containing cps sequences were blasted against this database using BLAST+ tools suite in an automated in-house Python scripts. Serotypes were attributed considering hits with the highest bit scores. When multiple hits had high bit score and close identity values (<0.3% compared to best hit) for an equivalent HSP length, they were all retained for serotype attribution. FASTQ reads were deposited in the NCBI Sequences Reads Archive and are accessible under accession numbers SRR5962910-SRR5962997 (Bioproject accession number PRJNA398497). Scripts have been deposited in the GitHub database and are available ([email protected]:Mauffrey/serotyping.git).
PneumoCaT (Pneumococcus Capsular typing Tool), a serotyping designed workflow, was also used for serotype identification [bib_ref] Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets..., Kapatai [/bib_ref]. Automation of PneumoCaT was performed using a shell command based script. PneumoCaT uses a two-step pipeline for serotype attribution. The first step aligns raw reads with a stored cps sequence library to determine potential serotypes for an isolate. In a second step variant analysis is called only if several serotypes belonging to the same genogroup are returned from step 1 [bib_ref] Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets..., Kapatai [/bib_ref]. Briefly, reads are mapped against specific genogroup databases and functions are called for the detection of presence/absence of genes, allelic variants, single nucleotide polymorphisms (SNPs) and loss-of-function mutations. A serotype is returned at this point if a unique CPS locus is identified that is not a predefined genogroup, otherwise capsular loci variant analysis occurs for serotype attribution.
Isolates misidentified with the assembly-based strategy were subjected to further investigations. The cps locus was extracted from the corresponding contig according to the best hit coordinates and aligned with cps reference sequences of both best hit and expected serotype, for comparison. Alignments were done using the Artemis Comparison Tool (ACT) v6 and WebACT [bib_ref] ACT: the Artemis comparison tool, Carver [/bib_ref].
The impact of coverage and N50 on the serotyping quality between samples identified at the serotype level and samples identified as serogroup, subset or misidentified was evaluated with a Wilcoxon-Mann-Whitney test with an alpha value of 0.05 (see .
## Serotype identification levels
For all the methods tested in this study, sample identification was classified as follows: 1) "Serotype" when a concordance was found with the Quellung and molecular identification methods, 2) "Serogroup" when a concordance was found with the Quellung identification as well as with other serotype(s) from the same serogroup, 3) "Subset" when a concordance was found with the Quellung identification as well as with other serotype(s) from a different serogroup, 4) "Misidentified" when no concordance was found with the Quellung and molecular identification methods and 5) "Not determined (N.D.)" when no amplification occurred in PCR multiplex reactions or when cpsB was not amplified in the sequetyping method. When isolates of the same serotypes had different identification levels with the same method, they were classified as inconsistent results when results per serotype were considered.
# Results
## Sequential multiplex pcr
Among all existing S. pneumoniae serotypes, the CDC sequential multiplex PCR protocol is able to detect 74 different serotypes. cpsA amplification ensures the presence of S. pneumoniae DNA in each reaction. In our experiments, cpsA amplification product was present in all reactions except for isolates of serotypes 25F and 38. The absence of amplification in those serotypes has been previously documented by Carvalho et al., . Moreover, no cpsA amplification occurred with S. pseudopneumoniae and S. mitis isolates.
In this study, 130 isolates were tested with multiplex PCR method, covering 83 serotypes. Of the tested isolates, 45/130 (35%) were identified at the serotype level, 42/130 (32%) were identified at the serogroup level, 22/130 (17%) were identified at the subset level, 19/130 (15%) were not determined, and 2/130 (1%) were misidentified [fig_ref] Table 1: Serotype identification results [/fig_ref]. All serotypes were not equally represented in our isolates selection, thus these results are not representative of the method efficiency concerning identification level. Nevertheless, all results were consistent when multiple isolates were tested for a same serotype, except for serotype 35A (1% of serotypes). Considering identification for each serotype, 22/83 (27%) were identified at the serotype level, 24/83 (29%) were identified at the serogroup level, 17/83 (20%) were identified at the subset level, 19/83 (23%) were not determined and 0/83 (0%) were misidentified [fig_ref] Table 1: Serotype identification results [/fig_ref]. One serotype showed inconsistent results (serotype 35A), which accounted for 1% of the serotypes.
Serotypes 34 and 35A showed unexpected results. Serotype 34 sample showed many amplicons, including a non-specific amplicon (250 bp) and the expected amplicon (408 bp), in the same reaction (multiplex PCR 7). Six more serotype 34 isolates were selected and subjected to identification with sequential multiplex PCR and the same non-specific amplification was present in 3 out of 6 reactions. The expected amplification product at 280 bp was not present in the multiplex PCR 7 with serotype 35A and 6 other serotype 35A isolates were further selected. For 5 out of 6 isolates, the expected amplicon was detected but a non-specific amplicon at 250 bp was also visible. It should be noted that expected amplicons bands are very well defined and have high intensity compared to non-specific amplicons bands which are generally less bright.
Non-specific amplification products were present in many PCR reactions. They seemed to occur randomly and did not depend on the isolate serotype. Only 4 different sizes non-specific amplicons were observed during this study, a non-specific bands at 500 bp in multiplex PCR 2, a non-specific band at 677 bp in multiplex PCR 3, a non-specific band at 850 bp in multiplex PCR 6 and a non-specific band at 250 bp in multiplex PCR 7. Except for the band at 677 bp in the multiplex PCR 3, these non-specific products did not correspond to expected product sizes in their respective multiplex PCR and were easily identified as non-specific. However, the amplification product at 677 bp in multiplex PCR 3 corresponds to the expected size for serotype 35B and is hardly identifiable as non-specific. Many non-specific amplicons were also present for S. pseudopneumoniae and S. mitis in most of the multiplex PCR.
## Sequetyping
Of the 124 S. pneumoniae isolates subjected to sequetyping, 118 (95%) were positive for cpsB amplification (1061 bp). No cpsB amplification was obtained for serotypes 25F, 37, 38, 39 and 43 which was in accordance with results from [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] as these serotypes were predicted in silico to be non-amplifiable. However, no cpsB amplification was obtained with serotype 29 although it was expected to be amplifiable according to [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref]. Therefore, 6 other serotype 29 isolates were selected and subjected to sequetyping. All 6 samples led to cpsB amplification. After sequencing and assembling, the average sequence length was 890 bp which is longer than the 732 bp region used by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] to test all their serotypes. Comparison of molecular serotyping methods for S. pneumoniae
One hundred eighteen sequences representing 78 serotypes were subjected to BLAST for identification. Two different databases were chosen for the analysis: the exhaustive NCBI Gen-Bank database and a more restrained but specific cpsB database created by [bib_ref] Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae..., Jin [/bib_ref]. Using the GenBank database, 61/124 (49%) were identified at the serotype level, 20/124 (16%) were identified at the serogroup level, 14/124 (11%) were identified at the subset level and 23/ 124 (19%) were misidentified. Using the Jin cpsB database, 65/124 (52%) were identified at the serotype level, 20/124 (16%) were identified at the serogroup level, 12/124 (10%) were identified at the subset level and 21/124 (17%) were misidentified [fig_ref] Table 1: Serotype identification results [/fig_ref]. Inconsistent results were obtained for some serotypes (6B, 6C, 19F and 23F) when using the GenBank database but not using the Jin cpsB database. Considering only serotypes, identification with the Gen-Bank database resulted in 35/83 (42%) identifications at the serotype level, 12/83 (14.5%) identifications at the serogroup level, 12/83 (14.5%) identifications at the subset level and 14/83 (17%) misidentified. With the Jin cpsB database, 38/83 (46%) were identified at the serotype level, 14/83 (17%) were identified at the serogroup level, 12/83 (14%) were identified at the subset level and 13/83 (16%) were misidentified. Results were slightly better with the Jin cpsB database [fig_ref] Table 1: Serotype identification results [/fig_ref] , in particular for inconsistent results.
The majority of misidentifications were due to the attribution of closely related serotypes of the same genogroup [bib_ref] Genetic Relatedness of the Streptococcus pneumoniae Capsular Biosynthetic Loci, Mavroidi [/bib_ref]. For example, one serotype 9A isolate was identified as serotype 9V, one serotype 11F isolate was identified as serotype 11C and one serotype 42 isolate was identified as serotype 35B/35C see S2 supplemental material for a complete and detailed list). For some misidentifications, there was no association between the determined serotype and the expected one. This was the case for one serotype 15C isolate identified as serotype 24F, one serotype 19F isolate identified as serotype 10A and one serotype 17A isolate was identified as serotype 10A. Serotype 29 isolates were all misidentified as serotype 35B/35C. Although these serotypes are genetically close, the percent similarity of our serotype 29 cpsB sequence compared with the serotype 29 reference sequence was only 83%.
Only one S. pseudopneumoniae isolate led to the amplification of cpsB. This sequence was associated with serotype 20 with 96% similarity which was the lowest score across all isolates.
## Whole genome sequencing
The number of paired-end reads obtained varied between 100 065 and 1 153 346 with an average of 542 388 (S3 . Assembling coverage varied from 14X to 296X and assembly's length varied from 1 982 679 bp to 2 285 405 bp. Three strains presented unexpected features. LSPQ4282 and SC0268 assembly's length were 6 793 942 bp and 2 698 601 bp, respectively, which is higher than the expected 2.0-2.2 Mbp length for S. pneumoniae. BLAST confirmed that Bacillus subtilis contamination occurred in LSPQ4282. After removal of B. subtilis associated contigs, assembly's length dropped to 1 721 249 bp and coverage dropped from 36x to 6x. Assembly with and without B. subtilis sequences were both submitted to the serotyping pipeline and results were identical. SC0268 did not present any contamination with non-Streptococcus strains. However, it is possible that the sample consisted in a mix of several S. pneumoniae strains, leading to an assembly length longer than expected. MA080904 presented an unlikely mean coverage of 1012x. After investigation, it appeared that some small contigs had a very high coverage value, leading to a non representative mean coverage. After filtration of contigs with coverage > 1000x, a more representative coverage value of 37x was found.
Serotype identification was mainly based on sequence identity level and HSP length (S4 . For 53 of 88 isolates (60%), the serotype corresponded to that determined by Quellung without any ambiguity. The serogroup was determined for 25 of 88 isolates (28%), 6 of 88 (7%) were determined at the subset level and 4 of 88 isolates (5%) were misidentified. Considering serotypes, concordance with Quellung identification was found for 29 of 53 serotypes (55%), concordance at the serogroup level was found for 13 of 53 serotypes (24.5%), 6 of 53 (11%) were determined at the subset level and 3 of 51 (5.5%) were misidentified. Inconsistent results were obtained for isolates of serotype 6B and 7F, representing 4% of the serotypes tested. For some isolates, BLAST results could not discriminate between two different serotypes because of their high degree of genetic similarities or due to the existence of DNA polymorphism among single serotypes [bib_ref] Evolution of the Capsular Regulatory Genes in Streptococcus pneumoniae, Varvio [/bib_ref]. This was the case for 15B/15C, 22A/22F, 7A/7F, 11A/11D, 25A/ 25F, 32A/32F, 33A/33F, 9A/9V, 12A/46, 12F/44, 18B/18C and 35A/35C/42. Statistical analysis showed that coverage and N50 seemed to have no significant impact on the quality of the serotyping results.
The cps locus sequence of misidentified isolates (serotypes 6D, 7F and 29) were aligned with the corresponding best hit reference sequence given by the in-house serotyping method and with the expected serotype sequence . No significant hit with 18B reference sequence was found for the misidentified serotype 18B isolate. Therefore, the cps locus was aligned with the best hit reference sequence .
The cps locus alignment of our serotype 29 isolate resulted in fragmented hits with low identity compared with the serotype 29 reference sequence. The region 1174-2915 bp of our serotype 29 isolate sequence did not match with both serotype 29 and serotype 35B reference sequences and coded for a tnp transposase. It appeared that the cps locus of the serotype 29 isolate was located at the end of the corresponding contig and may be incomplete, resulting in a 1303 bp shorter sequence compared to the serotype 29 reference sequence. A very poor alignment was also obtained for our serotype 7F isolate cps locus sequence compared with the serotype 7F reference sequence, with less than 50% of the cps locus sequence correctly aligned. For the serotype 6D isolate, the major difference between the 2 alignments was the absence of a match with the serotype 6D reference sequence in the 5170-6608 bp region coding for the glycosyl transferase wciN.
PneumoCaT was also used for serotype attribution using the same set of WGS data (reads data). If a capsular typing variant analysis occurred, the serotype resulting from this analysis was retained for the serotype prediction. Sixty-one of 87 isolates (70%) were successively identified at the serotype level and the remaining isolates (30%) were misidentified. Considering only serotypes, 31 of 52 serotypes (59.5%) were identified at the serotype level and 19 of 52 (36.5%) were misidentified. Inconsistent results were obtained for serotypes 7F and 11A, representing 4% of the serotypes tested. Half of the misidentifications were due to an incorrect capsular typing variant analysis, resulting in this high proportion of misidentified isolates. One isolate (47A) could not be serotyped due to a too low number of reads due to contamination with B. subtilis, drastically decreasing the number of S. pneumoniae reads.
# Discussion
S. pneumoniae serotyping has become critical since the release of the different PCV for the monitoring of putative emergent NVT. Unfortunately, the gold standard Quellung method is expensive and laborious and can lead to interpretation errors. The implementation of a new and reliable serotyping method is needed, especially for surveillance programs such as the provincial surveillance held at the Laboratoire de santé publique du Québec.
In this study, 3 different molecular based serotyping methods (sequential multiplex PCR, sequetyping and WGS) were compared in order to evaluate their efficiency in serotype attribution for S. pneumoniae invasive isolates. This is the first comparison between these 3 methods on a common set of isolates.
PCR methods are very powerful, reliable and easy to perform. Multiplex PCR is an even more efficient technique since one single reaction allows the simultaneous detection of more than one gene and/or allele. The CDC sequential multiplex PCR method gave the expected results, with 27%, 29% and 20% identifications at the serotype, serogroup and subset level, respectively. This was also the method presenting the least misidentified isolates (1%). However, serotypes among a serogroup are inevitably revealed under the same signal in the current protocol due to their high level of genetic homogeneity. For example, primer pair 6A/6B/6C cps sequence alignments. Alignment of cps loci of serotype 29 isolate (A), serotype 7F isolate (B), serotype 6D isolate (C) and serotype 18B isolate (D) with reference cps sequence and best hit cps sequence according to WGS identification. Alignment was generated with Artemis Comparison Tool (http://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act).
https://doi.org/10.1371/journal.pone.0189163.g002
Comparison of molecular serotyping methods for S. pneumoniae 6D in reaction 1 is simultaneously specific to four different serotypes. This is the most important limit for the efficiency of this method because no better results can be expected. Moreover, a significant number (23%) of serotypes were not detectable by this method, representing another limitation from a surveillance perspective. It also seems that small genetic variations in some isolates (serotype 35A) could determine the presence or absence of amplicon [bib_ref] Quantitative effects of position and type of single mismatch on single base..., Wu [/bib_ref]. It is possible that the isolates tested were genetic variants of the CDC isolates of serotype 35A and that the primers 35A/35C/42 were unable to match these isolates. This finding would mean that the method efficiency could vary from one geographic region to another depending on the genetic distance with the isolates used for primer design. Another important aspect is the specificity of the method for S. pneumoniae. Indeed, it is not uncommon to confuse S. pneumoniae with other Streptococcus spp. due to their high degree of similarity, especially S. pseudopneumoniae [bib_ref] Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and..., Arbique [/bib_ref]. Here, the internal control (cpsA) allowed differentiation between S. pneumoniae and S. pseudopneumoniae or S. mitis. However, 2 serotypes (25F and 38) were also negative for cpsA amplification making this discrimination not fully reliable. Finally, non-specific amplifications occurred during the study, as specified by the CDC (https://www.cdc.gov/ streplab/pcr.html). Although most of the non-specific products did not match with expected amplifications, some of them could lead to misidentification.
Sequetyping is not limited to the number of detectable serotypes as cpsB sequences of almost all serotypes are present in regularly updated public database. Nevertheless, cpsB is not amplifiable in all serotypes, making these serotypes not identifiable with this method. This was the case for serotypes 25F, 37, 38, 39 and 43 in our study. Sequences for serotypes 39 and 43 were predicted to be non-amplifiable by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] even though they were amplified in their study. However, they did not obtain any amplification for serotype 25F or 38, which is consistent with our results. Finally, serotype 37 cpsB sequence was predicted to be amplifiable but was not tested in vitro in their study.
We decided to use the local cpsB sequence database created by [bib_ref] Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae..., Jin [/bib_ref] instead of the database used by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] because this database was more comprehensive and covered more serotypes. Overall, we obtained more identification at the serotype level and less misidentifications using the local cpsB database as compared to the GenBank database. Significant differences were obtained for serotypes 6B, 6C, 19F and 23F where results between isolates of the same serotype were concordant with the cpsB database but not with GenBank database. Only well characterized sequences with full-length cpsB were chosen for this database and can explain these results. Indeed, slight variations in the cpsB sequence could have a major influence on serotype attribution when the GenBank database is used due to a lot of cpsB sequences presenting nucleotide variations not representative of the serotype. In contrast, the use of a local cpsB database with few but representative sequences avoided these mistakes. Apart from serotypes 12F, 17A, 18C, 24F, 29 and 35A, no equivalent data are available in [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] for the other misidentified serotypes we observed in this study. For serotype, serogroup, and subset levels identification, our results are generally the same as the ones obtained by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref]. However, Comparisons are not always possible since 38 of our serotypes are missing in the [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] study. Most of misidentified serotypes had some nucleotides of difference (from 1 to 59) with the best hit sequence, usually of the same serogroup or genogroup [bib_ref] Genetic Relatedness of the Streptococcus pneumoniae Capsular Biosynthetic Loci, Mavroidi [/bib_ref]. This is caused by intra-serotype variation [bib_ref] Evolution of the Capsular Regulatory Genes in Streptococcus pneumoniae, Varvio [/bib_ref] in the cps regulatory region and can lead to identification in the wrong serogroup. This issue has already been observed by [bib_ref] Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy, Leung [/bib_ref] with one 19F isolate identified as a serotype 1. Furthermore, some serotypes may have identical cpsB sequences as it is the case with some 6A and 6B isolates [bib_ref] Sequence Diversity within the Capsular Genes of Streptococcus pneumoniae Serogroup 6 and..., Elberse [/bib_ref]. Moreover, for our serotypes 17A and 29 isolates, no significant hits were obtained with serotype 17A and 29 cpsB sequences, respectively. S. pneumoniae genome diversity may be high between geographically distant locations, leading to divergence between serotype 17A and 29 cpsB sequences present in the databases and sequences obtained in this study. However, this appears to be very unlikely [bib_ref] Comparative Genomic Analyses of Seventeen Streptococcus pneumoniae Strains: Insights into the Pneumococcal..., Hiller [/bib_ref]. Our evaluation of the sequetyping approach has demonstrated that this serotyping method is not always able to correctly identify serotype probably due to short DNA sub region of a large locus used in this analysis. Of the 6 other non-S. pneumoniae isolates tested, only one S. pseudopneumoniae led to a cpsB amplicon. This was not expected as it has been reported that S. pseudopneumoniae cps locus is not complete compared to S. pneumoniae and does not contain cpsB. However, the low identity of the best HSP (96%) could help to discriminate this isolate. A recent method based on sequetyping including a second analysis step for homologous strains allowed to obtain more accurate results for these strains. Such protocol could putatively help to obtain better results and make sequetyping more attractive.
Two different approaches were used for serotype identification using WGS method. Our in-house workflow consisted in assembling contigs from sequencing data and to BLAST them with a cps loci sequence database. Eighty-two percent of serotypes were identified at the serotype or serogroup level, demonstrating the efficiency of this strategy. Regarding unresolved serotypes (7A/7F, 9A/9V, 11A/11D, 12A/12F/44/46, 18B/18C, 22A/22F, 25A/25F, 32A/32F, 33A/33F and 35A/35C/42), these were all identified as another serotype belonging to the same genogroup as defined by [bib_ref] Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets..., Kapatai [/bib_ref]. More sensitive genetic analysis methods would be required to make a more accurate identification such as the capsular variant analysis integrated in PneumoCaT (see below).
Interestingly, serotype 22F isolates matched serotypes 22F/22A but with two separate HSPs. This unexpected BLAST result is caused by the high divergence of two genes (wcwA and wcwC) in the cps locus of those isolates compared to their orthologous sequences in serotype 22F. Similar finding were reported for isolate 1772-40b (GenBank accession HE651318; Salter et al., 2012), a 22F serotype which matches perfectly with our 22F isolates [bib_ref] Variation at the capsule locus, cps, of mistyped and non-typable Streptococcus pneumoniae..., Salter [/bib_ref].
A serotype 29 isolate was misidentified with WGS and identified as serotype 35B. Serotype 35B and 29 are known to be genetically related, leading to cross-reactivity in antisera reactions [bib_ref] Structures of Capsular Polysaccharide Serotypes 35F and 35C of Streptococcus pneumoniae Determined..., Bush [/bib_ref]. However, no significant hit with serotype 29 was found in BLAST results, meaning that no relevant alignment could be made. These results were in agreement with sequetyping results obtained for serotype 29 isolates. Alignment with serotype 29 reference sequence (isolate 34373, [bib_ref] Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes, Bentley [/bib_ref] showed low identity although the serotype was confirmed by Quellung. Transposase coding region (tnp) was found downstream the dexB gene in the serotype 29 isolate. According to, those regions may contribute to the vertical exchange of the cps locus between pneumococcal isolates and hence to their molecular evolution and adaptation, which could explain the low identity with serotype 29 reference sequence. Serotypes 6D and 6B belong to the same genogroup. However, the glycosyl-transferase wciN is present in the 6B cps locus and not in the 6D cps locus, distinguishing those [bib_ref] Comparison of Capsular Genes of Streptococcus pneumoniae Serotype 6A, 6B, 6C, and..., Song [/bib_ref]. This gene was present in the studied serotype 6D isolate, which explains the misidentification with serotype 6B. It has been suggested that serotype 6D could have emerged from recombination between serotypes 6B and 6C but [bib_ref] Comparison of Capsular Genes of Streptococcus pneumoniae Serotype 6A, 6B, 6C, and..., Song [/bib_ref] highlighted the implausibility of this event because of a high genetic distance between these serotypes. Therefore, this gene acquisition was probably due to homologous recombination events or horizontal genetic transfers. The misidentification of serotype 7F (SC0218) isolate with serotype 14 and serotype 18B (SC0049) with 7B were surprising as these 2 serotypes belong to different genetic clusters [bib_ref] Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets..., Kapatai [/bib_ref]. These misidentifications cannot be attributed to sequencing quality as these 2 samples showed very good assembly's length, coverage and N50 values (see . Moreover, no cross-reactivity reactions are known between those serotypes (Statens Serum Institut, 2013, Key to pneumococcal factor antiseria. Accessed October 18, 2017. Available from http://www.ssidiagnostica.com/-/ media/Admin/Diagnostica-Downloads/Downloads-UK/Brochures/BrochurePneumococcalfactor-antisera-key-18058.ashx).
PneumoCaT is the second approach we used for WGS serotyping and totally integrates a capsular variant analysis step in its workflow. Single Nucleotide Polymorphisms (SNPs) analysis, allelic variations or presence/absence of genes are analyzed when more than one locus is matched or if the match corresponds to a defined genogroup [bib_ref] Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets..., Kapatai [/bib_ref]. Although the first step gave results similar to the results obtained with the assembly-based approach, the variant-based step identifications did not match with Quellung identification for half of the serotypes tested. However, concordance between PneumoCaT and Quellung identifications were found for 8 serotypes (7A, 9V, 12A, 12F, 15C, 22A, 22F and 33F) which were only identified at the serogroup level or subset with the assembly-based approach. S. pneumoniae is in constant evolution, resulting in the apparition of genetic variants [bib_ref] Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype..., Coffey [/bib_ref]. Recent publications have highlighted genetic variations present in the cps genes of certain serotypes leading to potential regional allelic differences [bib_ref] Spectrum of Pneumococcal Serotype 11A Variants Results from Incomplete Loss of Capsule..., Calix [/bib_ref] [bib_ref] Streptococcus pneumoniae Serotype 9A Isolates Contain Diverse Mutations to wcjE That Result..., Calix [/bib_ref] [bib_ref] Discovery of Streptococcus pneumoniae Serotype 6 Variants with Glycosyltransferases Synthesizing Two Differing..., Oliver [/bib_ref] [bib_ref] Genetic, Biochemical, and Serological Characterization of a New Pneumococcal Serotype, 6H, and..., Park [/bib_ref]. CPS allelic variants in our isolates may explain the misidentifications occurring at the capsular typing variant analysis step. The possibility of local genetic variations between serotype-specific genes may require regular updating of the Pneu-moCaT databases to prevent these misidentifications.
Another automated serotyping pipeline for S. pneumoniae, SeroBA, was recently developed and used a hybrid assembly and mapping approach. Although the authors proved that it was faster and less computational-intensive than PneumoCaT, serotype identification efficiency was equivalent between the two pipelines. Further comparative studies are required to fully evaluate the pipeline.
The aim of this study was to evaluate 3 DNA-based S. pneumoniae serotyping methods which could eventually replace the current Quellung gold standard method. Above all, none of the methods tested showed enough efficiency to be able to completely replace the Quellung method in surveillance programs. Indeed, identifications at the serogroup level were obtained with all of them but more particularly with sequential multiplex PCR. Though WGS produces reliable serotyping results, currently this method is still costly and time consuming. Nevertheless, with the automation of bioinformatic pipelines and the constant drop of reagent costs, this method could become very attractive for monitoring invasive S. pneumoniae serotypes. Moreover, the great amount of information generated with WGS can be easily valued with, for example, the analysis of molecular evolution of the isolates, the identification of putative vaccine targets in addition to the detection of antibiotic resistance and virulence genes. The sequential multiplex PCR and sequetyping strategy unlike WGS have specifically been developed to improve the serotyping response time and to reduce the associated costs. PCR has the inconvenience of requiring an adaptation to the local epidemiology of circulating serotypes. Simply changing the sequential order of the reaction may be sufficient but more often reviewing the combination of primers in the reaction mixture is needed.
In this study, we have demonstrated that WGS was the most reliable method among the 3 methods tested for serotyping of S. pneumoniae. However, serotype validation with Quellung is still required as some serotypes cannot be clearly distinguished with the cps sequences. Sequential multiplex PCR and sequetyping have the advantage to be cheaper than WGS and could also serve as a guide for Quellung method. But these methods have drawbacks making them less attractive. It is important to note that rare untypeable isolates, due to their lack of capsular polysaccharide, may generate a positive result with DNA based method [bib_ref] Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae, Kaijalainen [/bib_ref]. In such cases, the final serotype identification would be in disagreement with the Quellung reaction which would produce a negative result. Conversely, the sequetyping or multiplex PCR approach may be used when the capsular swelling of the Quellung reaction is difficult to observe through microscopic examination. Finally, a total replacement of the Quellung reaction by a molecular method seems not possible yet. Nevertheless, WGS appears to be a very promising tool and could replace the Quellung method in the near future with its extensive use and the development of databases.
Supporting information S1
[fig] Fig 1: S. pneumoniae serotype distribution. Serotype distribution in the province of Québec in 2016. Grey bars represent serotypes tested by WGS in this study. https://doi.org/10.1371/journal.pone.0189163.g001 Comparison of molecular serotyping methods for S. pneumoniae PLOS ONE | https://doi.org/10.1371/journal.pone.0189163 December 13, 2017 [/fig]
[table] Table 1: Serotype identification results. https://doi.org/10.1371/journal.pone.0189163.t001 [/table]
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Protocol to generate and characterize biofouling transformants of a model marine diatom
ProtocolProtocol to generate and characterize biofouling transformants of a model marine diatomDiatoms are a major group of microalgae that initiate biofouling by surface colonization of human-made underwater structures; however, the involved regulatory pathways remain uncharacterized. Here, we describe a protocol for identifying and validating regulatory genes involved in the morphology shift of the model diatom species Phaeodactylum tricornutum during surface colonization. We also provide a workflow for characterizing biofouling transformants. By using this protocol, gene targets such as GPCR signaling genes could be identified and manipulated to turn off diatom biofouling.
# Materials and equipment
For efficient extraction of total RNAs from diatom samples, plastic applicator sticks (with a length of appropriately 14 cm), which fit the 1.5 mL Eppendorf microtubes, are used to grind and break down frozen cell pellets into powders.
# Step-by-step method details
Preparing and cultivating two different morphotype cultures
Timing: 2 weeks 1. Grow diatoms on solid plates a. Make up a fresh medium according to the f/2+Si medium recipe. b. Autoclave for 25 min (at 121 C with a pressure of 15 psi or 1.03 3 10 5 Pa) and cool the mediumagar mix to 55 C. c. Add any antibiotics if needed (for example, to prepare agar plates with 100 mg/mL zeocin for growing zeocin-resistant diatom transformants) and pour the plates with 20 mL for each plate in a 100 mm diameter plate, and then cool down the plates to room temperature (22 G 2 C) and dry the plates in the safety cabinet (laminar flow hood) for one hour after the agar solidifies. 3. Extract RNA from two different morphotype cultures for RNA-seq a. Collect diatom samples from fresh cultures. For both solid culture with oval cells > 75% of the population, collect cells from plates and resuspend them in deionized water; for liquid culture with fusiform cell > 90% of the population, take 30-50 mL cultures for each sample (approximately 10-20 mg dry weight for next steps 1b-1d).b. Pellet cells in 50 mL tube by centrifugation at 4000 3 g at 20 C for 5 min, use the pipette to remove the supernatant and wash the cell pellets using deionized water; repeat the step for 2-3 times, and then resuspend cell pellets in 1 mL of deionized water. c. Transfer the resuspended cells to a centrifugation tube, pellet cells by centrifugation at 10000 3 g at 20 C for 1 min and use the pipette to remove the supernatant. d. Freeze cell pellets in liquid nitrogen or dry ice and grind the cell pellets in 1.5 mL microtube into powder with a clean plastic applicator stick by repeating the freeze and thaw cycle five to ten times. e. Extract total RNA by using the MagMAX-96 Total RNA Isolation kit (AM1830, Thermo Fisher Scientific Inc.) with all related reagents according to the manufacturer's instruction, including the steps of lysis and binding, initial nucleic acid purification, DNase treatment, and final RNA cleanup. f. Assess the quality of the extracted RNA samples using a Bioanalyser (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA) to measure RNA Integrity Numbers (RIN). Quantify RNA samples with specific concentration, purity, and integrity values by using the Qubit RNA assay kit. A minimum RIN score of 8 is preferred for proceeding to RNA-seq.
CRITICAL: The diatom cells must be ground thoroughly to break down cell walls by repeating the freeze and thaw cycle and also kept at low temperature (ice bath) conditions during the whole process to avoid any RNA degradation. In addition, the Lysis/Binding Solution for processing diatom samples must be freshly prepared and used on the same day.
4. Perform RNA-seq on RNA isolated from the WT P. tricornutum strain in liquid and on solid media a. Take the samples of total RNA with high quality after quality control using a bioanalyzer (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). The use of two or more technical replicates is recommended for each biological sample. b. Prepare mRNA library using Illumina TruSeq V2 RNA sample Prep Kit (San Diego, CA) according to the manufacturer's protocol (https://www.illumina.com/products/by-type/sequencing-kits/libraryprep-kits/truseq-rna-v2.html). c. Purify polyadenylate-containing mRNA by oligo(dT) magnetic beads from 1.0 mg of total RNA sample, fragment it, and reversely transcribe the first-strand cDNA using random primers. d. Then use the first-strand cDNA to synthesize second-strand cDNA by using DNA polymerase I and ribonuclease H. Treat the fragments after second-strand cDNA synthesis with end-repair, the addition of a single A base, and TruSeq adapter-index ligation to cDNA libraries. e. Amplify the fragment samples by PCR for 12 cycles to produce fragments with a size of 350-400 bp, including adapters, to create the final cDNA library for the next step 2f. f. Confirm fragment sizes and purity of the libraries with positive control by using a Bioanalyzer 2100 (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). g. Determine the quantities of the libraries required for RNA-seq by real-time qPCR using a KAPA library quantification kit (Kapa Biosystems, Roche) for the Illumina platform. h. Sequence the enriched cDNA libraries using the Miseq (Illumina) or HiSeq 2500 system (Illumina) platform with 2 3 150-base pair read length. i. Process data using the standard Illumina processing pipeline to segregate each multiplexed sample's reads. j. Take the RNA-seq data for further analysis and deposit the raw data to the National Center for Biotechnology Information (NCBI) database.
## Preparing materials and reagents for diatom transformation
Timing: 3 weeks to months 5. Identify a group of 61 signaling genes based on a total of 2,468 up-regulated genes in the solid wild type when compared with the liquid wild type cells using the KEGG Automatic Annotation Server (http://www.genome.jp/tools/kaas/) from 44 potential signaling pathways based on KEGG database with gene annotations of the model diatom P. tricornutum. 6. Select 14 cDNAs encoding candidate signaling genes including several GPCR signaling genes from the 61 signaling genes with a focus on GPCR signaling genes as GPCRs are on the top level of signal transduction hierarchy. 7. Send the design request of the nucleotide sequence to the company to synthesize and clone these signaling genes in the shuttle vector pPha-NR with a nitrate reductase promoter. 8. Receive the host E. coli strains with the designed plasmids and extract the plasmids for diatom transformation experiments.
## Transformation and selection of targeted morphotype strains
Timing: 2-4 weeks
For the transformation of different gene constructs to the diatom P. tricornutum, the electroporation protocolwas used with slight modifications for ease of implementation and optimization. A NEPA21 electroporator (Nepa Gene Co. Ltd.) was used for the electroporation experiment.
9. Transform different gene constructs to the diatom P. tricornutum a. Collect diatom samples from the fresh liquid culture at the exponential growth phase (OD 600 =0.3-0.5). Take 20-30 mL cultures for each sample. b. Pellet cells in 50 mL tube by centrifugation at 700 3 g at 20 C for 4 min, use the pipette to remove the supernatant and wash the cell pellets using 0.77 M mannitol; repeat the step for 2-3 times, and then resuspend cell pellets in 1 mL of 0.77 M mannitol. c. Linearize 3-5 mg of each plasmid construct by NdeI for 4 h at 37 C in a total reaction volume of 50 mL. d. Use DNA Clean & Concentrator TM -25 to recover linearized plasmids by following the manufacturer's instructions. e. Mix 3-5 mg of linearized plasmids with an aliquot of 0.15 mL cell suspension on ice and then transfer the mixture to an electroporation cuvette with a 0.2 cm gap on ice. f. Determine the impedance of the mixture in the electroporation cuvette and adjust its impedance by using 0.77 M mannitol solution to a value between 0.4 and 0.6 kU. g. Set the electroporator and do the transformation with the following parameters: square electric poring pulses at 300V (pulse duration, 5 ms; 8 pulses; interval 50 ms; 10% decay rate), and transferring pulses at 8V (pulse duration, 50 ms; 5 pulses; interval 50 ms; 40% decay rate).
Place the cuvettes with cell suspensions on ice immediately. CRITICAL: The diatom cells must be collected from the exponential growth phase for highly efficient transformation. Moreover, the impedance of mixed cells and plasmid DNA solution in the cuvette for electroporation must be adjusted to get a tradeoff between cell damage and DNA delivery efficiency.
10. Select successful transformants with targeted morphotypes a. Check the presence of single colonies on the selective agar plates after ten days to 2 weeks. b. Pick up single colonies and subculture them into liquid selective medium, respectively, for 1-2 weeks. c. Pellet cells from 1-2 mL liquid cultures by centrifugation at 10000 3 g for 1 min, use the pipette to remove the supernatant and wash the cell pellets using deionized water; repeat the step two times, and then resuspend cell pellets in 50 mL 5% Chelex-100. d. Vortex for 10 s and incubate at 98 C-100 C for 10 min. e. Cool down on the ice for 1 min and vortex for 10 s. f. Spin at 10000 3 g for 1 min and use 1 mL of supernatant for each of 20 mL PCR reaction. g. Screen and verify the incorporation of plasmid DNA into the genome by checking the PCR products with corresponding primers (primers of GPCR1A and GPCR4, as indicated in the Key Resource . h. Check the morphotypes of selected transformants under light microscopy and confirm the successful transformants are with targeted morphotypes.
## Preparing samples for characterization of selected transformants
Timing: days to weeks 11. Prepare diatom samples for light microscopy and scanning electron microscopy (SEM) experiments a. Cultivate and maintain seed cultures of both the wild type and the transformants under continuous cool white fluorescent lights of 50 mmol photons m À2 s À1 at 22 G 2 C. b. Subculture diatoms by inoculating seed cultures into the freshly prepared liquid medium (10%-20%, v/v) in 250 mL flasks with a total culture volume of 50 mL. c. Grow the liquid cultures under continuous cool white fluorescent lights of 50 mmol photons m À2 s À1 at 22 G 2 C to exponential growth phase (optical density OD 600 at 600 nm is between 0.1 and 0.2 in flasks) and confirm fusiform cells as the major morphotype in the wild type and oval cells as the major morphotype in the transformant liquid cultures. d. For the light microscopy experiment, dilute the samples to the desired cell density (between 10 5 and 10 6 cells/mL) and make a glass slide for microscopy observation directly; for the SEM experiment, dilute the samples to the desired cell density (between 10 5 and 10 6 cells/mL) for SEM observation and then remove salts by washing the samples with deionized water. 12. Prepare diatom samples for light stress, UV stress, and surface colonization experiments a. Cultivate and maintain seed cultures of both the wild type and the transformants under continuous cool white fluorescent lights of 50 mmol photons m À2 s À1 at 22 G 2 C. b. Subculture diatoms by inoculating seed cultures into the freshly prepared liquid medium (10%-20%, v/v) in 250 mL flasks with a total culture volume of 50 mL. c. Grow the liquid cultures under continuous cool white fluorescent lights of 50 mmol photons m À2 s À1 at 22 G 2 C to exponential growth phase and confirm fusiform cells as the major morphotype in the wild type and oval cells as the major morphotype in the transformant liquid cultures. d. Dilute the samples to the desired cell density (between 1310 5 and 5310 5 cells/mL) for each of these experiments. After dilution, samples need to be kept in the dark for one hour for the light stress experiment only.
## Characterization of selected transformants
Timing: 4 days
The morphotypes of selected transformants are determined with SEM imaging. Moreover, the transformants are characterized by the capability to respond to light stress, UV stress, and surface colonization compared to the wild type as a control.
13. SEM imaging a. Transfer the freshly diluted (using deionized water) samples (1-1.5 mL for each) from the previous preparation step (step 1 in the section of preparing samples for characterization of selected transformants) to microcentrifugation tubes. b. Dehydrate the samples by centrifuging at 5000 3 g for 1 min, removing the supernatant with a pipette, and washing the cell pellets using 20%, 40%, 60%, 80%, and 100% ethanol gradually. A centrifugation step at 5000 3 g for 1 min is used between washes. c. Vortex the cell samples in 100% ethanol. d. Deposit liquid solutions on Whatman track-etched membranes (VWR 514-0049). e. Dry the membranes and then sputter deposit approximately 2 nm of Au onto the membranes in a vacuum coater. f. Perform SEM imaging using a Thermo/FEI Quanta 450 Scanning Electron Microscope (or equivalent) with 5 kV accelerating voltage and a spot size (beam current) of 2 in high vacuum using an ET (Everhart-Thornley) secondary electron detector. g. Observe high-resolution images of individual diatom cells using SEM.
CRITICAL: The diatom cells must be dehydrated gradually to keep the natural structure of cells.
14. Light stress experiment a. Take the freshly prepared samples from the previous preparation step (step 2 in the section of preparing samples for characterization of selected transformants). b. Dilute the samples using f/2 + Si medium to a cell density of approximately OD 600 =0.05 c. Keep the samples in the dark for 1 h and vortex for 10 s after dark incubation. d. Set the PHYTO-PAM-II (Heinz Walz GmbH) instrument with constant white light stress of 220 mmol photons m À2 s À1 for 10-15 min. e. Determine the effective quantum yields under different light intensities of , and 588 mmol photons m À2 s À1 in a consecutive mode. f. Compare the difference between the transformants and the wild type in terms of quantum yields. 15. UV stress experiment a. Take the freshly prepared samples from the previous preparation step (step 2 in the section of preparing samples for characterization of selected transformants). b. Dilute the samples to a cell density of 1-3 3 10 6 cells/mL (appropriately at OD 600 of 0.1 to 0.
2) using f/2 + Si medium and transfer 3 mL of each sample to a 100 mm diameter plate c. Expose the samples in plates to UV light using a UV crosslinker with a UV exposure time of 60 s and energy of 0.1 J/cm 2 d. Incubate samples under a low light intensity of 30 mmol photons m À2 s À1 at room temperature (22 G 2 C) for over two days. e. Mix the samples thoroughly by pipetting the solution and count cells to get cell density after UV treatment.
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f. Calculate the survival rate by using the initial cell density and the cell density after UV treatment and compare the difference between the transformants and the wild type 16. Surface colonization to determine the adhesive differencebetween the transformants and the wild typea. Rinse glass microscope slides with deionized water, then soak the slides in 1M HCl for 24 h. b. Rinse the slides using deionized water and dry the slides. c. Take the freshly prepared samples from the previous preparation step (step 2 in the section of preparing samples for characterization of selected transformants) and dilute the samples to a cell density of 500,000 cells/mL (approximately at OD 600 of 0.03). d. Place each slide to a 100 mm diameter dish plate. e. Add 15 mL of diluted samples to each dish plate. f. Incubate the samples for 72 h at 22 G 2 C in growth chambers. g. Shake and rinse the slides gently to remove unattached cells on a shaker at 150 rpm. h. Count cells according to microscope images from 9 randomly sampled views. i. Compare the adhesive difference between the transformants and the wild type.
## Expected outcomes
The transcriptome analysis protocol will identify differentially expressed genes (DEGs) in the solid WT compared with liquid WT cells and the identification of overrepresented Gene Ontology (GO) terms for the DEGs from WT. The analysis will also allow us to identify the up-regulated genes involved in potential signaling pathways and predict their protein-protein interaction network. The ultimate expected outcome from the transcriptome analysis is to identify candidate GPCR signaling genes for transformation.
The transformation protocol will result in GPCR transformants. The selection of the transformants expressing the GPCR genes will help us sort and select for transformants that can shift cell morphotype from fusiform to oval forms when observed and compared to WT, as shown in.
The characterization of the selected GPCR transformants will show whether they can resist light and UV stress compared to WT. The surface colonization protocol will determine these transformants' capability to attach and colonize to the surface of glass slides compared to WT, as shown in.
## Quantification and statistical analysis
For the section of Transcriptome analysis of two different morphotype cultures, the CLC Genomics Workbench 11.0.1 (https://digitalinsights.qiagen.com) calculates the normalized Reads Per Kilobase per Million (RPKM) of genes expressed in the samples. The tool for obtaining the RPKM reads, RNA-seq Analysis, requires importing the fasta files of the sample-sequenced transcripts. A summary of the major steps is as follows: Import the fasta files using the ''Import tracks'' tool. Make sure that ''Batch mode'' is enabled when selecting the files to be imported. To run RNA-seq Analysis, go to Toolbox, Transcriptomic Analysis, then RNA-seq Analysis. Then there is a dialog box with steps to follow. Select the sequencing files imported previously and the reference annotation, P. tricornutum (2013-07-EBI-Phatr3), ASM15095v2, downloaded from EnsemblProtists ( http://protists. ensembl.org/index.html).
The detailed steps are in the manual of the CLC genomics (http://resources.qiagenbioinformatics. com/manuals/clcgenomicsworkbench). Using CLC genomics, it is optional to process RNAseq data of different samples to obtain differentially expressed genes (DEGs). Alternatively, you can use the DESeq2 R packagefor obtaining the DEGs.
To identify cellular functions enriched in the DEGs, use ClueGo plug-in (V2.5.0)in Cytoscape v3.6.0 ( https://cytoscape.org/). To do this:
In a Cytoscape session, install the ClueGO plug-in from App menubar. Select ClueGo App to start functional analysis and Gene Set Enrichment Analysis (GSEA) of the DEGs. In the control panel window, select the organism, P. tricornutum, and paste the list of the DEGs identifiers. Select the visual style, significance, the ontologies type, biological processes, and enable ''show only pathways with p value less than or equal to 0.05''.
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## Open access
Click ''Start'' to start the analysis. ClueGO determines the statistically significant Gene Ontology (GO) terms that are overrepresented in the DEGs. ClueGO calculates the p values using a twosided hypergeometric test and corrects them using the Benjamini-Hochberg false discovery rate (FDR).
When ClueGO finishes the analysis, the network pops up, visualizing the enriched GO terms represented in nodes. Each biological process has a specific node-color. The node size is proportional to the number of genes in a particular GO term.
## Photosynthetic efficiency
The WT and transformants' photosynthetic performance can be evaluated using the chlorophyll fluorometer PHYTO-PAM-II (Heinz Walz GmbH). The effective quantum yields (QY) of photosystem II (PSII) is used to assess the capability of the strain to respond to immediate white light stress (220 mmol photons m À2 s À1 over 10 min). The chlorophyll fluorometer uses equation (1) to calculate the maximum photosynthetic efficiency of PSII, where F 0 is the ground fluorescence in the dark-or low-light adapted cells and F m represents the maximal fluorescence.
Fv/Fm = (Fm À Fo)/Fm (Equation 1)
In Excel, calculate the average quantum yield of the triplicates using the function AVERAGE. Calculate the standard error using the function STDEV(range)/SQRT(3).
Plot the effective QY of PSII versus the photosynthetically active radiation (PAR, mmol m À2 s À1 ) and compare light stress response.
To identify DEGs in the various samples during surface colonization, use the DESeq2 R package. Using the negative binomial distribution, the DESeq2 method provides a differential analysis of RNA-seq count data. The package and reference manual are available at http:// www.bioconductor.org/packages/release/bioc/html/DESeq2.html.
To reduce the memory size of the data object, pre-filter each sample by removing the raw read counts <10. Using the single DESeq function implemented in DESeq2, perform differential gene expression analysis after normalizing the total read count. Note that the estimateSizeFactors function occurs within the DESeq function. By default, the geoMeans argument takes the geometric mean of the read counts across all samples, thereby scaling the read counts by a reference sample. Use the results function to generate a table of the log2 fold changes and p-values. The plotMA function implemented in DESeq2 can be used to visualize the log2 fold changes against the mean of normalized counts. Unless otherwise stated, a Student t-test should be performed to assess the statistical significance of physiological differences between samples. A p-value of less than 0.05 is used to reject the null hypothesis for comparisons between two groups.
# Limitations
While this protocol aims to identify key genes regulating the surface colonization process in the diatom P. tricornutum, it is possible that some regulatory genes are still not identified due to genome annotation limitations as well as the relatively low expression levels of some genes. Additionally, it is important to note that this protocol is primarily developed to manipulate the morphology shift of the model diatom P. tricornutum and the related surface colonization process. Therefore, we recommend following up this versatile protocol with an optimization workflow involving verifications of genome annotations as well as diatom RNA extraction methods to enable the successful application to other diatom species.
## Troubleshooting
Problem 1 Low RNA yield and RNA degradation (referring to the step: Transcriptome analysis of two different morphotype cultures).
## Potential solution
When working with RNA instruments, DNase and RNase-free materials should be used, including pipette tips, tubes, and corresponding solutions. The work area/bench should be cleaned with an RNase inactivating solution before starting with the RNA extraction.
RNase activity is reduced with temperature; thus, unless otherwise stated, work with samples on ice or at 4 C at all times, and set the needed devices/centrifuges at 4 C.
## Problem 2
Low transformation efficiency (referring to the step: Transformation and selection of targeted morphotype strains).
## Potential solution
It is crucial that the electroporation is performed on the diatom cells at their exponential growth phase OD 700 =0.2-0.4, 5.9 3 10 6 cells (detailed protocol). A slight modification was introduced to the protocol, adding a washing step and making sure the cells are active before the electroporation.
Ensure using good quality purified DNA. It is recommended to store plasmids at À20 C for longer periods and minimize freeze-thaw cycles to avoid DNA degradation in the longer-term. Linearize the plasmid with the corresponding restriction enzymes prior to the electroporation. A validation step can be added before the transformation, consisting of running gel electrophoresis for the obtained linear fragment in order to validate the expected fragment size.
## Problem 3
Integration (non-targeted integration) of the reporter genes in the diatom genome (referring to the step: Transformation and selection of targeted morphotype strains).
## Potential solution
To examine the integration of the genes in the P. tricornutum, it is essential that the transformed cells (colonies) are sub-cultured 2-3 times in media with 100 mg/mL of zeocin for antibiotic resistance selection. After isolating genomic DNA from transformed cells, PCR verification is conducted on the extracted DNA to detect the inserted fragment using specific primers and validate the cDNA integration in the diatom genome.
## Problem 4
Specificity related to oval/fusiform cells (referring to the step: Preparing and cultivating two different morphotype cultures).
## Potential solution
Use microscopy to ensure that majority of the cells (>60%) of required form for corresponding analyses (oval vs fusiform)
To analyze the transcription shifts between solid and liquid cultures, RNA sequencing is performed on RNA isolated from the wild type P. tricornutum strain grown in liquid and on solid media. Prior to RNA extraction, cells should be observed under a bright-field microscope to ensure that the expected fusiform and oval morphotypes are the dominant morphotypes in liquid and solid cultures, respectively.
## Problem 5
Slow growth of P. tricornutum after transformation on zeocin-selective solid plates (referring to the step: Transformation and selection of targeted morphotype strains).
## Potential solution
To obtain single colonies of transformants on zeocin-selective solid plates as expected, it's important to see diatom growth on plates within two weeks. The plates should be placed under relatively high humid (appropriately 80%) conditions to avoid drying out if cultivation duration is beyond two weeks. Alternatively, 1 mL of sterile f/2+Si medium could be added to the plates per week.
## Resource availability
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Weiqi Fu ([email protected]).
# Materials availability
All sequences of synthesized genes can be found in a recent publication.
Data and code availability RNA-seq data from this article can be found in the GenBank/NCBI data libraries (GenBank: PRJNA566271). The RNA-seq data have also been deposited in Dryad with a unique identifier (https://doi.org/10.5061/dryad.ns1rn8ppx).
# Acknowledgments
Financial support for this work was provided by New York University Abu Dhabi Faculty Research Funds (AD060) and NYUAD Institute grant (73 71210 CGSB9). W.F. was additionally supported by the Icelandic Technology Development Fund (163922-0611) and the Hundred Talents Program of Zhejiang University.
# Author contributions
W.F. and K.S.-A. conceived the research and wrote the paper. All authors contributed to the drafting and editing of the manuscript.
# Declaration of interests
The authors declare no competing interests.
## Ll
## Open access
STAR Protocols 2, 100716, September 17, 2021 |
Progressive subcutaneous emphysema. A rare finding: Pneumorrhachis
a b s t r a c tPneumorrhachis is a rare phenomenon which may be caused by trauma, intracraneal infection, pneumomediastinum or iatrogenic factors. Presence of air in the spinal canal is reported in most cases. In this article, we report a case with PR in the spinal canal without any neurological deficit, which developed secondary to subcutaneous emphysema.
# Introduction
Pneumorrhachis is a rare radiological finding which is characterized by presence of air in intraspinal compartments. It is a rare complication as a result of skull and spinal fractures, epidural abscess, epidural anesthesia and lumbar puncture and (traumatic pneumothorax and pneumomediastinum [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref]. In the majority of the cases, air has been reported in thoracic and lumbar region particularly in the cervical region, with the exception of two cases which had air extending in entire spinal canal [bib_ref] Cervicothoracolumbar pneumorachis. Case report and review of the literature, Chibbaro [/bib_ref]. We detected air along the entire spinal canal in our case which had no pneumomediastinum but which developed subcutaneous emphysema due to spontaneous pneumothorax. This study reports a rare case of etiological and radiological pneumorrhachis.
## Case report
A 31-year old asthenic male patient with no trauma history was admitted to the clinic with shortness of breath and chest pain. On physical examination, diminished air entry in left hemithorax and subcutaneous crepitations were identified. Following the posteroanterior chest graphics showing pneumotorax, tracheostomy tube and underwater seal drainage were performed. Upon the continuation of massive air drainage on the 2nd day of the follow up, we examined the case through thoracic CT with the diagnosis of lung bullae. Diffuse air was detected extending from cervical region to the lower lumbar region [fig_ref] Figure 1: Diffuse air deposition in to the spinal canal [/fig_ref]. It was initially thought that air might have entered in the spinal from cervical region with subcutaneous emphysema through vertebral foramen [fig_ref] Figure 2: Diffuse air deposition in to the spinal canal [/fig_ref]. CT scan showed that air in the spinal canal measures 9,7 mm at the deepest level [fig_ref] Figure 3: Subcutaneous emphysema extending towards the spinal canal through intervertebral foramen in the... [/fig_ref]. The case with no clear neurological symptom was examined by the neurosurgeon. No neurological deficit was detected in the case and we did not plan any surgical operation for pneumorrhachis. The patient underwent operation due to rupture and bullae in the left upper lobe of the lungs. The patient was discharged from the hospital on the 4th day. The follow up CT scan in the postoperative day 14 showed no subcutaneous emphysema and PR.
## Comment
The term pneumorrhachis was first used by Newbold et al. [bib_ref] Pneumothorax, pneumomediastinum, subcutaneous emphysema and pneumorrhachis as complications of common flu, Patel [/bib_ref]. Pneumorrhachis may cause air in different anatomical parts of the spinal canal depending on its mechanism. The majority of the pneumorrhachis cases reported in the literature have air bubbles in the spinal canal. In our case, intraspinal diffuse air has been detected along the entire canal and air measuring 9,7 mm in the cervical region has not been reported in the literature before, to our information.
Pneumorrhachis, which usually occurs after spinal trauma, may be caused by pneumothorax or similar conditions that produce high intrathoracic pressure, barotrauma, a recent iatrogenic manipulations during surgical, anaesthesiological and diagnostic interventions, malignancy and its associated therapy [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref]. Iatrogenic pneumorrhachis is mostly caused by epidural anesthesia [bib_ref] Pneumothorax, pneumomediastinum, subcutaneous emphysema and pneumorrhachis as complications of common flu, Patel [/bib_ref]. In the pneumorrhachis cases whose primary cause is pneumothorax, there is often accompanying pneumomediastinum and air penetration into the spinal canal takes place through posterior mediastinum. There is no facial barrier to prevent air penetration between posterior mediastinum or retropharyngeal space and epidural space. For this reason, in cases of mediastinal emphysema, air can easily enter into epidural space through neural foramina [bib_ref] Epidural emphysema associated with primary spontaneous pneumothorax, Aribas [/bib_ref]. The pneumorrhachis mechanism in our case was identified as subcutaneous emphysema extending towards the spinal canal through intervertebral foramen in the cervical region.
Pneumorrhachis can occur in two different locations, subdural and epidural. Subdural pneumorrhachis is usually associated with severe trauma. For differential diagnosis, malignancy, inflammation and infectious diseases by gas-forming organisms should be assessed [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref].
Pneumorrhachis is first of all a radiological diagnosis. CT is the most reliable and the fastest way of diagnosing pneumorrhachis. On the other hand, it might prove difficult to differentiate epidural and subdural in CT [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref]. Location and distribution of the air in the spinal canal entirely depends on the site of the air penetration, volume and rate. The air usually collects in the posterior epidural space where there is low resistance due to loose connective tissue as compared to the anterior space where there is high resistance due to rich vascular network [bib_ref] Pneumothorax, pneumomediastinum, subcutaneous emphysema and pneumorrhachis as complications of common flu, Patel [/bib_ref]. Presence of air in subdural space is usually associated with severe trauma. In cases of air in epidural space, the patient's clinical condition is usually better and pneumocephalous is not observed [bib_ref] Traumatic air in spinal canal (pneumorrhachis), El-Halabi [/bib_ref]. Intraspinal air does not tend to move. Pneumorrhachis is usually asymptomatic but pain and symptoms causing neurological deficits have been reported in a number of rare cases [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref].
There are no standards for the management of pneumorrhachis due to the rareness and different aetiologies of the cases. Nonsurgical treatment methods include dexamethasone, decompression and aspiration by a needle, high concentrations and hyperbaric oxygen [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref]. There are reports suggesting that asymptomatic patients and pneumorrhachis cases associated with can be treated conservatively with high-flow oxygen inhalation therapy [bib_ref] Pneumothorax, pneumomediastinum, subcutaneous emphysema and pneumorrhachis as complications of common flu, Patel [/bib_ref]. Intraspinal air, in particular in epidural pneumorrhachis cases, tends to resolve by itself in a few days. Thus a conservative approach is recommended for such cases [bib_ref] Pathogenesis, diagnosis and management of pneumorrhachis, Oertel [/bib_ref]. In our case, apart from surgical resection of lung bullae which caused pneumothorax and subcutaneous and surgical treatment of pneumothorax, no additional surgical treatment was undertaken for pneumorrhachis. The patient did not have any neurological symptom during the follow up period.
In general prophylactic management with antibiotics is not recommended in cases of epidural pneumorrhachis and in patients with subdural pneumorrhachis with no symptom of meningitis [bib_ref] Traumatic air in spinal canal (pneumorrhachis), El-Halabi [/bib_ref].
The management of patients with pneumorrhachis should be decided on an individual basis, and through a multidisciplinary approach. It should aim at identification of etiological factors and the causes and should aim at preventing morbidity and potential mortality.
[fig] Figure 1: Diffuse air deposition in to the spinal canal (Cervical and thoracic region). [/fig]
[fig] Figure 2: Diffuse air deposition in to the spinal canal. (Lumbar region). [/fig]
[fig] Figure 3: Subcutaneous emphysema extending towards the spinal canal through intervertebral foramen in the cervical region. [/fig]
|
HTLV-1 and HTLV-2: highly similar viruses with distinct oncogenic properties
# Introduction
Four types of HTLV have been described so far, the most prevalent types being HTLV-1 and HTLV-2 [bib_ref] HTLV-3/4 and simian foamy retroviruses in humans: discovery, epidemiology, crossspecies transmission and..., Gessain [/bib_ref].
HTLV-1 and HTLV-2 show considerable homology in terms of genome structure, replication pattern, and properties of the structural, regulatory, and accessory proteins. Their transmission follows the same route, by transfer of infected lymphocytes by perinatal transmission and breastfeeding and through blood transfusion, sexual contact, and use of intravenous drugs [bib_ref] Pathways of cell-cell transmission of HTLV-1, Pique [/bib_ref]. Both viruses utilize the GLUT-1 and NRP1 cellular receptors for their entry, although HTLV-1, but not HTLV-2, is dependent on heparan sulfate proteoglycans [bib_ref] Human T-cell leukemia virus type 1 HTLV-1 and HTLV-2 use different receptor..., Jones [/bib_ref]. Cell-to-cell transmission is essential for virus replication and occurs through the formation of a virological synapse [bib_ref] Human T-lymphotropic virus, type 1, tax protein triggers microtubule reorientation in the..., Nejmeddine [/bib_ref].
Despite these important analogies, HTLV-1 and HTLV-2 are strikingly different in terms of clinical impact, as only HTLV-1 is conclusively associated with neoplasia, namely adult T-cell leukemia/lymphoma (ATLL), which develops in up to 5% of infected individuals. A similar percentage of HTLV-1-infected persons develop a neurological disease termed HTLV-associated myeolopathy/tropical spastic paraparesis (HAM/TSP; [bib_ref] Tropical spastic paraparesis and HTLV-1 associated myelopathy: clinical, epidemiological, virological and therapeutic..., Gessain [/bib_ref]. In contrast, HTLV-2, though persistently associated with elevated lymphocyte and platelet counts [bib_ref] Long-term increases in lymphocytes and platelets in human Tlymphotropic virus type II..., Bartman [/bib_ref] and with an increase in overall cancer mortality [bib_ref] Increased all-cause and cancer mortality in HTLV-II infection, Biswas [/bib_ref] , does not cause hematologic disorders and is only sporadically associated with myelopathy [bib_ref] Human T-lymphotropic virus type II and neurological disease, Araujo [/bib_ref].
The two viruses also differ in their geographical distribution. HTLV-1 is endemic in Japan, sub-Saharan Africa, South America, and the Caribbean [bib_ref] Epidemiological aspects and world distribution of HTLV-1 infection, Gessain [/bib_ref] , whereas HTLV-2 is prevalent among the indigenous populations in Africa and the Indian-American tribes in Central and South America as well as among drug users in Europe and North America [bib_ref] HTLV-II infection in Italian drug abusers, Zella [/bib_ref] [bib_ref] The epidemiology and disease outcomes of human T-lymphotropic virus type II, Roucoux [/bib_ref]. Although their receptor usage allows HTLV-1 and HTLV-2 to be quite promiscuous for different cell types in vitro, they exhibit distinct cellular tropisms in vivo: HTLV-1 is mainly found in CD4+ T lymphocytes, whereas CD8+ T cells are the preferred target for HTLV-2 [bib_ref] In vivo cellular tropism of human T cell leukemia virus type II..., Ijichi [/bib_ref].
The following sections highlight differences in the biology of HTLV-1 and HTLV-2 that might provide clues to their distinct clinical outcomes. Emphasis is placed on the comparison of the regulatory proteins, kinetics of mRNA expression, clonal distribution patterns, and interaction with cellular factors and signal transduction pathways.
## The comparative anatomy of the htlv-1 and htlv-2 genomes
In their pioneering studies, [bib_ref] Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome..., Seiki [/bib_ref] noted the presence of a region in the HTLV-1 genome between the env open reading frame (ORF) and the 3 LTR that was not present in the previously described oncogenic retroviruses. This region, termed the "X region," is also present in HTLV-2 and codes for the regulatory and accessory proteins in partially overlapping ORFs named x-I through x-V. Expression of the complex arrangement of ORFs in such a compact genome is accomplished by a combination of ribosomal frameshifting, alternative splicing, and polycistronic translation [bib_ref] Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses:..., Ciminale [/bib_ref] [bib_ref] Expression and characterization of proteins produced by mRNAs spliced into the X..., Ciminale [/bib_ref] [bib_ref] Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus..., Koralnik [/bib_ref] as well as production of negative-strand transcripts that code for the HBZ (HTLV-1) and APH-2 (HTLV-2) proteins.
Like other retroviruses, the HTLVs produce an unspliced mRNA which codes for the Gag-Pol-Pro precursor protein and also serves as the viral genome and a singly spliced mRNA that codes for the Env surface glycoproteins. Peculiar to the HTLVs is the expression of the Tax and Rex regulatory proteins that play critical roles in driving expression from the 5 LTR promoter (Tax) and in enhancing the expression of partially spliced plus-strand mRNAs (Rex). The Tax and Rex ORFs (x-IV and x-III, respectively) are expressed from the same doubly spliced mRNA. In HTLV-1, www.frontiersin.org all the other X region proteins are coded by individual singly or doubly spliced transcripts, while some HTLV-2 transcripts express more than one ORF.
Recent studies of the temporal sequence of HTLV-1 gene expression in peripheral blood mononuclear cells (PBMCs) isolated from infected patients [bib_ref] Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ..., Rende [/bib_ref] revealed a "two-phase" kinetics with tax/rex mRNA expression preceding that of other viral transcripts. A similar analysis of HTLV-2 mRNA expression indicated a comparable pattern, although the relative abundance of some transcripts showed some intriguing differences among the two viruses [bib_ref] Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence..., Bender [/bib_ref].
The major differences between HTLV-1 and HTLV-2 gene products are summarized in and commented in the following sections.
## Tax proteins and their interactions with cellular pathways
The principal role of Tax during viral replication is to activate transcription of the LTR promoter through a process that involves the recruitment of CREB/ATF complexes on binding sites in the U3 region. Tax-1 and Tax-2 share this mechanism and are able to cross-activate each others' LTRs [bib_ref] HTLV-I and HTLV-II Tax: Differences in induction of micronuclei in cells and..., Semmes [/bib_ref].
Experiments performed in the early 1990s established a key role for Tax as the main viral oncoprotein necessary for the initial steps of T-cell transformation by HTLV-1 [bib_ref] Oncogenic transformation by the tax gene of human T-cell leukemia virus type..., Tanaka [/bib_ref]. Transgenic mouse models have consistently demonstrated that Tax expression causes tumors, including lymphoma and leukemia [bib_ref] Mice transgenic for HTLV-I LTR-tax exhibit tax expression in bone, skeletal alterations,..., Ruddle [/bib_ref] [bib_ref] Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic..., Hasegawa [/bib_ref]. Recent comparative studies on the transforming activities of HTLV-1 Tax (Tax-1) and HTLV-2 Tax (Tax-2) demonstrated that, in the absence of other viral proteins, both Tax-1 and Tax-2 immortalize human primary CD4+ T cells, but only Tax-2 is able to immortalize CD8+ T cells [bib_ref] Human T cell leukemia virus type 2 (HTLV-2) Tax2 has a dominant..., Imai [/bib_ref]. Furthermore, Tax-2 immortalizes human primary CD4+ T cells more efficiently than Tax-1 [bib_ref] Human T cell leukemia virus type 2 (HTLV-2) Tax2 has a dominant..., Imai [/bib_ref]. Although additional viral products have been demonstrated to be relevant for immortalization, the interactions between Tax and host proteins are still considered to be essential for the transformation process. Several studies of the oncogenic properties of Tax-1 highlighted its effects on DNA repair, cell cycle progression, cell death, and p53 function (reviewed by [bib_ref] New insight into the oncogenic mechanism of the retroviral oncoprotein Tax, Cheng [/bib_ref].
In addition to CREB/ATF proteins, Tax-1 interacts with many other host factors that influence cell proliferation and transformation, including components of the PI3K, AKT, MAPK, TGFβ, SRF, and NF-κB pathways . Tax-2 has been analyzed mainly for its effects on the NF-κB pathway, and was shown to activate the canonical pathway, but, unlike Tax-1, does not activate the noncanonical pathway [bib_ref] Identification of a novel motif responsible for the distinctive transforming activity of..., Shoji [/bib_ref] , suggesting that activation of this latter pathway might be a key element in HTLV-1-driven transformation in vivo. Although overall highly homologous, Tax-1 and Tax-2 present some distinct structural features. Tax-1 contains a PDZ binding motif (PBM) and leucine zipper domains that are absent in Tax-2. The C-terminal PBM domain is relevant for interaction of Tax-1 with host factors that regulate cell cycle progression and proliferation, such as the human homologue of the Drosophila melanogaster disk large tumor suppressor protein (hDLG; [bib_ref] Tax oncoprotein of HTLV-1 binds to the human homologue of Drosophila discs..., Suzuki [/bib_ref]. The leucine zipper regions present in the central region of Tax-1 are responsible for activation of the non-canonical NF-κB pathway [bib_ref] Distinct functions of HTLV-1 Tax1 from HTLV-2 Tax2 contribute key roles to..., Higuchi [/bib_ref]. The nuclear localization and nuclear export signals of Tax-1 and Tax-2 present significant differences which could explain the predominant nuclear localization of Tax-1 compared to Tax-2 [bib_ref] Localization of human T-cell lymphotropic virus type II Tax protein is dependent..., Turci [/bib_ref] [bib_ref] Association of HTLV Tax proteins with TAK1-binding protein 2 and RelA in..., Avesani [/bib_ref].
An additional difference between Tax-1 and Tax-2 that may play a role in NF-κB activation is the absence of interactions between Tax-2 and TNF receptor-associated factor TRAF6, an E3 ubiquitin ligase that is involved in ubiquitination of effectors of the NF-κB pathway. Both Tax-1 and Tax-2 are ubiquitinated and SUMOylated, but they differ in the pattern of modification [bib_ref] The importance of ubiquitination and sumoylation on the transforming activity of HTLV..., Zane [/bib_ref]. The role of ubiquitination, sumoylation, and acetylation of Tax in NF-κB activation and cellular transformation is still an open field of research [bib_ref] Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity:..., Turci [/bib_ref] [bib_ref] Acetylation at lysine 346 controls the transforming activity of the HTLV-1 Tax..., Lodewick [/bib_ref].
Activation of NF-κB by Tax is also connected to the deregulation of autophagy, an additional pathway that is altered in oncogenic signaling. Both Tax-1 and Tax-2 induce autophagosome accumulation, but their interactions with the components of the process differ. In fact, Tax-1 (but not Tax-2) directs the IKK complex to lipid rafts associated with autophagic molecules such as Beclin 1 and Bif-1 [bib_ref] HTLV-1 Tax is a critical lipid raft modulator that hijacks IκB kinases..., Huang [/bib_ref].
## Post-transcriptional regulation by rex
HTLV-1 Rex (Rex-1) and HTLV-2 Rex (Rex-2) share 60% homology at the amino acid level. Rex-1 and Rex-2 are phosphorylated proteins that actively shuttle between the nucleus and the cytoplasm [bib_ref] The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a..., Palmeri [/bib_ref] [bib_ref] Functional domain structure of human T-cell leukemia virus type 2 rex, Narayan [/bib_ref] and accumulate in the nucleus and nucleoli [bib_ref] Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein..., Nosaka [/bib_ref] [bib_ref] Expression and characterization of proteins produced by mRNAs spliced into the X..., Ciminale [/bib_ref] [bib_ref] Inhibition of human T-cell leukemia virus type 2 Rex function by truncated..., Ciminale [/bib_ref] [bib_ref] The post-transcriptional regulator Rex of the human T-cell leukemia virus type I..., Nosaka [/bib_ref] [bib_ref] Functional domain structure of human T-cell leukemia virus type 2 rex, Narayan [/bib_ref] , a property that is intimately linked to their ability to enhance the nuclear export of incompletely spliced viral mRNAs.
Rex-1 and Rex-2 share a similar domain structure which includes: (i) a nuclear localization signal (NLS; [bib_ref] Sequence requirements for nucleolar localization of human T cell leukemia virus type..., Siomi [/bib_ref] [bib_ref] Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein..., Nosaka [/bib_ref] , which mediates binding to the RXRE [bib_ref] In vitro binding of human T-cell leukemia virus rex proteins to the..., Grassmann [/bib_ref] located at the 3 end of all HTLV-1 transcripts and at the 5 end of the unspliced HTLV-2 mRNA [bib_ref] Identification of a cis-regulatory element involved in accumulation of human T-cell leukemia..., Ohta [/bib_ref] [bib_ref] Regulation of HTLV-II gene expression by Rex involves positive and negative cis-acting..., Black [/bib_ref] ; (ii) multimerization domains; and (iii) a leucine-rich sequence located near the middle of the protein (Rex-1 aa 79-99; Rex-2 aa 81-94), which functions as an activation domain (AD; [bib_ref] Definition of the human immunodeficiency virus type 1 Rev and human T-cell..., Weichselbraun [/bib_ref] and contains the nuclear export signal (NES; [bib_ref] Characterization of the nuclear export signal of human T-cell lymphotropic virus type..., Kim [/bib_ref] [bib_ref] The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a..., Palmeri [/bib_ref] that interacts with CRM1/exportin, which mediates the nuclear export of the Rex-viral mRNA complex [bib_ref] Identification of a novel cellular cofactor for the Rev/Rex class of retroviral..., Bogerd [/bib_ref] [bib_ref] Characterization of the nuclear export signal of human T-cell lymphotropic virus type..., Kim [/bib_ref] [bib_ref] The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a..., Palmeri [/bib_ref]. In addition a C-terminal domain unique to Rex-2 is a target for serine phosphorylation and may also contribute to efficient nucleocytoplasmic shuttling [bib_ref] Functional domain structure of human T-cell leukemia virus type 2 rex, Narayan [/bib_ref]. Although Rex is not required for cellular immortalization in vitro, it is necessary for infectivity and viral persistence in vivo [bib_ref] HTLV-1 Rex is required for viral spread and persistence in vivo but..., Ye [/bib_ref] , as it is required for the expression of the virion-associated structural proteins.
The dependence of HTLV mRNAs on Rex function is controlled by positively and negatively acting RNA sequences present Frontiers in Microbiology | Virology | Structural and functional differences between HTLV-1 and HTLV-2 gene products.
## Structural and functional properties viral proteins reference
## Htlv-1 htlv-2
Tax-1 on the primary transcript. Two major types of such RNA cis-acting elements have been described: (i) the Rex responsive element (RXRE) which, besides binding Rex, acts as an inhibitory sequence in the absence of Rex, and (ii) cis-acting repressive sequences (CRS) that determine poor stability and/or inefficient nucleocytoplasmic export. HTLV-1 contains a CRS which maps at the 5 end of the unspliced mRNA but is spliced out of the other transcripts [bib_ref] The U5 sequence is a cis-acting repressive element for genomic RNA expression..., Seiki [/bib_ref] [bib_ref] Nucleocytoplasmic transport of HTLV-1 RNA is regulated by two independent LTR encoded..., King [/bib_ref]. An additional CRS overlaps the RXRE and acts synergistically with the 5 -CRS. In contrast to the 5 -CRS, this 3 -CRS/RXRE is present at the 3 end of all viral transcripts. Both the 5 -and 3 -CRSs were shown to act mainly as nuclear retention sequences. The 5 -CRS does not bind Rex-1 [bib_ref] The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator binds directly..., Bogerd [/bib_ref] [bib_ref] The Rex regulatory protein of human T-cell lymphotropic virus type I binds..., Unge [/bib_ref] , and its inhibitory function is likely to www.frontiersin.org be mediated by other viral and/or cellular RNA-binding proteins. Other cis-acting inhibitory elements (CIEs) were mapped within the gag-pol and env regions of HTLV-1 [bib_ref] cis-Acting inhibitory elements within the pol-env region of human Tcell leukemia virus..., Saiga [/bib_ref]. The inhibitory effect of these regions is counteracted by binding of Rex-1 to the RXRE, although it is not clear whether they function mainly at the level of RNA stability or nucleocytoplasmic export. A 5 -CRS acting as a nuclear retention sequence was also mapped in the R-U5 region of HTLV-2 [bib_ref] Regulation of HTLV-II gene expression by Rex involves positive and negative cis-acting..., Black [/bib_ref].
## The accessory proteins
The "accessory" proteins were labeled as such because their ablation does not have apparent consequences on viral replication in vitro. However, studies performed in animal models indicate that some of these proteins are essential for efficient infectivity in vivo. Among the accessory proteins, p30Tof/p28, p12/p10, and p21Rex/tRex (in HTLV-1 and HTLV-2, respectively) are considered to be homologous based on their structure and functional properties, while p13 and p8 appear to be unique to HTLV-1, and p11 is peculiar to HTLV-2. p21Rex and tRex are truncated forms of Rex-1 and Rex-2, respectively, which lack the N-terminal arginine-rich NLS and are therefore incapable of binding the RXRE. HTLV-2 tRex is detected as four main isoforms of 22, 20, 18, and 17 kDa which differ in the initiation codon usage and phosphorylation status . The tRex proteins were shown to inhibit Rex-2 function , an activity that might favor latent infection. Although one study indicated that p21rex acts as a repressor of full-length Rex [bib_ref] Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism..., Heger [/bib_ref] , this finding was not supported by other studies [bib_ref] Nuclear export and expression of human T-cell leukemia virus type 1 tax/rex..., Bai [/bib_ref].
HTLV-1 p30Tof and HTLV-2 p28 are important for viral propagation in animal models [bib_ref] Human T-cell lymphotropic virus type 1 open reading frame II-encoded p30II is..., Silverman [/bib_ref] [bib_ref] Human T-cell leukemia virus type 2 post-transcriptional control protein p28 is required..., Yamamoto [/bib_ref] [bib_ref] Requirement of the human T-cell leukemia virus p12 and p30 products for..., Valeri [/bib_ref]. Both proteins sequester the tax/rex mRNA in the nucleus, an effect that may result in reduced viral expression and latency [bib_ref] HTLV-1-encoded p30(II) is a post-transcriptional negative regulator of viral replication, Nicot [/bib_ref] [bib_ref] Repression of human T-cell leukemia virus type 1 and type 2 replication..., Younis [/bib_ref]. p30Tof was also shown to interact with the RNA-binding domain of Rex and thereby interfere with its binding to the RXRE (Sinha- [bib_ref] Human T-cell lymphotrophic virus type I rex and p30 interactions govern the..., Sinha-Datta [/bib_ref] [bib_ref] HTLV-I p30: a versatile protein modulating virus replication and pathogenesis, Bai [/bib_ref] and to inhibit the expression of Toll-like receptor 4 [bib_ref] The HTLV-I p30 interferes with TLR4 signaling and modulates the release of..., Datta [/bib_ref] , suggesting a role in the innate immune response. These two properties have not been reported for p28. By interacting with the co-activator CBP/p300 [bib_ref] Human T-lymphotropic virus type 1 p30(II) functions as a transcription factor and..., Zhang [/bib_ref] , p30Tof also affects Tax-mediated viral expression and transcription of cellular genes involved in T-cell activation and apoptosis [bib_ref] Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively..., Michael [/bib_ref] [bib_ref] Genome wide analysis of human genes transcriptionally and post-transcriptionally regulated by the..., Taylor [/bib_ref]. Interestingly, p28 does not appear to affect CBP/p300-mediated transcription. Both p30Tof and p28 are targeted to the nucleus, but only p30Tof shows evident accumulation in the nucleoli [bib_ref] Expression and characterization of proteins produced by mRNAs spliced into the X..., Ciminale [/bib_ref] [bib_ref] The human T-cell lymphotropic virus type 1 Tof protein contains a bipartite..., D'agostino [/bib_ref]. Interestingly, HTLV-2 p28 and tRex are expressed at higher levels compared to their HTLV-1 counterparts p30/Tof and p21Rex, suggesting a higher propensity of HTLV-2 for latency compared to HTLV-1 [bib_ref] Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence..., Bender [/bib_ref].
p13 corresponds to the C-terminal 87 amino acids of p30Tof [bib_ref] Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus..., Koralnik [/bib_ref] , and it is localized mainly in the mitochondrial inner membrane [bib_ref] Mitochondrial targeting of the p13II protein coded by the x-II ORF of..., Ciminale [/bib_ref] [bib_ref] Mitochondrial alterations induced by the p13 II protein of human T-cell leukemia..., D'agostino [/bib_ref] and in part to the nucleus [bib_ref] Redox regulation of T-cell turnover by the p13 protein of human T-cell..., Silic-Benussi [/bib_ref] [bib_ref] Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein..., Andresen [/bib_ref]. Using a rabbit animal model, [bib_ref] Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13(II) is required for viral..., Hiraragi [/bib_ref] showed that p13 is required for viral infectivity in vivo. p13 increases mitochondrial permeability to K + and activates the electron transport chain, resulting in increased mitochondrial reactive oxygen species (ROS) production [bib_ref] Modulation of mitochondrial K(+) permeability and reactive oxygen species production by the..., Silic-Benussi [/bib_ref]. While p13 has a mitogenic effect in normal resting T cells, which have low ROS levels, the protein induces death of transformed T-cells, which are characterized by a high ROS setpoint [bib_ref] Mitochondrial targeting of the p13II protein coded by the x-II ORF of..., Ciminale [/bib_ref] [bib_ref] Mitochondria as functional targets of proteins coded by human tumor viruses, D'agostino [/bib_ref] [bib_ref] Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells..., Hiraragi [/bib_ref] [bib_ref] Suppression of tumor growth and cell proliferation by p13II, a mitochondrial protein..., Silic-Benussi [/bib_ref]. So far no HTLV-2 homologue of HTLV-1 p13 has been identified.
HTLV-1 p12 and its HTLV-2 homologue p10 are coded by the x-I ORF. p12 localizes in the endoplasmic reticulum (ER) and in the Golgi apparatus, where it reduces the expression of the β and γ c chains of the interleukin-2 receptor (IL-2R, [bib_ref] The human T-cell leukemia/lymphotropic virus type 1 p12I proteins bind the interleukin-2..., Mulloy [/bib_ref] and of MHC-I, thus hindering lysis of HTLV-1-infected cells by CTL . p12 also activates STAT-5, which provides a mitogenic signal to T cells and interacts with calreticulin and calnexin , resulting in incresased Ca 2+ release from the ER and activation of NF-AT, a mitogenic pathway in T-cells [bib_ref] A conserved calcineurin-binding motif in human T lymphotropic virus type 1 p12I..., Kim [/bib_ref]. Within the ER, p12 is cleaved into an 8-kDa protein (p8) which trafficks to the immunological synapse and favours T-cell anergy. p8 also increases cell-to-cell viral transmission through the formation of intercellular conduits . In analogy to p12, HTLV-2 p10 was shown to bind the MHC heavy chain; however, p10 does not bind the IL2R β chain or the 16-kDa subunit of the vacuolar H+ ATPase [bib_ref] The MHC class I heavy chain is a common target of the..., Johnson [/bib_ref]. No homologue of p8 has been described in HTLV-2.
A recent study from [bib_ref] Requirement of the human T-cell leukemia virus p12 and p30 products for..., Valeri [/bib_ref] , showed that p12 is required for in vivo propagation in macaques but not in rabbits.
The x-V ORF of HTLV-2 codes for p11 from a doubly spliced transcript that also codes for p10 [bib_ref] Expression and characterization of proteins produced by mRNAs spliced into the X..., Ciminale [/bib_ref]. Aside from its ability to bind to MHC heavy chain [bib_ref] The MHC class I heavy chain is a common target of the..., Johnson [/bib_ref] , nothing is known about the function of this protein. HTLV-1 also possesses an x-V ORF, but it does not appear to produce a p11 homologue. The function of p11 is still unclear.
## Hbz and aph-2
The structure and function of HBZ and APH-2 were recently reviewed [bib_ref] Functional comparison of antisense proteins of HTLV-1 and HTLV-2 in viral pathogenesis, Barbeau [/bib_ref]. HBZ is a nuclear transcriptional factor [bib_ref] The complementary strand of HTLV-1 RNA genome encodes bZIP transcription factor that..., Gaudray [/bib_ref] , able to interact with ATF/CREB proteins through its basic zipper (bZIP) domain causing the inhibition of its DNA-binding activity [bib_ref] A modified version of a Fos-associated cluster in HBZ affects Jun transcriptional..., Hivin [/bib_ref] ; this effect also influences the ability of Tax-1 to activate HTLV-1 transcription. APH-2 is likewise able to inhibit Tax-2-mediated transcription but its repressive activity is weaker than that of HBZ. This difference may be ascribed to the presence in HBZ but not in APH-2, of a transcriptional activation domain within its N -terminal region that mediates its interaction with the KIX domain of p300/CBP, thus competing for its binding to Tax-1 [bib_ref] An interaction between the human T cell leukemia virus type 1 basic..., Clerc [/bib_ref].
HBZ also deregulates several cellular pathways including c-Jun and JunD, FoxP3, NF-κB, TGF-β and Wnt [bib_ref] Functional comparison of antisense proteins of HTLV-1 and HTLV-2 in viral pathogenesis, Barbeau [/bib_ref]. In addition, HBZ, may enhance its own expression by controlling Frontiers in Microbiology | Virology the transcriptional activity of JunD [bib_ref] Human T-cell leukemia virus type 1 (HTLV-1) bZIP factor requires cellular transcription..., Gazon [/bib_ref]. Very little is known about the regulation of APH-2 expression in infected cells. However, unlike HBZ, APH-2 enhances the transcriptional activity of c-Jun family proteins [bib_ref] Interplay between the HTLV-2 Tax and APH-2 proteins in the regulation of..., Marban [/bib_ref].
The role of HBZ in T-cell transformation is supported by the finding that while Tax-1 expression is often repressed in ATL cells and appears to be dispensable for the late stages of leukemogenesis, HBZ is constitutively expressed in most ATL cases [bib_ref] HTLV-I basic leucine zipper factor gene mRNA supports proliferation of adult T..., Satou [/bib_ref]. Transgenic mice expressing HBZ in CD4+ T cells develop T-cell lymphomas and systemic inflammation that are reminiscent of ATL and HAM/TSP.
Although HTLV-2 is not causally linked to leukemia or lymphoma, it has been associated with lymphocytosis in infected patients [bib_ref] Long-term increases in lymphocytes and platelets in human Tlymphotropic virus type II..., Bartman [/bib_ref]. This is consistent with the observation that APH-2 is detected in most PBMCs of HTLV-2-infected patientsand that its expression is well correlated to proviral DNA load [bib_ref] HTLV-2 APH-2 expression is correlated with proviral load but APH-2 does not..., Douceron [/bib_ref]. Interestingly, it was observed that the APH-2 mRNA, similarly to HBZ, accumulates in the nucleus [bib_ref] Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence..., Bender [/bib_ref]. [bib_ref] Functional comparison of antisense proteins of HTLV-1 and HTLV-2 in viral pathogenesis, Barbeau [/bib_ref] have recently proposed a model to explain the different effects of HBZ and APH-2 on T-cell proliferation. Both HBZ and of APH-2 can suppress Tax expression, thus favoring evasion of the immune response. In addition, HBZ can stimulate its own expression, inhibit Tax-1-dependent viral expression and induce T-cell proliferation, which can lead to ATL. APH-2 is unable to induce cell proliferation and only partially down-regulates Tax-2 expression.
Although dispensable for the HTLV-1 infection and immortalization of T lymphocytes in vitro, studies conducted on a rabbit animal model suggest that HBZ enhances HTLV-1infectivity and persistence in vivo [bib_ref] Enhancement of infectivity and persistence in vivo by HBZ, a natural antisense..., Arnold [/bib_ref]. Furthermore, HBZ transgenic mice develop systemic inflammation and a CD4+ T-cell neoplasm that is reminiscent of ATL. Interestingly, in contrast to HBZ, HTLV-2 APH-2 appears to be dispensable for viral infection and persistence in a rabbit animal model , suggesting a functional divergence of the in vivo function of the HTLV-1 and HTLV-2 antisense proteins.
## Tropism and clonality
The preferential cellular tropism of HTLV-1 for CD4+ T cells and of HTLV-2 for CD8+ T cells is still not clearly understood. It appears that the two viruses make different use of the heparan sulfate proteoglycans to enter T cells [bib_ref] Human T-cell leukemia virus type 1 HTLV-1 and HTLV-2 use different receptor..., Jones [/bib_ref] , although recent evidence obtained in vivo rabbit model indicates that the apparent tropism for CD4+ or CD8+ cells mainly reflects preferential clonal expansion of infected cells . Both HTLV-1 and HTLV-2 are capable of inducing clonal expansion of infected cells in vivo [bib_ref] Clonal expansion of human T-cell leukemia virus type I-infected cells in asymptomatic..., Wattel [/bib_ref] [bib_ref] Clonal expansion of human T-cell leukemia virus type II in patients with..., Cimarelli [/bib_ref]. A detailed analysis in asymptomatic carriers, TSP/HAM and ATLL patients revealed that the genomic integration site and transcriptional orientation of the provirus are important factors for determining clonal abundance in vivo [bib_ref] The host genomic environment of the provirus determines the abundance of HTLV-1-infected..., Gillet [/bib_ref].
A very recent study demonstrated that, contrary to previous hypotheses, HTLV-2-infected individuals have a small number of highly expanded CD8+ T-cell clones, suggesting that HTLV-2 may be subjected to a more strict clonal selection than HTLV-1 in healthy carriers. These data suggest that selective clonal proliferation is more directly responsible for determining the viral burden of HTLV-2 than it does for HTLV-1 [bib_ref] Clonality of HTLV-2 in natural infection, Melamed [/bib_ref]. In contrast to observations made for HTLV-1, the environment surrounding the integration sites does not seem to have a substantial impact on the expansion of HTLV-2-infected clones [bib_ref] Clonality of HTLV-2 in natural infection, Melamed [/bib_ref].
The presence of few very abundant HTLV-2-infected clones is apparently reminiscent of the profile found in ATLL patients. However, the fact that HTLV-2 is not causally linked to a T-cell malignancy suggests that clonal abundance and heterogeneity may not per se constitute a determinant of malignant transformation and clinical outcome of HTLV infection [bib_ref] Clonality of HTLV-2 in natural infection, Melamed [/bib_ref].
# Conclusions
From the comparative analysis on the functional properties of HTLV-1 and HTLV-2 reported above, the following major differences can be outlined: (i) HTLV-2 is characterized by a more abundant expression of gene products that may favor viral latency (i.e., p28 and tRex); (ii) Tax-1 presents a PDZ binding motif, which is absent from Tax-2 and allows interaction with host factors regulating the cell cycle and proliferation; in addition, Tax-2 is unable to activate the non-canonical NF-κB pathway; (iii) Cis-acting inhibitory elements acting at the level of RNA stability are present within HTLV-1 whereas they have not been described in HTLV-2; (iv) while the truncated forms of Rex-2 were shown to inhibit Rex function, the HTLV-1 homologue p21Rex might lack this activity; (v) HTLV-1 expresses p13 and p8, which have not been described in HTLV-2; (vi) HTLV-2 expresses p11, which does not seem to have a homologue in HTLV-1; (vii) unlike APH-2, HBZ presents a basic zipper domain, as well a transcriptional activation domain, which mediate its capacity to enhance its own expression and deregulate several cellular pathways; (viii) HTLV-2 shows in vivo tropism for CD8+ T cells and induces expansion of a relatively small number of highly abundant clones.
In spite of the evidence accumulated so far on the similarities and differences between HTLV-1 and HTLV-2, it is not yet clear why only HTLV-1 causes a T-cell malignancy. While Tax-1 and HBZ induce T-cell lymphomas when expressed as transgenes in animal models, the in vivo transforming activities of Tax-2 and APH-2 have not been investigated. Aside from the NF-κB pathway, not much information is available regarding the interactions of Tax-2 with cellular pathways known to be engaged by Tax-1. The fact that HTLV-2, contrary to HTLV-1, is characterized by an oligoclonal proliferative distribution in asymptomatic hosts clearly indicates that the etiology of malignant transformation by HTLV-1 cannot be uniquely attributed to this phenomenon.
# Acknowledgments
The authors thank Luigi Chieco-Bianchi and Donna D'Agostino for discussions. Research on the HTLVs performed by Vincenzo Ciminale and Maria G. Romanelli was supported by grants from the Associazione Italiana per la Ricerca sul Cancro www.frontiersin.org (AIRC) -Cariverona and the Universities of Padova and Verona.
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Conflict of Interest Statement:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Received |
A critical review of the Online Safety Bill
## Introduction: online regulation in the uk
The internet has become increasingly integrted into our individual and communal lives: over 90% of UK citizens are now online. Indeed, with this, existing social maladies have found a new, and complex, expression: child abuse, terrorist propaganda, and the harassment of women and minority groups are among the phenomena that have been transformed by the internet, and that have rendered it an unsafe place for many of its netizens.To cite but a few statistics to this effect: the Internet Watch Foundation confirmed 153,383 cases of Child Sexual Abuse Material (CSAM) in the UK in 2020 2 ; the UK government claimed that all five domestic terrorist incidents in 2017 had online elements, including radicalization by international groups, such as ISIS; 21% of women in the UK have been victim to misogynistic abuse online; two in every three Britons are concerned about the proliferation of fake news. The internet seems a conducive environment for these harms: it is a space that sprawls jurisdictions, it develops at a faster pace than regulation, and it allows great degrees of anonymity and secrecy for those wanting to commit wrongdoing with impunity. Despite reporting concerns about their safety and the paucity of protective measures,Britons still enter online spaces-largely because of the benefits of internet services, but also out of a perceived lack of plausible alternatives, and because of an increasing truism that the internet is effectively an extension of the public square.
It is against this background that the British Parliament is now considering the Online Safety Bill (previously drafted with the title of ''The Online Harms Bill'').The purpose of the bill is to create ''a new regulatory regime to address illegal and harmful content online.''Key among the stipulated objectives of the legislation are the following:
THE BIGGER PICTURE This critical perspective makes a timely contribution to the tech policy debate concerning the monitoring and moderation of online content. Governments globally are currently considering a range of legislative interventions to limit online abuse, disinformation, and the dissemination of illegal content on social media platforms. These interventions will significantly impact online free speech, competition between platforms, and the democratic function of online platforms. By investigating the UK's Online Safety Bill, comparing it with similar interventions, and considering the political impact of different digital tools for moderation, this perspective aims to inform the current policy debate by combining technical and political insight. It indicates the need for further research into the comparative efficacy of different methods of content monitoring and moderation. d A free, open and secure internet: 1 individuals must be able to use the internet without restriction, except where limited and proportionate restriction is necessary for protecting individuals' rights and interests. d Freedom of expression online: each person has the right to express themselves freely and to receive information from others, unless where such expression is prohibited by law, as in the case of hate speech or terrorist propaganda. This right includes the expression and transmission of information online. d An online environment where companies take effective steps to keep their users safe, and where criminal, terrorist, and hostile foreign state activity is not left to contaminate the online space: 1 recent controversies-including allegations of Russian social media interference in elections, and terrorist recruitment online by ISIS-have emphasized the need to protect users on social media sites from bad-faith actors. d Rules and norms for the internet that discourage harmful behavior: 1 features of user-to-user services (including the possibility of anonymity) seem to encourage antisocial behavior. d The UK as a thriving digital economy, with a prosperous ecosystem of companies developing innovation in online safety: 1 user-to-user and search services are sites of innovation and growth. Accordingly, they are of great importance to the development of the digital economy. The UK has also been party to world-leading safety innovation previously, in the form of General Data Protection Regulation (GDPR). d Citizens who understand the risks of online activity, challenge unacceptable behaviors and know how to access help if they experience harm online, with children receiving extra protection: 1 user-to-user services potentially expose vulnerable people-particularly children-to bad-faith actors. It is therefore important to ensure that there is sufficient protection to make their internet use secure. d Renewed public confidence and trust in online companies and services: 1 controversies involving user-to-user and search services have undermined public trust in their reliability as news sources and service providers.
Given the extent of such harms, such policy objectives are commendable; in particular, because they are cognisant ofand indeed affirm-the need for a safe, free, open, and thriving digital environment. In this paper, we critically analyze the mechanisms and rationales for the regulation that the British government has proffered, arguing that the proposed legislation fails to meet the government's own desiderata. The effect of this legislation, in our opinion, will be a less open and free internet, one in which British companies have less access to a thriving digital economy, and in which the most effective steps toward preventing harm are neglected. Per our analysis, the government has not done enough to justify the need for its intervention, and has crafted a regulatory framework that leaves open the possibility of counterproductive and undemocratic interference.
To motivate our opinion, we offer a selected overview of the proposed legislation, where our selection of emphasis is presented with a view to offering commentary on those specific el-ements. Recognizing that this space is of critical public concern and presents genuinely novel forms of policy interaction (from duty of care; protection of the vulnerable; digital and algorithmic justice; privacy; respect, dignity, and decency in society; freedom of expression; etc.), readers will notice that our interventions highlight points of contestation, which, at a high level, represent calls for further evidence and greater consultation with relevant stakeholders.
We forward our critical review with two main sections: the first is an overview, where we summarize the key components of the Bill and its preceding White Papers.Here, we survey the following: Our intended readership are those with an interest in the regulation of digital services, both in industry and in policymaking. This is not limited to policymakers: we argue that choices at a technical level (i.e., what tools to use to make the platforms safer) have important moral and political implications, and so it is important that those in data science and machine learning understand the consequences of these tools. We intend to contribute to an increasing debate with the hope of improving the UK's digital regulation. The arguments we present here reflect our critical opinion, rather than an exposition of scientific fact, and so we invite critical reply to our claims here. We begin with an overview of the legislation. Those interested in our commentary should move directly to the section ''Measuring the Bill through proportionality and necessity.''
## An overview of the online safety bill
In 2020, the UK government issued the Online Harms White Paper with the purpose of creating a safer internet ecosystem and repairing public trust in digital platforms and search services. The White Paper has subsequently led to the Online Safety Bill, a draft piece of legislation giving more regulatory substance to the ambitions set out in the original White Paper. In this section, we offer a selected overview of the proposed legislation, where our selection of emphasis is presented with a view to offering commentary on those specific elements.
The Bill's approach is to place a duty of care on internet service providers of both user-to-user services in which users interact with each other online, such as the manner in which users typically interact on platforms like Facebook and Twitter, and search services that index information and allow users to navigate the internet, such as Google and Bing. The duty of care is framed in broad terms in the Bill, but it is composed of three distinct duties:1. To protect users from illegal content (section 9): although the production and dissemination of CSAM and terrorist propaganda are already illegal, the purpose of the Bill is to place a duty of care upon service providers to control the digital space with a view to limiting the potential spread of illegal content. It is unclear what the Bill is able to accomplish in this space that is not already possible under the existing legal landscape. 2. To take additional protective measures to make their site safe for children, if their service is likely to be used by children (section 10). Children on the internet are vulnerable to grooming, cyberbullying, encouragement to self-harm, and harm to their mental health. The purpose of the Bill is to place a duty of care on service providers to ensure the safety of children on their platforms. The litmus test for ''a service likely to be used by children'' is not well defined.
## 3.
To take additional measures to protect all users from content that is harmful without being illegal, if the service is of a sufficient reach and magnitude (section 11). Some online interactions, while legal, can nevertheless be harmful. This includes online campaigns of harassment that are not covered by existing criminal law, and the proliferation of disinformation (fake news). The purpose of the Bill is to place a duty of care upon service providers to ensure that their users are protected not only from unlawful harm, but also from lawful and harmful content.
In the sections below, we highlight key features of the legislation and the duty it places on service providers, with a focus on those features that we argue are problematic. The duty is justified with reference to the role of user-to-user and search services in proliferating harm, and so it is ultimately a duty owed to the users of internet services (albeit arbitrated by the government and regulator). The duty requires services to comply with Codes of Practice, and to report their compliance to the regulator to evidence their execution of their duty. Concomitantly, the regulator, Ofcom, is empowered to enact and enforce the Codes of Practice, although the Minister has the power to direct this enactment.
## Rationale for duty
In its initial White Paper on the subject (then under the auspices of the Online Harms Bill), the government presented both evidence of the extent of online harms and a precis of existing efforts to regulate online harms. We survey these reasons and highlight potential lacunas here by way of exposition before our critical analysis in section 2, which assesses whether the Bill's rationale as given demonstrates that tighter regulation of service providers is necessary for reducing harm.
The government presents the following online harms as the crucial impetus for the Bill: d Child sexual exploitation and abuse online: the White Paper cites the Internet Watch Foundation's statistics regarding the circulation of CSAM. The White Paper notes that, of the 80,319 cases of CSAM confirmed by the IWF, 43% of the children involved were between 11 and 15, 57% were under 10, and 2% were under 2. 2 d Terrorist content online: the government's concern is that the internet allows the proliferation of terrorist propaganda. The White Paper repeats the Rt Hon Amber Rudd's claim that five of the terrorist incidents in the UK in 2017 included internet elements, implying that they were radicalized by international groups, such as ISIS and Daesh. 3 d Content illegally uploaded from prisons: the White Paper claims (without citation) that there is an increase in the amount of content transmitted illegally from prisons. d The sale of opioids online: the White Paper cites the National Crime Agency's statistics that there have been at least 146 opioid-related deaths in the UK since 2016. It claims (without reference) that opioids are sold on ''several well-known social media sites.'' d Cyberbullying: the White Paper cites National Health Service data that one in five children aged 11-19 has experienced cyberbullying. Of those who experienced cyber-bullying, the White Paper claims that the propensities of victims towards social anxiety, depression, suicidality, and self-harm were all higher than corresponding cases of ordinary bullying. d Self-harm and suicide: the White Paper cites academic research that showed that 22.5% of young adults reported suicide and self-harm-related internet use; 70% of young adults with suicidal intent reported related internet use; approximately a quarter of children who presented to hospitals following self-harm, and of those who died of suicide, reported suicide-related internet use. 8 d Underage sharing of sexual imagery: the White Paper cites statistical evidence that suggests that between 26% and 38% of teenagers have sent sexual images to partners, whereas 12% to 49% of teenagers have received sexual images from partners. d Online disinformation: the White Paper cites a Reuters report showing that 61% of people want the government to do more to distinguish between real and fake news. The White Paper does not provide statistical evidence as to the prevalence of disinformation, but cites the Russian state's use of disinformation as an example. d Online manipulation: the government's concern is that the introduction of artificial intelligence will increase the prevalence and effectiveness of psychological manipulation. The government cites the need to replicate the regulation of manipulation in other media, such as the Broadcasting Act 1990, which prohibited subliminal advertising. d Online abuse of public figures: the government cites the disproportionate amount of abuse received by female public figures. It cites an international survey that found that two in every three female journalists were harassed online, including receiving death threats, cyberstalking, and obscene messages.
Where these activities are already illegal (as in the case of CSAM or terrorist recruitment and propaganda), the purpose of the legislation is not to prohibit the act itself but to establish the duties of online services to protect users from illegal content. This deviates from the existing regime, established by the EU's e-Commerce Directive, which exempts services from liability for illegal content, unless they know about the unlawfulness of the content or have sufficient information to conclude that it is unlawful and do not act expeditiously to remove the content. We cover the form of this duty in the next two subsections.
Where content is legal, the White Paper argues that it falls in a regulatory lacuna, since legal (but harmful) content is already subject to statutory regulation (typically overseen by Ofcom) when it is presented on other platforms, including live television, catch-up television, and subscription services. The Bill, therefore, fills the regulatory gap by extending similar regulatory control over the dissemination of content on user-to-user services.
## Scope of the duty
The scope of illegal content is, of course, specified already by preceding legislation, and includes material that promotes terrorism or that is classified as CSAM. However, the Online Safety Bill adds a new layer of liability to these activities because it requires that services adhere to an additional layer of compli-ance measures and empowers the government to hold named directors accountable for their services' failures to comply.
Ordinary sensibilities: what is more novel is the Bill's focus on harms that are not illegal. Citing online harassment in its White Paper, the government has suggested that there is a need to regulate behavior that is not prohibited or regulated otherwise, including speech that does not fall within the remit of hate speech. The Bill requires that service providers not only protect their users from illegal content but also from this species of content that would otherwise be legal, as long as it can reasonably be construed as potentially harmful to children or adults of ''ordinary sensibilities.''
The scope of this content is potentially very wide. According to the Bill, this duty should be triggered if:
the provider of the service has reasonable grounds to believe that the nature of the content is such that there is a material risk of the content having, or indirectly having, a significant adverse physical or psychological impact on an adult of ordinary sensibilities.
The Bill, therefore, uses the ''reasonableness'' standard that pervades English common law. This standard sets a variable threshold that focuses on the perspective of an epistemically responsible agent. This definition is further supplemented by the following stipulation in the Bill:
[A] risk of content ''indirectly'' having a significant adverse physical or psychological impact on an adult is a reference to a risk of either of the following-(a) content causing an individual to do or say things to a targeted adult that would have a significant adverse physical or psychological impact on such an adult; (b) content causing an adult to act in a way that-(i) has a significant adverse physical or psychological impact on that adult, or (ii) increases the likelihood of such an impact on that adult. This framing is noteworthy both because it includes in its scope a responsibility on the part of services for content that is not illegal otherwise, but also because it provides such wide interpretive scope depending on how one chooses to construe ''reasonableness.'' We return to this issue later in this paper.
Enforcing the duty The Bill and White Paper assert that service providers have, in principle, a duty of care to protect their users; to discharge this duty, the Bill stipulates that services must abide by Codes of Practice. The Bill itself does not specify the content of the Codes of Practice. Instead, it delegates this authority to the industry regulator, the Office of Communications (Ofcom). Ofcom is an independent industry regulator, although its chair is politically appointed.
While Ofcom has the power to set the Codes of Practice, the Bill vests the Minister of State with the power to veto the Codes of Practice, or to order Ofcom to modify the codes so that they align with ''government policy.'' The Bill, in effect, grants the Minister of State wide powers to direct the Codes of Practice.
Parliament, by contrast, has relatively little oversight over the Codes of Practice. The Bill adopts a negative form of parliamentary oversight with regard to the Codes of Practice: Parliament is assumed to have consented to the Codes of Practice unless its ll OPEN ACCESS members propose and pass a vote to reject the Codes. Scrutiny of the Codes of Practice, therefore, is the exception, rather than the default position of Parliament.
Assessing the Bill In this section, we have surveyed the government's own rationale for the Bill, as well as assessing the scope and content of the duties of care that the Bill imposes. Our focus for the remainder of this paper will be on:
(1) the causal claims in the government's rationale for the Bill and whether they justify the necessity of the legislation; (2) the authority that the Bill vests in the Minister and in Ofcom; (3) the wide scope of content that is included in service providers' duty of care per the Bill; and (4) the Bill's reliance on Codes of Practice to be prescribed by Ofcom in conjunction with the Minister.
In the sections that follow, we argue that: the government's rationale for the Bill provides insufficient justification for the necessity of this intervention, and that more consultation and justification are needed; the Bill grants far-reaching powers of interference to the executive; the scope of content covered by the Bill is worryingly broad; the emphasis on Codes of Practice is inapt; and that the Bill creates potential obstacles for small and medium enterprises.
## International comparisons
It is worth comparing the Bill with its international equivalents, since legislators in a number of jurisdictions have sought to regulate content-moderation on social media platforms. These proposed legislative interventions provide us with a useful set of benchmarks against which to measure the Safety Bill.
The Parliament of the European Union is currently considering the proposed Digital Services Act (DSA) to address content moderation in the EU.Like the OSB, the DSA is aimed at protecting the human rights of citizens of the EU online. However, the proposal differs in several important regards. The DSA stipulates more detailed, design-based duties with regard to legal but harmful content: user-to-user services must contain clear and accessible terms and conditions, content-reporting procedures, and appeal procedures following content or user removal, as well as requiring large platforms to cooperate with ''trusted flaggers'' (drawn from expert and professional institutions) who report harmful content (section 3). The DSA also requires very large platforms to perform assessments of their systemic risks, including systemic design features that threaten the exercise of fundamental rights, to declare the parameters of their recommender systems, and to evidence their mitigation strategies for minimizing system risk (section 4). Therefore, with regard to legal but harmful content, the DSA is concerned only with systemic design features of user-to-user services.
To help enterprises, the DSA empowers the European Commission to issue guidelines for the fulfilment of their duties stipulated in the Act (article 27). This differs from the Codes of Conduct in three important respects. First, the Commission is obliged to compose the guidelines in collaboration with the ser-vices affected and civil society organizations representing stakeholders. Second, the guidelines are meant to represent best practice in the industry, but enterprises can deviate from the guidelines with sufficient reason. Third, the guidelines do not create new duties: rather, they are meant only to help enterprises easily navigate the duties already established in the Act.
The DSA takes a comparable approach to the Platform Accountability and Consumer Transparency Act (PACT Act), which has been proposed in the Senate of the United States.Concerning legal but harmful content, The PACT Act, like the DSA, focuses on design features of user-to-user services: the Act stipulates transparency and process requirements for acceptable use, complaints, and content moderation, requiring services to submit transparency reports (section 5).
By contrast, the national legislatures in Brazil 11 and India 12 have both considered much stricter regulation of content monitoring online. The Brazilian executive issued a Provisional Measure 1068 to restrict content removal by social media platforms, limiting removal only to cases of nudity, violence, narcotics, and incitement to crime, thereby preventing social media platforms from removing disinformation (such as President Jair Bolsonaro's COVID-19 disinformation removed by Facebook, Twitter, and YouTube).The Indian government has similarly issued a number of regulations, including the Information Technology Actand Information Technology (Intermediary Guidelines and Digital Media Ethics Code) Rules of 2021, 12 which direct userto-user services to remove a wide range of content, including material that threatens the sovereignty of the Indian state, to use algorithmic systems to monitor and remove harmful content, and to trace encrypted messages to limit online anonymity. Activist groups have claimed that these measures are aimed at curbing dissent against the government, resulting in what they call ''digital authoritarianism.''These comparators are useful in framing the different degrees to which governments have chosen to interfere with services' content monitoring and moderation. The US and EU models are focused on design choices that empower users by making the terms and procedures of user-to-user services transparent and accessible. The Indian and Brazilian models, by contrast, are focused much more explicitly on directing the content that is permissible on user-to-user services. The UK government has intimated its inclination toward the former approach, but this remains relatively underdeveloped in the Bill itself, as we discuss in the following sections.
## Measuring the bill through proportionality and necessity
The Online Safety Bill will necessarily limit individuals' right to freedom of expression, and place costly positive duties on entrepreneurs that limit their free enjoyment of their property (not to mention downstream effects on their competitiveness). These rights are enshrined in the European Convention on Human Rights (article 10) and the subsequent Paris Protocol (article 1). However, we are concerned here not with the legal right-particularly since the UK parliament reserves the right to enact legislation that is incompatible with its commitment to the Convention. Rather, we are concerned with the normative right that underpins the aforementioned legal creations. As our point of departure, we assume that individuals are vested with natural rights to free expression and use of their property.
These rights, of course, are not absolute or insurmountable: it is, by way of exception, permissible to infringe upon these rights in the presence of sufficient countervailing justification. Rights provide ''moral breakwaters'' 16 that protect individual and collective interests, but the breakwaters can always be overcome with sufficient justification. However, since rights function as breakwaters, they cannot be limited anytime that infringement causes a net good. Rather, rights are only defeasible when the costs of respecting the rights is significantly greater than the cost of transgression. [bib_ref] Justifying harm, Rodin [/bib_ref] In other words, rights establish a default position, and deviation from this default requires significant and extraordinary reason. This sets the standard of evidence that the government should (normatively, albeit not legally) adduce to support the infringement of others' rights: their intervention should not simply produce a net good; rather, the net good must be sufficiently weighty to offset the cost of intentional rights infringement.
In normative theory, rights infringements are subject to the requirements of necessity and proportionality. [bib_ref] The sources and status of just war principles, Mcmahan [/bib_ref] Necessity permits rights infringements only as a last resort: rights cannot be transgressed if there are less harmful means available to achieve the same end. Proportionality permits rights infringement only if the expected outcome of the infringement is commensurate with the cost of the infringement-in other words, there must be an apt ''fit'' between means and ends.
What we want to suggest here is that the evidence presented by the government in justifying the Online Safety Bill (and the infringements it entails) does not meet the thresholds of necessity and proportionality. Given the stringency of the rights affected, it is critical that the government should adduce clear and convincing evidence that its proposal satisfies these requirements. To this end, we suggest that the government's rationale for the Bill raises several general concerns that cast doubt upon the proportionality and necessity of this intervention. We list these concerns below.
## Proportionality issues
In several instances, the White Paper cites genuine concerns about the contributions of user-to-user and search services. However, if we investigate these claims more closely, there is a mismatch between the putative justification of the White Paper and the remit of the legislation. In brief, the problem can be framed as follows: d the interventions in the Bill do relatively little to resolve the concerns raised by the White Paper; d to resolve the concerns thoroughly would require interfering with legitimate internet use.
This is a problem of proportionality, because the expected benefit of the intervention does not fit the magnitude of the rights infringement entailed by the intervention.
Consider, first, the problem of bullying. The government is right to want to address the problem of cyberbullying: one in five children report being subject to cyberbullying.However, the Bill only addresses a fractional part of this problem, because 90% of cyberbullying occurs in private messages between schoolmates.The Bill, as it is, is unlikely to resolve the problem that the White Paper outlines, since private communications fall outside of the scope of the Bill. Of course, the government could resolve the problem more effectively by expanding the remit of the Bill to include private communications, but this would clearly constitute an unacceptable breach of individual rights, and would require services to break the encryption of private messaging, which would have extremely deleterious privacy costs.
A similar problem plagues the government's ambitions concerning suicidality and self-harm. The White Paper is correct in emphasizing the correlation between internet use (particularly of search and user-to-user services) and suicidality, depression, and self-harm. However, it is again unclear whether the Bill's interventions present an effective and proportionate solution to the problem. First, although internet use is strongly correlated with suicidality and depression, this correlation is predominantly due to the effects of sleep loss and private cyberbullying, rather than exposure to suicide-related content (which has a very low correlation with suicidality). [bib_ref] Social media, internet use and suicide attempts in adolescents, Sedgwick [/bib_ref] [bib_ref] Testing the Proclaimed affordances of online support groups in a nationally representative..., Deandrea [/bib_ref] Second, where individuals experiencing suicidality have accessed suicide-related content prior to self-harming, the preponderance of this content has not been user-to-user content, but rather fact-based websites. [bib_ref] Social media, internet use and suicide attempts in adolescents, Sedgwick [/bib_ref] It is unclear, therefore, whether the Bill as it is will be able to resolve much of the problem of suicidality and self-harm. Again, the government would have to make much further-reaching interventions to get to the real causes of the problem, but this again would be at the cost of interfering with individual liberties.
## Necessity issues
The second point of concern is that, even where the Bill's interventions are effective in preventing or mitigating harm, there are interventions available to the government that would interfere less with individual rights. This would render the interventions in the Bill unnecessary, since they do not constitute the least costly means of addressing the intended harm.
For the purposes of measuring necessity, it is useful here to benchmark the interventions in the Bill to similar interventions in comparable legislation, such as the DSA. The DSA does not focus on content monitoring or moderation by platforms themselves, but rather focuses on setting out clear and easily accessible mechanisms for users to register complaints and to flag content that contravenes the terms and conditions of the service (sections 3 and 4). This approach causes less interference with the rights of individual users, because it means that they are not monitored by default, as well as imposing less burdensome duties on service providers, since they do not have to develop monitoring mechanisms (which we cover later in this paper). This approach also reduces interference by diminishing the possibility of removing non-harmful content, since only content flagged by users (rather than by monitoring AI) will be picked up.
The DSA's approach also avoids the Bill's more costly solution of de-anonymizing user-to-user services. The Bill proposes limiting the ability of individuals to use user-to-user services anonymously as a means of harm prevention. While the restriction of the ability of individuals to be anonymous might have benefits in terms of holding individuals more accountable for their actions, which could promote less toxic interactions online, the limiting of anonymity might be damaging to those who rely on ll OPEN ACCESS the lack of identification online to access support. [bib_ref] Virtuous or Vitriolic: the effect of anonymity on civility in online newspaper..., Santana [/bib_ref] Chat rooms and support groups can be beneficial for those experiencing mental health issues and one of the features that can increase their effectiveness is the ability to be anonymous. Individuals are able to access support without their identity being revealed, meaning they are free to discuss their experiences without being concerned about their employer or family finding out, for example. It is unclear how the Bill would balance minimizing harm facilitated by anonymity while avoiding bringing harm to those who rely on anonymity to access support. Relying instead on user reporting is a means of empowering individuals against online harm without limiting their ability to interact freely and anonymously online when it is beneficial to their wellbeing.
## Democratic deficit
Our second concern relates to the enforcement of the duties set out in the Bill. We argue that the structure of the Bill grants undue power to the executive, and deprives the public of the opportunity to exercise democratic oversight of the Bill's content.
In designing the structure of its regulation, the Bill assigns to Ofcom the power to issue Codes of Practice that will determine how user-to-user and search services are to fulfil the more abstract duties set out in the legislation. As mentioned in section 1.3, the Bill also grants the Minister the power to interfere with the Codes of Practice by exercising a veto power or by directing Ofcom to align the Codes of Practice with government policy.
Given that the government will be able to punish services and individuals who are derelict in their duties, and will inform what information can be shared and received on the internet, the Codes of Practice have significant implications for the rights of internet users and services. The Codes of Practice can make information more or less difficult to communicate. Our concern is that the stringency of these restrictions is dependent upon the regulator and ultimately the Minister, with little oversight from Parliament or the public (although the Department has suggested it will consult stakeholders). Since the Minister can direct the Codes of Practice, they are effectively granted the authority to determine how user-to-user services control speech on their platforms.
Our concern is that this creates the possibility of a democratic deficit in the Bill: the Minister retains sweeping powers to interfere with the limits and regulation of speech on the internet's key platforms, with Parliament playing only a minimal negative oversight role. This power is sweeping since the remit of the Bill is wide (particularly in defining the ''harmful but legal'' content for which services are responsible). This means that the Minister has significant power to interfere with an important set of rights (including free speech and free press) without the particulars of their interventions being vetted by Parliament or subject to public scrutiny. We suggest that this amount of power is susceptible to abuse, and does not accord with the Bill's vision of a free internet.
## New powers and duties
The Bill empowers Ofcom to enforce both services' duties concerning illegal content, as well as their duties concerning ''legal but harmful'' content. We have particular concerns about the duty placed upon services to control ''legal but harmful'' content, given the breadth of the definition of what counts as harmful.
Here, we argue that the breadth of the content covered by the Bill would be better addressed by ethics-by-design procedural mechanisms, rather than content-specific regulation.
The Bill defines harmful content in Part 2, Chapter 6, Section 46, repeating its formulation in defining content that is harmful to children. The formulation in this section has a few noteworthy features. The first is that it includes content that causes ''indirect'' harm and extends the remit of harm to psychological (and not just physical) harm. The second, more worrying, feature of the formulation is that it defines harm in terms of the reasonable understanding of a person of ''ordinary sensibilities.'' The introduction of the reasonableness element imports an interpretive element into the duty without inserting clear boundaries delineating the scope of the duty, which-as we explain in this section-is troublesome for a top-down approach to regulating harm.
Our concern here is not with the cogency of a wider definition of harmful speech as such. It is plausible, we think, to extend the remit of ''harmful speech'' beyond the remit of what counts in law as ''hate speech.'' However, in our opinion, the Online Safety Bill does not specify this remit with sufficient clarity for the purpose for which it is deployed. This formulation has the capacity to include a vast sweep of material that would be undesirable to limit. Whether an individual piece of content counts as ''harmful'' on this definition will depend significantly on the context of its use. It is this matter of interpretation that, we think, opens the possibility of over-censorship when enforced by a top-down approach in which the government specifies a list of harmful content or algorithmic systems are used to detect harmful content (as in the Brazilian and Indian cases).
Consider two examples that pose particular interpretive difficulty: manipulation and disinformation: d Manipulation: ''nudging'' refers to features of choice architecture that alter an individual's behavior in a predictable way [bib_ref] Nudge: Improving Decisions about Health, Thaler [/bib_ref] and, although disputed, can be said to be a form of manipulation of the choices people make.Nudging can take simple forms, including the placement of products in the supermarket, with branded products being placed at eye level to encourage consumers to spend more.Nudging has also been used in public health interventions, including in reducing meat consumption 26 and the encouragement of healthier food purchasing, [bib_ref] Does shelf space management intervention have an effect on calorie turnover at..., Adam [/bib_ref] as well as in pro-environmental behavior. [bib_ref] Nudging pro-environmental behavior: evidence and opportunities, Byerly [/bib_ref] Evidently, nudging (and manipulation) is present in analog settings and is not novel to online, algorithmic-driven settings, such as social media. Indeed, traditional information flow theory can be adapted to algorithmic nudging. [bib_ref] Algorithmic personalization and the two-step flow of communication, Soffer [/bib_ref] This is not to say that more analog forms of manipulation and algorithmic manipulation, or algorithmic personalization as it is commonly referred to in the literature, are exactly equivalent. Algorithmic manipulation can be more covert than human manipulation, [bib_ref] Algorithmic personalization and the two-step flow of communication, Soffer [/bib_ref] which, in part, is due to the often black box nature of algorithms. [bib_ref] Thinking outside the black-box: the case for ''algorithmic sovereignty'' in social media, Reviglio [/bib_ref] activity enables highly accurate profiles to be generated about them. [bib_ref] Five fears about mass predictive personalization in an age of surveillance capitalism, Yeung [/bib_ref] However, again targeted advertising, which falls within the remit of nudging/manipulation, [bib_ref] Advertising: strongly persuasive or nudging?, Barnard [/bib_ref] can occur offline, although with less personalization; television advertisements are targeted at the intended audience of the station, including targeting to children, [bib_ref] Exposure to food and beverage advertising on television among Canadian adolescents, Czoli [/bib_ref] [bib_ref] Food and beverage advertising to children and adolescents on television: a baseline..., Pinto [/bib_ref] and the placement of billboards enables targeting to specific demographics. [bib_ref] Alcohol availability and targeted advertising in racial/ ethnic minority communities, Alaniz [/bib_ref] [bib_ref] Smoke signs: patterns of tobacco billboard advertising in a metropolitan region, Luke [/bib_ref] It is not clear, in the context of the Bill, how we are to distinguish meaningfully between legitimate techniques of persuasion and bad faith manipulation. d Disinformation: the White Paper provides seemingly contradictory directives with regard to limiting disinformation. The White Paper claims, simultaneously, that user-touser services have an obligation to ''improve how their users understand'' the ''trustworthiness'' of news (7.29), but it also confirms that the purpose of regulation should be ''protecting users from harm, not judging what is true or not'' (7.31). It is not clear how these two imperatives can be squared. If ''harm'' is simply content that is already illegal (including hate speech, defamation, unlawful political interference), then it is unclear what additional protection the Bill will contribute. However, if ''harmful'' is construed more widely, then the Bill will invariably have to set parameters for the kind of information that counts as disinformation (rather than simply misinformation). This is a delicate interpretive task that depends upon the context in which information is disseminated, and so requiring stricter monitoring will require tradeoffs in which services will have to limit expression.
Our concern here is that the top-down monitoring of content (either by the government or by AI deployed by services) -given these interpretive difficulties-will increase the risk of excessive censorship. Whether an individual piece of content constitutes ''harm'' by the definition above will be highly sensitive to the context of its use. The context sensitivity of this definition suggests important technical difficulties for enterprises. Enterprises will presumably have to develop tools to scan content for a number of harms. Automatic detection of harm is an open problem far from being solved. [bib_ref] Current limitations in cyberbullying detection: on evaluation criteria, reproducibility, and data scarcity, Emmery [/bib_ref] The academic community researching automated detection of cyberbullying, for example, has made appeals for more universal and specific criteria concerning cyberbullying definitions, more consistent evaluation methods, and better-quality datasets. [bib_ref] Current limitations in cyberbullying detection: on evaluation criteria, reproducibility, and data scarcity, Emmery [/bib_ref] [bib_ref] Automatic cyberbullying detection: a systematic review, Rosa [/bib_ref] It is easy to see how larger companies with access to more data and highly skilled technical personnel would be better placed to solve the task, whereas smaller firms will struggle to meet this serious technical task.
Given the importance of the issue for the safety and human rights of users, we endorse the research community's call for a clearer set of criteria for ''harm.'' We also recommend supporting the creation of universal tools, which could be achieved by collecting or sharing datasets and existing technologies and would remove the burden from small and medium enterprises. However, more generally, it is our opinion that an ethics-by-design approach would mitigate much of this concern, because it would empower users to inform the moderation themselves with the help of the appropriate procedural mechanisms.
## Codes of practice
Our final concern relates to the Bill's focus on Codes of Practice deferred to Ofcom as the main regulatory mechanism. Our concern here is that the legislation's open-ended references to Codes of Practice opens the possibility of inappropriate regulatory tools. As we intimate in the previous section, our concern here is that the Codes of Practice leave open the possibility that regulation will restrict particular pieces or kinds of content. This would, of course, place an unduly onerous burden on service providers, and hold them responsible for activities on their sites for which they should not be held liable.
We note that the GDPR and the DSA include a similar mechanism that permits regulators to establish Codes of Conduct or best-practice guidelines.However, it is important to note that the Codes in these cases do not establish new rules that are not grounded in the legislation: rather, it provides efficient means for enterprises to comply with their duties established by complex legislation. Our concern is that the Online Safety Bill is sufficiently open-ended that the Codes of Practice will, in this case, amount to the creation of new rules, since the duties in the Bill are multiply realizable and open to a wide range of interpretations. This is because the Bill outlines only in broad terms the duties that services have to protect users, but does not prescribe (as the DSA and GDPR do) which features of their platforms are in the scope of the regulation (i.e., whether they have a duty to monitor and moderate specific pieces of content, or whether they only have a duty to adjust the design features of their services).
The most sensible approach, we argue, would be to adopt an ethical design approach that (1) focuses on the ethical features of the design process and (2) provides services with sufficient space to adopt flexible and innovative solutions to the social problems present on their platforms. A Code of Practice runs the risk of focusing less on design, and rigidifying the solutions that providers can use to solve problems.
In a recent memorandum on the topic, the Department of Digital, Culture, Media, and Sport has indicated that they will focus their Codes of Practice on design and process features of userto-user and search services.However, we would appeal to Ofcom, Parliament, and the DCMS to concretize this commitment to assuage concerns about content-specific censorship. It is important for the purposes of clarity that this be confirmed.
Compared with those jurisdictions that have taken a contentspecific approach to regulation, the Online Safety Bill is less stringent and specific. Indeed, India's Information Technology Rules,which echo the sentiment of the Online Safety Bill, defines content that must be reviewed under the rules, listing discrimination, psychotropic substances and smoking, imitable behavior, such as content depicting self harm and offensive language (such as expletives, nudity, sexual content, and violence). The Rules also require the appointment of a Chief Compliance Officer in social media companies to ensure compliance and cooperation, and identification of the first poster of the unacceptable content in some cases. Likewise, the Russian law On Information, Information Technologies and Information Protection requires social media sites to monitor and restrict content related to material concerning the advertising of alcohol and online casinos, disrespect for society, information on drug synthesis and production, and suicide. Violations are required to be registered with the Federal Roskomnadzor register.
## Concluding remarks
We share the government's concerns about the potential hazards of the internet, particularly with regard to vulnerable groups, particularly children. However, this is not the only imperative at stake: it is also important that the government foster an open internet, on which free speech and innovation can flourish. We accept that the state cannot fully satisfy all of these imperatives simultaneously: the state will necessarily have to make tradeoffs between safety, liberty, and innovation.
Insofar as we have been critical of the Online Safety Bill, it has been because we think it has not yet achieved an optimal balance between these imperatives. First, we argue that the Department must do more to justify this legislative intervention: there is a paucity of justificatory evidence for the scope of the Bill in the current White Papers issued in its support. Second, we have argued that the mechanisms of the Bill do not do enough to protect the liberties of platforms and their users, because it effectively defers much of the power to regulate platforms to the Minister. Third, we argue that the Bill imposes overly wide duties on platforms that can be deleterious to smaller enterprises and increase government intervention. Fourth, we argue that it is imperative for the government to commit to an ethical-by-design approach to the duty of care.
It is our opinion that it is possible for the government to correct the problems with the Bill and the White Papers we suggest here without having to make significant sacrifices to its strategic aims. We suggest that these changes-while seemingly small-will have a significant effect on making the internet freer, more open, and more innovative-as well as making it safe.
[table] d Rationale: drawing on the government's White Paper, we reconstruct their explanation for the necessity of the Bill, including the social problems it aims to correct and the regulatory lacuna it aims to fill. d Scope: we survey the wide range of unlawful and lawful harms on digital services that the Bill intends to regulate. d Enforcement: we survey the mechanisms through which the Bill enforces regulation, particularly by empowering the regulator, Ofcom, and the Minister. d International comparisons: we survey similar regulatory proposals from other jurisdictions aimed at resolving the same set of issues. Secondly, we offer our critical commentary on these features of the Bill. In our critical discussion, our main conclusions are the following: d Further evidence needed: we argue that the government's White Papers for the Online Harms Bill and Online Safety Bill do not provide sufficient evidence for the necessity or efficacy of regulatory intervention. Despite the prevalence of harms online, it is not clear from the White Papers why extensive government interference-with its concomitant limitations on freedom and individual rights-is a necessary or proportionate resort in resolving these issues. d Possible democratic deficit: we argue that the Online Safety Bill suffers a possible democratic deficit because it delegates extensive authority to Ofcom in its capacity as the industry regulator for digital media, and to the Minister. In assigning Ofcom the power to determine the Code of Practice for digital platforms, the Online Safety Bill empowers Ofcom with sweeping powers to enact rules for the internet with little democratic scrutiny by Parliament and no consultation. d Duties too wide: we are concerned about the potential [/table]
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Association between full monitoring of biomedical and lifestyle target indicators and HbA1c level in primary type 2 diabetes care: an observational cohort study (ELZHA-cohort 1)
# Abstract
Objective Management of type 2 diabetes mellitus (T2DM) requires frequent monitoring of patients. Within a collective care group setting, doubts on the clinical effects of registration are a barrier for full adoption of T2DM registration in general practice. We explored whether full monitoring of biomedical and lifestyle-related target indicators within a care group approach is associated with lower HbA 1c levels. Design Observational, real-life cohort study. setting Primary care data registry from the Hadoks (EerstelijnsZorggroepHaaglanden) care group. Exposure The care group provides general practitioners collectively with organisational support to facilitate structured T2DM primary care. Patients are offered quarterly medical and lifestyle-related consultation. Main outcome measure Full monitoring of each target indicator in patients with T2DM which includes minimally one measure of HbA 1c level, systolic blood pressure, LDL, BMI, smoking behaviour and physical exercise between January and December 2014; otherwise, patients were defined as 'incompletely monitored'. HbA 1c levels of 8137 fully monitored and 3958 incompletely monitored patients were compared, adjusted for the confounders diabetes duration, age and gender. Since recommended HbA 1c values depend on age, medication use and diabetes duration, analyses were stratified into three HbA 1c profile groups. Linear multilevel analyses enabled adjustment for general practice. Conclusions/interpretation This study shows that in a care group setting, fully monitored patients had significantly lower HbA 1c levels compared with incompletely monitored patients. Since this difference might have considerable clinical impact in terms of T2DM-related risks, this might help general practices in care group settings to overcome barriers on adequate registration and thus improve structured T2DM primary care. From population health management perspective, we recommend a systematic approach to adjust the structured care protocol for incompletely monitored subgroups.
# Introduction
Type 2 diabetes mellitus (T2DM) is a typical lifestyle-related disease. [bib_ref] Clinical impact of lifestyle interventions for the prevention of diabetes: an overview..., Howells [/bib_ref] The course of T2DM and potential complications are influenced by smoking behaviour, 2 3 BMI 4 and physical exercise. [bib_ref] Interrupting prolonged sitting in type 2 diabetes: nocturnal persistence of improved glycaemic..., Dempsey [/bib_ref] Adopting a healthier lifestyle, for example cessation of smoking or weight loss, is known to be very demanding for individual patients. It has been established that attention for non-conscious motivational factors affecting an individual's behaviour is important to realise sustained behavioural change. [bib_ref] Health goal priming as a situated intervention tool: how to benefit from..., Papies [/bib_ref] In addition, to avoid relapse and maintain long-term behavioural change, follow-up support for lifestyle-related themes is recommended. Accordingly, in the Netherlands, a nationally acknowledged strengths and limitations of this study ► The observational real-life design of this study prevented any interference with daily routines of GP practices, thus contributing to good reliability and representativeness of our findings. ► Because the availability of patients' data on age, medication use and diabetes duration allowed to conduct our analyses-in correspondence with professional GP guidelines-for specific HbA 1c threshold groups, the findings are relevant and useful for clinical practice. ► Taking into consideration that a missing registration does not necessarily reflect a lack of care but might be caused by technical or practical problems instead, the associations found in this study might be underestimated.
Open access scientific council of general practitioners (GPs) has determined professional guidelines for diabetes primary care. [bib_ref] NHG-Standaard Diabetes mellitus type 2 (derde herziening), Rutten [/bib_ref] In correspondence with the NICE guidelines,it is recommended to monitor HbA 1c levels and also the biomedical target indicators systolic blood pressure and LDL as well as lifestyle-related indicators at least once a year. However, for an average GP, providing structured primary diabetes care with sufficient attention for both biomedical monitoring and lifestyle adaptation 15 is reported to be challenging. [bib_ref] Barriers to effective management of type 2 diabetes in primary care: qualitative..., Rushforth [/bib_ref] Therefore, in many Western countries, varying from the USA and Europe to New Zealand, [bib_ref] The financial impact of clinical task substitution between practice nurses and GPs..., Hefford [/bib_ref] an increasing number of GPs has delegated the regular structured primary diabetes care to nurse practitioners.
It is known that implementing structured primary diabetes care and delegation of tasks to a nurse practitioner has considerable impact on the organisation of the GP practice. For example, in the USA, an evaluation of the recent Comprehensive Primary Care (CPC) programme revealed a need to refine practice workflows, to incorporate new staff roles and to overcome incompatibility of health technology systems. [bib_ref] Two-year costs and quality in the comprehensive primary care initiative, Dale [/bib_ref] To improve the delivery of structured primary diabetes care in the Netherlands, most GPs have joined together in local 'care groups'. [bib_ref] Experimenteren met de keten-dbc diabetes: de eerste zichtbare effecten, Struijs [/bib_ref] Care groups negotiate collective structured diabetes care protocols with the funding institutions of Dutch primary care, namely local health insurance companies. For GPs, participation in a care group is voluntary. However, the logistic and quality support to individual GP practices, which is part of the care group approach, might be seen as an incentive for care group participation. That is, the agreements between care groups and health insurance companies on structured diabetes care protocols enable GPs to offer high-quality intensive primary diabetes care. To illustrate, on an annual basis, four consultations at the GP practice with an explicit focus on lifestyle support as well as complementary allied health (eg, annual screening of fundus and feet) are facilitated. All patients who receive diabetes care in GP practice are eligible for participation in the structured care protocol. It is known that providing a structured diabetes care protocol is associated with better monitoring of patients. [bib_ref] Sex differences in the quality of diabetes care in the Netherlands (ZODIAC-45), Hendriks [/bib_ref] In addition, adequate registration of the diabetes-related patient health indicators is associated with improvement of the care process. [bib_ref] Impact of clinical registries on quality of patient care and clinical outcomes:..., Hoque [/bib_ref] The costs of this protocol are fully covered by health insurance companies. For patients, participation is free of charge.
According to a recent study, care group participation is associated with improvement of the proportion of patients with full monitoring of biomedical and lifestyle-related target indicators.However, a review on chronic care programmes in primary care reported that doubts among care providers on the clinical effects of an intervention are a barrier for adoption. [bib_ref] Facilitators and barriers of implementing the chronic care model in primary care:..., Kadu [/bib_ref] To our knowledge, little is known about the relationship between full monitoring of biomedical as well as lifestyle-related target diabetes indicators in a care group setting and clinical health outcomes. The HbA 1c level is established as a key diabetes health indicator. [bib_ref] Work-family life courses and BMI trajectories in three British birth cohorts, Lacey [/bib_ref] Therefore, this study aims to investigate the association between full monitoring of biomedical and lifestyle-related diabetes target indicators and HbA 1c level in patients with T2DM who receive a structured diabetes care protocol facilitated by a care group.
rEsEArCh DEsIgn AnD MEthODs study design and population Data were used of T2DM patients from the observational Eerstelijns Zorggroep Haaglanden (ELZHA) cohort, which is based on primary care registry data from a care group in the western part of the Netherlands. In January 2015, the care group numbered 168 GP practices (n=24 459 patients with T2DM). On a periodical basis, GP members share an overview of their patients' monitoring data with the care group. In February 2017, all GP practices were informed in writing and, based on an opt-out procedure, GP members were invited to participate in this cohort. For the present study, pseudonymised data on monitoring of diabetes target indicators and HbA 1c levels from patients were used from the calendar year 2014. Patients receiving continuously structured primary diabetes care from January 2014 through December 2014 at the same GP practice were included. At least one registration of HbA 1c in 2014 was necessary for inclusion. Since systolic blood pressure and LDL guidelines are specified for patients aged ≤80 years, patients aged ≥80 years were excluded. Patients were also excluded in case of missing data on age, gender or disease duration. Finally, since missing of data on medication use was partly caused by technical problems, patients without registration of medication prescription were also excluded.
## Exposure
Details of the ELZHA cohort study have been described previously (Van Bruggen et al, submitted). In short, within a care group setting, GPs are able to invite all their T2DM patients with primary care treatment for this structured care protocol. During a standard diabetes consultation or at time of diagnosis, patients are informed about this care protocol. Patients who provide consent to be enrolled can join the structured primary care protocol. The protocol includes a quarterly diabetes consultation, in which diabetes-related target indicators are checked and lifestyle education is provided, combined with complementary allied health such as an annual foot check, fundus screening and dietician's counselling. To facilitate the organisation and quality control of this protocol, GP practices receive practical and logistical support, including a computerised system to improve the care process and outcomes. Measurement of the diabetes target indicators (HbA 1c level, systolic blood pressure, LDL level, BMI, smoking behaviour and physical exercise) took place in 2014 at the end of each quarter. In the present study, patients were regarded as 'fully monitored' when each target indicator was registered at least once between January and December 2014. If one or more target indicators were not registered minimally one time in calendar year 2014, patients were defined as 'incompletely monitored'.
## Outcomes
The outcome of this study was HbA 1c level and this was computed in two steps. First, for each quarter, a mean HbA 1c value was calculated based on all available HbA 1c measures in that quarter. Based on the mean HbA 1c levels of all quarters, a mean was computed for the whole calendar year. HbA 1c level is presented in mmol/mol and %.
# Analysis
For patient's characteristics, categorical variables were reported as numbers and percentages. Continuous variables were reported as means with SD or, when non-normally distributed, as medians with IQR. Baseline characteristics of excluded patients were, if available, compared with the study population. Linear multilevel analyses were conducted to compare HbA 1c levels of fully monitored and incompletely monitored patients. Multilevel analyses allowed to adjust the individual observations (level 1) for GP practice (level 2). In addition, the analyses were adjusted for patient's age, duration of diabetes and gender, which are relevant possible confounders with regard to HbA 1c outcomes.
Tailored on specific key patient's characteristics (age, intensity of medication treatment and disease duration), professional Dutch GP guidelines recommend differentiated HbA 1c targets for three different patient profile groups based on age and prescribed medication. Details on the scientific determination of these target values are presented in the guidelines. [bib_ref] NHG-Standaard Diabetes mellitus type 2 (derde herziening), Rutten [/bib_ref] To summarise, (1) for patients aged <70 years and for older patients with a mild treatment regime (only metformin monotherapy prescription or lifestyle coaching), a target HbA 1c value of 53 mmol/mol (7.0 %) is recommended; (2) for patients aged ≥70 years who need more intensive treatment and were diagnosed with diabetes <10 years previously, a target HbA 1c value of 58 mmol/mol (7.5 %) is recommended;
(3) for patients aged ≥70 years who need more intensive treatment and were diagnosed with diabetes ≥10 years previously, a target HbA 1c value of 64 mmol/mol (8.0 %) is recommended. In the present study, since missing data on medication might reflect administrative omissions rather than absence of medication treatment, patients without data on medication were excluded.
In view of the relevance for clinical practice, separate multilevel analyses were conducted and reported for each of these HbA 1c profile groups. In addition, in a non-stratified multilevel analysis, we tested whether the magnitude of the effect found in HbA 1c profiles 2 and 3 differed significantly from HbA 1c profile 1. A p value<0.05 was considered statistically significant; for interaction, a p value<0.1 was considered statistically significant.
Descriptive statistics were analysed using SPSS, V.24.0. Multilevel analyses were performed using ML WiN (V.2.28).
## Patient and public involvement
Since this study was targeted on a GP-supporting approach of structured primary diabetes care, patients were not actively involved.
# Ethical considerations
Since the pseudonymised patients' data contained only age and gender, the data could be aggregated without enabling investigators to identify individual patients. Due to the high number of patients, informed consent of individual patients was not required.
# Results
This study included 167 GP practices (99%) with a total of 24 198 patients with T2DM; of these, 12 095 patients met the inclusion criteria (for a detailed flowchart of inclusion, see [fig_ref] Figure 1: Flow chart of patient inclusion [/fig_ref]. By definition, in this population, HbA 1c was always monitored, as not having an HbA 1c measure available was an exclusion criterion for the present study. Comparing characteristics of the excluded patients (n=12 103 patients) with the study population (n=12 095 patients, see online supplementary table 1), in excluded patients mean HbA 1c level (50.32 mmol/ mol, SD=12.8 mmol/mol; 6.76%, SD=3.32%, 7.535 registrations missing) was slightly lower than in the study population (52.5 mmol/mol, SD=1.07 mmol/mol; 6.95%, SD=3.16%). Comparing the median diabetes duration of excluded patients (5 years, IQR: 3-9, 63 registrations missing) to the study population (6 years, IQR: 3-10), no substantial differences were found. Regarding median age, excluded patients (71 years, IQR: 60-82, 2917 registrations missing) were older than included patients (median: 64 years, IQR: 56-71 years) and slightly more often women (50% [n=4251; 3530 registrations missing] vs 45% [n=5477]). More detailed characteristics of our study population, classified by HbA 1c profile and monitoring completeness, are presented in table 1. Of patients who were incompletely monitored, information on physical exercise was most often missing, followed by smoking, BMI, LDL and systolic blood pressure (figure 2).
Compared with incompletely monitored patients, fully monitored patients had lower mean HbA 1c levels in all three HbA 1c profiles. In addition, fully monitored patients had a longer duration of diabetes than incompletely monitored patients.
The crude analysis showed that compared with incompletely monitored patients, the mean HbA 1c of fully monitored patients was significantly lower in the first profile
## Open access
# Discussion
This study explored whether monitoring completeness of biomedical and lifestyle-related diabetes target indicators in a care group setting is associated with HbA 1c level. In all HbA 1c profile groups-defined based on patient age, intensity of medication treatment and disease duration-we found that fully monitored patients had lower HbA 1c levels than incompletely monitored patients; the differences ranged from 1.89 mmol/mol (0.17 %) to 3.36 mmol/mol (0.31 %), indicating that adequate diabetes monitoring of biomedical and lifestyle indicators in primary care is associated with better HbA 1c levels. To our knowledge, this is the first study to analyse the association between systematic diabetes monitoring in primary care and HbA 1c levels. Apart from one longitudinal Dutch study on structured primary diabetes care in a care group setting that reported a sharp decrease in the proportion of patients with a HbA 1c level ≥53 mmol/mol, 24 research on absolute HbA 1c differences is scarce and findings appear to be somewhat inconsistent. [bib_ref] Disease management programs for type 2 diabetes in Germany: a systematic literature..., Fuchs [/bib_ref] [bib_ref] Nurse case management improves blood pressure, emotional distress and diabetes complication screening, Gabbay [/bib_ref] [bib_ref] Impact of disease management programs on HbA1c values in type 2 diabetes..., Kostev [/bib_ref] [bib_ref] Effect of the Disease Management Program on HbA1c value in type 2..., Wiefarn [/bib_ref] Therefore, caution is required when comparing our findings with any earlier studies. However, for each 1% (10.9 mmol/mol) reduction in mean HbA 1c , a significant decrease in health risks has been reported, ranging from 21% for any endpoint related to diabetes including deaths to 14% for myocardial infarction and 37% for microvascular complications. [bib_ref] Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes..., Stratton [/bib_ref] Further, our finding that registration of physical exercise was most often lacking is in line with an earlier small-sized study in which only 19% of patients with T2DM reported 'being guided properly' with regard to physical exercise. [bib_ref] Conflict between diabetes guidelines and experienced counselling in sports and physical activity...., Stuij [/bib_ref] Our finding that compared with incomplete monitoring, full monitoring of patients is associated with a lower HbA 1c level might be explained by continuity of care in several ways. First, if patients are monitored at least once a year, an increasing HbA 1c level might be noticed at an early stage, resulting in fast and adequate treatment. Second, periodic monitoring and coaching of patients with regard to weight loss, smoking cessation and physical exercise contributes to enduring lifestyle adaptation, which may lead to lower HbA 1c levels. 35
## Open access
Since fully monitored patients with T2DM have significantly lower HbA 1c levels, their risk of any diabetes-related health complication is lower compared with incompletely monitored patients. Thus, in general, incomplete monitoring of a patient should be interpreted as an important sign of diabetes-related health risks-especially since incomplete records might be caused by no-show and also by low patient motivation, missing of prescribed lab tests and limited overall adherence to diabetes treatment. As reported by others, 36 a tailored approach based on data registry and adjusted to patients' characteristics (eg, monitoring completeness) is recommended. This might encourage awareness in GP practice regarding adequate diabetes management and might help GPs to overcome barriers on full adoption of the care group monitoring approach. In addition, the present findings might be relevant for other structured diabetes primary care settings that focus on frequent monitoring and adequate registration of diabetes-related health outcomes such as the CPC Plus programme in the USA. [bib_ref] Medicare's vision for advanced primary care: new directions for care delivery and..., Sessums [/bib_ref] The present study is characterised by several strengths. First, in our view, an important strength of this study is the design: although randomised clinical trials might help to eliminate bias, adequate powering and generalisability are familiar problems, [bib_ref] An observational study goes where randomized clinical trials have not, Frakt [/bib_ref] whereas observational studies allow to include large study populations. For example, in this study, all patients participating in a structured primary diabetes care programme were enrolled, thereby contributing to high representativeness of our study population. Second, generally, since our study design did not interfere with the daily routine of GP practices, we assume adequate reliability of our findings. Thus, the observational real-life setting in our study reflects the reality of diabetes monitoring and HbA 1c levels in primary care. Our design is in line with other studies that also used a pragmatic approach to conduct diabetes-related studies in primary care. [bib_ref] Diabetes treatments and risk of heart failure, cardiovascular disease, and all cause..., Hippisley-Cox [/bib_ref] [bib_ref] Metabolic effects associated with ICS in patients with COPD and comorbid type..., Price [/bib_ref] [bib_ref] Prescribing quality and prediction of clinical outcomes in patients with type 2..., Smits [/bib_ref] Third, since patients were included if they participated for at least 1 year at the same GP practice, bias caused by intermediate moving or referral to hospital diabetes care was avoided, which contributes to the stability and, thus, the validity of our findings. Finally, conducting separate analyses for each HbA 1c profile group allowed adjustment for the variety in the recommended HbA 1c target values.
Nevertheless, this study is also subject to some limitations that need to be mentioned. First, since no control group was included, no causal relationship between monitoring completeness and HbA 1c level can be proven. Second, a missing registration does not necessarily mean that the care has not been provided. For example, missings might be caused by technical problems or lack of time for registration. Patients being considered erroneously as 'incompletely monitored' might have underestimated the associations found, although we did correct our analyses for age, diabetes duration, gender and GP practice.
For future research, it might be useful to analyse the context of diabetes target monitoring and explore whether the association that we found reflects a causal relationship between monitoring completeness and HbA 1c level. In addition, from the GP perspective, examining potential barriers to complete monitoring, including potential benefits such as an increase of the proportion of patients with HbA 1c levels within recommended values, might provide keys to improvement of the monitoring process. To ameliorate the primary diabetes care of incompletely monitored patients, exploration of their preferences and needs is suggested. In addition, an evaluation of financial costs and benefits of this care approach is recommended.
To summarise, in patients with T2DM within a care group setting, full monitoring of biomedical and lifestyle target indicators is associated with lower HbA 1c levels compared with incomplete monitoring. These differences might be expected to have a considerable clinical impact in terms of diabetes-related risks. We recommend a systematic approach to analyse the needs of incompletely monitored patient groups and to adjust the structured care protocol for these subgroups in terms of population health management.
[fig] Figure 1: Flow chart of patient inclusion. Pts, patients. [/fig]
[fig] Figure 2: Overview of registered indicators in incompletely monitored patients within HbA 1c profile. HbA1c, Haemoglobin A 1c . [/fig]
[table] Table 1: Characteristics of the study population, classified by HbA 1c profile and monitoring completeness [/table]
[table] Table 2: Multilevel analyses evaluating the HbA 1c difference of fully monitored patients compared with incompletely monitored patients, stratified for HbA 1c profile *Crude analysis. †Multilevel analysis adjusted for age, diabetes duration and gender. [/table]
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The Rare Association of Moyamoya Disease and Cerebral Arteriovenous Malformations: a Case Report
A 36-year-old man was diagnosed with a right temporal lobe grade II cerebral arteriovenous malformation (cAVM) and was treated with radiosurgery. At nine months after the cAVM radiosurgery, the patient began to develop bilateral focal narrowing at the M1 segments of the bilateral middle cerebral arteries. The narrowing progressively deteriorated as was demonstrated on longitudinal serial follow-up MR imaging. X-ray angiography performed at 51 months after radiosurgery confirmed that the cAVM was cured and a diagnosis of moyamoya disease. To the best of our knowledge, this is the first case of cAVM-associated moyamoya disease that developed after radiosurgery. Given the chronological sequence of disease development and radiation dose distribution of radiosurgery, it is proposed that humoral or unknown predisposing factors, rather than direct radiation effects, are the cause of moyamoya disease associated with cAVM. lthough a relatively uncommon vascular disorder, a cerebral arteriovenous malformation (cAVM) is the major cause of hemorrhagic stroke in young and middle-aged patients, second only to the rupture of arterial aneurysms. A variety of cAVM related dysplastic phenomena affecting the feeding arteries and draining veins, including flow-related aneurysms, arterial stenosis and occlusion as well as and venous stenosis, stricture and occlusion, have been reported (1, 2). Among these features, an association of a cAVM and moyamoya disease has been rarely observed. Moreover, the longitudinally sequential development of a cAVM and moyamoya disease association is also extremely rare.Gamma-knife radiosurgery (GKRS) is an established treatment alternative for cAVM with clearly defined benefits and risks. The risks include adverse radiation effects and a risk of intracranial hemorrhage that develops during the latency period after GKRS (3). We present here a case of moyamoya disease that developed after cAVM radiosurgery. To the best of our knowledge and from a literature review, this is the first report of cAVM-associated moyamoya disease developed after radiosurgery.CASE REPORTA 36-year-old man suffered from intermittent episodes of dizziness and complex visual hallucinations for 2 3 years of irregular duration and interval. Due to a newly developed numbness sensation over the right temporal region for one month, the patient arrived at our hospital and asked for assistance. No loss of consciousness was found for each attack and the patient was neurologically intact. The cerebral angiography(Fig. 1A)showed a Spetzler grade II cAVM located at the right anterior temporal lobe that received an arterial supply mainly from the anterior temporal branches of the right
## The rare association of moyamoya disease and cerebral arteriovenous malformations: a case report
A 36-year-old man was diagnosed with a right temporal lobe grade II cerebral arteriovenous malformation (cAVM) and was treated with radiosurgery. At nine months after the cAVM radiosurgery, the patient began to develop bilateral focal narrowing at the M1 segments of the bilateral middle cerebral arteries. The narrowing progressively deteriorated as was demonstrated on longitudinal serial follow-up MR imaging. X-ray angiography performed at 51 months after radiosurgery confirmed that the cAVM was cured and a diagnosis of moyamoya disease. To the best of our knowledge, this is the first case of cAVM-associated moyamoya disease that developed after radiosurgery. Given the chronological sequence of disease development and radiation dose distribution of radiosurgery, it is proposed that humoral or unknown predisposing factors, rather than direct radiation effects, are the cause of moyamoya disease associated with cAVM. lthough a relatively uncommon vascular disorder, a cerebral arteriovenous malformation (cAVM) is the major cause of hemorrhagic stroke in young and middle-aged patients, second only to the rupture of arterial aneurysms. A variety of cAVM related dysplastic phenomena affecting the feeding arteries and draining veins, including flow-related aneurysms, arterial stenosis and occlusion as well as and venous stenosis, stricture and occlusion, have been reported [bib_ref] Association of cerebral arteriovenous malformations and spontaneous occlusion of major feeding arteries:..., Enam [/bib_ref]. Among these features, an association of a cAVM and moyamoya disease has been rarely observed. Moreover, the longitudinally sequential development of a cAVM and moyamoya disease association is also extremely rare.
Gamma-knife radiosurgery (GKRS) is an established treatment alternative for cAVM with clearly defined benefits and risks. The risks include adverse radiation effects and a risk of intracranial hemorrhage that develops during the latency period after GKRS (3). We present here a case of moyamoya disease that developed after cAVM radiosurgery. To the best of our knowledge and from a literature review, this is the first report of cAVM-associated moyamoya disease developed after radiosurgery.
## Case report
A 36-year-old man suffered from intermittent episodes of dizziness and complex visual hallucinations for 2 3 years of irregular duration and interval. Due to a newly developed numbness sensation over the right temporal region for one month, the patient arrived at our hospital and asked for assistance. No loss of consciousness was found for each attack and the patient was neurologically intact. The cerebral angiography [fig_ref] Figure 1: confirmed cure of the right anterior temporal cAVM and the presence of... [/fig_ref] showed a Spetzler grade II cAVM located at the right anterior temporal lobe that received an arterial supply mainly from the anterior temporal branches of the right middle cerebral artery (MCA) and the right posterior cerebral artery (PCA) and showed early draining mainly into the right superficial middle cerebral vein. No focal stenosis of the intracrainal vessels was found at the initial presentation. The patient underwent GKRS with a maximum target dose of 31.25 Gy and a minimum dose of 17.5 Gy to the periphery of the cAVM nidus at a 56% isodose level of the maximum target dose (average dose of 23.6 Gy). The cAVM nidus volume was estimated as 20.7 ml. The supraclinoid segment of the right internal carotid artery and proximal MCA and anterior cerebral artery (ACA) received as dose less than 6.25 Gy (20% isodose level) [fig_ref] Figure 1: confirmed cure of the right anterior temporal cAVM and the presence of... [/fig_ref]. The arteries on the contra-lateral side received far less than this radiation dose.
Continuous regression of the cAVM was observed on serial follow-up brain MRI. Clinically the frequency of seizure attacks decreased. However, focal narrowing at the M1 segments of the bilateral MCA was found on follow-up brain MR angiograms obtained at nine months and 34 months after radiosurgery [fig_ref] Figure 1: confirmed cure of the right anterior temporal cAVM and the presence of... [/fig_ref] with progressive deterioration. The patient suffered from near-fainting and slurred speech at 51 months after GKRS. Acute infarcts at the right corona radiatae were found on brain MRI. A cerebral angiogram [fig_ref] Figure 1: confirmed cure of the right anterior temporal cAVM and the presence of... [/fig_ref]
# Discussion
To the best of our knowledge and from a review of the clinical literature, only 14 cases (mean age, 34 years; age range, 11 50 years; male to female ratio 1:1) of combined cAVM and bilateral moyamoya disease has been reported . Most of the cases (12 cases) had both neurovasuclar anomalies at the first presentation (1, 4 7). Another two cases had de novo development of a cAVM at a nine years and four years follow-up of moyamoya disease [bib_ref] Arteriovenous malformation associated with moyamoya disease, Fuse [/bib_ref]. In two large retrospective reviews of occlusive vascular disease associated with cAVMs, twenty cases of cAVM with occlusion or focal stenosis of the major feeding arteries of a cAVM were reported, respectively accounting for 3% and 1.3% in each patient group [bib_ref] Association of cerebral arteriovenous malformations and spontaneous occlusion of major feeding arteries:..., Enam [/bib_ref]. Nine out of the 20 cases had a unilateral supraclinoid ICA occlusion with a variety of moyamoya collaterals.
According to the reports mentioned above, two plausible hypotheses were proposed to describe the pathogenesis and relationship between an associated cAVM and moyamoya disease. First, it was proposed that the intimal layers of feeding arteries proliferated to accommodate the high-flow stress that stemmed from a cAVM. The inherited protective mechanism eventually resulted in total occlusion of the supraclinoid ICA and the accompanying changes in moyamoya collateral vessels [bib_ref] Cerebral arteriovenous malformations associated with moyamoya phenomenon, Montanera [/bib_ref]. It was also speculated that the blood demand of brain tissue in moyamoya disease might reactivate the pre-quiescent angiogenesis process and finally contribute to a de novo anomalous arterio-venous connection [bib_ref] Arteriovenous malformation associated with moyamoya disease, Fuse [/bib_ref]. For both hypotheses, angiogenetic factors might play an important role. One may speculate that the radiation delivered in GKRS might induce the development of moyamoya disease. However, as shown in [fig_ref] Figure 1: confirmed cure of the right anterior temporal cAVM and the presence of... [/fig_ref] , the occluded right supraclinoid ICA, ACA and MCA received approximately a dose of 6.25 Gy during GKRS and the arteries in the contra-lateral side received far less than this dose. It is reasonable to speculate that the humoral angiogenetic factors or unknown predisposing factors from the irradiated cAVM may have been involved in the final development of moyamoya disease in this patient. Apart from the two cases of acquired unilateral moyamoya syndrome after cAVM radiosurgery that were presented at the 1997 Annual Meeting of the Congress of Neurological Surgeons, this is the first report of cAVM-associated moyamoya disease that developed after radiosurgery.
Successful staged bypass surgery and radiosurgery for patients with concurrent cAVM and moyamoya disease was reported by Seol et al. [bib_ref] Radiosurgical treatment of a cerebral arteriovenous malformation in a patient with moyamoya..., Seol [/bib_ref]. Our reported patient developed moyamoya disease after cAVM GKRS. It seems simplified that these two anomalies could be managed in two separate stages. However, the question about which disorder should be treated first for patients with a concurrent cAVM and moyamoya disease might be answered by the following hypothesis. Radiosurgery mitigates the hypoperfusion in the adjacent brain tissues of cAVM by the steady and progressive obliteration of cAVM nidus while avoiding the risk of sudden interruption of the flimsy moyamoya vessels. However, hemorrhagic events and ischemic changes in the time window between the administration of both therapies and before complete nidus obliteration of a cAVM are possible.
In summary, we have presented the first case of bilateral moyamoya disease developing after cAVM radiosurgery. Based on the chronological sequence of disease development and the distribution of the radiation dose in radiosurgery, it is proposed that humoral angiogenetic factors or unknown predisposing factors, rather than direct radiation effects, are the cause of moyamoya disease associated with a cAVM.
[fig] Figure 1: confirmed cure of the right anterior temporal cAVM and the presence of moyamoya disease with occlusion of the bilateral supraclinoid internal Moyamoya disease and concurrent cerebral arteriovenous malformation in 36-year-man. A. Composite anteroposterior (AP) view of bilateral carotid angiogram shows right temporal cerebral arteriovenous malformation. B, C. Stereotactic MRI with dose plan show that 17.5 Gy (yellow isodose line) is prescribed to perihphery of cerebral arteriovenous malformation nidus (magenta line). Supraclinoid segment of right internal carotid arteries and proximal middle cerebral artery received dose of 6.25 Gy (green isodose line). D, E. Nine months (D) and 34 months (E) after gamma-knife surgery. Time-of-flight (TOF) MR angiograms show progressive focal stenosis at bilateral middle cerebral artery. F. At 51 months after gamma-knife radiosurgery, bilateral carotid angiogram confirms cure of cerebral arteriovenous malformation and occlusion of bilateral supraclinoid internal carotid arteries, proximal middle cerebral artery and anterior cerebral artery with moyamoya collateral vessels. A1 segments of the ACA and collateral vessels in the bilateral basal ganglia. [/fig]
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Emerging novel targets for nonalcoholic fatty liver disease treatment: Evidence from recent basic studies
Nonalcoholic fatty liver disease (NAFLD), a leading chronic disease worldwide, affects approximately a quarter of the global population. Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD and is more likely to progress to liver fibrosis than simple steatosis. NASH is also identified as the most rapidly growing cause of hepatocellular carcinoma. Although in the past decade, several phase II/III clinical trials have shown promising results in the use of novel drugs targeting lipid synthase, farnesoid X receptor signaling, peroxisome proliferatoractivated receptor signaling, hepatocellular injury, and inflammatory signaling, proven pharmaceutical agents to treat NASH are still lacking. Thus, continuous exploration of the mechanism underlying the pathogenesis of NAFLD and the identification of novel therapeutic targets remain urgent tasks in the field. In the current review, we summarize studies reported in recent years that not only Wang GY et al. Basic study for NAFLD treatment WJG https://www.wjgnet.com 76 January 7, 2023 Volume 29 Issue 1provide new insights into the mechanisms of NAFLD development but also explore the possibility of treating NAFLD by targeting newly identified signaling pathways. We also discuss evidence focusing on the intrahepatic targets involved in the pathogenesis of NAFLD as well as extrahepatic targets affecting liver metabolism and function.Core Tip: Because of the urgent need to develop therapeutic approaches to treat nonalcoholic fatty liver disease (NAFLD), a large body of basic research has focused on the mechanisms of NAFLD to explore the possibility of new approaches to treat the disease. The current review summarizes studies reported in recent years that not only provide new insights into the mechanisms of NAFLD development but also explore the possibility of treating NAFLD by targeting newly identified signaling pathways. Evidence focusing on the intrahepatic targets involved in the pathogenesis of NAFLD as well as extrahepatic targets affecting liver metabolism and function are discussed. Citation: Wang GY, Zhang XY, Wang CJ, Guan YF. Emerging novel targets for nonalcoholic fatty liver disease treatment: Evidence from recent basic studies. World J Gastroenterol 2023; 29(1): 75-95Lipid accumulation in hepatocytes is a major feature of NAFLD. Disturbance of lipid and glucose metabolism causes metabolic stress in the liver. Therapeutic candidates directly regulating fatty acid metabolism that are currently in clinical trials include ACC, fatty acid synthase and stearoyl-CoA desaturase-1 inhibitors [6,9]. In addition to the targets acting on these lipid metabolic enzymes, in-depth basic research has uncovered multiple mechanisms underlying the metabolic disturbance during NAFLD, including but not limited to lipid, glucose, lactate and fructose metabolism, and has provided novel targets for NAFLD treatment. Sterol regulatory element-binding proteins (SREBPs), including SREBP1a, SREBP1c and SREBP2, are key regulators mediating free fatty acid, triglyceride (TG) and cholesterol synthesis. 25-Hydroxanoanosterol (25-HL) is a newly identified SREBP inhibitor that induces the SCAP-INSIG interaction, which retains SREBPs in the endoplasmic reticulum (ER). 25-HL reduces hepatic TG and cholesterol levels and improves liver inflammation and fibrosis in western diet-fed mice [10]. Orosomucoid (ORM) 2, a secreted protein, has been shown to inhibit SREBP1c by activating AMP-activated protein kinase (AMPK). Recombinant ORM2 protein or stabilized ORM2-FC fusion protein inhibits lipogenesis and improves steatohepatitis [11].Citrate regulates lipid metabolism as the substrate for lipogenesis. In the cytoplasm, citrate is catabolized by ATP-citrate lyase (ACLY) to generate acetyl-CoA and oxaloacetate. Acetyl-CoA is then converted to malonyl-CoA by ACC to fuel lipogenesis. The mitochondrial citrate carrier Slc25al plays an important role in regulating cytoplasmic and mitochondrial citrate pools. Tan and colleagues revealed that an inhibitor of Slc25a1, CTPI-2, inhibits the lipogenic pathway and prevents hepatic lipid accumulation in high-fat diet (HFD)-fed mice. Additionally, CTPI-2 also showed beneficial effects on liver inflammation [12]. ACLY, the enzyme mediating the production of acetyl-CoA from citrate, has been identified to be upregulated in the transition from simple steatosis to NASH [13]. Hepatocyte-specific deletion of ACLY reduces liver fatty acid and sterol synthesis, increases fatty acid oxidation, and attenuates glucose intolerance, liver steatosis, and ballooning [14]. Consistent with the observations following genetic inhibition, bempedoic acid, a pharmacological inhibitor of ACLY, also reduces fibrosis [14]. During the de novo lipogenesis process, nicotinamide adenine dinucleotide phosphate (NADPH), as an electron donor, aids in the reduction of acetyl-CoA. Thus, fine-tuning the NADPH pool can regulate lipogenesis. One-carbon units come largely from serine catabolism by the enzyme serine hydroxymethyltransferase (SHMT) [15]. Zhang et al [16] demonstrated that SHMT1-driven serine catabolism is a substantial NADPH source in the liver. They further found that inhibition of serine catabolism by Shmt1 gene knockout or pharmacological inhibition of SHMT1/2 enzymes significantly decreased hepatic lipogenesis [16]. Moreover, in sucrose-induced fatty liver, lipogenesis also requires NADPH from serine catabolism, which can be inhibited by the SHMT1/2 inhibitor SHIN2 IV. Therefore, SHMT1/2 inhibition has the potential to treat NAFLD by decreasing de novo lipogenesis.As an energy source and an important glycolysis product, lactate is increased in the plasma and liver of NAFLD individuals. A recent study showed that the acetylation of lactate dehydrogenase B (LDHB) is markedly increased in the livers of mice with NAFLD, which leads to decreased LDHB activity and increased hepatic lactate levels. The authors further found that P300/CBP-associated factor (PCAF)mediated LDHB acetylation at K82 exacerbates lipid accumulation and inflammatory responses in HFDfed mice. Consistently, embelin, an inhibitor of PCAF, significantly improves hepatic steatosis and inflammation in NASH mice [17]. In addition, increasing evidence has demonstrated that overconsumption of fructose is associated with NAFLD. Fructose is metabolized to fructose-1-phosphate by ketohexokinase. PF-06835919 is a ketohexokinase inhibitor that showed protective effects against fructose-induced liver steatosis. Moreover, the safety of PF-06835919 has been verified in a phase I clinical trial [18].Mitochondrial and ER functions are crucial in lipid and glucose metabolism, and mitochondrial dysfunction and ER stress are closely related to NAFLD [19]. Methylation-controlled J (MCJ) protein is located at the inner mitochondrial membrane and restrains mitochondrial respiration. Its expression level is found to be increased in NAFLD patients. MCJ deficiency reduces hepatic steatosis and improves liver fibrosis in methionine-and choline-deficient (MCD) diet-fed mice. In a recent study, lipid-nanoparticle-encapsulated (LNP) siRNA was employed to target MCJ in vivo. LNP-siMCJ increased the β-oxidation of fatty acids and ameliorated lipid accumulation and liver fibrosis in MCD diet-or high-fat/high-fructose diet-induced NASH mice. To further specifically target MCJ in hepatocytes, N-acetylgalactosamine (GalNAc)-modified siMCJ was used to treat NASH mice. Consistent with LNP-siMCJ, GalNAc-siMCJ also reduced liver steatosis and fibrosis [20]. In addition, low-dose sorafenib, the first small-molecule multi-kinase inhibitor, was reported to induce mitochondrial uncoupling and subsequently suppress free fatty acid-induced lipid accumulation and inflammation by activating AMPK in hepatocytes. Low-dose sorafenib protects high-fat/highcholesterol diet-fed mice against liver steatosis, inflammation and fibrosis and prevents the onset of NASH-associated HCC in mice. Notably, the beneficial effects of low-dose sorafenib were also confirmed in NASH monkeys [21]. Moreover, cyclophilin D is located in the mitochondrial matrix and mediates mitochondrial permeability transition pore opening. Cyclophilin D is upregulated in HFD-fed mice, which induces mitochondrial stress and hepatic steatosis. Cyclosporine A, an inhibitor of Wang GY et al. Basic study for NAFLD treatment WJG https://www.
# Introduction
Nonalcoholic fatty liver disease (NAFLD) has become a leading chronic disease worldwide, affecting approximately a quarter of the global population. Nonalcoholic steatohepatitis (NASH), the advanced form of NAFLD, is closely related to liver fibrosis and even cirrhosis [bib_ref] NASH limits anti-tumour surveillance in immunotherapy-treated HCC, Younossi [/bib_ref]. NASH has also been identified as the most rapidly growing cause of hepatocellular carcinoma (HCC) in liver transplant candidates in the United States . Multiple factors, including disturbed lipid homeostasis, insulin resistance, and inflammation, lead to metabolic stress in hepatocytes and subsequent hepatocyte injury in NAFLD. Hepatocyte injury further triggers the wound-healing response, which involves immune cells, liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs) and cholangiocytes, eventually leading to liver inflammation and fibrosis .
Despite the increasing prevalence of NASH, the United States Food and Drug Administration (FDA) has not yet approved any pharmaceuticals to treat patients with NASH. The mainstay of the current clinical recommendation for patients with NASH is lifestyle modification, with optimization of dietary structure, control of excessive consumption of fructose and fats and increased physical exercise, which may alleviate the progression of NASH to a certain extent . In addition, bariatric surgery may be indicated for some patients with NASH suffering from severe obesity . Regarding the pharmacotherapy for NASH, several biological pathways critical for glycolipid and bile acid metabolism, inflammation, hepatocellular damage (oxidative stress) and liver fibrosis have been explored as drug targets. Pharmaceutical agents, including modulators of farnesoid X receptor, peroxisome proliferator-activated receptors (PPARs), fibroblast growth factor, acetyl-CoA carboxylase (ACC), and apoptosis signalregulating kinase 1 (ASK1), have been shown to exhibit some positive effects against NAFLD/NASH with various limitations in multiple clinical trials . Thus, there is still an unmet clinical need to identify and validate novel targets for the treatment of NAFLD/NASH.
Recently, numerous novel therapeutic targets for NAFLD have been explored through basic research. These studies have focused on different pathophysiologic processes in NAFLD, including metabolic stress, liver inflammation, liver fibrosis and NASH-associated HCC. Diverse liver cells have been studied, such as hepatocytes, liver immune cells, HSCs and LSECs . As a metabolic disorder, crosstalk between the liver and extrahepatic organs, including the gut, adipose tissue, and skeletal muscle, largely contributes to the pathogenesis of NAFLD . Thus, targets outside the liver broaden potential approaches to ameliorate NAFLD . The drugs in phase II/III clinical trials were extensively discussed in a recently published review . In the present review, we summarize recent findings that not only provide new insights into the mechanisms of NAFLD development but also explore the possibility of treating NAFLD by targeting novel signaling pathways. cyclophilin D, decreased liver TG levels in HFD-fed mice by decreasing SREBP1c expression . ER stress also plays important roles in NAFLD. The expression of forkhead box A3 (FOXA3) is induced by ER stress, and FOXA3 mediates ER stress-induced liver steatosis. As expected, targeting FOXA3 via a siRNA-based approach attenuates liver steatosis in HFD-fed mice .
It has been previously reported that the expression of DEAD-box protein 5 (DDX5), an ATPdependent RNA helicase, is reduced in the livers of patients and mouse models with NASH, as well as in rodent models with NASH-HCC . Hepatic DDX5 improves lipid metabolism by inhibiting mammalian target of rapamycin complex 1 (mTORC1) activation via recruitment of the tuberous sclerosis complex (TSC)1/2 complex to mTOR . Through screening a natural compound library, hyperforcinol K was found to upregulate DDX5 expression by blocking tripartite motif protein 5mediated ubiquitinated degradation of DDX5. In an animal study, hyperforcinol K was found to be effective in attenuating lipid accumulation in the livers of NASH mice .
Sterile 20-type kinase serine/threonine kinase 25 (STK25) has been demonstrated to play important roles in liver lipid partitioning and systemic glucose and insulin homeostasis . The level of STK25 protein is positively correlated with the development of NASH in patients . Moreover, STK25 deficiency can protect against liver steatosis, inflammation, and fibrosis in mouse models of NASH-HCC . STK25 antisense oligonucleotides (ASOs) significantly improved the NASH phenotype, and this preclinical validation of the effective metabolic efficacy of pharmacological inhibition of STK25 merits recognition .
Many nuclear receptors play critical roles in metabolic regulation. In current phase II and III trials, PPARs, farnesoid X receptor, and thyroid hormone receptor-β are promising candidate targets for NASH treatment[6]. Based on recent basic studies, modulation of retinoic acid receptor-related orphan receptor alpha (RORa) provides a novel target to treat NASH. RS-2982, a newly identified agonist of RORa, exhibited beneficial effects on reducing body weight and hepatic steatosis in HFD-fed mice. Furthermore, RS-2982 decreased alanine aminotransferase (ALT) and aspartate aminotransferase levels and reduced liver inflammation and fibrosis in mice fed an atherogenic diet. Mechanistically, an increase in miR-122 expression may account for these beneficial effects of RS-2982 on obesity and NAFLD/NASH .
Arachidonic acid (ARA) is an ω-6 long-chain polyunsaturated fatty acid (PUFA) that can be catalyzed into a series of bioactive eicosanoids via cycloxygenases (COX-1 and COX-2) and microsomal prostaglandin E synthases (mPGES-1 and mPGES-2). It has been recently reported that mPGES-2 deficiency exhibits significant protective effects against diet-induced NASH-associated phenotypes, including hepatic steatosis, inflammation and fibrosis. However, the beneficial effect of mPGES-2 deficiency against NAFLD is dependent on decreased cytochrome P450 4A14 (CYP4A14) and increased acyl-CoA thioesterase 4 levels but not PGE2 . Mechanistically, mPGES-2 binds with heme, which is released after mPGES2 inhibition and in turn activates the heme receptor nuclear receptor subfamily 1 group D member 1 (NR1D1) to upregulate CYP4A14 and acyl-CoA thioesterase 4 expression[31]. CYP4A14 catalyzes omega-hydroxylation of medium-chain fatty acids and ARA in mice. Its expression was previously found to be significantly increased in the livers of patients and mice with NAFLD. Loss of CYP4A14 function also markedly attenuated liver damage, inflammation, and fibrosis in MCD dietinduced NASH . Moreover, both the mPGES-2 inhibitor SZ0232 and the CYP4A inhibitor TS-011 are capable of ameliorating the NASH phenotype in MCD diet-fed mice . Taken together, these findings demonstrate that ARA metabolic enzymes play critical roles in liver lipid homeostasis and represent attractive targets for developing therapeutic drugs for NAFLD/NASH.
Emerging evidence suggests that novel therapeutic targets can be developed based on the discovery of genetic variants related to liver lipid metabolism, such as PNPLA3, TM6SF2, MBOAT7 and HSD17B13 [34-37]. It has been reported that phosphorylation of HSD17B13 at serine 33 by PKA promotes lipolysis. Reproterol, a β2 agonist used for treating asthma, protects against the NASH phenotype via PKAmediated Ser33 phosphorylation of 17β-HSD13 . Most recently, a variant in the pleckstrin and Sec7 domain-containing 3 (PSD3) gene, rs71519934, was reported to reduce susceptibility to fatty liver disease, consistent with the finding that the expression level of PSD3 is increased in patients with NAFLD and that knockdown of PSD3 by siRNA decreases TG synthesis in hepatocytes. Furthermore, specific downregulation of PSD3 in hepatocytes by GalNAc-conjugated ASOs resulted in decreased hepatic lipid content and plasma ALT levels and improved liver fibrosis in NASH mice , suggesting that PSD3 may be a potential therapeutic target for the treatment of NAFLD/NASH.
## Strategies protecting hepatocytes from injury during nash development
Lipid accumulation in hepatocytes can cause cytotoxicity (lipotoxicity) in NAFLD . Hepatocyte injury is a key event during the development of NASH, and ballooned hepatocytes are an essential feature to diagnose NASH. In phase II/III clinical trials, pancaspase inhibitors and ASK1 inhibitors are currently under evaluation as therapeutic agents directly targeting apoptosis[6]. Studies focused on developing targets to prevent hepatocyte injury have also shown therapeutic effects in NASH.
Mitochondrial stress is a major factor driving hepatocyte injury and promotes the progression from simple steatosis to NASH. Mitochondrial matrix caseinolytic protease P (ClpP) is a protease component of the caseinolytic protease complex that maintains protein homeostasis in mitochondria. Its expression level is frequently downregulated in NASH, which induces mitochondrial stress and inflammation in hepatocytes. The ClpP activator A54556A is capable of greatly ameliorating the NASH phenotype in high-fat/high-fructose diet-fed mice . SH3 homology-associated BTK binding protein (SAB) was originally identified as a phosphorylated c-Jun N-terminal kinase (p-JNK) docking protein and a substrate of JNK in the mitochondrial outer membrane. The interaction of JNK with SAB leads to increased mitochondrial reactive oxygen species (ROS) production and promotes liver injury. In established NASH, hepatocyte-targeted GalNAc-Sab-ASO treatment can reverse steatohepatitis and fibrosis by abrogating the adverse effects of the JNK-SAB-ROS activation loop . Sirtuin activation plays an important role in maintaining mitochondrial homeostasis and shows protective effects on NAFLD . Increased NAD + levels can activate sirtuin. The enzyme α-amino-β-carboxymuconate-εsemialdehyde decarboxylase controls NAD + levels, and its inhibitor TES-991 boosts de novo NAD + synthesis and protects mice against liver injury and steatosis by improving mitochondrial function These findings demonstrate that strategies that increase Nrf2 expression and activity may be attractive strategies to limit the development of NAFLD/NASH by attenuating hepatocyte lipotoxicity and injury.
As mentioned above, apoptosis plays an important role in hepatocyte injury or loss during the development of NAFLD/NASH. BCL-2 belongs to the BCL-2 family, which is antiapoptotic. Through screening a small molecule library, acridone derivative A22 was found to increase BCL-2 expression by stabilizing the BCL-2 promoter i-motif. As expected, A22 can attenuate HFD-induced hepatocyte injury, hepatic steatosis and liver fibrosis . In addition, necroptosis, another form of programmed cell death, was found to be increased in NASH mouse models and the livers of NAFLD patients. Receptorinteracting protein 3 (RIP3) and receptor-interacting protein kinase (RIPK) 1 are key mediators of necroptosis. Liver RIP3 deficiency can attenuate MCD diet-induced liver injury, steatosis, inflammation and fibrosis . Similarly, the RIPK1 inhibitor RIPA-56 can reduce liver inflammation and fibrosis in HFD-fed mice by abrogating necroptosis. In addition, RIPA-56 is able to ameliorate hepatic steatosis by suppressing mixed lineage kinase domain-like protein levels .
Iron overload leads to hepatic oxidative stress and hepatocellular ballooning injury and plays a multifactorial role in the pathogenesis of NASH . Approximately one-third of patients with NAFLD show interrupted iron homeostasis . Ferroptosis is a nonapoptotic programmed cell death process characterized by iron-dependent and lipid peroxidation-associated cell death . The ferroptosis inhibitors Trolox and deferiprone can protect hepatocytes from cell death and suppress the subsequent initiation of inflammation in fatty liver .
In contrast to hepatocytes, agents that induce HSC apoptosis show beneficial effects against NASHrelated liver fibrosis[61]. Thus, when using pharmacological agents to prevent hepatocyte injury, strategies more accurately targeting hepatocytes may strengthen the therapeutic effects of these agents against NASH with limited side effects.
## Approaches targeting the inflammatory pathway to treat nafld/nash
Inflammatory responses mediated by various immune cells and hepatocytes promote the onset of NASH and liver fibrosis. Infiltration of neutrophils and macrophages is the main pathological feature of NASH. Neutrophil depletion attenuates diet-induced NASH[62]. CXC chemokine receptor 2 (CXCR2) signaling is considered to play a pivotal role in the entry of neutrophils into peripheral tissues. Leslie and colleagues demonstrated that human and mouse livers with NASH-HCC have more CXCR2 + neutrophils . The expression of CXCR2 in neutrophils is induced in NASH in an autocrine manner involving the upregulation of neutrophil-derived lipocalin 2[64]. AZD5069 is a small-molecule inhibitor of CXCR2. AZD5069 can significantly improve liver pathology in NAFLD, with reduced lipid content and hepatic neutrophil accumulation and improved insulin sensitivity . In addition, AZD5069mediated CXCR2 inhibition induces reprogramming of the tumor immune microenvironment, which promotes immune checkpoint inhibition in NASH-HCC[63], suggesting that blocking infiltrating neutrophil CXCR2 can restore sensitivity to immunotherapy in the NASH liver[66].
It is well known that the expression of macrophage scavenger receptor 1 (MSR1) mediates lipid uptake in macrophages and subsequently induces inflammatory activation via the JNK pathway. MSR1 was found to be correlated with liver inflammation in NAFLD patients . Notably, therapeutic inhibition of MSR1 with an anti-MSR1 antibody improved hepatic steatosis in a mouse model of NASH. The number of F4/80-positive cells and the expression level of tumor necrosis factor alpha (TNF-α) were also found to be reduced by the anti-MSR1 antibody therapy .
It has long been recognized that the NOD-like receptor protein 3 (NLRP3) inflammasome is activated in NASH livers, which leads to increased inflammation and programmed cell death. MCC950, an NLRP3 inhibitor, can improve NAFLD pathology and fibrosis in obese diabetic mice [bib_ref] Role of XBP1 in regulating the progression of non-alcoholic steatohepatitis, Wang [/bib_ref]. X-box binding protein-1 (XBP1) is a key factor regulating the unfolded protein response. Macrophage XBP1 is upregulated in NASH livers, which activates macrophage NLRP3 signaling and promotes hepatocyte steatosis and HSC activation. As expected, the XBP1 inhibitor toyocamycin exhibits protective effects against NASH [bib_ref] Critical Role of NFκB in the Pathogenesis of Non-alcoholic Fatty Liver Disease:..., Franceschetti [/bib_ref].
Nuclear factor-kappaB (NF-κB) is an essential transcription factor that mediates the inflammatory response and proinflammatory cytokine production in NASH [bib_ref] MicroRNA-378 promotes hepatic inflammation and fibrosis via modulation of the NF-κB-TNFα pathway, Zhang [/bib_ref]. miR-378 markedly facilitates the NF-κB-TNF-α axis in NASH development by directly targeting the Prkag2 gene, which encodes AMPK-γ2. Similarly, downregulation of miR-378 by ASO is also capable of alleviating NASH development [bib_ref] Immunomodulatory liposomes targeting liver macrophages arrest progression of nonalcoholic steatohepatitis, Maradana [/bib_ref].
In addition to neutrophils and macrophages, other immune cells, including dendritic cells (DCs) and lymphocytes, play pivotal roles in NASH . DC recruitment to the liver promotes the inflammatory response in NAFLD progression. Reportedly, delivery of liposomal curcumin or calcitriol to lipid-rich inflammatory DCs shifted their inflammatory profile toward a regulatory phenotype and improved hepatic steatosis, inflammation and fibrosis in a NASH mouse model . Inflammatory CX 3 CR1 + monocyte-derived inflammatory DCs (moDCs) are found to contribute to sustained inflammation and liver injury during NASH. Treating MCD-fed mice with the hydrogen sulfide donor i.e., sodium hydrosulphide, prevented the accumulation of CX 3 CR1 + moDCs and ameliorated parenchymal injury [bib_ref] Toll-Like Receptor-7 Signaling Promotes Nonalcoholic Steatohepatitis by Inhibiting Regulatory T Cells in..., Roh [/bib_ref]. Moreover, Toll-like receptor 7 (TLR7) signaling can induce proinflammatory cytokine production in Kupffer cells and DCs, subsequently suppressing regulatory T cells (Tregs) and leading to steatohepatitis. Notably, treatment with IRS-661, a TLR7 antagonist, could ameliorate NASH development [bib_ref] Blocking integrin α(4)β(7)-mediated CD4 T cell recruitment to the intestine and liver..., Rai [/bib_ref]. It has been reported that α4β7-mediated homing of CD4 T cells to the intestine and liver facilitates NASH development, and α4β7 blockade by neutralizing monoclonal antibody can attenuate hepatic inflammation and fibrosis and improve metabolic dysfunction associated with NASH [bib_ref] Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic..., Wang [/bib_ref]. In addition, a high level of Tregs reportedly promotes the initiation and progression of cancer in NASH livers. In a NASH-HCC model, anti-CD25 antibodies, capable of alternatively depleting Tregs, decreases the tumor burden and increased survival time . The interaction of B2 Lymphocytes with T cells contributes to NASH progression, B-cell activating factor-neutralizing monoclonal antibody Sandy-2 prevented hepatic B2 cell response and ameliorated the evolution of NASH in mice [bib_ref] Editorial: The Role of Bioactive Lipids in Homeostasis and Pathology, Wang [/bib_ref].
Bioactive lipids are important nodes in lipid metabolism and tissue homeostasis networks . An imbalance between protective and deteriorative bioactive lipids contributes to NASH progression . Pharmacological targeting of the synthesizing enzymes or receptors of these bioactive lipids has shown beneficial effects in NASH. Leukotriene B4 (LTB4), a proinflammatory metabolite derived from the ω-6 PUFA ARA, is significantly increased in patients with NAFLD . Both in vivo and in vitro experiments have shown that LTB4 can promote hepatocyte lipogenesis, which is dependent on the RNase activity of IRE1α through leukotriene B4 receptor 1 (Ltb4r1). Furthermore, LTB4/Ltb4r1 stimulation increases intracellular cAMP and then promotes IRE1α Ser724 phosphorylation by PKA . The Ltb4r1 inhibitor CP-105696 significantly alleviated ER stress and dyslipidemia in NAFLD mice . Unlike ω-6 PUFAs, ω-3 PUFAs and their metabolites show anti-inflammatory effects. Fat-1 mice, a transgenic animal model in which tissues are endogenously enriched with ω-3 PUFAs[81], are protected from diet-induced metabolic dysfunction and fibrosis . However, they are vulnerable to lipid peroxidation, which limits their clinical applications . Fraser et al developed a structurally modified ω-3 fatty acid, icosabutate, which was designed to resist oxidation and incorporation into hepatocytes and possesses the potential to activate free fatty acid receptor 4. They found that icosabutate, but not the ω-3 PUFA eicosapentaenoic acid, can ameliorate liver inflammation and fibrosis in NASH rats. Moreover, icosabutate treatment decreases liver injury in patients at high risk of NASH and cardiovascular disease [84].
## Approaches targeting hsc activation to prevent nash-related liver fibrosis
In clinical trials for NASH drug development, improvement of liver fibrosis has been frequently employed as a primary or secondary endpoint. Given the central role of HSCs in liver fibrosis, targeting HSC activation has long been proposed as a therapeutic strategy to prevent NASH-related fibrosis progression.
The Notch, Hedgehog, Hippo and WNT/β-catenin signaling pathways in hepatocytes play important roles in NASH-related liver fibrosis by modulating the hepatic microenvironment. The production of osteopontin (OPN), as a paracrine factor, from hepatocytes or cholangiocytes increases in NASH, which leads to the activation of HSCs[85-87]. Increased Notch signaling activity has been found to be responsible for the induction of OPN . Thus, several studies have targeted Notch signaling to treat NASH, including studies on Nicastrin ASO and nanoparticle-mediated delivery systems targeting Notch antagonism [bib_ref] Targeting NFATc4 attenuates non-alcoholic steatohepatitis in mice, Du [/bib_ref]. In addition, nuclear factor of activated T-cell 4 (NFATc4) was also reported to induce OPN expression by negatively regulating the transcriptional activity of PPARα. As expected, inhibition of NFATc4 decreased lipid content and improved inflammation and fibrosis in NASH mice [bib_ref] Hepatocyte TAZ/WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis, Wang [/bib_ref]. Hepatocyte transcriptional co-activator with PDZ-binding motif (TAZ) was found to promote NASH-related liver fibrosis by increasing Indian hedgehog, which activates HSCs [bib_ref] A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in..., Wang [/bib_ref]. A recent study reported that stabilized GalNAc-siRNAs targeting hepatocyte TAZ can significantly ameliorate liver inflammation and fibrosis in mice with established NASH[91]. Furthermore, WNT1-inducible signaling pathway protein 1 (WISP1), a member of the cellular communication network family, has recently been identified as an extracellular activator of the myocardin-related transcription factor-cytoskeleton pathway in HSCs. Activation of the WISP1-MRIF signaling pathway induces HSC migration and promotes liver fibrosis progression. Notably, an anti-WISP1 antibody significantly ameliorated liver fibrosis in NASH mice [bib_ref] Inhibiting Interleukin 11 Signaling Reduces Hepatocyte Death and Liver Fibrosis, Inflammation, and..., Widjaja [/bib_ref].
Lnterleukin-11 (IL-11) has been demonstrated to activate HSCs. Deletion of IL-11 receptor subunit alpha (IL-11RA) protects mice from NASH diet-induced hepatocyte death, liver inflammation and fibrosis. Widjaja et al [bib_ref] Ruxolitinib suppresses liver fibrosis progression and accelerates fibrosis reversal via selectively targeting..., Song [/bib_ref] developed a neutralizing anti-IL-11 antibody and neutralizing anti-IL-11RA antibody and found that both of them greatly decreased ALT levels and significantly improved hepatic steatosis and liver fibrosis in mouse models of NASH [bib_ref] Ruxolitinib suppresses liver fibrosis progression and accelerates fibrosis reversal via selectively targeting..., Song [/bib_ref].
Activation of the JAK/signal transducer and activator of transcription (STAT) pathway in HSCs promotes liver fibrosis. Ruxolitinib, an effective small-molecule JAK1/2 selective inhibitor, has been approved by the FDA for myelofibrosis treatment. Recently, it has been reported that ruxolitinib can block HSC activation and attenuate liver fibrosis progression [bib_ref] Janus kinase 2 inhibition by pacritinib as potential therapeutic target for liver..., Torres [/bib_ref]. In addition, the JAK2 inhibitor pacritinib affords protection against NAFLD-related liver fibrosis by inhibiting HSC activation [bib_ref] Blas-García A. Rilpivirine attenuates liver fibrosis through selective STAT1-mediated apoptosis in hepatic..., Martí-Rodrigo [/bib_ref]. Rilpivirine, a nonnucleoside reverse transcriptase inhibitor, is widely used to treat HIV infection. Rilpivirine can also ameliorate liver fibrosis, possibly through selective STAT1-dependent induction of apoptosis in HSCs, and suppress HFD-and CCl4-induced liver fibrosis. In addition, rilpivirine enhances STAT3-dependent proliferation in hepatocytes as an effect secondary to its pro-apoptotic effect in HSCs .
Protease-activated receptor-2 (PAR2) is an emerging new target for NASH that regulates liver injury, inflammation and fibrosis and plays a critical role in regulating hepatic cholesterol and glucolipid metabolism [bib_ref] PAR2 promotes impaired glucose uptake and insulin resistance in NAFLD through GLUT2..., Shearer [/bib_ref] [bib_ref] PAR2 controls cholesterol homeostasis and lipid metabolism in nonalcoholic fatty liver disease, Rana [/bib_ref] [bib_ref] Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease, Hammoutene [/bib_ref]. Pharmacological inhibition of PAR2 with pepducin PZ-235, a full antagonist of PAR2, not only protects against the activation of HSCs and fibrosis but also promotes hepatocellular viability by inhibiting mitochondrial ROS production induced by PAR2 stimulation [bib_ref] PAR2 promotes impaired glucose uptake and insulin resistance in NAFLD through GLUT2..., Shearer [/bib_ref].
## Approaches targeting lsecs and cholangiocytes to treat nafld
LSECs are highly specialized endothelial cells with fenestrae and lack a basement membrane. LSECs interact with hepatocytes, liver immune cells and HSCs and play important roles in regulating liver function. LSEC dysfunction, including LSEC capillarization, disturbed nitric oxide release from LSECs, and increased expression of adhesion molecules, all contribute to NAFLD pathogenesis [bib_ref] Lipidinduced endothelial vascular cell adhesion molecule 1 promotes nonalcoholic steatohepatitis pathogenesis, Furuta [/bib_ref]. Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule upregulated in LSECs in NASH mouse livers. Anti-VCAM1 antibody attenuates hepatic inflammation in NASH mice [bib_ref] Notch-triggered maladaptation of liver sinusoidal endothelium aggravates nonalcoholic steatohepatitis through endothelial nitric..., Fang [/bib_ref]. Endothelial nitric oxide synthase (eNOS) expression in LSECs is decreased in NASH mice, which is likely the result of Notch activation. Pharmacological inhibition of Notch signaling using DAPT and LY3039478, as well as the eNOS activator YC-1, improved the NASH phenotype in MCD diet-fed mice [bib_ref] Intrahepatic paracrine signaling by cardiotrophin-like cytokine factor 1 ameliorates diet-induced NASH in..., Liu [/bib_ref]. Although the role of LESCs in NAFLD has been increasingly emphasized, the underlying mechanisms and therapeutic approaches targeting LESCs need more exploration.
Cholangiocytes also play an important role in NASH development. In addition to hepatocytes, OPN is strongly expressed in cholangiocytes in NASH livers. Thus, it is highly possible that cholangiocytederived OPN can promote liver fibrosis by activating HSCs[87]. Cardiotrophin-like cytokine factor 1 is another cholangiocyte-derived paracrine factor that exerts beneficial effects in NASH. Downregulation of a subunit of its receptor complex leukemia inhibitory factor receptor contributes to the progression of NASH [bib_ref] Dietary carbohydrates and fats in nonalcoholic fatty liver disease, Yki-Järvinen [/bib_ref]. Compared with other nonparenchymal cells, there are fewer studies addressing the effects of cholangiocytes on NASH, and a related therapeutic approach is still lacking.
## Potential targets outside the liver
It is well-established that multiple organs form a network to regulate whole-body energy metabolism.
Extrahepatic organs influence liver function in an endocrine manner by releasing hormones or inflammatory factors or by regulating systemic metabolic homeostasis such as energy expenditure and insulin sensitivity. Thus, targeting these extrahepatic organs provides additional options for treating NAFLD/NASH.
## Targets affect systemic energy metabolism
Diets with reduced contents of sugars, refined carbohydrates and saturated fat are recommended to treat NAFLD [bib_ref] The Role of Exercise in the Interplay between Myokines, Hepatokines, Osteokines, Adipokines,..., Gonzalez-Gil [/bib_ref]. Moreover, exercise is considered as an effective strategy for preventing and treating NAFLD via metabolic pathways and interorgan crosstalk [bib_ref] Regulatory effects mediated by ulvan oligosaccharide and its zinc complex on lipid..., Chi [/bib_ref]. Glucagon-like peptide (GLP)-1 receptor agonists, which enhance insulin secretion and reduce food intake, show beneficial effects against NASH in clinical trials . In addition, fibroblast growth factor 21 analogs improved systemic energy metabolism and demonstrated potential in NASH therapy . In a recent basic study, a novel Zn supplement, ulvan oligosaccharide (UO)-Zn, can activate AMPK, leading to improved metabolic pathways in HFD-fed mice [bib_ref] The BCKDH Kinase and Phosphatase Integrate BCAA and Lipid Metabolism via Regulation..., White [/bib_ref]. The increased hepatic expression of branched chain ketoacid dehydrogenase kinase (BDK) partly induced high levels of branched-chain amino acids (BCAA) in metabolic diseases [bib_ref] Branched-chain amino acids in disease, White [/bib_ref] [bib_ref] Gut Microbiota-A Future Therapeutic Target for People with Non-Alcoholic Fatty Liver Disease:..., Forlano [/bib_ref]. Moreover, 3,6-dichlorobrenzo(b)thiophene-2-carboxylic acid, a small-molecule allosteric inhibitor of BDK, lowered levels of circulating BCAAs, reduced hepatic steatosis, and improved glucose tolerance in rodent models[107,108].
## The gut-liver axis
The crosstalk between the gut and liver is regarded as an important pathway in regulating hepatic functions. Intestinal inflammation and dysfunction of intestinal lipid and bile acid absorption, epithelial cell permeability and the microbiome affect liver function and contribute to NAFLD progression. The relationship between gut microbiota and potential therapeutic targets has been recently discussed in depth elsewhere [bib_ref] Role and effective therapeutic target of gut microbiota in NAFLD/NASH, Liu [/bib_ref] ; thus, we only focus on other aspects of the gut-liver axis.
Serum ceramide levels are positively related to NAFLD, and intestine-derived ceramide has been demonstrated to promote NAFLD . Intestinal hypoxia-inducible factor 2α (HIF-2α) and myelocytomatosis oncogene (MYC) affect NAFLD by regulating ceramide metabolism. Intestinal HIF-2α , but not HIF-1α, is activated in obese patients and mice. Intestine-specific HIF-2α knockout improves hepatic steatosis by decreasing intestine-derived ceramide levels. Mechanistically, HIF-2α transcriptionally upregulates the expression of Neu3, a key enzyme in the ceramide salvage pathway. Targeting HIF-2α with its inhibitor PT2385 significantly attenuated hepatic lipid accumulation and decreased plasma ALT levels in HFD-fed mice . Moreover, oral administration of PT2385 contributes to reduced body weight and improved insulin sensitivity . Intestinal MYC expression is positively related to body mass index and serum ALT levels in humans. Genetic ablation of MYC in the intestine suppressed obesity and hepatic steatosis and led to decreased ceramide levels in HFD-fed mice. Ceramide synthase 4 has been further demonstrated to be a target gene of MYC. In line with this finding, pharmacological inhibition of MYC with 10058-F4 was found to ameliorate the metabolic disorders and liver fibrosis induced by HFD feeding [bib_ref] Gut bacteria alleviate smoking-related NASH by degrading gut nicotine, Chen [/bib_ref]. In addition, increased intestinal ceramide also meditates the acceleration of NASH induced by nicotine .
Gut-derived 5-hydroxytryptamine (5-HT) has been reported to promote lipogenesis in the liver. Moreover, liver-specific knockout of the 5-HT receptor 2α (Htr2α) gene significantly lowered liver lipid content in HFD-fed mice and decreased the expression of genes involved in lipogenesis. To evaluate the potential of targeting the gut-5-HT-liver Htr2α pathway as a therapeutic strategy for NAFLD, the 5-HT receptor HTR2A antagonist sarpogrelate was used to treat HFD-fed mice. The results showed that sarpogrelate greatly ameliorates hepatic steatosis in NAFLD mice [bib_ref] Intestinal peroxisome proliferatoractivated receptor α-fatty acid-binding protein 1 axis modulates nonalcoholic steatohepatitis, Yan [/bib_ref].
Yan et al [bib_ref] Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective..., Montagner [/bib_ref] recently clarified that gut PPARα activation promotes NASH development by inducing fatty acid binding protein 1 expression, thereby aggravating fatty acid absorption in the small intestine [bib_ref] Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective..., Montagner [/bib_ref]. GW6471, a PPARα-specific antagonist, was found to accumulate in the small intestine at much higher levels than in the liver. Intestine-specific PPARα deficiency and gut PPARα antagonism by GW6471 both improve NASH phenotypes [bib_ref] Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective..., Montagner [/bib_ref]. The pleotropic roles of PPARα in different tissues in modulating NASH are worth discussing. Hepatocyte PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD [bib_ref] MGAT2 inhibitor decreases liver fibrosis and inflammation in murine NASH models and..., Cheng [/bib_ref]. Therapeutic strategies to prevent or treat NAFLD by activating hepatocyte PPARα and/or inhibiting gut PPARα may help guide drug delivery. Monoacylglycerol acyltransferase 2 (MGAT2) mediates the conversion of monoacylglycerol to diacylglycerol and is critical for dietary fat absorption. In the liver, MGAT2 regulates TG synthesis. BMS-963272, a potent MGAT2 inhibitor, improves the NAFLD activity score and liver fibrosis in NAFLD/NASH mice [bib_ref] A GLP-1/GLP-2 receptor dual agonist to treat NASH: Targeting the gut-liver axis..., Kim [/bib_ref]. Of note, its safety has been confirmed in phase 1 clinical trials [bib_ref] A GLP-1/GLP-2 receptor dual agonist to treat NASH: Targeting the gut-liver axis..., Kim [/bib_ref].
In addition, a long-acting dual agonist of GLP-1 and GLP-2 receptors, GLP1/2-Fc, has been found to greatly alter the microbiome composition and exhibits positive effects on gastrointestinal volume and the intestinal barrier. More importantly, GLP1/2-Fc can significantly ameliorate NASH phenotypes, including hepatic fat accumulation, inflammation, fibrosis and insulin tolerance[120], and its effects are superior to those of liraglutide, which has minimal efficacy on inflammation or fibrosis[121]. January 7, 2023
Volume 29 Issue 1
## The adipose tissue-liver axis
Recently, several novel adipokines have been identified and verified to play important roles in NASH development and progression by mediating the crosstalk between adipose tissue and the liver. Isthmin-1 (ISM1) was identified as an adipokine that can activate phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (Akt) signaling independent of insulin. ISM1 gene expression is positively correlated with body mass index, and its deficiency leads to glucose intolerance via inhibition of PI3K-Akt signaling. In addition, ISM1 suppressed de novo lipogenesis in the liver. Recombinant ISM1 protein exhibited beneficial effects on both glucose tolerance and hepatic steatosis in NAFLD mice .
Recently, Liu et al [bib_ref] The Novel Adipokine Gremlin 1 Antagonizes Insulin Action and Is Increased in..., Hedjazifar [/bib_ref] demonstrated that secreted protein acidic and rich in cysteine-like protein 1 (Sparcl1), which is a white adipose tissue-secreted protein, is correlated with hepatic pathological features in NASH patients. The authors further found that sparcl1 can induce liver inflammation by increasing C-C motif chemokine ligand 2 expression in the liver. To further determine the role of sparcl1 in the pathogenesis of NASH, the authors developed a neutralizing antibody against sparcl1 and found that the sparcl1-neutralizing antibody markedly ameliorated liver inflammation and liver fibrosis in NASH mice [bib_ref] The Novel Adipokine Gremlin 1 Antagonizes Insulin Action and Is Increased in..., Hedjazifar [/bib_ref]. Gremlin 1, a newly identified adipokine, is positively related to insulin resistance and NAFLD/NASH. Treatment with recombinant Gremlin 1 protein impairs insulin sensitivity in various cell types, including human primary adipocytes, skeletal muscle cells, and liver cells. The insulinsensitizing effect of the neutralizing anti-Gremlin 1 antibody indicates its beneficial effects on insulin resistance and NAFLD/NASH [bib_ref] Neuregulin 4 suppresses NASH-HCC development by restraining tumor-prone liver microenvironment, Zhang [/bib_ref]. Neuregulin 4 (NRG4) is an adipose tissue-derived endocrine factor that has been demonstrated to suppress NASH-associated HCC by restraining the tumor-prone liver immune microenvironment. A recombinant fusion protein comprising amino acids 1-55 of human NRG4 and the Fc domain of immunoglobulin G 1 was found to suppress HCC induced by a NASH diet plus oncogene overexpression[125]. SWELL1/LRRC8a, a leucine-rich repeat-containing transmembrane protein, functionally encodes an ion channel signaling complex on the adipocyte plasma membrane [bib_ref] SWELL1 is a regulator of adipocyte size, insulin signalling and glucose homeostasis, Zhang [/bib_ref]. Adipocyte SWELL1 is dispensable for adipose development . SWELL1 protein is reduced in the adipocytes of type 2 diabetic animals. The small molecule SN-401, which binds at a constriction point within the SWELL hexamer, can increase adipocyte SWELL1 protein expression and SWELL1-dependent insulin signaling. Similarly, SN-401 can normalize glucose tolerance by augmenting tissue glucose uptake, suppressing hepatic glucose production, increasing serum fibroblast growth factor 21 levels, and reducing hepatic steatosis and hepatocyte ballooning in obese type 2 diabetic mice . Taken together, these findings demonstrate that targeting the adipose tissue-liver axis may be an attractive strategy to treat NAFLD/NASH.
## The skeletal muscle-liver axis
The crosstalk between skeletal muscle and the liver also impacts NAFLD progression. As one of the energy metabolism organs, skeletal muscle can affect liver lipid and glucose metabolism by regulating systemic metabolic homeostasis. In addition, decreased skeletal muscle mass and altered myokine secretion are associated with exacerbation of NAFLD progression [bib_ref] Brief-reports: elevated myostatin levels in patients with liver disease: a potential contributor..., García [/bib_ref]. Myostatin signaling negatively regulates muscle mass and is positively related to liver fibrosis [bib_ref] Irisin is inversely associated with intrahepatic triglyceride contents in obese adults, Zhang [/bib_ref]. Irisin (encoded by fibronectin type III domain containing 5, Fndc5), a myokine induced by exercise, is also expressed in adipose tissue and the liver. Serum irisin levels are inversely associated with TG content in the livers of obese adults [bib_ref] NAD(+)-boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine..., Li [/bib_ref]. Recombinant Fndc5/irisin significantly improved the NASH phenotype in HFD-fed mice . The administration of nicotinamide riboside can alleviate obesity and steatosis by increasing irisin levels in HFD-fed mice . Collectively, therapeutic strategies to improve skeletal muscle quantity and quality or target myokine signaling may provide promising options to treat NASH, but basic studies to collect more evidence are warranted.
# Conclusion
Because of the urgent need to develop therapeutic approaches to treat NAFLD/NASH and the complexity of the pathophysiologic process of NAFLD, a large body of basic research has focused on the mechanisms of NAFLD to explore the possibility of applying new approaches to treat the disease . Metabolic stress-induced hepatocyte injury is an initial factor driving NASH development. Thus, in hepatocytes, strategies for treating NASH aim to improve metabolic disturbance and protect hepatocytes from injury. Alteration of the liver microenvironment and the interaction between diverse liver cells promote liver inflammation and fibrosis. Therefore, the inflammatory response of hepatocytes and immune cells in the liver is also an important target for NASH treatment. Strategies targeting HSC activation, proliferation and migration show beneficial effects on liver fibrosis and may become a novel way to attenuate NASH outcomes . As an indispensable part of hepatic metabolic regulation, extrahepatic organs play an essential role in liver lipid homeostasis, and their dysfunction contributes to the development and progression of NAFLD/NASH. The gut, adipose tissue and skeletal muscle can functionally affect liver or whole-body energy metabolic homeostasis via various endocrine factors and [fig_ref] Table 2: Novel extrahepatic targets explored by recent basic study [/fig_ref]. January 7, 2023
Volume 29 Issue 1 With the rapid development of research techniques, single-cell sequencing and spatial transcriptomics, single-cell proteomics and metabolomics have been widely used and will undoubtedly accelerate the discovery of novel NAFLD/NASH mechanisms and therapeutic targets. Drug libraries enable us to quickly find effective small molecules to target specific pathways identified in the basic research field. Endocrine factors that mediate the crosstalk between the liver and extrahepatic tissues provide an ideal target for developing neutralization antibodies or recombinant proteins for the treatment of NAFLD/NASH. In addition to traditional small molecules, neutralizing antibodies and recombinant proteins, nucleic acid-based therapy has become an attractive approach for the development of drugs for NAFLD/NASH patients. Nucleic acid-based therapeutics include ASOs, siRNAs, miRNAs and mRNAs with modifications to improve their stability, immunogenicity, and diversity [bib_ref] Novel antisense inhibition of diacylglycerol O-acyltransferase 2 for treatment of non-alcoholic fatty..., Loomba [/bib_ref]. Through nucleic acid-based approaches, therapeutic target genes can be specifically silenced or overexpressed in the liver. As an example, the safety and efficiency of HSD17B13 RNAi and DGAT2 ASOs have been examined in a few clinical trials, which showed a positive impact on liver function and stiffness in patients with NASH [bib_ref] An integrin-based nanoparticle that targets activated hepatic stellate cells and alleviates liver..., Li [/bib_ref].
To avoid side effects, efforts have been made to better target the liver or more accurately target specific hepatic cell types by exploring novel drug delivery systems. Based on the hepatocyte-specific expression of asialoglycoprotein receptor, conjugation of GalNAc with nucleic acids or nanoparticles can provide better targeting to hepatocytes. To specifically target HSCs, nanoparticles integrated with vitamin A, cyclic peptides, mannose 6-phosphate, and antibodies against synaptophysin have shown high affinity for HSCs [bib_ref] Therapeutic targets, novel drugs, and delivery systems for diabetes associated NAFLD and..., Kumar [/bib_ref] [bib_ref] A 3D Human Liver Model of Nonalcoholic Steatohepatitis, Duriez [/bib_ref]. By using these approaches, drugs can be more precisely delivered to HSCs to alleviate NASH-related liver fibrosis. For hepatic macrophages, nanoparticles modified with the phospholipid serine to mimic apoptotic cells can enhance uptake of the nanoparticles by hepatic macrophages [bib_ref] A 3D Human Liver Model of Nonalcoholic Steatohepatitis, Duriez [/bib_ref]. However, challenges remain in targeting other cell types.
Finally, novel preclinical and in vitro models are required for drug screening for NASH. A 3D human liver model constructed via coculture of hepatocytes with non-parenchymal cells in a 3D collagen matrix has been shown to mimic NASH features when treated with free fatty acids and TNF-α[138]. Liver organoids from pluripotent stem cells or induced pluripotent stem cells also provide unique preclinical platforms to fill the gap between animal studies and clinical trials for therapeutic target validation and
[table] Table 2: Novel extrahepatic targets explored by recent basic study [/table]
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The Overexpression of IQGAP1 and β-Catenin Is Associated with Tumor Progression in Hepatocellular Carcinoma In Vitro and In Vivo
The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with β-catenin and regulates β-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and β-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and β-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease β-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and β-catenin contribute to HCC progression by promoting cell proliferation and migration.
# Introduction
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and its prognosis depends on the tendency to metastasize [bib_ref] Differential effects of inactivated Axin1 and activated beta-catenin mutations in human hepatocellular..., Zucman-Rossi [/bib_ref] [bib_ref] Angiogenesis in liver disease, Fernandez [/bib_ref]. Although significant advances have been made in the treatment of metastatic HCC, a rapid recurrence and poor survival of HCC is still a challenging problem. Therefore, a thorough understanding of the molecular mechanisms of HCC metastasis is urgently required to identify effective diagnosis and therapeutic targets, which facilitates early treatment of HCC patients with a high risk of metastasis to improve the survival [bib_ref] Angiogenesis in liver disease, Fernandez [/bib_ref].
The IQ-domain GTPase-activating proteins 1 (IQGAP1) is a scaffold protein that regulates distinct cellular processes including cell adhesion, cell migration, extracellular signals through interacting with numerous proteins [bib_ref] IQGAP1 regulation and roles in cancer, Johnson [/bib_ref] [bib_ref] IQGAP1 a key regulator of adhesion and migration, Noritake [/bib_ref]. Multiple studies have shown that IQGAP1 is up-regulated in many human malignancies, such as lung cancer [bib_ref] Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern, Miyoshi [/bib_ref] , ovarian cancer [bib_ref] Overexpression and diffuse expression pattern of IQGAP1 at invasion fronts are independent..., Dong [/bib_ref] , colon cancer [bib_ref] Immunohistochemical analysis of IQGAP1 expression in human colorectal carcinomas its overexpression in..., Nabeshima [/bib_ref] , breast cancer [bib_ref] IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells, Jadeski [/bib_ref] [bib_ref] The interaction of IQGAP1 with the exocyst complex is required for tumor..., Sakurai-Yageta [/bib_ref] , melanoma [bib_ref] Genomic analysis of metastasis reveals an essential role for RhoC, Clark [/bib_ref] and HCC [bib_ref] Development of hepatocellular carcinoma in Iqgap2-deficient mice Is IQGAP1 dependent, Schmidt [/bib_ref]. And IQGAP1 plays a critical role in cancer cell invasion. The overexpression of IQGAP1 in colon cancer cell correlates with poor prognosis [bib_ref] Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis-critical role..., Hayashi [/bib_ref] , and cell motility was increased by overexpressing IQGAP1 in breast cancer [bib_ref] Rho isoform-specific interaction with IQGAP1 promotes breast cancer cell proliferation and migration, Casteel [/bib_ref]. IQGAP1 can directly interact with CDC42 and Rac1 to regulate E-cadherin mediated cell-cell adhesion [bib_ref] Role of IQGAP1, a Target of the Small GTPases Cdc42 and Rac1,..., Kuroda [/bib_ref]. IQGAP1 can also regulate nuclear localization of β-catenin, which also involves in cell transformation and migration in WNT signaling [bib_ref] IQGAP1 protein regulates nuclear localization of beta-catenin via importin-beta5 protein in Wnt..., Goto [/bib_ref]. However, the biological associations and effects between IQGAP1 with β-catenin are not discovered in HCC.
In this study, we focused the protein expression level of IQGAP1 and β-catenin in HCC cells and tissues and their associations with tumor malignancy degree, as well as their roles in cell proliferation and migration ability. Our findings have discovered that the expression of β-catenin can be affected by the IQGAP1 level, and their overexpression and interaction improve cell proliferation and migration ability in HCC progressing processes.
# Methods
## Cell culture
Human HCC cell lines HuH7 and HepG2 were obtained from the American Type Culture Collection. Human HCC cell lines MHCC-97H and MHCC-97L were established in the Liver Cancer Institute of Fudan University [bib_ref] Establishment of cell clones with different metastatic potential from the metastatic hepatocellular..., Li [/bib_ref] , and these cells were generously endowed for our research. MHCC-97H and MHCC-97L cells were originally established from the same parent cell line MHCC-97, with orthotopic inoculations of MHCC-97H and MHCC-97L to recipient nude mice respectively, with a spontaneous pulmonary metastasis occurred in 100% and 40% [bib_ref] Establishment of cell clones with different metastatic potential from the metastatic hepatocellular..., Li [/bib_ref]. The normal liver cell line LO2 was ordered from Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences. All these cells were maintained in the Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (16000-044, Gibco), 100 U/ml penniciline, and 100ug/ml streptomycin. Cells were incubated in a 37°C incubator with 5% CO 2 and 95% air.
## Hcc samples
Thirty-three pairs of HCC tissues (HCTs) and patients' autologous para-cancerous liver tissues (PLTs) were surgically resected to collect in West China Hospital, Sichuan University (Chengdu, P. R. China). Informed written consents were obtained from the patients. This project was approved by the Institutional Ethics Committee of State Key Laboratory of Biotherapy, West China Hospital of Sichuan University.
All tissues were immediately frozen after surgical operation in liquid nitrogen prior to experiments. The patients' gender, age and histological type of tumor differentiation were obtained from the surgical and pathological records. And the histological type of tumor differentiation was classified into high differentiation group and low differentiation group. Each case was identified through pathologic biopsy.
## Rna interference
Small interfering RNA (siRNA) against IQGAP1 were synthesized as the following sequences [bib_ref] Dia1 and IQGAP1 interact in cell migration and phagocytic cup formation, Brandt [/bib_ref] : IQGAP1, 5'-UUA UCG CCC AGA AAC AUC UUG UUG G-3'; Negative control oligonucleotides (NC) were 5'-UUC UCC GAA CGU GUC ACG U-3'. And the RNA interference experiments were performed with the reagent Lipofectamine 2000 following the manufacturer's protocols.
## Plasmids and cell transfection
The IQGAP1 cDNA (GenBank gi57242794) was cloned into pCMV6 plasmids by PCR-based cloning method, and the recombinant plasmid pFlag-IQGAP1 was obtained. The β-catenin cDNA (GenBank gi 148233337) was cloned into pEZ-M13 plasmids by PCR, and the recombinant plasmid was named pFlag-β-catenin. Cell transfections of pFlag-IQGAP1 plasmids were performed using Lipofectamine LTX reagent (15338030, Life Technologies), and the transfections of pFlag-β-catenin plasmids were performed with the reagent Lipofectamine 2000 (11668-019, Life Technologies) following the manufacturer's protocols. The amount of transfected plasmids was 2.5μg for each well of a 6-well plate, and the IQGAP1-specific siRNA was used 100nM for one well of a 6-well plate. In the co-transfection of pFlag-β-catenin plasmids and IQGAP1-specific siRNA, 2.5μg plasmids and 100 nM siRNA was used for one well of a 6-well plate at the same time.
## Rna extraction and real-time pcr
Total RNA was extracted using Trizol reagent (Cat. #15596-026, Invitrogen). The first-strand cDNA was generated using a cDNA synthesis kit (Cat. #170-8891, Bio-Rad). The real-time PCR was performed to measure the relative expression using the Supermix-Bio-Rad kit (Cat. #172-5261, Bio-Rad). The GAPDH mRNA level was taken as a comparison base for the target RNA expression. The relative RNA expression was calculated with the comparative CT method, which was normalized to the internal references. The primers were designed as follows. IQGAP1: forward primer 5 0 -TCC ATT ACT TAG GAA AGA GTG GAA ACT-3 0 and reverse primer 5 0 -CAA ACA CCA AAG CTT ACA ATA TAG TAC TGC-3 0 ; forward primer 5 0 -AAG CTC TTA CAC CCA CCA TCC-3 0 and reverse primer 5 0 -GTG CAT GAT TTG CGG GAC AAA-3 0 for β-catenin; forward primer 5 0 -CGT CTC CAC ACA TCA GCA CAA-3 0 and reverse primer 5 0 -TCT TGG CAG CAG GAT AGT CCT T-3 0 for c-myc; forward primer 5 0 -TGT TCG TGG CCT CTA AGA TGA AG-3 0 and reverse primer 5 0 -AGG TTC CAC TTG AGC TTG TTC AC-3 0 for Cyclin D1; forward primer 5 0 -GGG CCA CTT TAA AGA GCA G -3 0 and reverse primer 5 0 -CCT TCA TAC ATC GGG AGC A -3 0 for Axin2; forward primer 5 0 -TGG AAG GAC TCA TGA CCA CA-3 0 and reverse primer 5 0 -TTC AGC TCA GGG ATG ACC TT-3 0 for GAPDH.
## Luciferase reporter gene assay
The promoter fragments of β-catenin (from -1018 to +241) were cloned into pGL3 Basic luciferase reporter plasmid (Promega), and the pGL3-catenin plasmid was obtained. HepG2 cells were transfected with 0.5μg or 2μg pFlag-IQGAP1 plasmids to incubate for 24 hours, then transfected with 1μg pGL3-catenin plasmids or the empty pGL3-basic plasmids to incubate for 24 hours. Cells were harvested to assess the luciferase activity according to a commercial Dualluciferase reporter assay system (Cat. #E1910, Promega). 100 ng pRL-TK (Promega) renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. All experiments were performed in triplicate from independent cell cultures.
## Transwell migration assay
Cell migration ability was assayed based on a transwell chamber apparatus (Millipore). After being transfected with pFlag-IQGAP1 plasmids or IQGAP1-specific siRNA for 48h, cells were seeded in the upper chamber of a transwell, and the bottom of the chambers was filled with 600μl of DMEM containing 10% FBS. 1×10 4 cells in 200ul serum-free DMEM were cultured in the upper wells for 24 hours. The migrated cells were fixed and stained with 1% crystal violet. Images were captured using an inverted microscope (Olympus), and the migrated cells were counted manually. The percentage of migrative cells on the condition of IQGAP1 overexpression or knockdown was calculated by comparison with the control transfection with the empty plasmids or non-targeting siRNA.
## Cell viability
Cell viability was measured using MTT assay. After IQGAP1 overexpression or knockdown for 48h, 5×10 3 cells/well were seeded in one 96-well plate in DMEM supplemented with 10% FBS to incubate for 24h. And 20ul of MTT solution (5 mg/ml, Sigma) was added to each well to incubate for 2-4 h at 37°C, the formazan crystals were dissolved with 150ul Dimethyl Sulfoxide (Sigma). Absorbance was determined at 570 nm on Multiskan MK3 (Thermo Scientific) immediately. Each assay was separately performed for three replicates and all experiments were repeated at least three times. The cell viability percentage was estimated as follows.
Cell viability (%) = absorption test/ absorption control ×100% Meanwhile, in order to discriminate if cell growth inhibition was only due to changes in cell proliferation or due to cell death, cells under IQGAP1-specific siRNA treatment were detected cell death by flow cytometry analysis using BD Bioscience Annexin V/PI staining kit (556547, BD Biosciences). After cells were transfected with siRNA for 48h, cells were harvested to stain with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) for 15 min at room temperature in the dark. Then cell suspensions were immediately evaluated by flow cytometry (NovoCyte, ACEA Biosciences). The apoptotic cells were analyzed based on the percentage of Annexin V-positive cells, and the necrotic cells were analyzed based on the percentage of Annexin Vnegative and PI-positive cells. Cell death included the apoptotic cells and necrotic cells.
## Immunoprecipitation
HepG2 cells were harvested to dissolve with a co-immunoprecipitation (co-IP) buffer containing 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100 and protease inhibitor cocktail (05892970001 Roche). 1-2μg of anti-IQGAP1 antibodies (SC-81906, Santa Cruz Biotechnology) was incubated with 50μl slurry of protein A+G conjugated sepharose (P2012 Beyotime) to pull down protein complexes from cellular lysates (500μg per sample). And 1-2μg of mouse IgG (A7028 Beyotime) was taken as a control. After washing 4 times, the protein complexes were eluted with SDS-PAGE sample-loading buffer (P0015 Beyotime) and loaded onto PAGE gel for western blot detection.
## Western blot
Cells were lysed with RIPA buffer (50 mM Tris base,1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF). For protein subfractionation, nuclear and cytoplasmic proteins were respectively extracted from HepG2 cells transfected with pFlag-IQGAP1 plasmids by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (#78833, Pierce). Proteins were separated by 10% SDS-PAGE electrophoresis, and transferred from the gel onto the PVDF membrane (Amersham Biosciences) to detect protein expression level. The PVDF membrane was respectively incubate with anti-IQGAP1 (ab133490, Abcam) and anti-β-catenin monoclonal antibody at a dilution of 1:1000 at 4°C overnight, followed by three 15-min washes in phosphate-buffered saline within 0.1% Tween-20. The membranes were then incubated with HRP-conjugated secondary antibodies at 37°C for 60 min. Detection was performed with Western blot reagent ECL (Amersham Biosciences). The GAPDH detection against its rabbit monoclonal anti-GAPDH antibodies with being diluted 1:5000 [EPR16891] (ab181602, Abcam) was taken as a control. The nuclear protein LMNB1, as a nuclear housekeeping control, was detected against anti-LMNB1 antibodies with a dilution 1:1000 [ag3631] (12987-1-AP, Proteintech). The Image J software was used to quantify the Western blot result.
## Immunohistochemistry
The HCTs, PLTs were paraffin-embedded and cut into sections with 5μm-thickness for hematoxylin-eosin and immunohistochemistry (IHC) analysis mainly according to our previous protocols [bib_ref] Gene expression and methylation status of 14-3-3sigma in human renal carcinoma tissues, Liang [/bib_ref]. The anti-IQGAP1 antibody was diluted 1:500 (ab133490, Abcam), and the anti-β-catenin antibody was used a dilution of 1:400 (ab32572, Abcam). The second antibody was a biotinylated IgG to incubate 40 min at 37°C. Finally, the tissue slices were visualized by the 3, 3-diaminobenzidine solution and counterstained with hematoxylin. Substitution of the primary antibody with phosphate-buffered saline was served as a control for IHC.
The IHC scoring analysis of tissue slices was evaluated using light microscopy by two pathologists blinded for patient outcome to minimize variability. The estimated percentage of staining was determined by calculating average staining cells of 3-4 microscopic fields under 400-fold magnification. According to general evaluation standards [bib_ref] Overexpression of ETV4 is associated with poor prognosis in prostate cancer: involvement..., Qi [/bib_ref] , the proportion of positive staining cells was scored with 0-4. The positive staining below 5% was defined as 0, 6-25% positive staining was set as 1, and 26-50% positive staining was scored 2. The staining with 51-75% was defined 3, and staining ratio over 75% was scored 4. Meanwhile, staining intensity was graded 4 levels as follows: 0, negative; 1, weak; 2, moderate; 3, strong. Meanwhile, the immunoreactivity score for each slice, ranging from 0 to 12, was measured as immunostaining intensity multiplied by the percentage of positive cells. The score of 0 was defined as a negative expression (-); scores of 1-3 were accepted as a weakly positive expression (+), while 4-6 scores were defined as a moderately positive expression (++), and 7-12 scores were accepted as a strongly positive expression (+++), namely overexpression (scoring > 7).
# Statistical analysis
All data were determined as mean ± standard error of the mean (SEM). Comparisons between two groups were performed by Student's t test. Differences among multiple groups were assessed by one-way ANOVA analysis. And Spearman correlation was used to analyze the correlation of data. p<0.05 was considered to be statistically significant.
# Results
## Iqgap1 interacts with β-catenin in hcc cells
We have found that the endogenous IQGAP1 interacts with β-catenin in HepG2 cells by co-IP analysis [fig_ref] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression [/fig_ref]. In order to verify the expression association between IQGAP1 and β-catenin, the protein expression changes of β-catenin was compared between IQGAP overexpression and knockdown in HepG2 cells. The β-catenin expression level was greatly increased to 3 times when IQGAP overexpression was up to 1.67 fold by transiently transfecting pFlag-IQGAP1 plasmids [fig_ref] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression [/fig_ref]. Consistently, about 50% IQGAP1 knockdown by IQGAP1-specific siRNA (si-IQGAP1) induced β-catenin downregulation to 78% [fig_ref] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression [/fig_ref]. IQGAP1 regulates β-catenin expression in HCC cells. Generally, either the endogenous or the exogenous expression of βcatenin can be regulated by IQGAP1 level.
For purpose of investigating the biological effects of the expression level of IQGAP1 and βcatenin in HCC progression, we further compared their expressions in several HCC cell lines with different metastasis abilities. As expected, IQGAP1 expression was gradually increased in HepG2, MHCC-97L and MHCC-97H cells than the normal liver cell line LO2 [fig_ref] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression [/fig_ref]. Especially it was greatly overexpressed in those with metastatic potentials, MHCC-97L and MHCC-97H cells. Corresponding to the higher expression of IQGAP1, β-catenin was respectively up-regulated to over 2, 3.2 and 4.8 folds among HepG2, MHCC-97L and MHCC-97H cells than LO2. There is an obviously positive correlation between the expression levels of the two proteins with HCC cell metastasis potentials.
Overexpression of IQGAP1, β-catenin in HCC tissues and their association with HCC clinic characteristics Furthermore, the associations of IQGAP1 and β-catenin expression were confirmed to play important roles in liver cancer progress from clinic immunohistochemistry analysis for liver cancer tissues as follows.
The expression profiling of IQGAP1 and β-catenin in vivo were examined in 33 pairs of HCTs and patients' autologous PLTs by immunostaining. Each tissue information and protein IHC scoring were summarized in the S1 and S2 Tables. The expression correlation of protein IQGAP1 and β-catenin was analyzed with a Spearman correlation. The association of these two proteins exhibited a significantly positive correlation of IQGAP1 versus β-catenin (Spearman r = 0.7030; p<0.001, n = 33). Out of all 33 cancer samples, IQGAP1 and β-catenin were overexpressed (strong expression) [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] in 36.36% and 33.33% HCTs, averagely with immunoreactivity scoring over 10 [fig_ref] Table 1: Protein immunoreactivity between HCTs and PLTs [/fig_ref] , which was far more than either the overexpression level or the percentage compared with the PLTs (p<0.05). Among the PLTs, IQGAP1 overexpression [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] with 8.33 scores was detected in 9.09% samples, and β-catenin overexpression [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] averagely with 8.25 scores was in 12.12% cases based on same scoring criteria. In 33 HCTs, except the non-expression [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] and weak expression of IQGAP1 [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] , 8 cases (24.24%) showed moderate staining [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] , which was no significant difference with the 30.33% moderate expressing PLTs [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref]. The expression distribution of IQGAP1 in HCTs was located in cytoplasm and cell membrane [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref]. Our statistically significant data supported that IQGAP1 was obviously overexpressed in HCC.
Similarly, except 9 cases (27.27%) showed weak expression of β-catenin, the other 19 HCTs (57.57%) was obviously detected in expression, including 8 cases (24.24%) with moderate staining and 11 cases (33.33%) with overexpression in HCTs [fig_ref] Table 1: Protein immunoreactivity between HCTs and PLTs [/fig_ref]. The expression distribution of β-catenin in HCTs was mainly located in cell membrane [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] , cytoplasm and nucleus [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref]. While in PLTs, β-catenin was observed in cell membrane [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] and cytoplasm [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref]. Based on the same criteria, 25 cases (75.76%) of PLTs had β-catenin expression, including 36.36% (12 cases) weak expression, 27.27% (9 cases) moderate staining and 12.12% (4 cases) strong expression. And the other 8 PLTs showed no expression of β-catenin. For HCTs, 4 groups of negative, weak, moderate and strong expression were representatively showed in [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] , and the corresponding staining in PLTs were showed in [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref]. Generally, 11 cases (33.33%) of HCTs showed overexpression of β-catenin with average scores 10.45, whereas only 4 cases (12.12%) PLTs showed β-catenin overexpression with average scoring 8.25 (p<0.05). Furthermore, the association of protein expression with HCC clinic information was also analyzed. The clinicopathological characteristics of HCC samples included patients' gender, age and tumor differentiation level. The average IHC score of IQGAP1 and β-catenin expression was 4.58 and 4.17 respectively, a moderate expression, among 24 HCC samples with a high differentiation , while an overexpression level for IQGAP1 and β-catenin, with IHC scoring 8.67±0.99 and 8.00±1.26 respectively, exited in the other 9 HCC tissues with a low differentiation level (p<0.05). Therefore, the overexpression level of the two proteins, IQGAP1 and β-catenin, is associated with tumor low differentiation degree. On the other hand, the expression difference of the two proteins is neither related with patient's age nor with human gender.
## Iqgap1 prompts transcription and translocation expression of β-catenin
Whether the regulation of IQGAP1 over beta-catenin is at the transcriptional or post-transcriptional level? Firstly we detected the mRNA level of IQGAP1 and β-catenin upon IQGAP1 overexpression and knockdown by real-time PCR. As results, when IQGAP1 mRNA was overexpressed to 2.9 times, the mRNA level of β-catenin was up-regulated to 2.34 times [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref]. On the contrary, when IQGAP1 mRNA was knockdown to 35%, β-catenin mRNA was also decreased to 55% [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref]. So far IQGAP1 can regulate β-catenin at the mRNA level. Furthermore, a luciferase β-catenin promoter analysis was consistent with the transcriptional regulation. As shown in the [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref] when HepG2 cells were co-transfected with 1μg pGL3-catenin plasmids and 0.5μg pFlag-IQGAP1, the luciferase activity exhibited 1.8 times than control cells which were co-transfected with 1 μg pGL3-catenin plasmids and 0.5μg pCMV empty plasmids. And when HepG2 cells were co-transfected with 1μg pGL3-catenin plasmids and 2 μg pFlag-IQGAP1, the luciferase activity was increased to 3.98 times than control cells which were cotransfected with 1 μg pGL3-catenin plasmids and 2μg pCMV empty plasmids. In addition, the luciferase activity had no obvious changes in two groups of control cells co-transfected with 1μg pGL3 empty plasmids and 0.5μg or 2 μg pFlag-IQGAP1 plasmids. These results indicated IQGAP1 can activate β-catenin transcription. Next, we evaluated whether the upregulated β-catenin induced some target gene changes upon IQGAP1 overexpression. Response for regulations of β-catenin and IQGAP1, the mRNA expression level of three target genes, including c-myc, cyclin D1 and Axin2 were measured by real-time PCR [bib_ref] Enrichment of the beta-catenin-TCF complex at the S and G2 phases ensures..., Ding [/bib_ref]. The upregulation of beta-catenin induced a higher expression of its direct target genes [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref]. Response for a 1.34-fold upregulation of β-catenin mRNA in HepG2 cells transiently transfected with pFlag-IQGAP1 plasmids [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref] , the mRNA level of c-myc, cyclin D1 and Axin2 was respectively increased to 1.53, 1.60 and 1.62 folds [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref]. Furthermore, in order to determine cellular distribution changes of IQGAP1 and β-catenin, we separately detected protein expression profiling in cytoplasm and nuclear through protein subfractionation analysis. Besides an increase of cytoplasmic β-catenin upon IQGAP1 overexpression, nuclear IQGAP1 and beta-catenin was respectively up-regulated to 1.92 and 2.12 times in IQGAP1-overexpressing cells [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref] , which indicated IQGAP1 upregulation improved the transcription and expression of β-catenin, and the increased β-catenin could translocated to the nucleus.
## Upregulation of iqgap1 and β-catenin regulates cell proliferation and migration
Based on our findings, IQGAP1 and β-catenin are usually overexpressed in vitro and in vivo, and their overexpression levels are associated with tumor malignancy degree of HCC. Therefore we hypothesized that IQGAP1 may play a role in mediating cell proliferation and cell migration.
Response to IQGAP-overexpression [fig_ref] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression [/fig_ref] and knockdown [fig_ref] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression [/fig_ref] , cell proliferation was significantly increased to 132% or decreased to 86% (n = 3, p<0.001) in cell number [fig_ref] Fig 4: IQGAP1 regulates cell proliferation and migration ability [/fig_ref]. Meanwhile cell migration ability was also assayed using a transwell-cultured chamber. Cells transiently transfected with pFlag-IQGAP1 plasmids exhibited a mean 2.18fold of cell migration numbers compared to that transfected with empty plasmids (n = 3, p<0.01) [fig_ref] Fig 4: IQGAP1 regulates cell proliferation and migration ability [/fig_ref]. Whereas the knockdown of IQGAP1 by IQGAP1-specific siRNA in HepG2 cells showed a low migration ability compared with the mock group cells transfected with non-target control oligonucleotides at a ratio of 0.44 (n = 3, p<0.01) [fig_ref] Fig 4: IQGAP1 regulates cell proliferation and migration ability [/fig_ref].
Moreover we repeated the experiments in another HCC cell line HuH7, and the results also indicated high expression level of IQGAP1 promoted cell proliferation and migration ability (S1 The conclusion was same as the data from HepG2 cells [fig_ref] Fig 4: IQGAP1 regulates cell proliferation and migration ability [/fig_ref]. In IQGAP1-overexpressing HuH7 cells, cell proliferation was significantly increased to 125% (n = 3, p<0.001) approximately detected by MTT, and cell migration ability was enhanced to 1.33-fold compared to the transfected with empty plasmids (n = 3, p<0.01). On the contrary, when IQGAP1 knockdown by IQGAP1-specific siRNA in HuH7 cells, cell growth was decreased to 79% (n = 3, p<0.01) in cell quantity, and a lower cell migration with 0.70-fold downregulation was observed (n = 3, p<0.05) compared with mock cells transfected with non-target control oligonucleotides.
Flow cytometry analysis was performed to eliminate the decreased cell proliferation effects caused by cell death (S2 . Corresponding to the IQGAP1 knockdown by IQGAP1-specific siRNA in the [fig_ref] Fig 4: IQGAP1 regulates cell proliferation and migration ability [/fig_ref] after HepG2 cells were transfected with IQGAP1-specific siRNA for 48h, we harvested cells and stained with Annexin V-FITC/PI to determine if the proliferation rate was decreased due to cell death. The cell death ratio was 5.17% ± 0.42%, 6.78% ± 0.50% respectively for cells transfected with non-target control oligonucleotides (si NC) and those transfected with IQGAP1-specific siRNA. It was shown cell death had no significant differences after IQGAP1 knockdown (p>0.05), which indicated that the observed effects are only due to changes in proliferation rate.
In order to investigate the functional associations of IQGAP1 with β-catenin, a rescue experiment was performed by overexpressing β-catenin in the condition of IQGAP1 knockdown. When the endogenous IQGAP1 in HepG2 was greatly inhibited by IQGAP1 siRNA [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref] , the downregulation of β-catenin was induced both at mRNA [fig_ref] Fig 5: β-catenin regulates cell growth and migration [/fig_ref] and protein level [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref] , the middle), following the targeting genes (c-myc, cyclin D1 and Axin2) were reduced at mRNA level [fig_ref] Fig 5: β-catenin regulates cell growth and migration [/fig_ref]. Furthermore, cell growth and migration was also inhibited to 57.3% and 82.6% [fig_ref] Fig 5: β-catenin regulates cell growth and migration [/fig_ref] compared to the control. On the other hand, compared with IQGAP1 knockdown, the overexpressing β-catenin under IQGAP1 knockdown resulted in a recovery increase of the downstream genes, and cell proliferation and migration was obviously upregulated too. The β-catenin protein level was almost 2.5 times than that in the IQGAP1 knockdown group and the mRNA level was increased to 5.45-fold. As expected, the c-myc, cyclin D1 and Axin2 mRNA level was increased to 1.52, 1.71 and 2.51 times compared with IQGAP1-knockdown group, so as cell migration was increased to 1.35 times and proliferation ability was increased to 143%. These results indicated that the alteration of β-catenin level is due to the gain or loss of IQGAP1 expression, in which their expressions and associations have effects on cell growth and migration.
In combination with the protein expression level changes of IQGAP1 and β-catenin and cell growth, we can conclude that the overexpression of IQGAP1 and β-catenin in vitro and in vivo promotes cell proliferation and migration ability in HCC, while their downregulation reduces cell growth and migration. The Overexpression of IQGAP1 and Beta-Catenin in HCC IQGAP1 and β-catenin interacting network discovered by bioinformatics analysis Due to the multiple binding partners of IQGAP1 [fig_ref] Fig 6: Fig 6 [/fig_ref] based on the online software STRING, it has been indicated that IQGAP1 lies in the central position to interact with different proteins, including β-catenin, cell division cycle 42 (CDC42), E-cadherin (CDH1) and adenomatous The Overexpression of IQGAP1 and Beta-Catenin in HCC polyposis coli (APC) to promotes cell motility and invasion. In the protein interaction map, several proteins, including CDC42, E-cadherin and APC dynamically involve in the interactions with IQGAP1 and β-catenin. For example, the activated CDC42 positively regulates Ecadherin-mediated cell-cell adhesion by inhibiting the interaction of IQGAP1 with β-catenin [bib_ref] Cdc42 and Rac1 regulate the interaction of IQGAP1 with β-Catenin, Fukata [/bib_ref]. The different ratio of E-cadherin-β-catenin-IQGAP1 complex to E-cadherin-β-cateninα-catenin complex would result in different adhesion type and cell-cell dissociation [bib_ref] IQGAP1 regulation and roles in cancer, Johnson [/bib_ref]. Under these conditions, IQGAP1 does not bind to β-catenin and cannot dissociate α-catenin from the cadherin-catenin complex, leading to strong adhesion. By contrast, IQGAP1 is freed from CDC42/Rac1 complex and interacts with β-catenin to dissociate α-catenin from the cadherincatenin complex, which results in weak adhesion and promotes cell migration [bib_ref] IQGAP1 a key regulator of adhesion and migration, Noritake [/bib_ref]. The β-catenin, APC, GSK3B, AXIN1, LEF1, and TCF7L2 are all parts of the WNT signaling [bib_ref] Wnt/beta-Catenin Signaling: Components, Mechanisms, and Diseases, Macdonald [/bib_ref]. And IQGAP1 is reported to take part in WNT signaling pathway [bib_ref] RSPO-LGR4 functions via IQGAP1 to potentiate Wnt signaling, Carmon [/bib_ref]. So far, we estimate that IQGAP1 interacts with β-catenin to take part in WNT signaling pathway to regulate cell proliferation and cell migration.
# Discussion
In eukaryotic cells, scaffold proteins play crucial roles in many important signaling pathways [bib_ref] Scaffold proteins: hubs for controlling the flow of cellular information, Good [/bib_ref] [bib_ref] Scaffolds: interaction platforms for cellular signalling circuits, Zeke [/bib_ref]. As a scaffold protein, IQGAP1 could interact with a number of proteins which could lead to oncogenesis. The alteration of IQGAP1 expression and localization correlate with cancer progression in several human primary tumors [bib_ref] Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern, Miyoshi [/bib_ref] [bib_ref] IQGAP1 modulates activation of B-Raf, Ren [/bib_ref] [bib_ref] Identification of differentially expressed genes in human lung squamous cell carcinoma using..., Sun [/bib_ref] [bib_ref] Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM..., Dong [/bib_ref] [bib_ref] Targeted knockdown of IQGAP1 inhibits the progression of esophageal squamous cell carcinoma..., Wang [/bib_ref]. Our studies discovered that IQGAP1 interacts with β-catenin, and both of their overexpression level regulates cell proliferation and cell migration in HCC.
We have demonstrated that the overexpression of IQGAP1 can upregulate the expression of β-catenin. In several hepatocellular cell lines, the overexpression level of IQGAP1 and its The interacting proteins with IQGAP1 and β-catenin analyzed by a bioinformatics software STRING. The functional partners were predicted by different methods, which were shown in different line colors. black (\, coexpression), means genes co-expressed in same or in other species; purple (\, experiments), shows a significant protein interaction from literatures; light blue (\, database), shows a significant protein interaction group gathered from curated databases; yellow (\, textmining), shows protein interaction groups extracted from scientific literatures; grey blue (\, homology), shows a protein interaction group gathered from homology. The predicted functional partners include as follows. APC, adenomatous polyposis coli; CDC42, cell division cycle 42; CDH5, cadherin 5; CDH2, cadherin 2, type 1, N-cadherin; CDH1, cadherin 1, type 1, Ecadherin; AXIN1, axin 1; GSK3B, glycogen synthase kinase 3 beta; LEF1, lymphoid enhancer-binding factor 1; PSEN1, presenilin 1; TCF7L2, transcription factor 7-like 2.
doi:10.1371/journal.pone.0133770.g006
The Overexpression of IQGAP1 and Beta-Catenin in HCC binding protein β-catenin have a positive correlation with cell metastasis potentials due to their contributions for cell proliferation and migration. And a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues; especially their overexpression is clinically correlated with tumor malignancy or differentiation degree. The aberrant accumulation of β-catenin is observed at high frequency in many cancers [bib_ref] Nuclear beta-catenin is increased in systemic sclerosis pulmonary fibrosis and promotes lung..., Lam [/bib_ref]. This accumulation correlates with either mutational activation of β-catenin or mutational inactivation of APC and Axin1 genes in some tumors [bib_ref] Wnt signaling through inhibition of beta-catenin degradation in an intact Axin1 complex, Li [/bib_ref] [bib_ref] Somatic mutations of the betacatenin gene are frequent in mouse and human..., De La Coste [/bib_ref]. However, not all the β-catenin accumulation contacted with the absence of a mutation in these genes [bib_ref] Wnt/beta-catenin signaling pathway in hepatocellular carcinomas cases from Colombia, Suarez [/bib_ref]. Thus, there must be additional sources for aberrant β-catenin accumulation in cancer cells. Here, we confirmed that the overexpression of β-catenin is regulated by IQGAP1 to promote cell growth and migration in HCC.
Due to multiple interacting partners of IQGAP1 [fig_ref] Fig 6: Fig 6 [/fig_ref] , IQGAP1 and its interacting protein β-catenin can involve in different signal pathways to regulate cell proliferation and mobility. βcatenin plays an important role in cell-cell adhesion at the plasma membrane and in transactivation of specific genes via TCF/LEF transcription factors in the nucleus [bib_ref] The many ways of Wnt in cancer, Polakis [/bib_ref]. In addition, the nuclear accumulation of β-catenin can also promote cell migration and cell transformation [bib_ref] Involvement of the Wntbeta-catenin pathway in invasion and migration of oral squamous..., Iwai [/bib_ref] [bib_ref] Critical roles of p53 in epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma..., Wang [/bib_ref]. In our study, the cellular distribution of β-catenin is partially shifted to nucleus in HCC samples [fig_ref] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs [/fig_ref] compared with the normal PLTs, and the overexpression of IQGAP1 promotes β-catenin translocation to nucleus in HepG2 cells [fig_ref] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus [/fig_ref] , which both are consistent with the previous reports. In this paper, we discovered that IQGAP1 can regulate β-catenin expression and nuclear accumulation, meanwhile their upregulation and association induce βcatenin-targeting gene expression and promote cell migration.
# Conclusions
The overexpression of IQGAP1 and β-catenin is associated with tumor progression in HCC in vitro and in vivo due to their contributions for cell proliferation and migration.
Supporting Information S1
[fig] Fig 1: IQGAP1 interacts with β-catenin and regulates β-catenin expression. (A) Endogenous IQGAP1 interacts with β-catenin in HepG2 cells. 500ug of HepG2 cell lysate was immunoprecipitated with anti-IQGAP1 antibodies, and bound proteins were analyzed by immunoblotting with the indicated antibodies. 5% of total lysate was loaded as a control (input). (B) The IQGAP1 overexpression improved the expression of βcatenin. Transfection with pCMV6 empty plasmids was taken as a control and the untreated HepG2 cells were taken as a mock. flag-IQ: pFlag-IQGAP1 plasmids. (C) The IQGAP1 knockdown by IQGAP1 siRNA (si-IQ) reduced β-catenin expression. Transfection with non-target siRNA was taken as a control (si-NC), and untreated HepG2 cells were taken as a mock. (D)The expression of IQGAP1 and β-catenin was gradually increased in several HCC cell lines. LO2 was a normal liver cell line. 97L: MHCC-97L; 97H: MHCC-97H. doi:10.1371/journal.pone.0133770.g001 The Overexpression of IQGAP1 and Beta-Catenin in HCC PLOS ONE | DOI:10.1371/journal.pone.0133770 August 7, 2015 [/fig]
[fig] Fig 2: Different expression level of IQGAP1 and β-catenin in HCTs and PLTs. The negative, weak, moderate and strong staining activity of IQGAP1 was respectively shown in HCTs (A-D) and PLTs (I-L). IQGAP1 located in cytoplasm and cell membrane in HCTs (D), and IQGAP1 was observed in cytoplasm in PLTs (L). The β-catenin with negative, weak, moderate and strong staining activity was respectively detected in HCTs (E-H) and PLTs (M-P). The expression of β-catenin located in cell membrane (G), cytoplasm and nucleus (H) in HCTs, while it mainly located in cell membrane (O) and cytoplasm (P) in PLTs. Scale bar represents 50 μm (original magnification×400). HCTs: hepatocellular carcinoma tissues. PLTs: paracancerous liver tissues. doi:10.1371/journal.pone.0133770.g002 The Overexpression of IQGAP1 and Beta-Catenin in HCC PLOS ONE | DOI:10.1371/journal.pone.0133770 August 7, 2015 [/fig]
[fig] Fig 3: IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus. (A) IQGAP1 overexpression by transfecting with pFlag-IQGAP1 plasmids (flag-IQ) upregulated β-catenin mRNA level. Transfection with empty pCMV6 plasmids was taken as a control. And the mRNA level of c-myc, cyclin D1 and Axin2 upon IQGAP1 overexpression was shown in (D). (B) IQGAP1 knockdown, by specific siRNA for IQGAP1 (si-IQ), decreased β-catenin mRNA level. Transfection with non-target siRNA was taken as a control (si NC). (C) IQGAP1 activates β-catenin transcription by a luciferase promoter analysis. (E) The overexpression of IQGAP1 induced β-catenin translocation from cytoplasm to nucleus. LMNB1 was a nuclear protein control. GAPDH was a cytoplasmic protein control. ** Student's t test p<0.01, *** Student's t test p<0.001. doi:10.1371/journal.pone.0133770.g003 The Overexpression of IQGAP1 and Beta-Catenin in HCC PLOS ONE | DOI:10.1371/journal.pone.0133770 August 7, 2015 [/fig]
[fig] Fig 4: IQGAP1 regulates cell proliferation and migration ability. Overexpression of IQGAP1 in HepG2 cells enhanced cell proliferation (A) and cell migration (C, D). Knockdown of IQGAP1 decreased cell growth (B) and migration (E, F). Control: HepG2 cells transfected with empty pCMV6 plasmids; flag-IQ:HepG2 cells transfected with pFlag-IQGAP1 plasmids. siNC: HepG2 cells transfected with control siRNA; si-IQ: HepG2 cells transfected with IQGAP1 siRNA. Scale bar represents 100 μm (original magnification×200). doi:10.1371/journal.pone.0133770.g004 [/fig]
[fig] Fig 5: β-catenin regulates cell growth and migration. (A) β-catenin mRNA exhibited a recovery increase in IQGAP1-knockdown HepG2 cells re-transfected β-catenin plasmids. The upregulation of β-catenin induced the mRNA expression of c-myc, cyclin D1 and Axin2 (B), and enhanced cell proliferation (E) and migration (C, D). Control: HepG2 cells transfected with empty vectors. flag-β-cat: HepG2 cells transfected with pFlag-β-catenin plasmids. si-IQ: HepG2 cells transfected with IQGAP1 siRNA; flag-β-cat+si-IQ: HepG2 cells transfected with pFlag-β-catenin plasmids on the condition of IQGAP1 knockdown. ** Student's t test p<0.01, *** Student's t test p<0.001. Scale bar: 100 μm (original magnification×200). doi:10.1371/journal.pone.0133770.g005 [/fig]
[fig] Fig 6: Fig 6. The interacting proteins with IQGAP1 and β-catenin analyzed by a bioinformatics software STRING. The functional partners were predicted by different methods, which were shown in different line colors. black (\, coexpression), means genes co-expressed in same or in other species; purple (\, experiments), shows a significant protein interaction from literatures; light blue (\, database), shows a significant protein interaction group gathered from curated databases; yellow (\, textmining), shows protein interaction groups extracted from scientific literatures; grey blue (\, homology), shows a protein interaction group gathered from homology. The predicted functional partners include as follows. APC, adenomatous polyposis coli; CDC42, cell division cycle 42; CDH5, cadherin 5; CDH2, cadherin 2, type 1, N-cadherin; CDH1, cadherin 1, type 1, Ecadherin; AXIN1, axin 1; GSK3B, glycogen synthase kinase 3 beta; LEF1, lymphoid enhancer-binding factor 1; PSEN1, presenilin 1; TCF7L2, transcription factor 7-like 2. [/fig]
[fig] S1: Fig. IQGAP1 regulated cell proliferation and migration of HuH7 cells. Cells were stained with Annexin V-FITC/PI. siNC: HepG2 cells transfected with control siRNA; si-IQ: HepG2 cells transfected with IQGAP1 siRNA. (TIF) S2 Fig. Cell death detected by flow cytometry. IQGAP1 regulated cell proliferation and migration ability of HuH7 cells. Overexpression of IQGAP1 enhanced cell proliferation (A) and cell migration (C, D) in HuH7 cells. IQGAP1 Knockdown decreased cell proliferation (B) and migration (E, F). Control: HuH7 cells transfected with empty pCMV6 plasmids; flag-IQ: HuH7 cells transfected with pFlag-IQGAP1. siNC: HuH7 cells transfected with control siRNA; si-IQ: HuH7 cells transfected with IQGAP1 siRNA. Scale bar represents 100 μm (original mag-nification×200). (TIF) [/fig]
[table] Table 1: Protein immunoreactivity between HCTs and PLTs.Table 2. The association of protein expression and HCC clinicopathological features. [/table]
[table] Table: Protein IHC scoring for hepatocellular carcinoma tissues. (XLSX) S2 Table. Protein IHC scoring for para-cancerous liver tissues. (XLSX) [/table]
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Chromatin-Associated Molecular Patterns (CAMPs) in sepsis
# Introduction
Sepsis is a complex inflammatory disorder initiated by a dysregulated host immune response to infection [bib_ref] The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), Singer [/bib_ref]. In the United States, approximately 1.7 million adults are affected each year causing more than 250,000 deaths [bib_ref] Hospital deaths in patients with sepsis from 2 independent cohorts, Liu [/bib_ref] [bib_ref] Prevalence, underlying causes, and preventability of sepsis-associated mortality in US acute care..., Rhee [/bib_ref]. The innate immune system is activated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering inflammation [bib_ref] Sterile inflammation: Sensing and reacting to damage, Chen [/bib_ref] [bib_ref] Inflammation 2010: New adventures of an old flame, Medzhitov [/bib_ref]. Intracellular proteins, nucleic acids, and lipids originating from the nucleus, cytoplasm, mitochondria, and granules fall into the broad category of DAMPs [bib_ref] Sterile inflammation: Sensing and reacting to damage, Chen [/bib_ref] [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref]. PAMPs can be further separated into MAMPs and NAMPs, containing the molecular patterns derived from microbes and nematodes, respectively [bib_ref] Neonatal intestinal dysbiosis in necrotizing enterocolitis, Denning [/bib_ref] [bib_ref] Arabidopsis leucine-rich repeat receptor-like kinase NILR1 is required for induction of innate..., Mendy [/bib_ref]. In addition, lifestyle-associated inflammatory diseases and their corresponding molecular patterns (LAMPs) have been distinguished from conventional DAMPs [bib_ref] DAMPs, PAMPs, and LAMPs in Immunity and Sterile Inflammation, Zindel [/bib_ref]. Self-associated molecular patterns (SAMPs) maintain cellular homeostasis and regulate innate immune cells when they become activated [bib_ref] Forearm circulation in man at high altitude, Weil [/bib_ref]. To simplify this broad area of DAMPs, the molecules derived from the nucleus or molecules associated with chromatin can be designated as chromatin-associated molecular patterns (CAMPs). Thus, by name, CAMPs refer to chromatin made up of nucleosomes containing DNA and histones. However, several nucleic acids and proteins, i.e., mitochondrial DNA, cell-free (cf) RNAs, microRNAs, telomeric repeat-containing RNA (TERRA), extracellular traps (ETs), and RNA-or DNA-binding proteins can also be included in this group, given their similar origins' contribution to inflammation and organ dysfunction in sepsis. Increased levels of CAMPs, i.e., cfRNA, cfDNA, histones, extracellular cold-inducible RNA-binding protein (eCIRP), high mobility group box 1 (HMGB1), and ETs in blood have been shown to correlate with disease severity in sepsis [fig_ref] Table 1: CAMPs in Experimental and Clinical Sepsis [/fig_ref] [bib_ref] Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis, Czaikoski [/bib_ref] [bib_ref] Prognostic utility and characterization of cell-free DNA in patients with severe sepsis, Dwivedi [/bib_ref] [bib_ref] HMG-1 as a late mediator of endotoxin lethality in mice, Wang [/bib_ref] [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref] [bib_ref] Ribonuclease 1 attenuates septic cardiomyopathy and cardiac apoptosis in a murine model..., Zechendorf [/bib_ref] [bib_ref] The Cold-Inducible RNA-Binding Protein (CIRP) Level in Peripheral Blood Predicts Sepsis Outcome, Zhou [/bib_ref].
CAMPs are released from cells through active and passive release pathways. Among the active release processes, exosomal, lysosomal, and gasdermin D (GSDMD) pores (pre-pyroptotic) comprise the primary mechanisms for CAMP release while keeping cells alive [bib_ref] Release mechanisms of major DAMPs, Murao [/bib_ref] [bib_ref] Exosome-Mediated eCIRP Release From Macrophages to Induce Inflammation in Sepsis, Murao [/bib_ref] [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref] [bib_ref] Gasdermin D plays a vital role in the generation of neutrophil extracellular..., Sollberger [/bib_ref]. By contrast, passive release mechanisms include secondary necrosis, necroptosis, pyroptosis, and NETosis [bib_ref] Release mechanisms of major DAMPs, Murao [/bib_ref] [bib_ref] Necroptosis: the release of damageassociated molecular patterns and its physiological relevance, Kaczmarek [/bib_ref] [bib_ref] Indirect regulation of HMGB1 release by gasdermin D, Volchuk [/bib_ref]. In septic conditions, once released from cells, CAMPs recognize their receptors, i.e., Toll-like receptor 4 (TLR4), TLR2, TLR7, TLR9, receptor for advanced glycation end products (RAGE), triggering receptor expressed on myeloid cells-1 (TREM-1), CD24, and cytosolic DNA sensors absent in melanoma 2 (AIM2), and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) to activate immune cells, causing inflammation and tissue injury. [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref] [bib_ref] AIM2 in health and disease: Inflammasome and beyond, Kumari [/bib_ref] [bib_ref] Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type..., Sun [/bib_ref] [bib_ref] Crosstalk between Cytoplasmic RIG-I and STING Sensing Pathways, Zevini [/bib_ref] Conversely, neutralizing antibodies or antagonists against those receptors have been shown to attenuate CAMP-induced inflammation [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref]. In this review we will discuss molecules related to CAMPs, how CAMPs are released from cells during inflammation, and the signal transduction pathways involving CAMP-mediated inflammation in experimental and clinical sepsis ; [fig_ref] Table 2: CAMPs Signal Transduction Pathways [/fig_ref]. Finally, we will emphasize targeting CAMPs to abrogate inflammation in preclinical conditions ; [fig_ref] Table 3: Targeting CAMPs in Sepsis [/fig_ref].
## Chromatin-associated molecular patterns (camps)
Chromatin is a structural complex of DNA with various basic and acidic proteins. Histones are basic, positively charged proteins that associate with negatively charged DNA, thereby organizing DNA into structures called nucleosomes [bib_ref] Histone and chromatin cross-talk, Fischle [/bib_ref] [bib_ref] Translating the histone code, Jenuwein [/bib_ref]. A nucleosome consists of a 147 bp DNA sequence wrapped around a set of eight histones called an octamer. Each histone octamer consists of two copies of each of the histone proteins, H2A, H2B, H3, and H4. The linker histones, H1, sit on the ends of DNA, keeping DNA correctly wrapped with core histones [bib_ref] Histone and chromatin cross-talk, Fischle [/bib_ref] [bib_ref] Translating the histone code, Jenuwein [/bib_ref]. Chromatin's primary function is to compress DNA into a compact unit that fits within the cell's nucleus. Chromatin increases genome stability and hinders the enzymes needed for gene transcription [bib_ref] Histone and chromatin cross-talk, Fischle [/bib_ref] [bib_ref] Translating the histone code, Jenuwein [/bib_ref] [bib_ref] Histones: Annotating chromatin, Campos [/bib_ref]. The phenomena of chemical modifications of histones and DNA, called epigenetic marks, change chromatin structure and expose regulatory elements for transcription factors to bind and impact gene expression [bib_ref] Histones: Annotating chromatin, Campos [/bib_ref] [bib_ref] Stability and flexibility of epigenetic gene regulation in mammalian development, Reik [/bib_ref]. Chromatin modifications link with various cell processes, including DNA replication, transcription, DNA repair, genetic recombination, and cell division [bib_ref] Histones: Annotating chromatin, Campos [/bib_ref] [bib_ref] Stability and flexibility of epigenetic gene regulation in mammalian development, Reik [/bib_ref] [bib_ref] Chromatin and DNA replication, Macalpine [/bib_ref]. Following stress, infection, injury, or other inflammatory stimuli, chromatin (and associated states of nucleic acids) are released from cells, acting as CAMPs to augment inflammation and tissue injury. CAMPs include but are not limited to extracellular DNA, histones, RNA, microRNA (miRNA), mitochondrial DNA, eCIRP, HMGB1, and ETs.
## Extracellular dna
During cell death, such as apoptosis, necrosis, or ETosis, DNA exits the cell. Extracellular or cell-free DNAs (cfDNAs), including pathogen-derived CpG, damaged cell-released nuclear or mitochondrial DNA, and ETs, have been reported to not only represent biomarkers of sepsis but also contribute to the length and severity of the inflammatory response in immune cells [bib_ref] The origin and properties of extracellular DNA: From PAMP to DAMP, Pisetsky [/bib_ref]. While bacterial DNA can activate the immune system through its CpG motifs, mammalian DNA is ordinarily inactive and only acquires activity once released extracellularly [bib_ref] The origin and properties of extracellular DNA: From PAMP to DAMP, Pisetsky [/bib_ref]. DNA can be associated with nuclear, cytoplasmic and serum proteins, which can promote its uptake intracellularly to stimulate internal DNA sensors [bib_ref] The cGAS-STING pathway as a therapeutic target in inflammatory diseases, Decout [/bib_ref].
The effect of DNA to serve as a CAMP necessitates the transfer of DNA from one cell to the extracellular space and then uptake into another cell [bib_ref] The Alarmin Properties of DNA and DNA-associated Nuclear Proteins, Magna [/bib_ref]. There are various mechanisms by which [bib_ref] Origin of Circulating Free DNA in Sepsis: Analysis of the CLP Mouse..., Hamaguchi [/bib_ref] [bib_ref] Early Dynamics of Plasma Dna in a Mouse Model of Sepsis, Lauková [/bib_ref].
Plasma cfDNA correlated with severity of septic patients and ICU mortality [bib_ref] Plasma DNA concentration as a predictor of mortality and sepsis in critically..., Rhodes [/bib_ref]. Serum levels of cfDNA are elevated and predict prognosis and ICU mortality in sepsis [bib_ref] Prognostic utility and characterization of cell-free DNA in patients with severe sepsis, Dwivedi [/bib_ref] [bib_ref] Cell-free DNA and outcome in sepsis, Rhodes [/bib_ref]. cfDNA levels correlated with sepsis severity and organ dysfunction [bib_ref] Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis, Czaikoski [/bib_ref]. cfDNA marker of sepsis severity and prediction of inflammatory second hit in ICU patients [bib_ref] Neutrophilderived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development..., Margraf [/bib_ref].
mtDNA mtDNA induces systemic and lung inflammation when administered intravenously in rats [bib_ref] Mitochondrial DNA induces inflammation and increases TLR9/NF-κB expression in lung tissue, Zhang [/bib_ref].
Elevated in the plasma of septic patients and correlated with disease severity [bib_ref] Comparison of the proinflammatory and procoagulant properties of nuclear, mitochondrial, and bacterial..., Bhagirath [/bib_ref].
## Ets
Neutrophil, basophil, eosinophil, and macrophage ETs have been implicated in pathologic inflammation [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref] [bib_ref] Macrophage Extracellular Traps: A Scoping Review, Doster [/bib_ref] [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref]. NETs are increased in alveolar spaces and microvasculature in murine LPS-induced endotoxemia [bib_ref] In vivo characterization of neutrophil extracellular traps in various organs of a..., Tanaka [/bib_ref].
Increased plasma levels of NETs correlated with organ dysfunction in septic patients [bib_ref] Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis, Czaikoski [/bib_ref].
Histones H3 is released in LPS-induced endotoxemia, and increased plasma levels are associated with severity of shock [bib_ref] Identification of citrullinated histone H3 as a potential serum protein biomarker in..., Li [/bib_ref].
Increased circulating levels in septic patients [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref].
exRNA exRNAs induce inflammatory responses in inflammatory models [bib_ref] Recognition of Endogenous Nucleic Acids by the Innate Immune System, Roers [/bib_ref]. Levels of exRNA are elevated in the serum of septic patients [bib_ref] Ribonuclease 1 attenuates septic cardiomyopathy and cardiac apoptosis in a murine model..., Zechendorf [/bib_ref].
## Mirna
In the serum of CLP mice, exosomes showed elevated levels of miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b [bib_ref] Profiling circulating microRNA expression in experimental sepsis using cecal ligation and puncture, Wu [/bib_ref].
Elevated circulating levels of miRNAs in patients with sepsis [bib_ref] Use of miRNAs as biomarkers in sepsis, Dumache [/bib_ref].
## Ecirp
Mediator of injury and inflammation in preclinical models of sepsis and shock [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref].
Serum levels are elevated in septic patients and predictive of sepsis severity and overall mortality [bib_ref] The Cold-Inducible RNA-Binding Protein (CIRP) Level in Peripheral Blood Predicts Sepsis Outcome, Zhou [/bib_ref].
## Hmgb1
Increased levels in LPS-induced endotoxemia [bib_ref] Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha,..., Lang [/bib_ref]. Significantly increased in CLP-induced sepsis and associated as a late mediator of inflammation with inflammatory cytokines [bib_ref] Reversing established sepsis with antagonists of endogenous high-mobility group box 1, Yang [/bib_ref].
Serum levels are elevated in human patients with bacteremia and sepsis-induced organ dysfunction [bib_ref] HMG-1 as a late mediator of endotoxin lethality in mice, Wang [/bib_ref]. Elevated in pneumonia-induced sepsis and associated with mortality [bib_ref] Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with..., Sundén-Cullberg [/bib_ref].
TERRA cfTERRA stimulates inflammatory cytokines when incubated with immune-responsive cells [bib_ref] Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes, Wang [/bib_ref].
DNA may then be recognized and trigger downstream signaling pathways that lead to inflammation. The three major receptors responsible for DNA-driven immune responses include toll-like receptor 9 (TLR9), absent in melanoma 2 (AIM2) and cyclic-GMP-AMP synthase (cGAS). TLR9 is expressed on the endosomal membrane. It functions as a DNA receptor that specifically recognizes hypomethylated CpG motifs and induces type-I interferon (IFN) as well as other inflammatory genes [bib_ref] Toll-like receptors and Type I interferons, Uematsu [/bib_ref]. The coupling mechanisms of cytosolic DNA to its downstream proinflammatory signaling cascades is multifold. Cytosolic DNA can drive the Nlrp3 inflammasome or PYHIN inflammasomes assembly and activation, including AIM2, and interferon-inducible protein 16 (IFI16). Upon inflammasome activation, DNA sensors recruit adaptors to activate caspase-1, leading to proteolytic cleavage and release of active forms of IL-1β and IL-18 [bib_ref] AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC, Hornung [/bib_ref] [bib_ref] The roles of TLRs, RLRs and NLRs in pathogen recognition, Kawai [/bib_ref]. Perhaps most significant in cytosolic DNA sensing is the DNA-binding protein, cGAS. cGAS is responsible for recognizing self-verse other sources of chromatin (i.e., CAMPs) and initiating a robust response to their accumulation in the cytosol through activation of STING [bib_ref] Phosphorylation and chromatin tethering prevent cGAS activation during mitosis, Li [/bib_ref] [bib_ref] DNA sensing by the cGAS-STING pathway in health and disease, Motwani [/bib_ref]. Recent work has highlighted the integral role of cGAS in distinguishing sources of DNA. For example, the binding of nuclear chromatin to histones H2A and H2B, and through selective suppression of cytosolic cGAS through the cell cycle have been shown to prevent aberrant activation of cGAS [bib_ref] Phosphorylation and chromatin tethering prevent cGAS activation during mitosis, Li [/bib_ref] [bib_ref] Structural mechanism of cGAS inhibition by the nucleosome, Pathare [/bib_ref]. For nonself-DNA in the cGAS-STING pathway, DNA binding activates cytosolic cGAS to generate the second messenger cyclic GMP-AMP (cGAMP), which binds to the endoplasmic reticulum-localized adaptor protein STING. After activation, STING translocates to the Golgi and recruits kinases such as TANK-binding kinase 1 (TBK1) and IkB kinase (IKK) which phosphorylate interferon regulatory factor 3 (IRF3) and the NFkB inhibitor IkBα. TBK1 acts as a convergence point for multiple PRR-driven pathways in IRF3 phosphorylation and eventual transcriptional activation of type-I IFN and related genes. Notably, besides acting as an adaptor for DNA sensing, STING is also capable of acting as a direct sensor for secondary messenger molecules including cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP), thereby serving as a strong inducer of type-I IFN [bib_ref] The cGAS-STING pathway as a therapeutic target in inflammatory diseases, Decout [/bib_ref]. Other known DNA-binding proteins may similarly mediate DNA-induced type-I IFN and pro-inflammatory cytokine production. These DNA-binding proteins include DNAdependent activator or IRFs (DAI) [bib_ref] DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune..., Takaoka [/bib_ref] , RNA polymerase III [bib_ref] RNA polymerase III detects cytosolic DNA and induces type I interferons through..., Chiu [/bib_ref] , IFI16 [bib_ref] IFI16 is an innate immune sensor for intracellular DNA, Unterholzner [/bib_ref] , oligodeoxynucleotides (including DHX36 and DHX9) [bib_ref] DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule..., Zhang [/bib_ref] [bib_ref] DHX9 pairs with IPS-1 to sense double-stranded RNA in myeloid dendritic cells, Zhang [/bib_ref] , and DDX41 [bib_ref] The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic..., Parvatiyar [/bib_ref]. Finally, additional DNA binding proteins may be involved in cytosolic DNA sensing leading to production of type 3 interferon and IFNβ, including Ku70 and leucine-rich repeat in flightless-I interacting protein (LRRFIP1), respectively [bib_ref] Molecular basis of DNA recognition in the immune system, Atianand [/bib_ref] [bib_ref] The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I..., Yang [/bib_ref] [bib_ref] Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type..., Zhang [/bib_ref].
In human sepsis, plasma levels of cfDNA are elevated, and high plasma DNA is linked to increased mortality in sepsis [bib_ref] Plasma DNA concentration as a predictor of mortality and sepsis in critically..., Rhodes [/bib_ref]. Endogenous DNases comprise the first line of defense against DNA functioning as a CAMP. Studies of mice lacking these enzymes demonstrated overexpression of type-I IFNs in macrophages and led to embryonic lethality in mice in STINGdependent manner, suggesting potential therapeutic strategies targeted at DNA as a CAMP in sepsis [bib_ref] STING manifests self DNA-dependent inflammatory disease, Ahn [/bib_ref]. Further, experimental models targeting components of these signaling pathways has been shown to have beneficial effect. For example, STING knockout mice were protected in CLP-induced sepsis through reduced coagulation [bib_ref] TMEM173 Drives Lethal Coagulation in Sepsis, Zhang [/bib_ref]. Similarly, models utilizing mice deficient in AIM2 [bib_ref] PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation, Xie [/bib_ref] , TLR9 [bib_ref] Toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis, Plitas [/bib_ref] , TLR4 [bib_ref] Knockout of Toll-like receptor 4 improves survival and cardiac function in a..., Zhou [/bib_ref] , IRF3 [bib_ref] Aged IRF3-KO Mice are Protected from Sepsis, Goswami [/bib_ref] , and related proteins were protected in experimental sepsis models, suggesting further therapeutic potential. Other strategies have investigated the use Release of CAMPs in sepsis. Septic insults, PAMPs, and other noxious stimuli activate immune cells (macrophages) to increase the expression and release of CAMPs, i.e., nuclear and mitochondrial DNAs, histones, RNAs, miRNAs, extracellular traps, HMGB1, and eCIRP. CAMPs are released through active processes like exosomes and GSDMD-mediated pores and passive release mechanisms like pyroptosis, necroptosis, ETosis, and secondary necrosis. PRR Pattern recognition receptor, GSDMD Gasdermin D, HMGB1 High mobility group box 1, eCIRP extracellular CIRP, CAMPs chromatin-associated molecular patterns, exDNA extracellular DNA, mtDNA Mitochondrial DNA, TERRA Telomeric repeat-containing RNA.
of~40 nm cationic nanoparticles (cNP) to scavenge cfDNA and inhibit the activation of primary monocytes [bib_ref] Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation, Liang [/bib_ref]. In the context of sepsis, nucleic acid-binding nanoparticles (NABPs) aiding cfDNA clearance exhibited beneficial outcomes [bib_ref] Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers, Dawulieti [/bib_ref]. This suggests a different direction of nanomedicine in treating inflammatory pathologies, including sepsis [bib_ref] Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation, Liang [/bib_ref] [bib_ref] Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers, Dawulieti [/bib_ref].
## Mitochondrial dna
Mitochondrial DNA (mtDNA) is another source of extracellular DNA that serves as a CAMP [bib_ref] Circulating mitochondrial DAMPs cause inflammatory responses to injury, Zhang [/bib_ref]. mtDNA released from the mitochondrial compartment to the cytoplasm sense intracellular DNA sensors to induce inflammation [bib_ref] Extracellular CIRP activates STING to exacerbate hemorrhagic shock, Chen [/bib_ref] [bib_ref] Mitochondrial DNA in inflammation and immunity, Riley [/bib_ref]. Human mitochondria have evolved from endosymbiotic bacteria and thus may express molecules that resemble bacterial products [bib_ref] The genome sequence of Rickettsia prowazekii and the origin of mitochondria, Andersson [/bib_ref]. Because of this resemblance, extracellular mtDNA whose CpG motifs are also unmethylated can serve as mediators of inflammation. As a result of cell death or cell activation, whole mitochondria (including proteins and DNA) can be released extracellularly in a process termed extrusion and thereafter elicit an immune response [bib_ref] The roles of mitochondrial damage-associated molecular patterns in diseases, Nakahira [/bib_ref] [bib_ref] Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense, Yousefi [/bib_ref]. mtDNA repair mechanisms are essential to cope with mtDNA damage. mtDNA degradation may contribute to the exaggerated innate immune response by fragmented mtDNA. Several components of the mtDNA replication machinery, such as DNA polymerase γ, helicase Twinkle, and exonuclease (EXOG, ENDOG, or MGME1), as well as a major DNA-packaging protein mitochondrial transcription factor A (TFAM) play critical roles in maintaining mtDNA integrity [bib_ref] Minimizing the damage: Repair pathways keep mitochondrial DNA intact, Kazak [/bib_ref].
Recent studies have further characterized the inflammatory cascade caused by mitochondrial extrusion, as it has been shown that mitochondria may act as a CAMP when released from cells undergoing necroptosis induced by TNFα [bib_ref] Mitochondria released by cells undergoing TNF-α-induced necroptosis act as danger signals, Maeda [/bib_ref]. For example, outside the cell, mtDNA causes mouse splenocytes to produce TNFα and bone marrow-derived macrophages to secrete IL-1β [bib_ref] Circulating mitochondrial DAMPs cause inflammatory responses to injury, Zhang [/bib_ref] [bib_ref] Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses, Collins [/bib_ref]. mtDNA can stimulate various PRRs, including TLR9 [bib_ref] Circulating mitochondrial DAMPs cause inflammatory responses to injury, Zhang [/bib_ref] [bib_ref] Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses, Collins [/bib_ref]. This endosomal receptor activates NF-κB-and IRF- mediated pro-inflammatory responses upon recognizing mtDNA and has been shown in animal models to induce inflammatory cytokine secretion by macrophages and neutrophil chemotaxis [bib_ref] Mitochondrial DNA induces inflammation and increases TLR9/NF-κB expression in lung tissue, Zhang [/bib_ref]. In addition to activation through TLR9, degraded intracellular mtDNA can also engage the cytosolic sensor, cGAS, and initiate STING signaling to trigger IFN responses [bib_ref] Extracellular CIRP activates STING to exacerbate hemorrhagic shock, Chen [/bib_ref] [bib_ref] Mitochondrial DNA stress primes the antiviral innate immune response, West [/bib_ref]. Furthermore, DNA has been shown to extend neutrophils lifespan. When stimulated with mtDNA, neutrophils have increased viability, which may contribute to excessive accumulation in tissues and initiate uncontrolled inflammation, causing poor outcomes in sepsis [bib_ref] Comparison of the proinflammatory and procoagulant properties of nuclear, mitochondrial, and bacterial..., Bhagirath [/bib_ref] [bib_ref] Neutrophils in development of multiple organ failure in sepsis, Brown [/bib_ref]. Engulfment of released mitochondria has also been shown to alter macrophage production of cytokines and lead to dendritic cell maturation [bib_ref] The Alarmin Properties of DNA and DNA-associated Nuclear Proteins, Magna [/bib_ref].
## Extracellular traps
Extracellular traps (ETs) refer to the process of chromatin reduction, breakdown of the nuclear envelope, and subsequent disruption of the extracellular membrane due to reactive oxygen species, resulting in the release of DNA structures [bib_ref] Neutrophil extracellular traps kill bacteria, Brinkmann [/bib_ref] [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref]. The most widely studied form of ET release is by neutrophils, the contents of which herein are referred to as neutrophil extracellular traps (NETs). One pathway involving NET formation by PAMPs and DAMPs is the activation of peptidyl arginine deaminase 4 (PAD4) via the TLR4 receptor [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref]. However, aside from TLR4/PAD4 pathway, NET formation is also mediated by Rho activation, cell cycle protein cyclin-dependent kinases 4 and 6 (CDK4/6) expression, and gasdermin D (GSDMD) pores [bib_ref] Gasdermin D plays a vital role in the generation of neutrophil extracellular..., Sollberger [/bib_ref] [bib_ref] Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps, Amulic [/bib_ref] [bib_ref] Extracellular CIRP and TREM-1 axis promotes ICAM-1-Rho-mediated NETosis in sepsis, Murao [/bib_ref]. Once released, various proteins can adhere to NETs, including CAMPs, such as histones, HMGB1, CIRP, myeloperoxidase (MPO), LL37, and over 30 components of primary and secondary granules, among which confer bactericidal activity [bib_ref] Extracellular cold-inducible RNA-binding protein regulates neutrophil extracellular trap formation and tissue damage..., Linders [/bib_ref] [bib_ref] Mineral particles stimulate innate immunity through neutrophil extracellular traps containing HMGB1, Peng [/bib_ref] [bib_ref] Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host..., Urban [/bib_ref].
ET's role in maintaining host homeostasis is twofold. On the one hand, they can protect hosts from infectious diseases; however, on the other hand, they may cause pathologic alterations to induce tissue injury [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref]. During sepsis for example, neutrophilendothelial cell interaction is crucial for promoting neutrophil infiltration into tissues; however, this interaction leads to increased NET formation [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated..., Gupta [/bib_ref]. Co-culture of neutrophils with endothelial cells has been shown to cause endothelial cell damage, which is attributed in part to excessive NETs, as NADPH oxidase inhibitors or DNase ameliorate endothelial dysfunction and cell damage [bib_ref] Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated..., Gupta [/bib_ref]. NETs have been shown to play a crucial role in the pathogenesis of various pro-inflammatory conditions, including the promotion of intravascular thrombosis in disseminated intravascular coagulation, which has been shown to increase the morbidity and mortality in sepsis [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis..., Mcdonald [/bib_ref]. In various murine models of acute lung injury (ALI), increased levels of NETs, as well as histones H3 and H4, were found in the bronchoalveolar fluid [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref]. Administration of extracellular histones contained in NETs has been shown to increase damage to alveolar epithelial cells and the magnitude of ALI [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] The role of extracellular histone in organ injury, Silk [/bib_ref]. Recent discovery has also shown NET-associated RNA to be a physiologically significant component of NETs. Specifically, RNA-LL37 sensing, subsequent cytokine release and self-propagating NET formation was shown to contribute to disease exacerbation in the inflammatory condition of psoriasis [bib_ref] Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis, Herster [/bib_ref]. Along with NETosis, enzymes are released and have detrimental effects in promoting inflammation. Neutrophil elastase, a key component of chromatin degranulation, has been shown to increase the permeability of alveolar epithelial cells, whereas inhibiting this enzyme is beneficial in animal models of inflammation and associated ALI [bib_ref] Extracellular histones are essential effectors of C5aR-and C5L2-mediated tissue damage and inflammation..., Bosmann [/bib_ref]. Serine proteases have also been shown to break down surfactants which are involved in the clearance of inflammatory cells and residual inflammation after ALI [bib_ref] The role of extracellular histone in organ injury, Silk [/bib_ref]. Finally, NETs can bind to the cytosolic DNA sensor, cGAS. Macrophages phagocytose NETs, and subsequently intracellular NETs' DNA activates cGAS and induces type-I IFN production as previously described [bib_ref] The cytosolic DNA sensor cGAS recognizes neutrophil extracellular traps, Apel [/bib_ref].
Akin [bib_ref] Targeting the eCIRP/TREM-1 interaction with a small molecule inhibitor improves cardiac dysfunction..., Denning [/bib_ref] miR130b-3p mimic CLP Attenuated systemic inflammation and acute lung injury in sepsis [bib_ref] Extracellular microRNA 130b-3p inhibits eCIRP-induced inflammation, Gurien [/bib_ref] HMGB1 Anti-HMGB1 neutralizing antibodies LPS, CLP Improved cytokine profile, increased resistance to secondary bacterial infections, and improved survival Decreased systemic inflammation and improved lethality Improved neutrophil function and reduced post-sepsis immunosuppression [bib_ref] HMG-1 as a late mediator of endotoxin lethality in mice, Wang [/bib_ref] [bib_ref] The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis, Deng [/bib_ref] [bib_ref] Frontline Science: HMGB1 induces neutrophil dysfunction in experimental sepsis and in patients..., Grégoire [/bib_ref] [bib_ref] Therapeutic targeting of HMGB1 during experimental sepsis modulates the inflammatory cytokine profile..., Stevens [/bib_ref] Anti-HMGB1 neutralizing antibodies Surgical induction peritonitis Improved survival and protected against organ injury [bib_ref] Reversing established sepsis with antagonists of endogenous high-mobility group box 1, Yang [/bib_ref] DNA-binding A box LPS, CLP Protected against organ injury and improved survival [bib_ref] Reversing established sepsis with antagonists of endogenous high-mobility group box 1, Yang [/bib_ref] RAGE antagonist, recombinant box A CLP Reduced systemic inflammation and improved survival [bib_ref] The anti-inflammatory activity of HMGB1 A box is enhanced when fused with..., Gong [/bib_ref] HMGB1 antagonist, Ethyl pyruvate LPS, CLP Reduced systemic inflammation and improved survival [bib_ref] Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic..., Ulloa [/bib_ref] HMGB1 antagonist, Nicotine LPS, CLP Attenuated clinical manifestations of endotoxemia and improved survival [bib_ref] Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis, Wang [/bib_ref] HMGB1 antagonist, Chloroquine LPS, CLP Inhibited cytokine activity and improved survival [bib_ref] Chloroquine inhibits HMGB1 inflammatory signaling and protects mice from lethal sepsis, Yang [/bib_ref] C.P. Nofi et al.
immune cells including eosinophils, basophils, and macrophages upon induction with various stimuli including PAMPs, DAMPs, and cytokines. These forms of ETosis similarly involve the pathways used for NETosis [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref] [bib_ref] Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation, Lee [/bib_ref] [bib_ref] Eosinophil extracellular traps drive asthma progression through neuro-immune signals, Lu [/bib_ref]. Granulocytes including eosinophils and basophils exhibit similar release mechanisms and contents as NETs. Extracellular DNA released from eosinophils, or eosinophil extracellular traps (EETs) have been shown to be triggered by clinically relevant allergens and amplify inflammation. Clinically, EETs have demonstrated to be elevated in bronchoalveolar lavage fluid and associated with asthma severity in patients and are likely involved in triggering of other immune responses [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref] [bib_ref] Eosinophil extracellular traps drive asthma progression through neuro-immune signals, Lu [/bib_ref]. Basophil extracellular traps (BETs) contain mitochondrial DNA, granule proteins and proteases and are released upon cytokine priming and complement activation [bib_ref] NADPH oxidase-independent formation of extracellular DNA traps by basophils, Morshed [/bib_ref]. Noting that BETs have been found in both human and mouse inflamed tissues, it is conceivable that they also play a role in inflammatory conditions [bib_ref] NADPH oxidase-independent formation of extracellular DNA traps by basophils, Morshed [/bib_ref].
Finally, macrophage extracellular traps (METs), have also been shown to entrap and kill microbes [bib_ref] Extracellular traps and macrophages: new roles for the versatile phagocyte, Boe [/bib_ref] [bib_ref] Statins enhance formation of phagocyte extracellular traps, Chow [/bib_ref] [bib_ref] Macrophage Extracellular Traps: A Scoping Review, Doster [/bib_ref]. METs contain nuclear and mitochondrial DNA, MPO, and lysozyme proteins similar to NETs, EETs, and BETs [bib_ref] Macrophage Extracellular Traps: A Scoping Review, Doster [/bib_ref] [bib_ref] Mannheimia haemolytica and its leukotoxin cause macrophage extracellular trap formation by bovine..., Aulik [/bib_ref] [bib_ref] Macrophage extracellular trap formation promoted by platelet activation is a key mediator..., Okubo [/bib_ref]. Although MET release has been demonstrated in murine primary macrophages and macrophage cell lines, the release and impact of METs in human conditions is largely underexplored, however similar to NETs, METs likely play a role in disease pathologies [bib_ref] Macrophage Extracellular Traps: A Scoping Review, Doster [/bib_ref].
Strategies to address excessive ET formation have demonstrated therapeutic potential in acute inflammatory conditions. For one, inhibition of NETosis via PAD4 deficiency or inhibition reduces the release of extracellular DNA, resulting in improved outcomes in sepsis [bib_ref] PAD4 deficiency leads to decreased organ dysfunction and improved survival in a..., Biron [/bib_ref]. PAD4 inhibitors, similar to chloroquine and APC, are early inhibitors targeting NET formation. On the other hand, late inhibitors of NETs, such as DNase or anti-histone antibodies have also been explored and demonstrated therapeutic potential. [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] PAD4 deficiency leads to decreased organ dysfunction and improved survival in a..., Biron [/bib_ref] [bib_ref] Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET..., Lewis [/bib_ref] [bib_ref] PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock, Martinod [/bib_ref] EXTRACELLULAR HISTONES Histones are cationic, intra-nuclear proteins that maintain the normal structure of chromatin [bib_ref] Histones: Annotating chromatin, Campos [/bib_ref] [bib_ref] Stability and flexibility of epigenetic gene regulation in mammalian development, Reik [/bib_ref] [bib_ref] Chromatin and DNA replication, Macalpine [/bib_ref]. In acute sterile organ injury, various toxic stimuli, including ischemic, traumatic, and hemorrhagic pathologies can result in cell death. Through this process and similar to DNA, histones and DNA-bound histones (nucleosomes) are released into the extracellular space by necrosis, apoptosis, and ETosis. Extracellular, DNA-free histones may be associated with histone chaperones or histoneassociated factors to maintain their DNA-free form. Once in the extracellular space, histones (and related components) act as CAMPs, thereby promoting inflammation. Although Xu et al. demonstrated that intravenous injection of histones was lethal in mice [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref] , incredibly, little distinction has been made between the different forms of extracellular histones. This is in large part due to the limitations of available techniques to differentiate between free versus DNA-bound forms of histones. Importantly, the cytotoxicity and proinflammatory signaling induced by free histones compared to nucleosome-associated histones differ [bib_ref] Release and activity of histone in diseases, Chen [/bib_ref] [bib_ref] Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation, Marsman [/bib_ref].
Inflammatory phenomena of extracellular histones have been explored in various experimental conditions. For one, the toxic effect of histones has been demonstrated when added to endothelial cells [bib_ref] The role of extracellular histone in organ injury, Silk [/bib_ref] [bib_ref] Release and activity of histone in diseases, Chen [/bib_ref]. In experimental models of murine sepsis, in vivo studies have shown intravenously injected histones were lethal, whereas anti-histone antibodies reduced mortality [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref]. Furthermore, sublethal doses of intravenously administered histones were pro-inflammatory, resulting in high levels of TNFα, IL-6, and IL-10 in a TLR4-dependent manner [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref]. In human sepsis, levels of histones are significantly increased [bib_ref] Impact of plasma histones in human sepsis and their contribution to cellular..., Ekaney [/bib_ref] , and consistent with experimental murine models, appear to cause cellular injury in a TLR4-dependent manner [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref] [bib_ref] Extracellular histones are mediators of death through TLR2 and TLR4 in mouse..., Xu [/bib_ref].
## Extracellular rna
Extracellular RNAs (exRNAs) are a heterogeneous group of ribonucleic acids, including messenger (m), ribosomal (r), micro (mi), long non-coding (lnc), and circular (circ) RNAs. These RNAs can be released from cells into the extracellular space in free form, bound to proteins or phospholipids, or in association with extracellular vesicles (EVs) [bib_ref] Extracellular RNA as a Versatile DAMP and Alarm Signal That Influences Leukocyte..., Preissner [/bib_ref] [bib_ref] Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic..., Valadi [/bib_ref] [bib_ref] Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection, Zernecke [/bib_ref]. Analyses of EV-associated exRNAs revealed that miRNAs together with rRNAs comprise the most prevalent form of exRNA; however, by weight, rRNAs are the most abundant type in human plasma [bib_ref] High Throughput Sequencing of Extracellular RNA from Human Plasma, Danielson [/bib_ref] [bib_ref] DAMPs and miRNA: Features of Stress Physiology and Immune Homeostasis, Fleshner [/bib_ref] [bib_ref] MicroRNAs are transported in plasma and delivered to recipient cells by high-density..., Vickers [/bib_ref] [bib_ref] Extracellular RNA in a single droplet of human serum reflects physiologic and..., Zhou [/bib_ref]. exRNAs associate with several proteins or ribonucleoprotein complexes and bind to high-density lipoproteins that protect them from degradation by extracellular RNases [bib_ref] RNA delivery by extracellular vesicles in mammalian cells and its applications, O'brien [/bib_ref]. More importantly, however, these protein interactions can impact the immunogenicity of exRNAs [bib_ref] RNA delivery by extracellular vesicles in mammalian cells and its applications, O'brien [/bib_ref] [bib_ref] Complex formation with nucleic acids and aptamers alters the antigenic properties of..., Jaax [/bib_ref]. Although in physiologic conditions only low levels of circulating exRNA can be detected in extracellular fluids, in acute states of cellular stress (such as in hypoxia, infection, and inflammation), the concentration of exRNAs is dramatically increased [bib_ref] Extracellular RNA in a single droplet of human serum reflects physiologic and..., Zhou [/bib_ref] [bib_ref] RNA delivery by extracellular vesicles in mammalian cells and its applications, O'brien [/bib_ref] [bib_ref] Cellular and extracellular miRNAs are blood-compartment-specific diagnostic targets in sepsis, Reithmair [/bib_ref]. While different forms of exRNAs, including miRNAs and lncRNAs have been implicated in influencing inflammatory processes at different levels, recent work has revealed the influence of ribosomal exRNA as an important DAMP on cellular processes for leukocyte recruitment [bib_ref] Extracellular RNA as a Versatile DAMP and Alarm Signal That Influences Leukocyte..., Preissner [/bib_ref] [bib_ref] Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome, Tosar [/bib_ref]. For example, ribosomal exRNA may induce vascular hyper-permeability and vasogenic edema. This may be accomplished through activation of the vascular endothelial growth factor (VEGF) receptor-2 system, as well as through recruitment of leukocytes to the inflamed endothelium through M1-type polarization of inflammatory macrophages, or through exRNA serving as a pro-thrombotic cofactor thereby promoting thrombosis [bib_ref] Extracellular RNA as a Versatile DAMP and Alarm Signal That Influences Leukocyte..., Preissner [/bib_ref] [bib_ref] Extracellular RNA mediates endothelial-cell permeability via vascular endothelial growth factor, Fischer [/bib_ref]. In addition to sterile inflammation, exRNAs also augment the linking of bacteria to host cells, facilitating microbial invasion [bib_ref] Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial..., Zakrzewicz [/bib_ref]. exRNAs function as CAMPs in a typical manner through recognition by membranebound PRRs including TLRs and RAGE as well as cytosolic receptors including retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein-5 (MDA-5) [bib_ref] RAGE Enhances TLR Responses through Binding and Internalization of RNA, Bertheloot [/bib_ref] [bib_ref] Recognition of Endogenous Nucleic Acids by the Innate Immune System, Roers [/bib_ref]. The binding of exRNAs by such PRRs leads to the induction of different signaling pathways, resulting in the activation of transcription factors like c-Jun-N-terminal kinase or NF-κB and release of pro-inflammatory cytokines [bib_ref] Pathogen recognition and innate immunity, Akira [/bib_ref] [bib_ref] Innate immune pattern recognition: A cell biological perspective, Brubaker [/bib_ref]. Interestingly, ribosomal exRNAs fulfill several additional extracellular functions independent of recognition by PRRs, including the progression of cardiovascular diseases [bib_ref] Extracellular RNA as a Versatile DAMP and Alarm Signal That Influences Leukocyte..., Preissner [/bib_ref].
miRNAs are small endogenous non-coding RNAs that play a critical role in post-transcriptional regulation of gene expression by binding to complementary target mRNAs [bib_ref] MicroRNAs: Genomics, biogenesis, mechanism, and function, Bartel [/bib_ref] [bib_ref] An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis..., Lau [/bib_ref]. miRNAs have been reported to affect immune processes, for example by inhibiting of NF-κB expression and modulating immune cell proliferation and differentiation [bib_ref] miR-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells, Jude [/bib_ref]. Although the focus of miRNAs has revolved around their intracellular role, miRNAs have been readily found in the blood, with changes associated with acute physiologic stressors [bib_ref] DAMPs and miRNA: Features of Stress Physiology and Immune Homeostasis, Fleshner [/bib_ref] [bib_ref] Circulating plasma extracellular vesicles from septic mice induce inflammation via MicroRNA-and TLR7-Dependent..., Xu [/bib_ref] [bib_ref] Dysregulation in microRNA expression in peripheral blood mononuclear cells of sepsis patients..., Zhou [/bib_ref]. Many circulating miRNAs are bound to protective proteins (high-density lipoprotein and argonuate protein) or packaged into protective microvesicles (exosomes), as unbound miRNAs are rapidly degraded in the bloodstream [bib_ref] DAMPs and miRNA: Features of Stress Physiology and Immune Homeostasis, Fleshner [/bib_ref]. Compared to non-exosome associated miRNA, circulating miRNA found in exosomes are more likely modulated by stressor exposure. Stress-induced exosomal miRNA reductions have been correlated with increases in inflammatory proteins, thereby suggesting stress-modulated exosomes may be immune-stimulatory [bib_ref] DAMPs and miRNA: Features of Stress Physiology and Immune Homeostasis, Fleshner [/bib_ref]. In a murine sepsis model, several miRNAs were found to be released into the blood via EVs. Compared to sham EVs, in septic EVs several miRNAs exhibited >1.5-fold increase. Specifically, these miRNAs included miR-126-3p, miR-122-5p, miR-146a-5p, miR-145-5p, miR-26a-5p, miR-150-5p, miR-222-3p, and miR-181a-5p. Furthermore, septic EVs were proinflammatory and increased IL-6, TNFα, IL-1β, and MIP-2 production via TLR7and MyD88-dependent pathways [bib_ref] Circulating plasma extracellular vesicles from septic mice induce inflammation via MicroRNA-and TLR7-Dependent..., Xu [/bib_ref].
In human sepsis, it has been demonstrated that miR-182, miR-143, miR-145, miR-146a, miR-150, and miR-155 were dysregulated in septic patients, and downregulation of specific miRNAs correlated with increased inflammatory cytokine production and monocyte proliferation [bib_ref] Dysregulation in microRNA expression in peripheral blood mononuclear cells of sepsis patients..., Zhou [/bib_ref] [bib_ref] Circulating microRNAs as Potential Biomarkers of Infectious Disease, Correia [/bib_ref]. However, recently Guerin et al. revealed an unconventional function of extracellular miRNA to neutralize the action of a CAMP, eCIRP, because CIRP has a housekeeping role in interacting with RNAs [bib_ref] Extracellular microRNA 130b-3p inhibits eCIRP-induced inflammation, Gurien [/bib_ref]. Extracellular miRNA 130b-3p mimic inhibited eCIRP-induced inflammation in experimental models of sepsis. Although the damaging effect of eRNA can be counteracted by endogenous circulating RNase1, under acute inflammatory states, only the administration of exogenous, non-toxic RNase1 provides an effective and safe therapeutic regimen. Thus, novel in vitro and in vivo strategies, including natural endonucleases or synthetic nucleic acid binding/ neutralizing polymers as antagonists, have been explored and show promise in combatting the destructive nature of eRNA [bib_ref] Extracellular nucleic acids in immunity and cardiovascular responses: between alert and disease, Preissner [/bib_ref] [bib_ref] Neutrophil Extracellular Traps: Villains and Targets in Arterial, Venous, and Cancer-Associated Thrombosis, Thålin [/bib_ref].
## Terra, glycornas, and extracellular ribosomes
Telomeres are the repetitive nucleotide regions found on chromosomal ends that protect DNA from decay. Telomeric repeat-containing RNA (TERRA), a lncRNA transcribed from telomeres, has been identified as a telomere-associated regulator of chromosome end protection [bib_ref] Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and..., Arnoult [/bib_ref]. Thus, intracellularly, TERRA plays a crucial role in telomere length homeostasis. A recent study reported that TERRA can be found in extracellular fractions in mouse tumor and embryonic brain tissue, as well as in human cell cultures that may stimulate the innate immune response [bib_ref] Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes, Wang [/bib_ref]. Cell-free TERRA (cfTERRA) is a shorter form (∼200 nucleotides) of cellular TERRA and copurifies with CD63-and CD83-positive exosome vesicles. cfTERRA can also be found as a complex with histone proteins. Incubation of cfTERRA containing exosomes with peripheral blood mononuclear cells stimulated the expression of TNFα, IL-6, and C-X-C chemokine 10 (CXCL10) [bib_ref] Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes, Wang [/bib_ref]. Although these findings with extracellular TERRA implicate a novel extrinsic function in tumor microenvironments, elucidation of extracellular TERRA in sepsis will direct a novel pathophysiology of inflammatory diseases.
Recently, highly conserved small lncRNAs, named glycoRNAs, were discovered [bib_ref] Small RNAs are modified with N-glycans and displayed on the surface of..., Flynn [/bib_ref]. These RNAs bear N-glycans in their structural backbone that are highly sialylated and fucosylated. GlycoRNAs are present in multiple cell types on the cell surface. They can interact with anti-dsRNA antibodies and members of the sialic-acid-binding immunoglobulin-like lectins (Siglecs) receptor family. Siglecs are expressed in various immune cells that recognize the sialic acid-containing ligands and initiate downstream signaling by activating Shp1 to negatively regulate TLR4 and B cell receptor (BCR) signaling pathways [bib_ref] The Role of Siglec-G on Immune Cells in Sepsis, Royster [/bib_ref]. Since glycoRNAs are exposed to the external environment of cells, their interaction with various proteins could be possible, pinpointing their novel role in the immune system. A recent study identified the presence of small ribosomal subunit 40 S by negative stain transmission electron microscopy and velocity sedimentation in sucrose gradients of concentrated extracellular fractions [bib_ref] Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome, Tosar [/bib_ref]. Improved understanding of extracellular ribosomes could possibly implicate them as damage-associated molecular patterns or subclassify them as CAMPs.
## Ecirp
The RNA chaperone protein, CIRP, plays a critical role in upregulating the inflammatory cascade when released from cells as eCIRP in acute inflammatory conditions, including sepsis. Elevated plasma levels of eCIRP have been independently correlated with worse prognosis in human sepsis [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref] [bib_ref] Extracellular microRNA 130b-3p inhibits eCIRP-induced inflammation, Gurien [/bib_ref]. Intracellular CIRP can be released outside the cell through various pathways. For one, CIRP can be released passively during necrotic cell death. In addition, in times of cellular stress, CIRP can be translocated from the nucleus to cytoplasmic stress granules and released extracellularly through exosome-mediated pathways, inflammasome-mediated GSDMD activation, pyroptosis, or necroptosis [bib_ref] Release mechanisms of major DAMPs, Murao [/bib_ref] [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref]. Once released extracellularly, eCIRP recognizes its cognate receptor TLR4/MD2 complex expressed in several cell types, activating downstream NF-κB pathways and stimulating the release of pro-inflammatory cytokines [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref] [bib_ref] Extracellular CIRP (eCIRP) and inflammation, Aziz [/bib_ref]. eCIRP has also been shown to stimulate the Nlrp3 inflammasome, leading to caspase-1 activation and subsequent expression of IL-1β and IL-18 and pyroptosis [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref] [bib_ref] Extracellular CIRP (eCIRP) and inflammation, Aziz [/bib_ref]. In addition to increasing proinflammatory cytokines, eCIRP has been shown to contribute to end-organ injury in sepsis and other acute inflammatory conditions [bib_ref] Extracellular CIRP (eCIRP) and inflammation, Aziz [/bib_ref].
In many cell types, including macrophages, lymphocytes, and neutrophils, eCIRP has been demonstrated to act as a CAMP in the context of cellular activation, cytokine and chemokine production, and NET formation. For example, injection of recombinant mouse (rm) CIRP leads to ALI in mice via macrophage, neutrophil, and endothelial cell activation and cytokine production in the lungs [bib_ref] Cold-inducible RNA-binding protein causes endothelial dysfunction via activation of Nlrp3 inflammasome, Yang [/bib_ref]. Furthermore, beneficial outcomes have been demonstrated through CIRP inhibition by using newly identified antagonists, C23 and M3, targeting its binding to TLR4 and TREM-1, respectively, or in CIRP knockout mice in various murine models of acute inflammatory conditions [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref] [bib_ref] Extracellular CIRP (eCIRP) and inflammation, Aziz [/bib_ref] [bib_ref] Extracellular CIRP as an endogenous TREM-1 ligand to fuel inflammation in sepsis, Denning [/bib_ref]. In sepsis, therapeutic potential has been demonstrated by using anti-CIRP antibodies or CIRP-derived inhibitory peptides (C23 and M3) to prolong survival and attenuate end-organ injury [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref] [bib_ref] Extracellular CIRP (eCIRP) and inflammation, Aziz [/bib_ref] [bib_ref] Extracellular CIRP as an endogenous TREM-1 ligand to fuel inflammation in sepsis, Denning [/bib_ref].
## Hmgb1
The nuclear nonhistone chromatin-binding protein, HMGB1, plays a critical role in many intracellular functions, including the DNA replication and repair, regulation of transcriptional activity, and nucleosome formation [bib_ref] High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal, Lotze [/bib_ref]. When mobilized from the nucleus to the cytoplasm and then released extracellularly, HMGB1 becomes pro-inflammatory [bib_ref] HMG-1 as a late mediator of endotoxin lethality in mice, Wang [/bib_ref] [bib_ref] The cytokine activity of HMGB1, Yang [/bib_ref]. The extracellular release of HMGB1 can occur actively through cytoplasmic vesicles or passively from necrotic cells (either alone or in complex with RNA, DNA, or nucleosomes) or through pyroptosis [bib_ref] HMG-1 as a late mediator of endotoxin lethality in mice, Wang [/bib_ref] [bib_ref] Indirect regulation of HMGB1 release by gasdermin D, Volchuk [/bib_ref] [bib_ref] The cytokine activity of HMGB1, Yang [/bib_ref] [bib_ref] Novel role of PKR in inflammasome activation and HMGB1 release, Lu [/bib_ref]. Doublestranded RNA-dependent protein kinase (PKR) induces inflammasome activation and subsequent release of HMGB1 [bib_ref] Novel role of PKR in inflammasome activation and HMGB1 release, Lu [/bib_ref]. Extracellular HMGB1 activates innate immune cells to propagate pro-inflammatory signaling cascades. This occurs through recruitment of neutrophils to the site of tissue injury and through HMGB1 binding of other PAMPs, (including DNA, LPS, and lipoteichoic acid), which serves to potentiate their inflammatory impact [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref] [bib_ref] The cytokine activity of HMGB1, Yang [/bib_ref] [bib_ref] Role of HMGB1 in apoptosis-mediated sepsis lethality, Qin [/bib_ref]. Furthermore, HMGB1 has been shown to bind to numerous cell surface receptors, including RAGE, TLR2, TLR4, TLR9, and TREM-1 [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref]. Binding of HMGB1 to these receptors leads to the activation of macrophages and endothelial cells and downstream production of pro-inflammatory chemokines, cytokines, and endothelial adhesion molecules [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref]. HMGB1 is markedly elevated in human sepsis and is widely known as a late mediator of sepsis, leading to greater morbidity, and mortality [bib_ref] High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal, Lotze [/bib_ref] [bib_ref] Circulating high-mobility group box 1 (HMGB1) concentrations are elevated in both uncomplicated..., Angus [/bib_ref]. HMGB1 has been shown to significantly attenuate erythropoietin (EPO)-mediated phosphorylation of the JAK2/STAT5 and mTOR signaling pathways, contributing to the chronic phase of anemia of inflammation [bib_ref] HMGB1-Mediated Restriction of EPO Signaling Contributes to Anemia of Inflammation, Dulmovits [/bib_ref]. As released extracellular HMGB1 can induce considerable inflammation and has demonstrated to cause detrimental effects globally in various disease states [bib_ref] The multifunctional alarmin HMGB1 with roles in the pathophysiology of sepsis and..., Diener [/bib_ref] , many therapeutic strategies have been employed, supporting that targeting HMBG1 can improve outcomes in sepsis (including neutralizing antibodies, HMGB1 antagonists, and small inhibitory peptides) [bib_ref] DAMPs and NETs in Sepsis, Denning [/bib_ref] [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref].
## Detection of camps in sepsis
Since CAMPs play a critical role in the immune response to infection, and elevated levels of CAMPs serve as diagnostic and prognostic markers in sepsis, assessment of CAMPs in biological fluids, i.e., the blood of experimental and clinical sepsis samples, is vital in determining the extent of the inflammation and tissue injury, monitoring disease progression, and elucidating potential treatment effects [fig_ref] Figure 3: Detection of CAMPs in sepsis [/fig_ref]. The levels of cfDNA in the plasma of sepsis patients are determined by real-time quantitative PCR (qPCR), quantitative PicoGreen fluorescence assay, and anti-dsDNA ELISA [bib_ref] Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with..., Maruchi [/bib_ref] [bib_ref] Plasma Microbial Cell-Free DNA Sequencing Technology for the Diagnosis of Sepsis in..., Wang [/bib_ref]. Moreover, next-generation sequencing technology may be used to identify pathogens from cell-free plasma DNA of septic patients to overcome the shortcomings of traditional bacterial culture [bib_ref] Nextgeneration sequencing diagnostics of bacteremia in septic patients, Grumaz [/bib_ref]. The presence of extracellular nuclear DNA (nDNA) and mtDNA in septic patients' plasma are determined by qPCR using specific primers for nDNA and mtDNA [bib_ref] Plasma Nuclear and Mitochondrial DNA Levels, and Markers of Inflammation, Shock, and..., Timmermans [/bib_ref]. Fragmented mtDNA in cells can be determined by staining cells with MitoTracker Red (mitochondria), TUNEL (fragmented DNA), and Hoechst (nucleus) [bib_ref] Extracellular CIRP activates STING to exacerbate hemorrhagic shock, Chen [/bib_ref]. Acknowledging that NETs contain DNA, citH3, and MPO, the plasma contents of NETs in sepsis and COVID-19 patients can be determined by PicoGreen fluorescence assay and ELISA by detecting citH3 and MPO using dsDNA Abs [bib_ref] Neutrophil extracellular traps in COVID-19, Zuo [/bib_ref]. Immunohistochemistry can also be used to reveal NETs in lung samples, as was demonstrated in autopsy lung samples of COVID-19 patients to be exaggerated [bib_ref] Targeting potential drivers of COVID-19: Neutrophil extracellular traps, Barnes [/bib_ref]. Circulating histones in septic patients' sera are determined by ELISA [bib_ref] Circulating Histones in Sepsis: Potential Outcome Predictors and Therapeutic Targets, Li [/bib_ref]. Although it is tricky to distinguish the free verse nucleosomal histones, subtracting the values of DNA containing histones (determined by using dsDNA ELISA assays) from the values of total histones may indirectly provide amounts of free histones in the blood of septic patients. Extracellular RNA, especially the miRNA as free form or in EV contained form, are mainly detected by qPCR and microarray after isolating the total RNA from plasma samples [bib_ref] Extracellular microRNA 130b-3p inhibits eCIRP-induced inflammation, Gurien [/bib_ref] [bib_ref] Methodological challenges in utilizing miRNAs as circulating biomarkers, Moldovan [/bib_ref]. TERRA can be determined by RNA in situ hybridization assay [bib_ref] Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes, Wang [/bib_ref]. Moreover, RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions extracted from normal and cancer patient blood plasma [bib_ref] Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes, Wang [/bib_ref]. It has also been reported that cfTERRA can be identified by cyro-electron microscopy and ChIP assays [bib_ref] Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes, Wang [/bib_ref]. The plasma levels of the DNA-and RNA-binding proteins HMGB1 and eCIRP in septic patients are measured by ELISA [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref] [bib_ref] Extracellular microRNA 130b-3p inhibits eCIRP-induced inflammation, Gurien [/bib_ref] [bib_ref] Reversing established sepsis with antagonists of endogenous high-mobility group box 1, Yang [/bib_ref].
## Scavenging camps to regulate inflammation
CAMPs may be released passively from necrotic cells. Since inefficient clearance of apoptotic cells may lead to necrosis and augmented CAMP release, molecules that contribute to apoptotic cells' phagocytic clearance may help reduce circulating CAMPs. Milk fat globule-EGF factor VIII (MFG-E8) plays a critical role in efferocytosis, and thus may be implicated in the regulation of CAMPs [bib_ref] Review: milk fat globule-EGF factor 8 expression, function and plausible signal transduction..., Aziz [/bib_ref]. Intracellular DNase and nucleic acid-binding polymers (NABPs) have also been shown to play a pivotal role in the degradation of DNA in apoptotic cells or engulfed cells [bib_ref] Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers, Dawulieti [/bib_ref] [bib_ref] Phagosome maturation during the removal of apoptotic cells: receptors lead the way, Zhou [/bib_ref]. Furthermore, other scavenging molecules, as have been described in their role for scavenging dead cells, debris, and DAMPs from the extracellular compartment, may also be implicated in the regulation and removal of CAMPs as means for ameliorating inflammation [bib_ref] Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers, Dawulieti [/bib_ref] [bib_ref] Review: milk fat globule-EGF factor 8 expression, function and plausible signal transduction..., Aziz [/bib_ref].
Other therapeutic strategies to target CAMP-induced inflammation have been extensively studied, as depicted in and summarized in [fig_ref] Table 3: Targeting CAMPs in Sepsis [/fig_ref]. For example, the neutralization of cfDNA through DNase and scavenger-molecule based approaches have been utilized. Previously, NABPs have been shown to scavenge proinflammatory nucleic acids and modulate inflammation at injured sites [bib_ref] Nucleic acid-binding polymers as anti-inflammatory agents: reducing the danger of nuclear attack, Pisetsky [/bib_ref]. Specifically, third generation polyamidoamine dendrimer (PAMAM-G3) is a widely studied NABP that has demonstrated ability to prevent TLR activation in target immune cells through scavenging nucleic acid and nucleic-acid proteins in C.P. Nofi et al. a multitude of acute inflammatory disease states, including liver failure, influenza infection, and cancer metastasis. To address the limitation of unwanted, in vivo cytotoxic effects of PAMAM-G3, a mesoporous silica nanoparticle functionalized with polyethylenimine (MSN-PEI), a nucleic acid-biding nanoparticle (NABN), was synthesized with the intent of improving toxicity profiles. Novel synthesized nanoparticles have similarly demonstrated the ability to scavenge cfDNA and ameliorate septic injury in experimental models of sepsis, including cecal ligation and puncture (CLP) [bib_ref] Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers, Dawulieti [/bib_ref]. Thus, further nanoparticulate NABNs-based scavenging approaches may provide a promising future therapeutic avenue for addressing cfDNA in lethal inflammatory disorders, including sepsis.
A different strategy that has been investigated is the use of membrane-coated cartridges to scavenge dead cells, pathogens, or specifically DAMPs from circulation. Early employment of this technique utilized polymyxin B immobilized to a polystyrenederived fiber to remove circulating LPS. Using this scavenger cartridge, blood if filtered outside the patient using an extracorporeal circuit, thereby detoxifying blood and removing nearly 90% of circulating LPS. This therapeutic strategy has been implemented in septic patients with little reported adverse events, however further studies are needed to determine true clinical efficacy in improving outcomes [bib_ref] Effect of Targeted Polymyxin B Hemoperfusion on 28-Day Mortality in Patients With..., Dellinger [/bib_ref]. Utilizing a similar strategy, certain types of NABPs, e.g., PANAM-G3, beta-cyclodextrincontaining polycation (CDP) and hexadimethrine bromide (HDMBr), immobilized onto an electrospun microfiber mesh were capable of capturing and removing extracellular DNAs as well as HMGB1 from circulation. NABP-immobilized mesh also neutralized the ability of DAMPs generated by ex vivo cell culture or DAMPs circulating in the blood of trauma patients to stimulate multiple TLRs in vitro and in vivo [bib_ref] Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis, Lee [/bib_ref]. Thus, therapeutic approaches utilizing membranes coated with CAMP-capturing polymers may be a promising strategy during hemofiltration, extracorporeal membrane oxygenation (ECMO) and continuous renal replacement therapy (RRT) to scavenge CAMPs and ameliorate CAMPinduced inflammation in septic patients.
A broad variety of proteins can promote internalization of harmful molecules with subsequent pro-and anti-inflammatory impacts. Previously, it has been shown that HMGB1 can bind LPS and target macrophage internalization and delivery to lysosomes via the RAGE receptor. Although HMGB1 is permeabilized in the acidic environment of the lysosome, the impact of cytosolic LPS in this mechanism results in the activation of caspase-11, pyroptosis, and cell death in endotoxemia and bacterial sepsis [bib_ref] The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis, Deng [/bib_ref]. DAMPs (such as HMGB1 and peroxiredoxins) are ligands for many other scavenger receptors that similarly promote internalization. For example, class A scavenger receptors (including MSR1) have been shown to facilitate macrophage internalization of HMGB1, but also that these receptors (MSR1 and MARCO) served as co-receptors for pro-inflammatory TLR4 signaling. In this same work, however, double scavenger receptor-(MSR1 and MARCO)-deficient mice still internalize HMGB1 efficiently, suggesting that other scavenger receptors or related molecules play a role in macrophage internalization [bib_ref] Role of scavenger receptors as damage-associated molecular pattern receptors in Toll-like receptor..., Komai [/bib_ref]. On the other hand, it has been reported that clearance of DAMPs by class A scavenger receptors may provide anti-inflammatory impacts. For example, scavenger receptor-mediated clearance of DAMPs in a murine ischemic cerebral stroke model, largely mediated by MSR1, served to attenuate DAMP-mediated inflammatory signaling, thereby improving cerebral pathology [bib_ref] MAFB prevents excess inflammation after ischemic stroke by accelerating clearance of damage..., Shichita [/bib_ref]. Broadly, the role of class A scavenger receptors in inflammation is controversial, and likewise the resulting sequelae of this receptor-ligand interaction of scavenger receptors to DAMPs is not fully understood. Regardless, better understanding of these interactions and their relationship to the clearance of CAMPs in acute disease states may promote discovery of new therapeutic strategies in regulating CAMPinduced inflammation. Along with these scavenging mechanisms, a summary of methods for targeting CAMPs to attenuate inflammation and ALI in sepsis is shown in [fig_ref] Figure 3: Detection of CAMPs in sepsis [/fig_ref] , and the preclinical evidence for each CAMP-specific therapeutic strategy is summarized in [fig_ref] Table 3: Targeting CAMPs in Sepsis [/fig_ref].
## Conclusions and future directions
Sepsis is a multifactorial inflammatory disease condition whose pathophysiology is enigmatic. Distinguishing CAMPs from the broad area of DAMPs may establish the notion that the source/ origin of DAMPs matters for the differential intensities of inflammation in sepsis, further directing source control to regulate the release of CAMPs during sepsis. Given that various infectious insults can contribute to the progression of inflammation to sepsis, elucidating the unique roles of CAMPs in other modes of inflammation, including sterile inflammation, bacterial and viralbased inflammation, and disease-specific inflammation, are worthy areas of continued research. Furthermore, the number of molecules considered to be DAMPs is increasing, as a recent study unveiled a myriad of intracellular molecules released during LPS stimulation of macrophages through active and pyroptotic pathways. Our approach of grouping DAMPs into the unique category of CAMPs will further stimulate the creation of other new subcategories based on the characteristics or size of released molecules. Studies on the release and functions of CAMPs are mainly focused on immune cells in terms of inducing cytokine production and cellular heterogeneity. Future studies focused on the role of CAMPs on non-immune cells may also reveal new directions on cell-type specific effects of CAMP release and their mode of action in sepsis.
Several post-translational forms of HMGB1 have been identified, relying on the extracellular environmental pH among other factors. Modified extracellular HMGB1 (the redox state of cysteines 23, 45, and 106) exhibit different functions compared to their parent form [bib_ref] Damage-associated molecular patterns as doubleedged swords in sepsis, Zhou [/bib_ref]. Future studies on whether other CAMPs (like eCIRP and histones) show similar post-translational modifications induced by external environments will be of great value. During transcription, several transcriptional factors bind to regulatory elements. As ETs or cfDNA are released, there is a possibility that these transcriptional factors may also be released along with bound DNA. Identification of extracellular transaction factors and their functions on the immune system may uncover greater understanding in the disease pathophysiology of sepsis. Furthermore, during inflammatory responses, epigenetic changes of DNA and histones in cells are altered. Epigenetically modified CAMPs may exhibit differential outcomes in sepsis pathophysiology. Finally, these identified CAMPs may interact with one another (and other proteins) to form complexes once released extracellularly, in addition to the crosstalk between their downstream signaling pathways. Regarding the interplay of CAMPs, it has previously been shown that targeting one CAMP could abrogate inflammation and tissue injury by inhibiting pro-inflammatory mediators and other CAMPs [bib_ref] Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis, Qiang [/bib_ref] -highlighting the importance of the interrelationship among various CAMPs in sepsis. As CAMP interactions may confer great inflammatory consequences, studying these interactions may provide new targets in preventing dangerous inflammatory cascades in sepsis. Unveiling the unique category of these important endogenous molecules, CAMPs, in sepsis defines the pathophysiology of inflammatory disease and provides new therapeutic avenues in preventing and treating uncontrolled inflammation.
## Data availability
There are no experimental datasets given that this is a review article that is prepared based on a literature review.
[fig] Figure 3: Detection of CAMPs in sepsis. For diagnostic and prognostic purposes, various samples of human and murine sepsis can be used to detect CAMPs using several immunological and molecular biological assay tools. CLP Cecal ligation, and puncture, NETs Neutrophil extracellular traps, eCIRP Extracellular CIRP, CAMPs, chromatin-associated molecular patterns; exDNA Extracellular DNA, mtDNA Mitochondrial DNA, TERRA Telomeric repeat-containing RNA. [/fig]
[table] Table 1: CAMPs in Experimental and Clinical Sepsis. [/table]
[table] Table 2: CAMPs Signal Transduction Pathways.cfDNA Cell-free DNA, TLR toll-like receptors, NF-kB Nuclear factor kappa B, IRF Interferon regulatory factor, AIM2 Absent in melanoma 2, IFI16 Interferoninducible protein 16, cGAS Cyclic guanosine monophosphate-adenosine monophosphate synthase, STING Stimulator of interferon genes, mtDNA Mitochondrial DNA, NETs Neutrophil extracellular traps, NLRP3 NLR family pyrin domain containing 3, exRNA Extracellular RNA, RAGE Receptor for advanced glycation end products, RIG1 Retinoic acid inducible gene I, MDA5 Melanoma differentiation-associated protein-5, miRNA micro-RNA, eCIRP Extracellular coldinducible RNA-binding protein, TREM-1 Triggering receptors expressed on myeloid cells-1, HMGB1 High mobility group box 1, TERRA Telomeric repeatcontaining RNA. [/table]
[table] Table 3: Targeting CAMPs in Sepsis. [/table]
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MicroRNAs as Biomarkers in Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is an incurable and fatal disorder characterized by the progressive loss of motor neurons in the cerebral cortex, brain stem, and spinal cord. Sporadic ALS form accounts for the majority of patients, but in 1-13.5% of cases the disease is inherited. The diagnosis of ALS is mainly based on clinical assessment and electrophysiological examinations with a history of symptom progression and is then made with a significant delay from symptom onset. Thus, the identification of biomarkers specific for ALS could be of a fundamental importance in the clinical practice. An ideal biomarker should display high specificity and sensitivity for discriminating ALS from control subjects and from ALS-mimics and other neurological diseases, and should then monitor disease progression within individual patients. microRNAs (miRNAs) are considered promising biomarkers for neurodegenerative diseases, since they are remarkably stable in human body fluids and can reflect physiological and pathological processes relevant for ALS. Here, we review the state of the art of miRNA biomarker identification for ALS in cerebrospinal fluid (CSF), blood and muscle tissue; we discuss advantages and disadvantages of different approaches, and underline the limits but also the great potential of this research for future practical applications.
# Introduction
Amyotrophic lateral sclerosis (ALS), the most common adult-onset neurodegenerative disorder, is an incurable and invariably fatal condition characterized by the progressive loss of motor neurons in the motor cortex, brain stem, and spinal cord [bib_ref] Amyotrophic lateral sclerosis, Rowland [/bib_ref]. Motor neurons are selectively affected by degeneration and death, however the collective evidence is that ALS is non-cell autonomous, but rather pathogenesis and disease progression depend on the active participation of non-neuronal neighboring cells such as microglia, astrocytes, muscle and T cells [bib_ref] Current hypotheses for the underlying biology of amyotrophic lateral sclerosis, Rothstein [/bib_ref] [bib_ref] Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond, Ilieva [/bib_ref]. Motor neuron degeneration causes progressive weakness of limb, thoracic, abdominal, and bulbar muscles.
During the early stages of the disease symptoms may vary depending on dysfunction of upper motor neurons (UMN) in the motor cortex (resulting in hyperreflexia, extensor plantar response, and increased muscle tone), or lower motor neuron (LMN) in the brainstem and spinal cord (leading to generalized weakness, muscle atrophy, hyporeflexia, fasciculations, and muscle cramps) [bib_ref] Amyotrophic lateral sclerosis, Rowland [/bib_ref]. Patients with bulbar onset ALS usually develop slurred and nasal speech and difficulty chewing or swallowing. Bulbar onset occurs less frequently than limb involvement, and accounts for about 25% of ALS cases. During the disease course, most cases show the presence of both LMN and UMN signs affecting spinal and brainstem regions [bib_ref] Amyotrophic lateral sclerosis, Wijesekera [/bib_ref]. Death, mainly due to bulbar dysfunction and respiratory insufficiency, occurs within 2-4 years of first symptoms; however, a small group of patients with ALS may survive for 10 or more years [bib_ref] Amyotrophic lateral sclerosis, Brown [/bib_ref].
## Epidemiology and genetic factors
The incidence of ALS is 2.1 per 100,000 persons per year, with an estimated prevalence of 5.4 cases per 100,000 population [bib_ref] Global epidemiology of amyotrophic lateral sclerosis: A systematic review of the published..., Chiò [/bib_ref]. Based on data collected by population-based registers, the incidence of ALS increases after the age of 40, shows a peak in the late 60s or early 70s, and then displays a fast decline [bib_ref] Descriptive epidemiology of amyotrophic lateral sclerosis: New evidence and unsolved issues, Logroscino [/bib_ref]. The reported male to female ratio varies widely with the age: a sex ratio of 2 or higher is observed for younger patients, while it appears to decrease towards 1 when the proportion of older patients increases [bib_ref] The sex ratio in amyotrophic lateral sclerosis: A population based study, Manjaly [/bib_ref]. Over the years, several environmental and lifestyle risk factors have been suggested as potential contributors to the cause of ALS. Nevertheless, no conclusive data are yet available, and further studies are required to identify exogenous risk factors of ALS [bib_ref] Descriptive epidemiology of amyotrophic lateral sclerosis: New evidence and unsolved issues, Logroscino [/bib_ref] [bib_ref] What we truly know about occupation as a risk factor for ALS:..., Sutedja [/bib_ref].
Most cases (around 90%) are classified as sporadic ALS (SALS), since they are not associated with a documented family history. In 1-13% of patients the disease is inherited and defined as familial ALS (FALS), most frequently with a Mendelian dominant inheritance and high penetrance, even though pedigrees with recessive inheritance or incomplete penetrance have been described [bib_ref] Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide dismutase gene, Andersen [/bib_ref]. The mean age of onset for FALS is 46 years and for SALS is 56 years. In familial ALS, age of onset displays a Gaussian distribution, whereas an age-dependent incidence characterizes sporadic ALS [bib_ref] Amyotrophic lateral sclerosis, Wijesekera [/bib_ref]. Disease with an onset prior to 25 years of age is defined as "juvenile ALS" [bib_ref] Conditions combining a bilateral pyramidal syndrome with limb and bulbar amyotrophy, Ben Hamida [/bib_ref]. Apart from the mean age of onset, sporadic and familial forms are clinically indistinguishable suggesting a common pathogenesis.
Several genes have been associated with pathogenesis of ALS. The most common ALS causative genes include chromosome 9 open reading frame 72 (C9orf72), Cu2+/Zn2+ superoxide dismutase (SOD1), TAR DNA-binding protein 43 (TARDBP), and RNA binding protein FUS (FUS) [bib_ref] Clinical genetics of amyotrophic lateral sclerosis: What do we really know?, Andersen [/bib_ref] [bib_ref] Frontotemporal lobar degeneration with TDP-43 proteinopathy and chromosome 9p repeat expansion in..., Bigio [/bib_ref] [bib_ref] Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis, Rizzo [/bib_ref] , but a lot of other genes have been associated with the disease [bib_ref] Current knowledge and recent insights into the genetic basis of amyotrophic lateral..., Volk [/bib_ref]. Notably, the mutated genes in ALS encode for proteins with very distinct functions in the cell. However, interestingly many ALS-linked genes, particularly TARDBP and FUS, are involved in RNA metabolism, including microRNA (miRNA) processing [bib_ref] TDP-43 promotes microRNA biogenesis as a component of the drosha and dicer..., Kawahara [/bib_ref] [bib_ref] TDP-43 and FUS/TLS: Emerging roles in RNA processing and neurodegeneration, Lagier-Tourenne [/bib_ref].
## Diagnosis and treatment
There is no objective laboratory test able to provide the diagnosis of ALS, which remains mainly based on clinical assessment, electrophysiological examinations, and exclusion of conditions that can mimic ALS. The certainty level of the diagnosis of ALS may be classified into different categories by clinical and laboratory assessments based on El Escorial criteria [bib_ref] El escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis, Brooks [/bib_ref].
Currently, riluzole and edaravone represent the only drugs approved by the FDA for ALS, providing however a limited improvement in survival [bib_ref] Amyotrophic lateral sclerosis, Brown [/bib_ref]. The most significant benefit of riluzole is observed after intervention in the early stages of the disease [bib_ref] Riluzole therapy for motor neurone disease: An early Australian experience, Zoing [/bib_ref]. Thus, an early diagnosis of ALS could provide the most effective results. Since diagnosis of ALS relies on clinical symptoms, and the time from the first symptoms to diagnosis is about 12 months, there is a delay hindering a successful therapy [bib_ref] Amyotrophic lateral sclerosis, Brown [/bib_ref]. This phenomenon underlies the importance of the development of screening tests able to detect the disease in early stages.
## Role of biomarkers in als
In the last years, research has been focused on the identification of potential biological markers to use in diagnostic procedure and clinical practice.
According to the National Institutes of Health Biomarkers Definitions Working Group, a biomarker is defined as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention". Biomarkers can be classified into three general categories: (1) diagnostic biomarkers, which are used for differential diagnosis; (2) prognostic biomarkers, which can differentiate a good or a bad outcome of the disease; and (3) predictive biomarkers, which are utilized for assessing whether a treatment may be effective for a specific patient or not.
In the case of ALS, biomarkers would allow an earlier and more accurate diagnosis, with the opportunity to start an earlier treatment able to modify the disease course. They could help the classification/stratification of ALS patients, monitor the disease progression and identify patients who will respond better to a particular drug. Biomarkers can also provide a valuable tool for the identification of new therapeutic approaches and drive patients' enrollment in clinical trials. Furthermore, they may represent a link between the results obtained in animal models and the human patients, providing insight on potential therapeutic targets.
Over the last two decades, intensive work has been carried out to find consistent biomarkers for ALS. Several candidates involved in excitotoxicity, oxidative stress, neuroinflammation, metabolic dysfunction, and neurodegeneration processes have been explored [bib_ref] Blood biomarkers for amyotrophic lateral sclerosis: Myth or reality?, Robelin [/bib_ref] , but, unfortunately, none of these biomarkers has been currently translated into a practical diagnostic tool.
## Mirnas as biomarkers
Recently, among the different categories of potential biomarkers, miRNAs have aroused great interest in several fields of research. miRNAs are short (about 22 nucleotides in length) non-coding RNA molecules that play an important role as endogenous regulators of gene expression acting at the post-transcriptional level. miRNAs are synthesized from primary miRNAs, which are transcribed in the nucleus. Primary miRNAs are processed into pre-miRNAs by Drosha and then exported to the cytoplasm. Pre-miRNAs are eventually processed by the Dicer complex, resulting in mature miRNAs, which form RNA-induced silencing complexes [bib_ref] Overview of microrna biogenesis, mechanisms of actions, and circulation, O'brien [/bib_ref]. miRNAs have a tissue-specific expression and this knowledge can help to better understand a normal and a disease development of the respective tissue [bib_ref] Distribution of miRNA expression across human tissues, Ludwig [/bib_ref]. miRNAs are known to play important roles in many physiological and pathological processes, including tumorigenesis [bib_ref] Oncomirs: From tumor biology to molecularly targeted anticancer strategies, Mocellin [/bib_ref] , metabolism [bib_ref] Roles of microRNAs beyond development-metabolism and neural plasticity, Aumiller [/bib_ref] , immune function [bib_ref] MicroRNAs: Novel regulators of immunity, Carissimi [/bib_ref] , and several neurodegenerative disorders [bib_ref] MicroRNAs in neurodegeneration, Bushati [/bib_ref] , such as Parkinson's disease, Alzheimer's disease, Huntington's disease [bib_ref] Macro roles for microRNAs in neurodegenerative diseases, Rajgor [/bib_ref] and also ALS [bib_ref] MicroRNA Metabolism and dysregulation in amyotrophic lateral sclerosis, Rinchetti [/bib_ref].
miRNAs have several intrinsic characteristics that make them promising as biomarkers. An ideal biomarker should display high sensitivity, specificity, and predictive power. miRNAs have been shown to have high specificity, and, in particular in cancer research, where a plethora of publications has been generated, it has been demonstrated that miRNA expression profiles differ among cancer types according to diagnosis and developmental stage of the tumor, with a better resolution than traditional gene expression analysis [bib_ref] MicroRNA expression profiles classify human cancers, Lu [/bib_ref]. Moreover, unlike other RNA classes, miRNAs are remarkably stable and therefore can be robustly measured in many biological body fluids including plasma, tears, saliva and cerebrospinal fluid [bib_ref] The microRNA spectrum in 12 body fluids, Weber [/bib_ref]. Indeed, miRNAs appear resistant to boiling, repeated freeze-thawing cycles, pH changes, and fragmentation by chemical or enzymes [bib_ref] Circulating microRNAs as stable blood-based markers for cancer detection, Mitchell [/bib_ref] [bib_ref] MicroRNAs in body fluids-the mix of hormones and biomarkers, Cortez [/bib_ref] [bib_ref] Cell-free circulating miRNA biomarkers in cancer, Mo [/bib_ref]. Furthermore, recent evidence indicates that miRNAs can be detected in biological fluids and can be used to "capture" changes in the cells of origin, including neurons [bib_ref] MicroRNAs as biomarkers for CNS disease, Rao [/bib_ref].
In addition to these general considerations, several findings suggest a specific involvement of miRNAs in ALS. For example, the loss of Dicer is sufficient to cause progressive degeneration of spinal motor neurons [bib_ref] MiRNA malfunction causes spinal motor neuron disease, Haramati [/bib_ref] ; in addition, a global down-regulation of miRNAs is a frequent molecular denominator for multiple forms of human ALS [bib_ref] Dysregulated miRNA biogenesis downstream of cellular stress and als-causing mutations: A new..., Emde [/bib_ref]. Moreover, a common theme for several ALS-related genes is a role in RNA processing pathways [bib_ref] The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS), Strong [/bib_ref]. FUS facilitates co-transcriptional Drosha recruitment to specific miRNA loci [bib_ref] Bozzoni, I. FUS stimulates microRNA biogenesis by facilitating co-transcriptional drosha recruitment, Morlando [/bib_ref] and TARDBP participate to miRNA biogenesis as a component of both Drosha and Dicer complexes [bib_ref] TDP-43 promotes microRNA biogenesis as a component of the drosha and dicer..., Kawahara [/bib_ref].
## Mirna detection
During the last decade, the development of methods for detecting miRNAs has risen to become a very attractive area of research. Although miRNAs have characteristics that made them suitable biomarkers, the detection of these molecules is challenging due to their intrinsic characteristics including small size, sequence similarity among various members, low level and tissue-specific or developmental stage-specific expression. Two approaches commonly used in the research of miRNAs as biomarkers, including studies in the area of neurodegenerative diseases and in particular in ALS, are reported below.
(1) Measurement of hundreds of miRNAs in specimens from patients with a pathology of interest and from control subjects using profiling methods, such as microarray, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)-based array, quantitative nCounter or Next Generation Sequencing (NGS), with subsequent validation of identified miRNAs by qRT-PCR;
(2) Analysis of selected miRNA(s) already known as related to specific tissues, cell types, or gene expression pathways. In this case, the number of miRNA(s) to be tested is limited, which makes the use of individual qRT-PCR appropriate, increasing sensitivity and reproducibility of the analysis.
Among the profiling methods, microarray is a powerful high-throughput widely used tool that screens large numbers of miRNAs analyzing simultaneously several samples processed in parallel in a single experiment [bib_ref] MicroRNA detection by microarray, Li [/bib_ref]. An alternative method is deep-sequencing, which relays on NGS machines that can process millions of sequence reads in parallel in just a few days [bib_ref] Next generation sequencing of miRNAs-strategies, resources and methods, Motameny [/bib_ref] [bib_ref] Discovering MicroRNAs from deep sequencing data using miRDeep, Friedländer [/bib_ref]. Sequence reads are processed by bioinformatics analysis, which identifies both known and novel miRNAs in the data sets, and perform a relative quantification using a digital approach [bib_ref] Expression profiling of microRNAs by deep sequencing, Creighton [/bib_ref]. Finally, qRT-PCR arrays can also be used to detect multiple miRNAs at the same time [bib_ref] High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA, Mestdagh [/bib_ref]. This approach is able to detect miRNAs in very low copy number [bib_ref] A high-throughput method to monitor the expression of microRNA precursors, Schmittgen [/bib_ref]. This is an important aspect, since large amounts of RNA from clinical samples can be difficult to obtain. Other advantages of qRT-PCR-based techniques used in routine diagnostic are sensitivity, specificity, speed and simplicity [bib_ref] Reliability of real-time reverse-transcription PCR in clinical diagnostics: Gold standard or substandard?, Murphy [/bib_ref]. Of note, potential biomarkers selected by array-based analysis need to be confirmed by qRT-PCR, due to high variability and low reproducibility of results obtained from these techniques [bib_ref] Detection methods for microRNAs in clinic practice, Planell-Saguer [/bib_ref].
A critical issue in qRT-PCR analysis is the data normalization approach. Normalization refers to adjusting for variations in data that are due to known factors (usually technical factors) and not related to the biological differences that are being investigated, and that could otherwise lead to inaccurate quantification. For this reason, stable normalizers are needed, but identifying such molecules is challenging, and it is often necessary to select them on a case-by-case basis [bib_ref] Data normalization strategies for microRNA quantification, Schwarzenbach [/bib_ref]. Normalization to reference invariant miRNAs [bib_ref] Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable..., Peltier [/bib_ref] is effective in many cases, but this approach requires that the reference miRNA is not influenced by the condition being studied. Exogenous spike-in controls added to samples during the miRNAs extraction may be used to compensate the variability caused by extraction efficiency and possible presence of inhibitors [bib_ref] Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse..., Kroh [/bib_ref]. The combined use of two or more normalizers usually allows reducing experimental variability and improving reliability of the analysis.
## Mirnas as biomarkers for als
The first paper about miRNAs as biomarkers for ALS in human samples was published by De Felice and colleagues in 2012 [bib_ref] A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis, De Felice [/bib_ref]. Since then, a large number of studies have been performed on cerebrospinal fluid (CSF), blood and muscle biopsies from ALS patients. See [fig_ref] Figure 1: MicroRNA [/fig_ref] for a schematic workflow of identification of miRNA-based biomarkers.
## Mirnas in cerebrospinal fluid
Cerebrospinal fluid (CSF) is the fluid that bathes the central nervous system (CNS) and, due to this direct interaction, represents a potentially ideal source for identifying biomarkers for ALS. miRNAs present in CSF can mirror CNS physiological and pathological conditions representing more sensitive biomarkers of brain changes than those present in other biofluids [bib_ref] MicroRNAs as biomarkers for CNS disease, Rao [/bib_ref]. The presence of miRNAs in CSF was first demonstrated by Cogswell and colleagues [bib_ref] Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative..., Cogswell [/bib_ref]. The authors reported that the amount of miRNAs secreted or excreted from other organs to CSF is very limited and that the major source of miRNAs detected in CSF are immune cells present in this biofluid. In addition, other studies showed that the miRNAs present in CSF derived also from neurons [bib_ref] microRNA (miRNA) speciation in Alzheimer's disease (AD) cerebrospinal fluid (CSF) and extracellular..., Alexandrov [/bib_ref].
CSF samples are obtained by lumbar puncture, a procedure used for diagnostic purposes to confirm ALS diagnosis and exclude other pathologies, as inflammatory nerve conditions. Lumbar puncture, however, represents an invasive procedure, that cannot be repeated during the disease course for ethical implications. Thus, analysis of miRNAs in CSF is not suitable to identify biomarkers to follow disease progression.
Up to date, five studies have been published about the identification of miRNAs as biomarkers in CSF from ALS patients. Results are shown in [fig_ref] Table 1: Deregulated microRNAs [/fig_ref].
## Mirnas in cerebrospinal fluid
Cerebrospinal fluid (CSF) is the fluid that bathes the central nervous system (CNS) and, due to this direct interaction, represents a potentially ideal source for identifying biomarkers for ALS. miRNAs present in CSF can mirror CNS physiological and pathological conditions representing more sensitive biomarkers of brain changes than those present in other biofluids [bib_ref] MicroRNAs as biomarkers for CNS disease, Rao [/bib_ref]. The presence of miRNAs in CSF was first demonstrated by Cogswell and colleagues [bib_ref] Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative..., Cogswell [/bib_ref]. The authors reported that the amount of miRNAs secreted or excreted from other organs to CSF is very limited and that the major source of miRNAs detected in CSF are immune cells present in this biofluid. In addition, other studies showed that the miRNAs present in CSF derived also from neurons [bib_ref] microRNA (miRNA) speciation in Alzheimer's disease (AD) cerebrospinal fluid (CSF) and extracellular..., Alexandrov [/bib_ref]. CSF samples are obtained by lumbar puncture, a procedure used for diagnostic purposes to confirm ALS diagnosis and exclude other pathologies, as inflammatory nerve conditions. Lumbar puncture, however, represents an invasive procedure, that cannot be repeated during the disease course for ethical implications. Thus, analysis of miRNAs in CSF is not suitable to identify biomarkers to follow disease progression.
Up to date, five studies have been published about the identification of miRNAs as biomarkers in CSF from ALS patients. Results are shown in [fig_ref] Table 1: Deregulated microRNAs [/fig_ref].
The first three studies in [fig_ref] Table 1: Deregulated microRNAs [/fig_ref] selected a limited set of miRNAs to analyze: 43 miRNAs found up-regulated in SOD1 spinal cord CD39+ microglia and splenic Ly6Chi monocytes [bib_ref] Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS, Butovsky [/bib_ref] , a group of TARDBP binding miRNAs [bib_ref] Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis, Freischmidt [/bib_ref] , or one selected miRNA, over-expressed in ALS blood leucocytes [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref] , respectively. The other two studies performed a miRNA expression profiling, using qRT-PCR [bib_ref] Identification of miRNAs as potential biomarkers in cerebrospinal fluid from amyotrophic lateral..., Benigni [/bib_ref] or small RNA sequencing (NGS) [bib_ref] Small RNA sequencing of sporadic amyotrophic lateral sclerosis cerebrospinal fluid reveals differentially..., Waller [/bib_ref]. In both profiling studies, results were validated by qRT-PCR for each single miRNA. While Benigni and colleagues found eight out of fourteen miRNAs as significantly deregulated, Waller and coworkers failed to confirm statistically significant differences in miRNA expression [bib_ref] Identification of miRNAs as potential biomarkers in cerebrospinal fluid from amyotrophic lateral..., Benigni [/bib_ref] [bib_ref] Small RNA sequencing of sporadic amyotrophic lateral sclerosis cerebrospinal fluid reveals differentially..., Waller [/bib_ref]. A common feature observed by the authors is an overall down-regulation of miRNAs in CSF samples from ALS patients [bib_ref] Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis, Freischmidt [/bib_ref] [bib_ref] Identification of miRNAs as potential biomarkers in cerebrospinal fluid from amyotrophic lateral..., Benigni [/bib_ref] [bib_ref] Small RNA sequencing of sporadic amyotrophic lateral sclerosis cerebrospinal fluid reveals differentially..., Waller [/bib_ref] , in agreement with other studies showing that the majority of deregulated miRNAs in tissues from ALS models and ALS patients are down-regulated [bib_ref] Amyotrophic lateral sclerosis: mechanisms and therapeutics in the epigenomic era, Paez-Colasante [/bib_ref]. This could suggest a general default in RNA metabolism in ALS [bib_ref] The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS), Strong [/bib_ref].
In general, however, these studies highlight a wide heterogeneity among miRNAs significantly deregulated. A possible explanation could be the variability in terms of experimental approach and technical procedures and the reduced number of CSF samples analyzed in each study.
Some authors evaluated the correlation between CSF and serum miRNA expression levels. A significant positive correlation between expression levels in CSF and serum from ALS patients was found for miR-338-3p [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref] and miR-143-3p [bib_ref] Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis, Freischmidt [/bib_ref]. However, the amount of most miRNAs was independently regulated between the two biofluids at individual level. This suggests that CSF miRNAs do not simply reflect the usually more abundant serum miRNAs, and changes in the serum do not necessarily reproduce alterations of CSF levels [bib_ref] Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis, Freischmidt [/bib_ref].
It should be noted that, among the different body fluids, the lowest abundance of miRNAs appears in CSF. Thus, it is possible that some potentially promising and informative miRNAs, identified both in vivo and in vitro ALS models, are below the limit of detection of the available methods of analysis. For example, the miR-218, a motor neurons-enriched miRNA, has been found increased in CSF of ALS rodent models: its expression correlates with the number of remaining spinal motor neurons and is responsive to motor neuron sparing therapy [bib_ref] MicroRNA profiling reveals marker of motor neuron disease in ALS models, Hoye [/bib_ref]. miR-218 could thus represent a potential biomarker to assess drug effects on motor neurons during clinical trials in ALS patients. However, at the present time, this miRNA is detectable only in some CSF samples, and thus a comparison between ALS patients and controls is not possible.
An approach to overcome the technical limits due to low abundance of several miRNAs in CSF could be to focus on those miRNAs found up-regulated in ALS patients. Among these, miR-338-3p seems to be very promising, since it has been reported as consistently upregulated in CSF, serum and leukocytes from ALS patients [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref]. miR-338-3p is involved in several molecular pathways and could contribute to ALS pathogenesis through different modalities, such as neurodegeneration and apoptosis. Recent evidence suggests that miR-338 participates in the control of neuroblast apoptosis and in neuroblastoma pathogenesis [bib_ref] A potential regulatory role for intronic microRNA-338-3p for its host gene encoding..., Kos [/bib_ref] and it is able to suppress neuroblastoma proliferation, invasion and migration [bib_ref] miR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a, Chen [/bib_ref]. Interestingly, also another miRNA found up-regulated in CSF from ALS patients, miR181a-5p, has been proposed as an anti-oncomir, which acts as a tumor suppressor in normal tissues, promoting growth inhibition and apoptosis [bib_ref] miR-21 and 221 upregulation and miR-181b downregulation in human grade II-IV astrocytic..., Conti [/bib_ref]. These findings suggest that these up-regulated miRNAs are involved in ALS pathogenetic process through apoptotic mechanisms responsible for cell death.
In order to increase diagnostic accuracy, up-regulated miRNAs can be used in combination with other miRNAs, identified as down-regulated. Benigni and colleagues reported that the ratios of miR-181a-5p/miR-15b-5p and miR-181a-5p/miR-21-5p considerably increased the specificity with a slight decrease in sensitivity compared with each individual miRNA [bib_ref] Identification of miRNAs as potential biomarkers in cerebrospinal fluid from amyotrophic lateral..., Benigni [/bib_ref]. A wider use of this strategy could allow improvements in the performance of identified biomarkers and should be taken into account for future studies, as further discussed in the following paragraphs.
## Circulating mirnas
The use of blood samples in the diagnostic routine presents several advantages. Blood specimens are easy to obtain, process and store, and the samples required for the analysis can be collected without using invasive procedures for the patients. The lack of ethical implications as compared with CSF and muscle biopsy makes it possible to repeat the blood draw during the disease progression. Since miRNAs circulate in the blood in a highly stable form, this may facilitate the procedure of storage and conservation and increase the flexibility of the analysis.
Blood-based biomarkers may originate from the CNS through a transfer between the blood and CSF at the blood-CSF barrier [bib_ref] The blood-cerebrospinal fluid barrier: Structure and functional significance, Johanson [/bib_ref] [bib_ref] A balanced view of the cerebrospinal fluid composition and functions: Focus on..., Spector [/bib_ref] , suggesting that the same biomarkers could be present in both biofluids. They may be generated also by other organs and tissues affected during ALS, such as degenerating muscles or peripheral blood cells. Therefore, blood can represent an excellent biofluid for discovery and validation of biomarkers for ALS [bib_ref] Fluid-based biomarkers for amyotrophic lateral sclerosis, Vu [/bib_ref]. On the other hand, miRNAs present in blood can reflect other pathophysiological conditions concurrent but not directly related to ALS disease (e.g. inflammatory status, response to pharmacological treatments, etc.), which may represent confounding factors.
Several studies on circulating miRNAs as potential biomarkers for ALS have been published. The findings from such studies are summarized in [fig_ref] Table 2: Deregulated circulating microRNAs [/fig_ref]. As reported in [fig_ref] Table 2: Deregulated circulating microRNAs [/fig_ref] , several studies have identified numerous potential miRNA biomarkers in peripheral blood from ALS patients, however their results rarely overlap with each other. This high discrepancy in the identified miRNAs is probably associated with the variability of quantification methods, miRNA normalizers used, number of samples included, clinical features of patients, and also with the differences in selected source of miRNAs (serum, plasma, leukocytes, and whole blood). Another possible reason for the poor reproducibility of results may be the high level of heterogeneity in miRNA profiles of SALS patients in comparison to FALS patients. Freischmidt and colleagues initially reported a signature of 22 miRNAs significantly down-regulated in FALS and presymptomatic mutation carriers [bib_ref] Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation..., Freischmidt [/bib_ref]. Subsequently, the same authors replicated the analysis of these miRNAs in a larger SALS sample group using identical technical procedures, and found only 2 miRNAs significantly down-regulated in all SALS patients. A more accurate analysis of results revealed that around 60% of SALS patients shared a serum miRNA fingerprint with genetic cases, while the remaining around 40% of patients were evenly distributed among control samples. The absence of FALS-like miRNA patterns in these patients may mirror a higher impact of exogenous factors and possibly a lower and/or different genetic influence in a subgroup of SALS patients [bib_ref] Serum microRNAs in sporadic amyotrophic lateral sclerosis, Freischmidt [/bib_ref].
Interestingly, the miRNA expression profiles derived from the study performed by Freischmidt and colleagues [bib_ref] Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation..., Freischmidt [/bib_ref] were re-elaborated applying principal component analysis (PCA)-based unsupervised feature extraction (FE), another analysis approach [bib_ref] Exploring microRNA biomarker for amyotrophic lateral sclerosis, Taguchi [/bib_ref]. The authors identified a total of 51 deregulated miRNAs, 27 down-regulated and 24 up-regulated in ALS patients in comparison with healthy controls. Applying the linear discriminant analysis (LDA) to these selected miRNAs, overall accuracy was 0.66 including healthy controls, ALS mutation carriers, FALS and SALS patients. Of note, excluding SALS patients, LDA was able to successfully discriminate healthy controls, ALS mutation carriers and FALS patients, with an accuracy rising up to 0.84, confirming as the heterogeneity of SALS group can introduce a wider variability in circulating miRNA profiles.
Among the studies published until now, a largely used approach is miRNA profiling on blood samples from ALS patients and controls, carried out by microarray [bib_ref] A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis, De Felice [/bib_ref] [bib_ref] Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation..., Freischmidt [/bib_ref] [bib_ref] Serum microRNAs in sporadic amyotrophic lateral sclerosis, Freischmidt [/bib_ref] [bib_ref] Identification of plasma microRNAs as a biomarker of sporadic amyotrophic lateral Sclerosis, Takahashi [/bib_ref] [bib_ref] Aberration of miRNAs expression in leukocytes from sporadic amyotrophic lateral sclerosis, Chen [/bib_ref] , PCR-array [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] [bib_ref] Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral..., Waller [/bib_ref] and NGS [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref]. Other studies performed analysis on specific miRNAs, selected from data previously reported in the literature [bib_ref] Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis, Freischmidt [/bib_ref] [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref] [bib_ref] circulating microRNAs as biomarkers of muscle differentiation and atrophy in ALS, Tasca [/bib_ref] [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref] [bib_ref] Differential expression of several miRNAs and the host genes AATK and DNM2..., Vrabec [/bib_ref]. In other cases, the first step of the research was a microarray analysis on samples from transgenic mice [bib_ref] Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS, Butovsky [/bib_ref] [bib_ref] MicroRNA-206: A potential circulating biomarker candidate for amyotrophic lateral sclerosis, Toivonen [/bib_ref] [bib_ref] Genome-wide circulating microRNA expression profiling reveals potential biomarkers for amyotrophic lateral sclerosis, Matamala [/bib_ref] or skeletal muscle biopsies from ALS patients [bib_ref] Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation..., Freischmidt [/bib_ref] , followed by validation of miRNAs found deregulated in the first step of analysis.
Only one study analyzed miRNA expression specifically in serum exosomes [bib_ref] Comparison of the extraction and determination of serum exosome and miRNA in..., Xu [/bib_ref]. Exosomes are double lipid vesicles secreted by a variety of cells and widespread in the peripheral body fluid. They can reflect physiological and pathological changes of the cells of origin, representing potential new biomarkers for disease diagnosis [bib_ref] Exosomes: New players in cell-cell communication, Bang [/bib_ref]. miRNAs are enriched in exosomes, and the exosome membrane structure can protect them from degradation by RNA enzymes. The authors investigated the expression of only miR-27a-3p, previously reported as present in myoblast-derived exosomes [bib_ref] Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration..., Cui [/bib_ref] , and found a down-regulation of this miRNA in ALS patients, suggesting that miRNA exosome analysis could represent a future perspective for ALS biomarker identification.
Despite a poor overlapping among the miRNAs identified as deregulated in ALS, some circulating miRNAs seem to be particularly promising as potential biomarkers in ALS patients. [fig_ref] Table 3: The most promising circulating microRNAs [/fig_ref] summarizes these miRNAs, reported as de-regulated in two or more papers.
As shown in the [fig_ref] Table 3: The most promising circulating microRNAs [/fig_ref] , some common pathways emerge: some miRNAs are involved in neurodegeneration and apoptosis (miR-338, miR-142, miR-183 and let-7d), other miRNAs act at muscle level (miR-206, miR-133a, miR-133b and miR-27a). In particular, miR-206, miR-133a and miR-133b are myo-miRNAs, molecules specifically expressed in striated muscle and involved in muscle proliferation, repair and regeneration. Their expression levels change during the process of myogenesis, development, atrophy, degeneration, and myopathies [bib_ref] Mega roles of microRNAs in regulation of skeletal muscle health and disease, Sharma [/bib_ref]. The more recurrent result is an up-regulation of circulating miR-206 in ALS patients. miR-206 is a human skeletal muscle-specific miRNA that promotes the formation of new neuromuscular junctions following nerve injury, and therefore plays a crucial role in the reinnervation process [bib_ref] MiR-206, a key modulator of skeletal muscle development and disease, Ma [/bib_ref]. In miR-206 knock-out mice, delayed and incomplete muscular reinnervation was observed in comparison to those animals that expressed miR-206. In addition, high expression levels of miR-206 were found in a mouse model of ALS, and its under-expression was associated with a faster progression of the disease [bib_ref] MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice, Williams [/bib_ref]. A consensus for higher expression levels of this miRNA in ALS patients compared to controls was reported by several authors [bib_ref] MicroRNA-206: A potential circulating biomarker candidate for amyotrophic lateral sclerosis, Toivonen [/bib_ref] [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] [bib_ref] circulating microRNAs as biomarkers of muscle differentiation and atrophy in ALS, Tasca [/bib_ref] [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref] [bib_ref] Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral..., Waller [/bib_ref] [bib_ref] Differential expression of several miRNAs and the host genes AATK and DNM2..., Vrabec [/bib_ref]. Although miR-206 seems to represent a valid circulating biomarker for ALS, it is still to define whether the elevated expression of this miRNA is the result of the disease or its cause. ↑ Myo-miRNA: muscle proliferation, repair and regeneration [bib_ref] circulating microRNAs as biomarkers of muscle differentiation and atrophy in ALS, Tasca [/bib_ref] [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] 142 ↑ miRNA predicted to target a specific set of genes associated to the pathophysiology of ALS, including TARDBP and C9orf72. [bib_ref] Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS, Butovsky [/bib_ref] [bib_ref] Genome-wide circulating microRNA expression profiling reveals potential biomarkers for amyotrophic lateral sclerosis, Matamala [/bib_ref] 183 ↓ miRNA involved in neurodegenerative signaling pathway, including PI3K-Akt and MAPK pathway. miR-183/mTOR pathway contributes to spinal muscular atrophy pathology [bib_ref] Aberration of miRNAs expression in leukocytes from sporadic amyotrophic lateral sclerosis, Chen [/bib_ref] [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref] 27a ↓ miRNA involved in muscle growth, myoblast proliferation acting on myostatin. It is present in myoblast-derived exosomes [bib_ref] circulating microRNAs as biomarkers of muscle differentiation and atrophy in ALS, Tasca [/bib_ref] [bib_ref] Comparison of the extraction and determination of serum exosome and miRNA in..., Xu [/bib_ref] let-7d ↓ Involvement in apoptosis by the Hippo signaling pathway [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref] Abbreviations: Ref., Reference; ↑/↓, up-regulated/down-regulated.
While all the works performed a comparison between samples from ALS patients and healthy controls, only a subset of them included also samples from patients affected by other neurological disorders. Neurological controls comprised Parkinson's disease [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref] [bib_ref] Identification of plasma microRNAs as a biomarker of sporadic amyotrophic lateral Sclerosis, Takahashi [/bib_ref] [bib_ref] Aberration of miRNAs expression in leukocytes from sporadic amyotrophic lateral sclerosis, Chen [/bib_ref] [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref] , Alzeihmer's Disease [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref] [bib_ref] Serum microRNAs in sporadic amyotrophic lateral sclerosis, Freischmidt [/bib_ref] [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref] [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] , Huntington's disease [bib_ref] MiR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic..., De Felice [/bib_ref] [bib_ref] Serum microRNAs in sporadic amyotrophic lateral sclerosis, Freischmidt [/bib_ref] [bib_ref] Aberration of miRNAs expression in leukocytes from sporadic amyotrophic lateral sclerosis, Chen [/bib_ref] , Multiple Sclerosis [bib_ref] Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS, Butovsky [/bib_ref] [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] and ALS-mimic conditions [bib_ref] Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral..., Waller [/bib_ref]. The use of neurological controls can help to discriminate whether identified miRNAs are really specific for ALS or are common features linked to neurodegenerative processes. For example, the comparison of miRNA expression between ALS and Parkinson's disease patients suggested that miR-183 might be specific for SALS, whereas miR-451 and miR-3935 might be more general biomarkers linked to neurodegenerative disorders [bib_ref] Aberration of miRNAs expression in leukocytes from sporadic amyotrophic lateral sclerosis, Chen [/bib_ref]. In addition, the inclusion of an ALS-mimic patients' group may contribute to identify miRNA biomarkers to use in the differential diagnosis in the early stages of the disease.
Only a part of the studies performed until now investigated the potential correlations among miRNA expression levels and ALS clinical features, sometimes in longitudinal studies, measuring miRNA levels in the same ALS patient over time [bib_ref] Identification of plasma microRNAs as a biomarker of sporadic amyotrophic lateral Sclerosis, Takahashi [/bib_ref] [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] [bib_ref] Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral..., Waller [/bib_ref] [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref]. In some case this analysis failed to find any association [bib_ref] MicroRNA-206: A potential circulating biomarker candidate for amyotrophic lateral sclerosis, Toivonen [/bib_ref] [bib_ref] Serum microRNAs in sporadic amyotrophic lateral sclerosis, Freischmidt [/bib_ref] [bib_ref] Differential expression of several miRNAs and the host genes AATK and DNM2..., Vrabec [/bib_ref] , in other cases specific correlations were reported. Some authors described associations of miRNA expression levels with ALS site of onset [bib_ref] Identification of plasma microRNAs as a biomarker of sporadic amyotrophic lateral Sclerosis, Takahashi [/bib_ref] [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref] [bib_ref] Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral..., Waller [/bib_ref] [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref] , ALS Functional Rating Scale-revised (ALSFRS-R) and/or vital capacity (VC) [bib_ref] Identification of plasma microRNAs as a biomarker of sporadic amyotrophic lateral Sclerosis, Takahashi [/bib_ref] [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] [bib_ref] Genome-wide circulating microRNA expression profiling reveals potential biomarkers for amyotrophic lateral sclerosis, Matamala [/bib_ref] [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref] , Medical Research Council (MRC) sumscore [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] and with the disease progression rate [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] [bib_ref] Dysregulation of MicroRNAs and target genes networks in peripheral blood of patients..., Liguori [/bib_ref]. Only two studies investigated the possible associations of specific serum miRNAs with riluzole treatment, failing to identify any correlation [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref] [bib_ref] Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral..., Waller [/bib_ref]. Such results must be anyway considered with caution, since the number of subjects included in every group is limited. They need to be confirmed in larger cohorts of ALS patients, to really define the role of miRNA expression in ALS clinical presentation and progression. From this perspective, it would be very important that, after the identification of potential miRNA biomarkers, more longitudinal studies were performed, to evaluate if these miRNAs could be used as prognostic indicators.
As already mentioned for CSF studies, also in serum the analysis of combinations of several miRNAs has shown a higher accuracy than single miRNAs in discriminating ALS from healthy controls or other neurological disorders [bib_ref] Aberration of miRNAs expression in leukocytes from sporadic amyotrophic lateral sclerosis, Chen [/bib_ref] [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref] [bib_ref] Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis, Raheja [/bib_ref]. A very interesting approach is reported by Sheinerman and colleagues, who developed a strategy based on miRNA pairs, consisting of one miRNA enriched in synapses of a brain region affected by the disease and another miRNA enriched in a different brain region or cell type. The use of the pair of miRNA derived from the same organ allowed decreasing potential overlap with pathologies of other organs and reducing also inter-individual variability. The authors demonstrated that, combining two or three effective miRNA pairs into a single miRNA classifier, they could achieve a greater accuracy in discriminating ALS both from healthy controls and patients affected by other neurological disorders [bib_ref] Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative..., Sheinerman [/bib_ref]. Thus, in the future studies it should be considered that, while the deregulation of a single miRNA can be a feature common to several neurological diseases, panels of deregulated miRNAs, or combinations of them, may result highly specific for ALS and represent a signature for this disease.
Finally, a relevant aspect of the use of miRNAs as ALS biomarkers is their potential of identifying the disease in very early stages, also before any clinical manifestation. In their work, Freischmidt and colleagues showed that a specific subset of miRNAs, reduced in the serum of patients with familial and sporadic ALS, was reduced also in presymptomatic carriers of pathogenic ALS mutations. Moreover, the down-regulation was largely independent of the underlying disease gene and was stronger in patients with familial ALS than in pre-manifest mutation carriers, suggesting that alterations of miRNA profiles could be progressive when comparing the pre-manifest and manifest phase of the disease [bib_ref] Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation..., Freischmidt [/bib_ref]. If confirmed, these findings may be of fundamental importance for the development of screening tests able to detect ALS in early asymptomatic stages and for future preventive therapeutic strategies before the occurrence of clinically evaluable symptoms.
## Mirnas in muscle biopsies
Skeletal muscle is another potential source for the identification of candidate miRNA biomarkers. In the last years, it has become evident that ALS does not affect only motor neurons but also other cell types, including striated muscle, which play an active role in the disease pathogenesis. Before the clinical onset and during the disease progression, the affected skeletal muscle of ALS patients attempts to restore function by futile cycles of reinnervation and denervation [bib_ref] The role of skeletal muscle in amyotrophic lateral sclerosis, Loeffler [/bib_ref]. Eventually, persistent muscle wasting exceeds the ability to repair and consequently the atrophy process starts. Due to the crucial role of the skeletal muscle in ALS pathogenesis, recent studies have focused their research on the identification of specific muscle miRNAs in ALS tissues, which could potentially be use as prognostic biomarkers of disease. Moreover, miRNAs identified in skeletal muscle of ALS patients could be used as biomarkers also in plasma or serum, where they can be released by the affected tissues. This strategy seems to be particularly interesting, since muscle biopsy is unfortunately an invasive practice and cannot be proposed for longitudinal studies to follow disease progression.
Several studies focused on analysis of myo-miRNAs, including miR-1, miR-133a, miR-133b, miR-206, miR-208a, miR-208b, miR-499, and miR-486 [bib_ref] Muscle-specific microRNAs in skeletal muscle development, Horak [/bib_ref]. Most of them explored the role of these miRNAs in mouse models (for a review see [bib_ref] Skeletal muscle MicroRNAs as key players in the pathogenesis of amyotrophic lateral..., Pietro [/bib_ref] , but only few studies investigated the role of these molecules as possible markers in muscle biopsies of patients with ALS, due to the rarity and difficulty to obtain this kind of samples. miRNAs found deregulated in muscle biopsies from ALS patients compared to healthy control subjects are shown in [fig_ref] Table 4: Deregulated miRNAs in skeletal muscle biopsies of amyotrophic lateral sclerosis [/fig_ref].
Most studies focused on the expression of myo-miRNAs [bib_ref] Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in..., Bruneteau [/bib_ref] [bib_ref] Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral..., Russell [/bib_ref] [bib_ref] Skeletal muscle remodelling as a function of disease progression in amyotrophic lateral..., Jensen [/bib_ref] [bib_ref] Micro-RNAs in ALS muscle: Differences in gender, age at onset and disease..., Pegoraro [/bib_ref] ; only in some cases also other miRNAs were included, for example miRNAs related to inflammation/angiogenesis [bib_ref] Micro-RNAs in ALS muscle: Differences in gender, age at onset and disease..., Pegoraro [/bib_ref] or selected by microarray [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] or NGS approaches [bib_ref] Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis, Si [/bib_ref] [bib_ref] Differential expression of microRNAs and other small RNAs in muscle tissue of..., Kovanda [/bib_ref]. Overall, results are sometimes contrasting and poorly reproducible. These non-concordant finding could be attributed to different types of muscle used for biopsy, discordance among the samples in terms of inclusion criteria of patients (age, gender, evolution of disease, onset) and different techniques and internal control molecules used to assess miRNA expression levels. Some authors performed also a correlation analysis among miRNA expression and ALS clinical features. [fig_ref] Table 5: Deregulated miRNAs in skeletal muscle biopsies of specific amyotrophic lateral sclerosis [/fig_ref] reports miRNAs altered in tissue of specific stratified ALS patients' groups analyzed in comparison to control subjects.
In addition, in other papers the associations with clinical variables were analyzed comparing groups of patients to each other. Stratifying ALS patients, an up-regulation of myo-miRNAs (miR -206, miR-133a, miR-133b and miR-27a) and of inflammatory miRNAs (miR-155, miR-146a and miR -221) was discovered in ALS patients with earlier age at onset (<55 years) and longer disease duration [bib_ref] Micro-RNAs in ALS muscle: Differences in gender, age at onset and disease..., Pegoraro [/bib_ref]. Moreover, significantly higher expression levels of the same myo-miRNAs and inflammatory miRNAs were detected in male than in female. This gender difference has been hypothesized to be related to a difference in hormonal regulation, implying a slower disease progression in women [bib_ref] Micro-RNAs in ALS muscle: Differences in gender, age at onset and disease..., Pegoraro [/bib_ref]. In another paper, miR-29c, miR-208b and miR-499 were reported as increased in patients with slow disease course. Expression data were analyzed in patients categorized into "early" and "late" based on disease duration at the moment of biopsy (more or less one year). miR-9 and miR-206 significantly increased in the early patients' group and, of note, miR-206 inversely correlated with the time from symptoms onset to muscle biopsy, indicating an early response to denervation in skeletal muscle. Although the results are often inconsistent among different studies, some trends in miRNAs deregulation seem to emerge. One of the most interesting miRNA is miR-133a, which was found to be up-regulated in human ALS tissues [bib_ref] Micro-RNAs in ALS muscle: Differences in gender, age at onset and disease..., Pegoraro [/bib_ref] [bib_ref] Differential expression of microRNAs and other small RNAs in muscle tissue of..., Kovanda [/bib_ref] , particularly in patients with slow disease progression and in biopsies obtained before one year from the symptom onset. At the same time, a significant reduction of this miRNA was present in a specific ALS patients' group with higher disease severity [bib_ref] Differential expression of microRNAs and other small RNAs in muscle tissue of..., Kovanda [/bib_ref] , suggesting changes in its expression during the disease progression. In contrast, however, other studies detected a down-regulation of miR-133a in human biopsies, as reported also in mice [bib_ref] Skeletal muscle remodelling as a function of disease progression in amyotrophic lateral..., Jensen [/bib_ref] [bib_ref] Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis, Si [/bib_ref]. At the moment, the strongest data are those concerning miR-206. Indeed, the mechanisms responsible for the increase of this miRNA seem to be conserved in the skeletal muscle of mouse models and in that from ALS patients, and the up-regulation described in both cases is an ALS-specific response to the denervation. miR-206 was found significantly up-regulated in muscle samples from ALS patients compared to control subjects [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] [bib_ref] Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in..., Bruneteau [/bib_ref] [bib_ref] Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral..., Russell [/bib_ref] [bib_ref] Micro-RNAs in ALS muscle: Differences in gender, age at onset and disease..., Pegoraro [/bib_ref] , similarly to what observed in blood samples, strengthening the role of this miRNA as potential biomarker for ALS. Of note, miR-206 showed an increased trend in muscle biopsies from long-term survivor patients, even though below the statistically significance [bib_ref] Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in..., Bruneteau [/bib_ref]. De Andrade and colleagues [bib_ref] MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral..., De Andrade [/bib_ref] reported that this miRNA was over-expressed both in plasma and skeletal muscle of patients with ALS, but the over-expression was not progressive during the follow-up. They supposed that miR-206 expression increased early in the disease course, reaches a plateau and then begins to fall. In agreement with this hypothesis, an up-regulation of miR-206 was described in muscle biopsies from ALS patients within one year from the clinical onset, becoming less evident as the disease progresses to a later stage. Finally, Si and collaborators reported a non-significant upward trend in miR-206 in muscle samples from ALS patients compared to controls. This result, however, was correlated to a high standard error for this miRNA due to the variability among samples. Moreover, the authors reported a significant inverse correlation between this miRNA and the muscle power of the biopsied muscle, hypothesizing that it could be a marker of disease activity. This finding highlights the importance to associate miR-206 levels with muscle-specific clinical assessment rather than overall clinical status [bib_ref] Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis, Si [/bib_ref].
Although a concordant miRNA signature have not been identified yet in ALS patient muscle biopsies, these findings show that miRNAs could be useful prognostic markers to better understand the course of disease. In particular, the identification of a specific muscular miRNA profile through multicenter studies, able to increase the statistical power of the analysis, could lead to a stratification of the patients in order to identify prognostic biomarkers to use as indicator of disease progression, facilitating the clinical management of patients.
## Conclusions and future perspectives
Despite the intense research activity of the last years, the use of miRNAs as biomarkers for diagnosis of ALS and clinical management of patients is still in an early stage of development. Several interesting data have been obtained so far, with important insights into the disease processes. However, results achieved in different studies are most of the time conflicting and poorly reproducible, making it difficult to unequivocally identify which miRNA(s) may be selected as biomarker in clinical practice. In order to overcome these limits, some improvements in the research approach should be taken into account.
First of all, one factor strongly complicating the comparison among data reported by different research groups is the wide range of methods used for the identification of potential miRNA biomarkers and the different techniques for miRNA measurement and data normalization. A common acceptance of certain guidelines, standard research protocols, and strong methods of statistical analysis of miRNAs will be important in the future to achieve reliable biomarkers.
Another critical issue is the relatively small number of patients included in the studies performed until now. Results are often interesting, but they need to be verified in larger cohorts of ALS patients. It would be really important that those miRNAs, which have shown initial promise, were validated in independent laboratories and/or in multicenter collaborations. In addition, since ALS is a highly heterogeneous disease, replication studies should increase the number of patients stratifying them based on clinical and genetic features, in order to obtain a better assessment of the potential associations among miRNAs and these variables.
Further, in several studies miRNA levels of ALS patients have been compared only to those of control subjects not affected by neurological disorders. This approach may bring to the identification of miRNAs able to successfully differentiate patients from healthy control subjects, but these miRNAs are often associated with common pathologic processes of neurodegeneration and are not specific for ALS. It will be of fundamental importance to extend the comparison to patients affected by other neurodegenerative diseases, in particular ALS-mimic disorders, to evaluate the specificity of deregulated miRNAs for ALS.
One of the more interesting approaches to miRNA biomarker identification is the use of a complex set of biomarkers, or combinations or ratios of biomarkers from different pathogenic pathways, rather than the employ of a single marker. This strategy has been shown to increase the sensitivity and/or specificity of potential ALS biomarkers and to contain more exhaustive diagnostic information, and should be more widely used in future researches.
At the same time, when possible, future studies should try to combine data obtained from multiple source of sample (blood, CSF, muscle) of the same patient. Up to date, only few studies have performed this kind of analysis, and their results are quite conflicting. However, an extensive analysis of correlations among different samples could be helpful to obtain more informative data and improve patients' stratification. In addition, for circulating miRNAs, it would be important to perform longitudinal studies on a large number of patients, in order to identify potential biomarkers of disease progression, and evaluate their role as prognostic indicators.
In conclusion, miRNAs constitute very promising biomarkers for ALS, but there is still much work to be done to validate and use them in clinical routine. The ultimate objective is to include these biomarkers in all phases of ALS management, from the diagnosis to the clinical trials, and, in perspective, to the identification of future therapeutic approaches.
[fig] Figure 1: MicroRNA (miRNA)-based biomarkers in amyotrophic lateral sclerosis (ALS) patients. Schematic workflow to identify possible miRNAs as biomarkers starting from ALS patients' sample using different quantitative approaches. The comparison among the common characteristics of miRNA detection platforms is summarized in the figure. Sensibility, specificity and throughput are classified as follows: +++ (very high), ++ (moderate), +/++ (moderate to low) and + (low). Abbrevations: qRT-PCR, quantitative Real-Time Polymerase Chain Reaction; NGS, Next Generation Sequencing. [/fig]
[table] Table 1: Deregulated microRNAs (miRNAs) in cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis (ALS) patients compared to healthy controls. [/table]
[table] Table 2: Deregulated circulating microRNAs (miRNAs) in amyotrophic lateral sclerosis (ALS) patients compared to healthy controls. [/table]
[table] Table 3: The most promising circulating microRNAs (miRNAs) detected as potential biomarkers in amyotrophic lateral sclerosis (ALS) patients. [/table]
[table] Table 4: Deregulated miRNAs in skeletal muscle biopsies of amyotrophic lateral sclerosis (ALS) patients compared to healthy controls. up-regulated/down-regulated; SALS, sporadic amyotrophic lateral sclerosis patients; FALS, familial amyotrophic lateral sclerosis patients; HCs, healthy controls; qRT-PCR, quantitative Real-Time Polymerase Chain Reaction; NGS, Next Generation Sequencing. [/table]
[table] Table 5: Deregulated miRNAs in skeletal muscle biopsies of specific amyotrophic lateral sclerosis (ALS) patients' groups analyzed in comparison to healthy controls.1 , ALS slow group (≥4 years of disease progression without requiring respiratory supports); 2 , ALS rapid group (<4 years of disease progression without respiratory supports or death occurring <4 years from symptoms onset); 3 , early stage group (less than one year from symptom onset to muscle biopsy); 4 , late stage group (more than one year from symptom onset to muscle biopsy); 5 , group of patients with higher disease severity. Abbreviations: Ref., Reference; ↑/↓, up-regulated/down-regulated; SALS, sporadic amyotrophic lateral sclerosis patients; FALS, familial amyotrophic lateral sclerosis patients; HCs, healthy controls; qRT-PCR, quantitative Real-Time Polymerase Chain Reaction; NGS, Next Generation Sequencing. [/table]
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Methodological quality of English-language genetic guidelines on hereditary breast-cancer screening and management: an evaluation using the AGREE instrument
Background: We examined the methodological quality of guidelines on syndromes conferring genetic susceptibility to breast cancer.Methods: PubMed, EMBASE, and Google were searched for guidelines published up to October 2010. All guidelines in English were included. The Appraisal of Guidelines, Research and Evaluation (AGREE) instrument was used to assess the quality of the guidelines, and their reported evidence base was evaluated.Results: Thirteen guidelines were deemed eligible: seven had been developed by independent associations, and the other six had national/state endorsements. Four guidelines performed satisfactorily, achieving a score of greater than 50% in all six AGREE domains. Mean ± SD standardized scores for the six AGREE domains were: 90 ± 9% for 'scope and purpose', 51 ± 18% for 'stakeholder involvement', 55 ± 27% for 'rigour of development', 80 ± 11% for 'clarity and presentation', 37 ± 32% for 'applicability', and 47 ± 38% for 'editorial independence'. Ten of the thirteen guidelines were found to be based on research evidence.Conclusions: Given the ethical implications and the high costs of genetic testing for hereditary breast cancer, guidelines on this topic should provide clear and evidence-based recommendations. Our analysis shows that there is scope for improving many aspects of the methodological quality of current guidelines. The AGREE instrument is a useful tool, and could be used profitably by guidelines developers to improve the quality of recommendations.
# Background
Breast cancer comprises 22.9% of all cancers in women, and an estimated 460,000 deaths from breast cancer occurred worldwide in 2008, representing around 14% of cancer deaths in women. Breast cancer represents a challenge for public health, and in spite of the extremely high incidence rates, secondary prevention is considered to have a major role in decreasing mortality rates and costs. However, this notion has been challenged by a recent Cochrane review [bib_ref] Screening for breast cancer with mammography, Gøtzsche [/bib_ref] , reporting that screening reduces breast-cancer mortality by around 15%, which corresponds to an absolute risk reduction of only 0.05%. Nevertheless, regardless of the real effect of screening on the mortality related to sporadic breast cancer, the current scientific evidence supports secondary prevention for individuals at a high genetic risk of developing breast cancer.
A considerable proportion of breast cancers presents with genetic recurrence patterns. The two genes most frequently involved in hereditary breast cancer are the tumor suppressor genes BRCA1 and BRCA2, which are mutated in approximately 25% of hereditary breast cancers and around 5% of all breast cancers. Woman carrying mutations in either BRCA1 or BRCA2 have an 80 to 90% lifetime risk of developing breast cancer and a 20 to 50% chance of developing ovarian cancer. Thanks to early multimodal screening, breast cancer in people carrying BRCA1 or BRCA2 mutations can be diagnosed at an early stage, with consequent favorable effects on their survival and quality of life, and also on costs for the health system. Additionally, carriers can benefit from specific tertiary prevention interventions, as the risk of ovarian, contralateral breast cancer, and of other associated carcinomas (such as prostate, pancreas, and colon) is considerable. It is therefore clear that the identification of mutation carriers of BRCA1/2 represents a key issue in public health for the potential implementation of specific prevention and management programs, such as intensive riskadjusted screening, counseling, and prophylactic treatments.
The probability that an individual is carrier of a BRCA1 or BRCA2 mutation can be estimated based on the frequency and age of onset of the disease in relatives and on the organs affected (breast, ovary). Several algorithms are available to estimate the risk of being a carrier of the mutations. However, genetic testing, however, is the ultimate tool for diagnosis; issues concerning who should be tested and in which context, and the management of test users, are not easily dealt with, and the tests are expensive, and require a great deal of human resources and expertise. There are also ethical and legal issues that need to be considered; genetic information is sensitive, and data protection is necessary. All these issues need to be clearly addressed by valid, reliable, independent, and easily applicable guidelines. The Appraisal of Guidelines, Research, and Evaluation (AGREE) instrument represents a tool for a thorough quality assessment of guidelines. AGREE is a validated tool produced by the PL96-3669 research program funded by the European Union. It has been developed by researchers and policy-makers from several European countries, as well as Canada, the USA, and New Zealand. Over the past few years, AGREE has become a benchmark in both the evaluation of existing guidelines [bib_ref] Postpublication external review of the Japanese guidelines for the management of stroke, Shinohara [/bib_ref] [bib_ref] Screening for HIV in health care settings: a guidance statement from the..., Qaseem [/bib_ref] [bib_ref] Quality of professional society guidelines and consensus conference statements in critical care, Sinuff [/bib_ref] and the development of new ones [bib_ref] for the International Consensus Upper Gastrointestinal Bleeding Conference Group: International consensus recommendations..., Barkun [/bib_ref] [bib_ref] European Hernia Society guidelines on the treatment of inguinal hernia in adult..., Simons [/bib_ref]. Application of AGREE has shown that the quality of clinical and preventive guidelines is generally poor [bib_ref] International assessment of the quality of clinical practice guidelines in oncology using..., Burgers [/bib_ref] [bib_ref] Quality evaluation of guidelines on genetic screening, surveillance and management of hereditary..., Simone [/bib_ref] , and that some aspects of their quality, such as their applicability and the involvement of stakeholders, are particularly unsatisfactory [bib_ref] Quality evaluation of guidelines on genetic screening, surveillance and management of hereditary..., Simone [/bib_ref] [bib_ref] A critical appraisal of the quality of 55 critical care pharmacotherapy clinical..., Gorman [/bib_ref]. The instrument has been applied to guidelines produced in virtually every field of clinical practice, focusing on therapies, treatments, and procedures, and was also recently applied to genetic guidelines on colorectal cancer [bib_ref] Quality evaluation of guidelines on genetic screening, surveillance and management of hereditary..., Simone [/bib_ref].
The aim of this study was to provide a critical evaluation, using the AGREE instrument, of the quality of guidelines focusing on the management of individuals at higher genetic risk of breast cancer.
# Methods
We searched for guidelines published up to October 2010 that aimed to provide recommendations on the genetic screening, surveillance, and management of people who have or are suspected to have a hereditary breast-cancer susceptibility syndrome. The MedLine, EMBASE and Google databases were searched through using the following terms: (Guidelines OR Recommendations) AND Breast AND Cancer AND Screening AND (BRCA$ OR Hereditary). Reference lists of the eligible papers were also searched manually. We included only guidelines published in English that provided explicit recommendations on the management of individuals who had or were at risk of having genetic forms of breast cancer. When more than one set of guidelines was produced by the same professional body, only the most recently issued was considered. All guidelines on breast-cancer screening reporting non-original (that is, referring to other sets of guidelines on the matter of hereditary forms of breast cancer) recommendations were excluded. For each guideline, we specified the target population and objectives. In particular, the target population was defined as the general population or specific subgroups. Recommendations on breast cancer in men were also reported.
Objectives were grouped as follows.
- Assessment of level of risk for breast cancer (low, average, high) of the target population.
- Definition of the criteria of appropriateness for genetic testing.
- Definition of the criteria for empirical diagnosis of susceptibility syndromes.
- Assessment of surveillance options for individuals with a diagnosis or suspicion of susceptibility syndromes.
- Evaluation of options for prophylactic or postdiagnosis treatments.
Three investigators (BS, EDF, NN) appraised all the selected guidelines using the AGREE instrument. AGREE provides criteria to assess the quality of the methods used for developing the guidelines and of their reporting. The instrument consists of 23 key items organized into 6 domains: 'scope and purpose', 'stakeholder involvement', 'rigour of development', 'clarity and presentation', 'applicability' and 'editorial independence'. Each domain is intended to capture a separate dimension of guideline quality. Items were evaluated independently by the three investigators using a four-point scale as indicated by the AGREE instructions (from 4 (strongly agree) down to 1 (strongly disagree)). The summary score of each domain is calculated by summing the scores of all of the individual items present in the domain, and successively by standardizing the total score as a percentage of the maximum possible score for that domain, as suggested by the authors of AGREE (range 0 to 100%). Item scores were discussed by the three appraisers, and large scoring discrepancies (defined as ≤2 points difference in the score assigned by the evaluators to the same item) were resolved by consensus.
According to the AGREE collaboration Group, based on the results for each of the six domains evaluated, a guideline can be 'strongly recommended', 'recommended with provisions', or 'not recommended'. The instrument does not provide criteria to formulate the overall assessment on the guideline, leaving it up to the discretion of the evaluator. We considered as satisfactory any guideline that scored at least 50% in all six of the domains as defined by AGREE. Guidelines were further classified based on whether they were developed by independent associations or by national/state-endorsed societies. The Mann-Whitney test was used to compare the median values of each of the 6 domain scores obtained by applying the AGREE instrument to the 17 guidelines, based on the presence or absence of an endorsement.
We also integrated the AGREE instrument by applying an additional system aimed at evaluating whether guidelines could be considered evidence-based. Following a scheme already proposed in the literature [bib_ref] Quality evaluation of guidelines on genetic screening, surveillance and management of hereditary..., Simone [/bib_ref] [bib_ref] Quality assessment of clinical practice guidelines in perioperative care: a systematic appraisal, Barajas-Nava [/bib_ref] , we defined three criteria for this purpose: the search strategy having been reported in at least one database, the quality of evidence classified, and the strength of recommendations reported.
# Results
## Literature search
The electronic databases search identified 215 results from MedLine, 188 from EMBASE, and over 302,000 from Google. After a first reading of the titles, any results that were not guidelines were excluded. Duplicates were also excluded, and the application of the inclusion and exclusion criteria [fig_ref] Figure 1: Flowchart of the guidelines selection process [/fig_ref] led to the final selection of 13 sets of guidelines (detailed in [fig_ref] Table 1: Description of the thirteen breast cancer screening guidelines included in the study [/fig_ref] [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] [bib_ref] Practice Issues Subcommittee of the National Society of Genetic Counselors' Familial Cancer..., Berliner [/bib_ref] [bib_ref] American Cancer Society guidelines for breast cancer screening: update 2003, Smith [/bib_ref]. All the selected guidelines were developed in English-speaking countries because of the restrictions used in the research (eight from the USA [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] [bib_ref] Practice Issues Subcommittee of the National Society of Genetic Counselors' Familial Cancer..., Berliner [/bib_ref] [bib_ref] American Cancer Society guidelines for breast cancer screening: update 2003, Smith [/bib_ref] , two from the UK , and one each from Canada, New Zealandand Singapore [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref]. Of the 13 guidelines, 7 were produced by independent professional scientific societies [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] [bib_ref] Practice Issues Subcommittee of the National Society of Genetic Counselors' Familial Cancer..., Berliner [/bib_ref] [bib_ref] American Cancer Society guidelines for breast cancer screening: update 2003, Smith [/bib_ref] , whereas six were developed with the endorsement of national/state authorities [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] [fig_ref] Table 1: Description of the thirteen breast cancer screening guidelines included in the study [/fig_ref].
## Target population and objectives of guidelines
The guidelines analyzed are relatively homogeneous in terms of target populations: they all begin by focusing on the general population and then provide specific recommendations on patients with high-risk syndromes. Regarding the objectives, surveillance recommendations are provided by all the guidelines, but not all give indications about how to perform a risk assessment [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] , criteria of appropriateness for genetic testing [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] , the definition of empirical diagnostic criteria of susceptibility syndromes [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] or the available treatment options. Apart from the BRCA1/2 syndromes, most guidelines also provide recommendations on, or at least mention, less common syndromes such as Li-Fraumeni, Peutz-Jeghers, and Cowden syndromes [fig_ref] Table 1: Description of the thirteen breast cancer screening guidelines included in the study [/fig_ref]. Although the main recommendations are focused on women, all the guidelines provide at least some recommendations on syndromic breast cancer in men.
## Appraisal of guidelines
Based on the criteria defined in the methods section, 10 (77%) of the 13 guidelines are evidence-based [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] [bib_ref] American Cancer Society guidelines for breast cancer screening: update 2003, Smith [/bib_ref] [fig_ref] Table 1: Description of the thirteen breast cancer screening guidelines included in the study [/fig_ref] , and apart from the 3 exceptions [bib_ref] Practice Issues Subcommittee of the National Society of Genetic Counselors' Familial Cancer..., Berliner [/bib_ref] , all guidelines stated, either in the text or in a clearly specified link, the methods used in the literature search, the quality of the evidence, and the strength of recommendations reported.
Application of the AGREE instrument produced six standardized scores for each guideline, pertaining to the specific domain [fig_ref] Table 2: Standardized scores [/fig_ref]. We deemed satisfactory the guidelines produced by the Institute for Clinical Systems Improvement (ICSI), The New Zealand Guidelines Group (NZGG), the UK National Health System (NHS) and the Scottish Intercollegiate Guidelines Network (SIGN), which all had a score of at least 50% in each of the six domains. All the other guidelines scored below 50% in at least one domain. The lowest scores were assigned to the Guidelines produced by the Ministry of Health of Singapore [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] , with five of the six domains scoring below 50%. Two more guidelinesalso achieved scores of below 50% in four of the six domains [fig_ref] Table 2: Standardized scores [/fig_ref].
As shown in [fig_ref] Table 2: Standardized scores [/fig_ref] , the highest score (100%) for domain 1 (scope and purpose) was given to the guidelines of the American College of Obstetricians and Gynecologists (ACOG)and the National Society of Genetic Counselors [bib_ref] Practice Issues Subcommittee of the National Society of Genetic Counselors' Familial Cancer..., Berliner [/bib_ref] , whereas the lowest score (7%) was assigned to the Singapore guideline [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref]. Scores for domain 2 (stakeholder involvement) were generally very low, ranging from 75% (NZGGand NHS to 8% (University of Michigan. The SIGN guidelinesgained the highest score (97%) for domain 3 (rigour of development), whereas the lowest (8%) was assigned to the guidelines from Towards Optimized Practice Alberta. The highest score (100%) assigned to domain 4 (clarity and presentation) was achieved by the NZGG, and the lowest (56%) by the American Cancer Society [bib_ref] American Cancer Society guidelines for breast cancer screening: update 2003, Smith [/bib_ref]. SIGNhad the best score (89%) in domain 5 (applicability), whereas the University of Michigan had the worst (0%). Finally, the top scores (100%) for domain 6 (editorial independence) were obtained by the National Cancer Comprehensive Networkand the NZGG, whereas four guidelines [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] scored 0% in this domain for not being explicit on conflicts of interest and on independence statements from funding bodies. details the overall mean scores for all the 23 items included in the 6 domains, and the overall mean standardized scores for each of the 6 domains from the 13 guidelines evaluated. The highest score was obtained for domain 1 (scope and purpose) with a value of 90 ± 9%, and domain 4 (clarity and presentation) with 80 ± 11%, whereas the lowest scores were for domain 5 (applicability) with 37 ± 32%, and domain 6 (editorial independence) with 47 ± 38%. Domains 2 (stakeholder involvement) and 3 (rigour of development) scored overall 51 ± 18% and 55 ± 27%, respectively .
Comparison between endorsed and non-endorsed guidelines showed that the former performed better in five of the six domains, although no statistical significance was attained for any domain.
# Discussion
Genetic forms of breast cancer are an issue for public health. Women with a family history of breast cancer, and especially women with genetically known forms of susceptibility, can benefit from appropriate prevention and treatment interventions. Outcomes for breast cancer are strongly associated with the stage and degree of disease progression at the time of diagnosis, and this also holds true for genetically determined forms. Because effective screening surveillance and adequate preventive measures are proven to have a dramatic effect on the survival and the quality of life of individuals with inherited breast-cancer syndromes [bib_ref] Screening for breast cancer with mammography, Gøtzsche [/bib_ref] , specific recommendations to define high-risk individuals and appropriate screening protocols should be provided. It is essential that, given the ethical implications of genetic testing, and also in consideration of the high costs related to their administration, guidelines should provide very clear and In this study, we aim to evaluate the quality of methodology of guidelines dealing with the issue of genetic testing for hereditary breast cancer, using the AGREE instrument. The application of AGREE allows evaluation of various aspects of guidelines quality: 'scope and purpose', taking into account whether the objectives, the clinical questions, and the target population are properly specified; 'stakeholder involvement', assessing which professional groups have been involved in the guideline development, and whether patients' views and preferences have been sought; 'rigour of development', with a list of key items focusing on the methods used by the developers, starting from the literature search up to the external review of the recommendations; 'clarity and presentation', focusing on how easily the user is able to find the key recommendations and the possible alternatives in the guideline; 'applicability', with three key items assessing how organizational barriers, potential cost implications, and patient monitoring/audit have been discussed; and 'editorial independence', assessing independence statements and records of potential conflicts of interest of the guideline developers.
The evaluation of the quality of the evidence which the guidelines build upon was beyond our objectives. We focused on the methods used in the course of development of the guidelines, which is the purpose of the AGREE instrument, based on the rationale that high methodological quality is fundamental in terms of credibility, reproducibility and transparency of guidelines. Furthermore, in the case of genetic susceptibility syndromes for breast cancer, as of today, there is a limited body of evidence focusing on the best screening and management options. All the guidelines considered in this review are based on the same studies, therefore the recommendations necessarily converge. The recommendations on the topic given by the guidelines analyzed are as follows.
- All individuals at high risk (individuals from known high-risk families, or with high scores on the BRCAPRO [bib_ref] Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2, Parmigiani [/bib_ref] or BOADICEA [bib_ref] Predicting the likelihood of carrying a BRCA1 or BRCA2 mutation: validation of..., Antoniou [/bib_ref] programs, or deemed at high risk based on clinical judgment) should be offered referral for information on genetic testing.
- Counseling from training personnel should be always available.
- If a mutation is identified in one individual from a high-risk family, predictive testing should then be offered to all adult at-risk family members.
- Known carriers of a BRCA1 or BRCA2 gene mutation should be offered counseling and the option of prophylactic mastectomy, and prophylactic salpingo-oophorectomy should also be discussed.
- Individualized screening strategies for known carriers of BRCA1 or BRCA2 gene mutations should be considered, such as earlier screening, shorter intervals between screens, and possibly annual MRI surveillance.
The most important difference between guidelines, however, and we believe it to be noteworthy, is how the different developers used the same evidence to produce the guidelines. The application of AGREE detected some major flaws in the development of the 13 guidelines on the topic, as some of the aspects investigated by AGREE were not included in these guidelines. With very few exceptions, the 13 guidelines all performed poorly with regard to 'stakeholder involvement' (domain 2) and 'editorial independence' (domain 6). Regarding stakeholder involvement, target users of the guideline (general practitioners, gynecologists, oncologists) remained generally undefined (key item 6), patient representatives were seldom involved (key item 5) in guideline development, and most guidelines were not piloted among end users (key item 7). Regarding editorial independence, explicit statements of independence from funding bodies (key item 22) were often not clearly stated, and did not allow the identification of possible conflicts of interest. The application of AGREE also showed that the methodological quality of the guidelines was suboptimal in terms of 'rigour of development' (domain 3) and 'applicability' (domain 5). Most guidelines lacked explicit statements on the criteria for selecting the evidence (key item 9), on whether they were externally reviewed before publication (key item 13), and on procedures for their update (key item [bib_ref] Quality assessment of clinical practice guidelines in perioperative care: a systematic appraisal, Barajas-Nava [/bib_ref]. Generally speaking, the AGREE instrument gave high scores for domains 1 (scope and purpose) and 4 (clarity and presentation), even though not all guidelines received fully positive evaluations.
Although there was a good degree of convergence between guidelines in terms of recommendations provided, our study does have implications for clinical practice as well. As mentioned above, the AGREE instrument provides six independent scores for six corresponding aspects of the guidelines; clinicians would be interested primarily in the 'applicability' domain. It is fundamental that recommendations are not only rigorous in method but also feasible when applied to a specific clinical setting. In this sense, we recommend clinicians should rely preferentially on the guidelines that performed better with regards to the 'applicability' domain , as those guidelines gave more consideration to issues related to overcoming possible organizational barriers when applying the recommendation (key item [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] , and to presenting criteria for monitoring and audit purposes (key item 20).
By applying the AGREE instrument to the 13 guidelines on genetic testing for breast cancer, we found that guidelines developed by the ICSI, the NZGG, the SIGNand the NHS scored above 50% in all six domains, with the NZGG, who acknowledged the adoption of AGREE in the guideline development, scoring above 70% in all domains. As for the other guidelines, twoyielded poor scores (below 50%) in one of the six domains, three [bib_ref] for the American Cancer Society Breast Cancer Advisory Simone et al, Saslow [/bib_ref] [bib_ref] Practice Issues Subcommittee of the National Society of Genetic Counselors' Familial Cancer..., Berliner [/bib_ref] [bib_ref] American Cancer Society guidelines for breast cancer screening: update 2003, Smith [/bib_ref] in two of the six domains, onein three of the six domains, twoin four of the six domains, and one [bib_ref] Ministry of health clinical practice guidelines: cancer screening, Lee [/bib_ref] in five of the six domains.
The guidelines produced by societies with an official endorsement tended to perform better with regard to all Mean scores for the 23 items and overall standardized scores for each domain from the 13 guidelines evaluated assessed with AGREE.
## Domain
Item Mean score (range)
## (scope and purpose)
- The overall objective(s) of the guideline is (are) specifically described 3.7 (2.3 to 4.0) - The clinical question(s) covered by the guideline is(are) specifically described 3.6 (2.7 to 4.0) - The patients to whom the guideline is meant to apply are specifically six the AGREE domains, however, a significant difference was not detected, probably due to the small sample size.
# Conclusions
The high number of guidelines with low methodological quality in the literature on genetic testing for hereditary breast cancer prompted us to evaluate their methodological quality scientifically. We also provided an insight on important factors that have been missed out of some guidelines, and which, in our opinion, should be considered. The whole objective of using the AGREE instrument is to provide a common ground on rigor and transparency of guideline development, and to suggest how to improve on the existing guidelines. In this sense, the most self-explanatory example is that of conflicts of interest; the AGREE instrument recommends that guidelines always report explicitly whether conflicts exist or not. The absence of an explicit statement does not necessarily mean that a conflict of interest exists, but rather that providing such a statement was not a standard procedure in the development of the guidelines. We would recommend that all future guidelines should always state explicitly that conflicts of interest do or do not exist. It is noteworthy that the results reported here are very similar to those reported for guidelines focusing on genetic forms of colorectal cancer [bib_ref] Quality evaluation of guidelines on genetic screening, surveillance and management of hereditary..., Simone [/bib_ref]. Although this study and the previous study do not cover the whole subject of genetic-testing guidelines, they certainly corroborate each other in the notion that there is much to be achieved and improved in terms of methodology and quality where genetic tests are concerned.
[fig] Figure 1: Flowchart of the guidelines selection process. based recommendations on who should be tested, based on their personal and family history and on clinical criteria. [/fig]
[fig] •••: The guideline development group includes individuals from all the relevant professional groups 3.of development) • Systematic methods were used to search for evidence 2.6 (1.0 to 4.0) • The criteria for selecting the evidence are clearly described 2.8 (1.0 to 4.0) • The methods used for formulating the recommendations are clearly described 3.0 (1.0 to 4.0) • The health benefits, side effects, and risks have been considered in formulating There is an explicit link between the recommendations and the supporting evidence 3.1 (1.3 to 4.0) • The guideline has been externally reviewed by experts before its publication 2.3 (1.0 to 4.0) • A procedure for updating the guideline is The different options for management of the condition are clearly The potential organizational barriers in applying the recommendations have been discussed 2.2 (1.0 to 4.0) • The potential cost implications of applying the recommendations have been considered 2.4 (1.0 to 4.0) • The guideline presents key review criteria for monitoring and/or independence) • The guideline is editorially independent from the funding body 2.2 (1.0 to 4.0) • Conflicts of interest of guideline development members have been [/fig]
[table] Table 1: Description of the thirteen breast cancer screening guidelines included in the study.Other syndromes: Bannayan-Riley-Ruvalcaba syndrome, Peutz-Jeghers syndrome, hereditary diffuse gastric cancer and ataxia telangectasia. [/table]
[table] Table 2: Standardized scores (%) on the Appraisal of Guidelines, Research and Evaluation (AGREE) instrument assigned to the 13 guidelines. [/table]
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The unintended consequences of sex education: an ethnography of a development intervention in Latin America
This paper is an ethnography of a four-year, multi-disciplinary adolescent sexual and reproductive health intervention in Bolivia, Nicaragua and Ecuador. An important goal of the intervention À and of the larger global field of adolescent sexual and reproductive health À is to create more open parent-to-teen communication. This paper analyzes the project's efforts to foster such communication and how social actors variously interpreted, responded to, and repurposed the intervention's language and practices. While the intervention emphasized the goal of 'open communication,' its participants more often used the term 'confianza' (trust). This norm was defined in ways that might À or might not À include revealing information about sexual activity. Questioning public health assumptions about parentÀteen communication on sex, in and of itself, is key to healthy sexual behavior, the paper explores a pragmatics of communication on sex that includes silence, implied expectations, gendered conflicts, and temporally delayed knowledge.
# Introduction
There is by now an extensive anthropological critique of the international development project, focusing on large infrastructure projects, the de-politicizing effects of development, the creation of neo-colonial relationships, and much else [bib_ref] Anthropology and its evil twin; development in the constitution of a discipline, Ferguson [/bib_ref]. We also see the increasing use of anthropological concepts and research methods in development work itself [bib_ref] Order and disjuncture: theoretical shifts in the anthropology of aid and development, Rossi [/bib_ref] [bib_ref] Anthropologists only need apply: challenges of applied anthropology, Sillitoe [/bib_ref] [bib_ref] The anthropology of international development, Mosse [/bib_ref]. This phenomenon is particularly visible in the field of global health where anthropologists have often been employed to understand the 'cultural barriers' to good healthcare delivery or to act as 'mediators' between healthcare institutions and communities [bib_ref] Three propositions for a critically applied medical anthropology, Scheper-Hughes [/bib_ref]. The use of anthropological research in development is itself controversial and has raised the question of whether such research simply serves as a 'rubber stamp' for topdown interventions, or whether it has much effect at all [bib_ref] The anthropology of international development, Mosse [/bib_ref] [bib_ref] Devoted to development and moral progress, ethical work, and divine favor in..., Pandian [/bib_ref] [bib_ref] The construction of the global poor: an anthropological critique, Gupta [/bib_ref]. What has been less studied, however, is how such *Corresponding author. Email: [email protected] Ó 2014 The Author(s). Published by Taylor & Francis. This is an Open Access article. Non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly attributed, cited, and is not altered, transformed, or built upon in any way, is permitted. The moral rights of the named author(s) have been asserted. anthropology works on the ground. How do the various social actors interpret and 'use' development interventions?
This paper is an anthropological 'meta-study' À an ethnography of a multi-disciplinary health intervention that included ethnographic fieldwork. Its focus is a fouryear, international project on adolescent sexual and reproductive health carried out in Cochabamba, Bolivia, Managua, Nicaragua, and Cuenca, Ecuador. 'Community-Embedded Reproductive Health Care for Adolescents' (CERCA) was conducted with European Commission funding, bringing together public health professionals, obstetrician-gynecologists, epidemiologists, economists, psychologists, statisticians, peer educators and anthropologists from Europe, North America and South America. The intervention embraced current 'progressive' ideas, ranging from the standard global health emphasis on healthcare rights and local capacity building to the use of anthropological fieldwork and the need to identify and respond to 'community' needs [bib_ref] Community embedded reproductive health interventions for adolescents in Latin America: development and..., Decat [/bib_ref].
The rationale to include anthropologists, as in many other development projects, was to study such community needs as well as the wider socio-cultural context shaping healthcare decisions. A key part of this context was communication between parents and teens. One of the CERCA project's guiding premises was that unilateral public health policies are not enough to improve adolescent sexual and reproductive health in the global South [bib_ref] Community embedded reproductive health interventions for adolescents in Latin America: development and..., Decat [/bib_ref]. Instead it aimed to take comprehensive actions targeting multiple 'levels': clinics and communities, behaviors and beliefs, individual adolescents, and crucially, families. Reflecting priorities first defined by the International Conference on Population and Development 20 years ago, parents were seen as key agents of change .
Open communication with parents might seem to be a self-evident 'good' since adolescents need sexual education from elders in order to access contraception or resist peer or partner pressures to have sex before they feel ready. In the US, the dominant public health narrative of the past two decades has linked actions to improve 'open communication' between parents and teens to positive adolescent health outcomes [bib_ref] Patterns of condom use among adolescents: the impact of mother-adolescent communication, Miller [/bib_ref] [bib_ref] Teenage partners' communication about sexual risk and condom use: the importance of..., Whitaker [/bib_ref] [bib_ref] Parent-adolescent communication about sex and birth control: a conceptual framework, Jaccard [/bib_ref] [bib_ref] The role of motherdaughter sexual risk communication in reducing sexual risk behaviors..., Hutchinson [/bib_ref] [bib_ref] Beyond the "big talk": the roles of breadth and repetition in parent-adolescent..., Martino [/bib_ref]. Outside the US, the global field of adolescent and reproductive sexual health has also viewed À although less prominently À improved family communication as an important goal in health interventions [bib_ref] Correlates of adolescent pregnancy in La Paz, Bolivia: findings from a quantitative-qualitative..., Liposvek [/bib_ref] [bib_ref] The psychosocial context of young adult sexual behavior in Nicaragua: looking through..., Rani [/bib_ref] [bib_ref] Parental influence on reproductive health behaviour of youths in Ibadan, Nigeria, Amoran [/bib_ref] [bib_ref] Mother-daughter communication about sexual maturation, abstinence and unintended pregnancy: experiences from an..., Crichton [/bib_ref] [bib_ref] Hidden love: sexual ideologies and relationship ideals among rural South African adolescents..., Harrison [/bib_ref] [bib_ref] Let's talk about sex: reflections on conversations about love and sexuality in..., Bochow [/bib_ref] [bib_ref] Research brief: sexual communication and knowledge among Mexican parents and their adolescent..., Gallegos [/bib_ref] [bib_ref] Intervenciones con padres de familia para modificar el comportamiento sexual en adolescentes, Atienzo [/bib_ref] [bib_ref] Because you're on birth control, it automatically makes you promiscuous or something:..., Caal [/bib_ref]. [bib_ref] A review of interventions with parents to promote the sexual health of..., Wight [/bib_ref] , however, have pointed to the considerable difficulties in measuring 'open communication' between parents and teens. In a more anthropological vein, [bib_ref] Secrecy as embodied practice: beyond the confessional imperative, Hardon [/bib_ref] discuss the moral dimension of pressures to be 'open' in health communication: '"Speaking is healing" is the confessional imperative that juxtaposes secrecy to truth telling. To not be open implies withholding the truth, a moral act. Hardon and Posel argue that this normative understanding informs many public health interventions today. And while the goal of open communication reflects laudable public health goals of combating stigma surrounding sexually transmitted diseases, it can paradoxically blind us to the social utility of secrecy: 'What we reveal and what we withhold are sites of negotiation, integral to the ways in which we inhabit the social world' [bib_ref] Secrecy as embodied practice: beyond the confessional imperative, Hardon [/bib_ref].
In this paper we analyze the CERCA project's efforts to foster open communication between parents and teens and how social actors interpreted, responded to, and sometimes repurposed the intervention's language and practices. While direct verbal communication may be the most obvious way to communicate knowledge of sex, it is far from the only way. Instead, we found distinct and sometimes conflicting social uses of this knowledge, for example knowledge that was shared, withheld, or saved for later use. While the CERCA project and public health discourse emphasized the goal of 'open communication,' the people targeted by the project more often used the term confianza (trust). The norm of confianza was defined in ways that might À or might not À include revealing information about sexual activity.
There were also important affective aspects of communication À shameful, shaming, or empowering À that subtly shaped efforts to achieve the moral norm of 'openness.' Whether or not teens claimed to have 'good', 'bad', plenty or no communication with adults, they were also exposed to the morality tales, gossip, family histories, half-whispered scandals, and contradictory messages about sex that adults speak. For adults, communication on sex was not a straightforward transmission of facts about contraception, but entailed a navigation of intergenerational conflicts and threats to their children's public reputation.
The intervention, including its anthropological component, was circumscribed by existing social relations but also created spaces for new dynamics to emerge. Different actors used the intervention for their own ends, only some of which dovetailed with the original public health aims. For example, participants used intervention spaces to shame a 'lying' husband, to share knowledge that should nevertheless not be 'acted upon,' or to find out who a son or daughter was dating. Other adults, while acknowledging the intervention's importance, insisted it was a 'woman's thing' or 'for gays.' Silence, shame, or simple lack of interest in speaking about sexual and reproductive health not only signified an absence of 'open communication,' but were themselves highly expressive of larger social contradictions and struggles to define moral norms.
## Research methods and sites
This paper draws on fieldwork primarily conducted in Cuenca (Nelson, 10 months total during the project's pre-intervention and intervention stages, between January 2011 and March 2013), shorter periods of intensive research in Managua and Cochabamba one month in each, January and February 2013), and rolling peer discussion groups (five rounds in total, . Fieldwork in all three cities during the intervention period consisted of in-depth semi-structured and unstructured interviews with adult and youth participants of CERCA, and with non-participants living in target communities (37 in Cuenca, 20 in Managua, 14 in Cochabamba); participant ethnographic research with young people and parents of young people peer-conducted interviews) using modified 'PEER' methods [bib_ref] Researching sexual and reproductive behaviour: a peer ethnographic approach, Price [/bib_ref] ; observation of intervention activities (school-based and clinic-based workshops, health fairs, movie forums, public outreach events); peer group discussions (18 in Cuenca, 13 in Cochabamba, 15 in Nicaragua); and two documentary films [bib_ref] When target groups talk back: at the intersection of visual ethnography and..., Nelson [/bib_ref]. The majority of in-depth interviews and peer-group discussions were transcribed in their original Spanish. The transcripts were subjected to holistic content analysis where key themes were identified and triangulated with field notes and observations. Intervention activities varied by city but included outreach campaigns, sex education workshops in schools, free text-message and Internet hotlines, 'adolescentfriendly communication' training for health providers, and mobile health clinics and condom dispensaries. The project also had a major focus on research, including 'deliverables' such as international journal publications (for example this paper). Anthropological research also aimed to create new spaces for dialogue. Regular discussion groups were organized in each site, held separately with teens and parents. The groups were named comit es comunitarios to reinforce the premise that discussions would function as non-hierarchical spaces in which all participants (facilitators included) held equally valid knowledge [bib_ref] Hidden love: sexual ideologies and relationship ideals among rural South African adolescents..., Harrison [/bib_ref] [bib_ref] Socioeconomic context and the sexual behaviour of Ugandan out-of-school youth, Bohmer [/bib_ref]. These 'community committees,' it was hoped, would reveal the barriers to more open communication between teens and parents, as well as provide a space where they could discuss solutions.
## 'open communication' or 'confianza'? interference and overlap between two moral norms
An important premise of the intervention strategy À anchored in the vast global literature on the importance of teenÀparent communication to sexual and reproductive health À was that it would foster 'open communication' where it had previously not existed. But in all three sites, young people as well as parents much more commonly referred to 'confianza' À having it (tenemos confianza) or lacking it (falta de confianza). There is a subtle difference between the terms confianza and 'open communication.' Confianza implies mutual trust and intimacy, but unlike open communication, does not necessarily entail a relationship where sex is talked about openly. In fact, the moral norm of open communication at times came into direct conflict with the moral norm of confianza.
It was the last peer discussion group in Cuenca, Ecuador, after almost three years of research. The young men and women, and one of the mothers, were by then well known to the ethnographer. Bruno, Julio Ã1 , Virginia à , Wilson, Bryan, Oscar, Laura and Ana Luc ıa had variously participated as peer researchers, peer group discussants, focus group discussants, interviewees and documentary film collaborators. Beyond their research participation, all but two were on posters promoting the project, which dotted the local health center and high school corridors: 'Quererse mutuamente es protegerse sanamente' (To love each other is to protect each other's health). During this period, some of the original peer group had graduated from high school, and were now outside the intervention's target population. Romances between participants had bloomed and withered, certain exes had been exiled. This small group of teenagers had at length analyzed, debated and dramatized love, sex, jealousy, gossip, peer pressures À and family pressures. So it was a surprise when Julio entered what was intended to be a 'peer space' accompanied by his mother.
Julio sat slumped, arms crossed and silent next to his mother. After some 20 minutes of listening to the teenagers critique the lack of trust between adults and young people, his mother raised her hand: I have a question for you who are so linked to this issue of sexuality. Does it seem right (conveniente) to you that a young man or woman, at an early age, is advised to have sexual relations? 2
The ethnographer knew Julio had a serious girlfriend, for she had seen them holding hands and cuddling. Now his friends looked at him with a mix of embarrassment and concern as he sank deeper into his chair. In a place where people talked about 'sexual relations' in code in a lyrical southern Sierras Spanish that circled and whirled and steered clear of vulgaridades, her directness came as a shock. Put on the spot, the ethnographer stumbled out a non-committal response, 'It is not a question of convenience or right, but just a matter of fact that lots of young cuencanos are already sexually active.'
His mother conceded, 'We can't put a knife to their back saying they can't do it.' She turned to Julio, 'This is my son. To motivate my son I have told him, "saying to someone 'I love you' doesn't mean that you are kissing in the street. I will beat you (te pego) if I see you kissing in public".' A debate ensued. Bruno countered that his grandfather was pleased that CERCA had given his grandson knowledge on how to use condoms. In response, Julio's mother claimed, 'If I ask my son with whom he has the most trust (confianza) he will say his mom.' For her, having confianza did not mean the ability of a teen to ask for contraceptive knowledge without fear of consequences or a parent's ability to provide information about contraceptives; it signified the strength and intimacy of the motherÀson bond.
In the days that followed, the grandmother of Julio's good friend Virginia à told the story of when Julio had arrived crying here one day, saying 'I'm going to die' because he had asked his mom 'how old does a person have to be to have sex?' And his mom grabbed a stick and beat him. The poor boy didn't know where to go so he came here.
In a separate conversation, a health professional involved in the project offered the unsolicited information that Julio had already come to the clinic, having text-messaged the free CERCA hotline to get emergency contraception for his girlfriend.
While the CERCA intervention sought to build confianza À usually interpreted as 'open communication' À between adolescents and parents, the ethnographic research process itself became a venue for a diverse set of social actors to contest the meanings and uses of confianza. For Julio's mother, confianza appeared to be the unquestioned basis of their close relationship and was not threatened by her ruling out any discussion of sex. For Virginia's grandmother, it meant providing young people with a place where sex could be discussed openly and without punishment. For Bruno (Julio's friend and peer), it meant adult family members supporting young people to learn about modern contraceptive methods. For the health professional, it meant adolescent-friendly contraception services that young people could use privately. For the ethnographer, it meant creating enough trust between those targeted by the intervention and those running it to be able to talk openly about sensitive subjects.
The moment Julio's mother walked into the 'peer' space, different interpretations of confianza came to a head. The intervention did not simply 'reveal' a pre-existing intergenerational conflict or a 'cultural barrier' (i.e. a beating) to open communication; the intervention also created new 'temporary socialities.' As Bochow (2012: S18) notes, conversations about 'love and sexuality are in themselves social acts that create a momentary, spontaneous sense of "being together".' Within the context of a health intervention dedicated to making sexuality a topic of familial conversation, this 'being together' was not spontaneous. Instead, multiple interpretations of the project's call for more open talk about sexuality constituted social forms of communication in and of themselves.
In the final period of fieldwork, the lead ethnographer brought together adults and adolescents (sometimes related, sometimes not) in joint discussion groups (they had previously been kept separate). The decision to do so was made partly because peer group discussants had, in anonymous 'comments slips,' asked for the chance to have facilitated conversations with adults. In the course of one workshop in Managua, discussants spoke about the differences between 'mandar' (to order around) and 'dialogar' (to discuss). Several of the mothers, having themselves gotten pregnant in adolescence, argued that knowing who their daughters were with made it possible to give 'buen consejo' (good advice) to prevent them from making the same 'mistakes.' But the young women complained that their mothers boss them around and nose around in their private lives (entrometerse en la vida privada). In a one-on-one interview the next day, one teenager said that a parent who gets 'up in your business' by asking direct questions about sexual intimacy is, despite speaking openly, displaying a lack of trust (no conf ıan en vos). Here again, 'open communication' means something quite different from confianza.
The complexities of confianza were reiterated by young people across the CERCA research sites. When asked how they talk about sex and sexuality with parents or significant adults, the initial answers fell into two categories. Either they claimed that 'no hay confianza' (there's no trust) or, conversely, 'tenemos confianza' (we have trust). These same claims were contradicted within the same interview as well as in instances where we had ongoing conversations and contact over the life of the project. Those who reported a lack of confianza would nonetheless demonstrate detailed awareness of their family's rules and expectations regarding who they should, or would be allowed to be romantically partnered with, when and in what context they would be allowed to begin having sex, etc. And those who claimed they did have confianza could nevertheless speak of precise boundaries to communication, which aspects of their sexual lives must be hidden, and which questions could not be asked.
## Sexual knowledge in conflict
The different interpretations of 'open communication' and 'confianza' do not simply reflect a translational discrepancy between public health discourse and everyday language. They instead point to larger power dynamics and changing moral and sexual norms that circumscribe what can be spoken and what must be silenced in parent-adolescent talk and within extended families. Some teens, as we have seen, contested their parents' 'prying' by complaining that such behavior reflected a lack of confianza. On the other hand, some parents understood the project as justifying a return to the socio-sexual practices of previous generations where adolescent partnerships would be subjected to family approval. In one instance, Patricio, the father of a seventeen-year old girl who had participated in the peer discussion groups, said:
If I am honest and sincere, the project has already given a result, because my daughter has a boyfriend and given the way things are these days I didn't even realize it. And some days ago the boy came to the house to talk to us. Why? So that we could give permission for her to go out with him . . . something very few young people do these days. And this is really great, doctor, if I'm not mistaken.
Other adults reiterated this father's praise of the workshops on open communication as a means to enhance parental control over adolescent relationships. In both Managua and Cuenca, adult project participants interpreted the emphasis on open communication as a license to encourage their children to make public relationships that might otherwise have been kept hidden. But as the story of Julio's mother illustrates, parental encouragement to bring a boyfriend or girlfriend forward for familial judgment did not entail approval of public displays of affection, nor did it equal a green light for the initiation of sexual relations. Patricio explained: 'You can't just let them run wild (dejarlas sueltas), saying you can do whatever you want, or go out with whomever you want at any hour of the day. Parental involvement in teen partner choice was asserted in the idiom of confianza, but spoke to issues that went beyond the focus on the parentÀchild dyad that is the norm in North American adolescent sexual and reproductive health programs. Young people spoke of receiving conflicting advice from older relatives, for example aunts versus mothers versus cousins, or uncles versus brothers versus fathers. Having confianza with young people was also important to some family members because they believed it was their duty to assess the socioeconomic status and seriousness of a young relative's potential partner À for example, through a face-to-face meeting.
Parental judgments also reflected each region's racial and economic hierarchies. For example, in Cuenca status is often judged according to perceived distance from a state of indigenousness, which in turn indexes both 'race' and class background (see. In the outer barrios of Managua, parents were more concerned by a young person's perceived proximity to 'the street' as a place of gang violence, sexual promiscuity and drug use. The monitoring of teens' reputations was not exclusively a parental concern. In many Cochabamban and Cuencan families, one or both parents were migrant laborers and the duty and right to protect young people and monitor their activities were distributed across extended family networks. Communication on sex thus went far beyond the transmission of information about contraception or the presence or absence of parentÀteen trust, but was shaped by the local semiotics of appearance, status hierarchies, and the making and unmaking of (especially female) 'reputation.'
The wrong amount of sex and involvement with the wrong partner were frequently the subject of morality tales, scandals and gossip (chisme or chuchicheo). Both young people and adult participants in all three countries used pejoratives to describe (perceived) sexually active young women ('roses without petals' and 'ball warmers') and sexually nonactive young men ('mama's boys' and 'future priests'). As one young cuencana explained, 'I've got to watch myself so I don't end up in the mouths of others.' Anthropologists [bib_ref] Gossip and scandal, Gluckman [/bib_ref] [bib_ref] Rethinking gossip and scandal, Merry [/bib_ref] have argued that gossip should not be seen as a degraded form of speech but as a dynamic act of meaning-making and constitutive of power relations within communities. Bochow points out that for young people in Kumasi, Ghana, speech is 'an act of proper social conduct' . In the CERCA intervention, gossip functioned as a parallel form of communication to that prescribed for health service provider interactions (client confidentiality) and the open communication demanded of parents (talk about sex openly) and adolescents (make relationships and sexual status public).
Some parents actively supported the project's goal of enabling teens to acquire knowledge about contraception and/or sexually transmitted diseases, but also insisted that such knowledge should not be 'used' but 'saved' for later. [bib_ref] Teen sexuality and the troubling discourse of readiness, Ashcraft [/bib_ref] argues that while 'the discourse of readiness' is widespread in the field of adolescent sexual and reproductive health, it can ignore the relational context of readiness that not only depends on the teen's self-perceived emotional state. One mother and nurse discussed how she instructed her teenage daughter:
You can have [sexual relations] when you desire them, when you want them, but right now you have to prevent it from happening. And she knows the methods. I show them to her: this is a carton of pills, this is a copper T, this is a condom. This mother's ostensible openness about sexual health is a complex form of communication where health-and rights-oriented sex talk mingles with subtle assertions of parental control. The daughter's 'desires' are seemingly validated yet she is also instructed not to have desires 'right now. While in some instances adults 'hijacked' the norm of open communication to support the goal of enforcing parental control, in others adults claimed the norm of openness for entirely different purposes. In the third meeting of an adult peer discussion group held on the outskirts of Cuenca, three mothers of adolescent children and one grandmother (Beatriz à ) and grandfather (Carlos à ) sat down to talk. The discussion centered on participants' understandings of the challenges young people face, and that they as adults have faced, in achieving a healthy sexual life.
'I think,' said Beatriz à , 'that young people, well nobody, really, is being truthful, especially men. "I'm like this, I'm like that" and it's not the case. It is all a lie.' It quickly transpired that Beatriz was using statements in the questionnaire about consent and honesty with partners to publically accuse her husband, Carlos, of infidelities. In attempting to divert the discussion back to adolescent concerns, the facilitator (Nelson) was blocked by Carlos, Beatriz, and a third member of the peer group, Marcia: Here, the imperative of 'open communication' central to the project became a platform on which Blanca could publically, and with the support of female peers, demand the truth about her husband's sexual improprieties. Similarly, Carlos used the language of the questionnaire to defend his reputation, only to be put back in his place by Marcia's accusations of machismo.
This exchange points to the subtle ways in which 'open communication' À as well as the talk and practices of the intervention and wider project of adolescent sexual and reproductive health À become gendered. For Julio's mother, talking about sexual and reproductive health was itself sexual or sexualizing. For some adult men, such talk was seen as feminizing and counter to 'healthy' (in the sense of normal) male sexual behavior. Miguel à , 15, from Managua, an active participant in the community committees, was reluctant to share how his older cousins talked to him about sex, except to say that it was 'vulgar' and that they had explicitly counselled him not to use condoms. He provided an example instead from his friend's father, who had been asked to participate in CERCA activities:
My dad's friend says, 'No way man, I'm not going to talk with you all. I'm not going to talk about this with you all. . .it's a woman's thing, a gay thing,' he says, 'Talking about sexuality, this and that, it's for gays and women. You all are crazy [vos sos loco].'
The idea that the CERCA project and its attendant research was 'for gays and women' was reiterated by other men. These views reflected perhaps the relative absence of male adults at parent-targeted activities (high school-based workshops, community outreach activities, health fairs). But they also pointed to a more widespread 'gendering' not just of sex talk, but of the very field of adolescent sexual and reproductive health. Adult men who did participate in the project shared stories of male relatives and neighbors who disagreed with the project's aims. Wives also told of husbands who claimed 'we women are learning things we shouldn't be learning about' or gave examples of fatherly and grandfatherly relationship advice counter to that given by wives, sisters and aunts.
In a small group discussion with three young men in Cochabamba (aged 15À16) in the final months of the intervention, one joked: The contradictory messages that this young man received from his parents were not due to a lack of 'open communication' or trust. On the contrary, both mother and father were speaking freely, revealing their own sexual histories. The machista attitudes of adult men might be considered by some public health campaigns as a 'cultural barrier' to reducing teen pregnancy. Yet sexual double standards for boys and girls were not limited to the 'community' but were also found among health providers and within more elite circles, for example when a male doctor expressed the opinion that 'libertine' sexual behavior was passed from mother to daughter, or a municipal representative spoke at a CERCA event about the 'natural sexual passivity' of young women and the sexual dominance of young men. Nor was there consensus in the 'community' as to what constituted normal or good behavior for male and female teens. Some young men were critical of what they called the machista attitudes of the adult men in their lives. Others requested discussions of gay rights, abortion rights and the porn industry, showing themselves to be more 'open' in talking about sex than many adults, including some health providers who had decided that these issues were beyond the project's concerns.
# Conclusion
Questioning public health assumptions that open parentÀteen communication on sex, in and of itself, is key to healthy sexual behavior, we explored a pragmatics of communication that included silences, evasions, implied expectations, temporally delayed knowledge, and gendered conflicts. The paper began by highlighting differences in how teens and adults interpreted confianza. These differences had significant effects on the intervention and have implications for the wider field of health promotion.
For one, the goal of creating a 'community-embedded' intervention was potentially undermined by community members having different understandings of what can and should be said about sex. Some parents, such as Julio's mother, thought the intervention encouraged not (only) talk about sex, but also inappropriate sex itself. Another way of putting this would be to say that, for her, talking about sex was already a kind of sex. Julio's mother's views might seem common-sensical to sociolinguists interested in the performative effects of language. For her, knowledge about sex naturally comes from having sex. In a sense, the intervention seems to reverse this 'natural' sequence of cause and effect: it says knowledge of sex should come before sex itself (to make sure it's done right, at the right time, with the right person). From her point of view, the intervention's view of sex À as well as 'communication' À might have seemed na€ ıve (or cynical), i.e. the assumption that language about sex can be safely quarantined from the activity itself. In this particular instance, Julio's mother happened to be right: Julio was asking questions about sex because in fact he was already having sex. She was also right that his sexual activity was linked to the intervention because he had gone there to obtain emergency contraception.
However, it would be mistaken to say that the intervention simply tried to impose a 'progressive' or global health norm of open communication about sex on a community norm of confianza (regardless of whether one would view this as a valid goal). While there were some instances of open conflict among project participants, often more subtle forms of persuasion were used À which depended as much on silence, evasion and implied threats as it did on 'open communication.' As argues, power struggles in the arena of international development are 'founded less on direct confrontation than on a kind of semiotic proselytism that appears in continuous negotiations over meanings and attempts to enroll others in one's interpretations of specific circumstances.' Parents and teens were not neatly divided along generational lines; there were important differences among parents and grandparents as well as among teens. Social actors also 'used' the intervention for different purposes À to expose infidelities, to gain access to contraception, or to resist or reinforce parental influence over young peoples' relationships.
The different ways social actors interpreted and used the intervention point to more than a problem of 'translating' between global public health and local norms À a problem that, say, an anthropologist could help resolve by providing a better translation. Rather, they also demonstrate the pragmatic, affective and performative aspects of language often missed in health promotion. Speech does not just convey useful information À on contraception, for example. It also constitutes and is made by social bonds. It incites, shames, and restrains. Speech in turn is restrained by social contradictions and double binds that cannot be directly 'named.' Summing up the polyphonic voices heard during the community committees would lead to several contradictory statements, such as: 'Teens have too much knowledge of sex; teens don't have enough knowledge of sex.' 'Teens should have knowledge of sex, but not yet.' 'Girls should not allow themselves to be "damaged" by having sex; boys should have a bunch now or else won't become men.' It is unlikely that these contradictions are the result of a lack of 'open communication.' On the contrary, they are a highly expressive form of communication. They 'say' what can be difficult to say directly: what is at stake in sex, power struggles, and conflicts in moral norms. This is not to say that change is not possible, but to question the paradigm that makes 'more communication' the antidote to the constructed problem of 'no communication. Project participants' communication practices reflected larger conflicts over sociosexual norms: family-sanctioned adolescent relationships or adolescent freedom to choose partners; public displays of affection by young unmarried couples or tight restrictions on bodily contact; communication on sex as inciting sexual practice or communication on sex as delaying sexual practice. Fostering 'open communication' brought some of these tensions to light, but it also underlined the difficulty of resolving them through more, or more open, communication. While the intervention did not always have the effects that public health planners had planned, neither was it ineffective. Various social actors À including the project staff À often effectively used its language and practices for different aims, demonstrating the polyphonic nature of the community.
[fig] C: : Figuring out what you want and don't want should be a mutual agreement between both people. . . B: It's not true. [Men] should be honest and true. Say, 'Listen, I don't love you.' They shouldn't lie! No lying! Marcia: They're machistas. It's not that they don't know what they want, they are machistas. There are even old men who are dishonest. [/fig]
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Safe On-Boat Resuscitation by Lifeguards in COVID-19 Era: A Pilot Study Comparing Three Sets of Personal Protective Equipment
Introduction: On-boat resuscitation can be applied by lifeguards in an inflatable rescue boat (IRB). Due to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2) and recommendations for the use of personal protective equipment (PPE), prehospital care procedures need to be re-evaluated. The objective of this study was to determine how the use of PPE influences the amount of preparation time needed before beginning actual resuscitation and the quality of cardiopulmonary resuscitation (CPR; QCPR) on an IRB. Methods: Three CPR tests were performed by 14 lifeguards, in teams of two, wearing different PPE:(1)Basic PPE (B-PPE): gloves, a mask, and protective glasses; (2) Full PPE (F-PPE): B-PPE þ a waterproof apron; and (3) Basic PPE þ plastic blanket (BþPPE). On-boat resuscitation using a bag-valve-mask (BVM) and high efficiency particulate air (HEPA) filter was performed sailing at 20km/hour. Results: Using B-PPE takes less time and is significantly faster than F-PPE (B-PPE 17 [SD = 2] seconds versus F-PPE 69 [SD = 17] seconds; P = .001), and the use of BþPPE is slightly higher (B-PPE 17 [SD = 2] seconds versus BþPPE 34 [SD = 6] seconds; P = .002). The QCPR remained similar in all three scenarios (P >.05), reaching values over 79%. Conclusion: The use of PPE during on-board resuscitation is feasible and does not interfere with quality when performed by trained lifeguards. The use of a plastic blanket could be a quick and easy alternative to offer extra protection to lifeguards during CPR on an IRB.
# Introduction
Resuscitation in case of drowning is considered a particular circumstance. [bib_ref] European Resuscitation Council Guidelines for Resuscitation 2015: Section 4. Cardiac arrest in..., Truhlář [/bib_ref] The two aspects that define the complexity of drowning cardiopulmonary resuscitation (CPR) are the asphyxial origin of the cardiac arrest 2 and the challenging environment that often delays the onset of CPR. [bib_ref] Effectiveness and safety of in-water resuscitation performed by lifeguards and laypersons: a..., Winkler [/bib_ref] In this context, "time is brain," and to quickly combat hypoxia, several studies on lifeboats have analyzed how on-boat resuscitation is feasible.
Prior to the appearance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2) which causes coronavirus disease 2019 (COVID-,certain studies had evaluated CPR and the use of automatic external defibrillators on inflatable rescue boats (IRB). [bib_ref] Can surf-lifeguards perform a quality cardiopulmonary resuscitation sailing on a lifeboat? A..., Barcala-Furelos [/bib_ref] [bib_ref] Circumstances, outcome and quality of cardiopulmonary resuscitation by lifeboat crews, Seesink [/bib_ref] [bib_ref] Moderate sea states do not influence the application of an AED in..., De Vries [/bib_ref] However, after the appearance of COVID-19, the CPR recommendations have been updated, proposing the use of the bag-valve-mask (BVM) with a high efficiency particulate air (HEPA) filter, handled by two rescuers. [bib_ref] European Resuscitation Council COVID-19 guidelines executive summary, Nolan [/bib_ref] In addition, as an anti-contagious measure, the use of personal protective equipment (PPE) has been emphasized, including at least: polycarbonate safety glasses, gloves, and a mask filtering facepiece (FFP)/N95,and for safer clinical practice, a short-sleeved apron for droplet precaution and/or a long-sleeved gown for airborne-precaution. [bib_ref] Occupational health recommendations for lifeguards in aquatic emergencies in the Covid-19 era:..., Barcala-Furelos [/bib_ref] Alternatively, either due to the absence of PPE, the low economic cost, or the peculiarities of the clinical intervention, the use of plastic blankets has been experimented with as an extra or alternative protection in the intra-hospital setting, [bib_ref] Clear plastic drapes may be effective at limiting aerosolization and droplet spray..., Matava [/bib_ref] [bib_ref] Barrier system for airway management of COVID-19 patients, Brown [/bib_ref] [bib_ref] Use of drape/patient covering during potentially aerosolizing procedures, Allen [/bib_ref] and in prehospital settings, as shown by a recent pilot study with a lifeguard resuscitation simulation on the beach. [bib_ref] Plastic blanket drowning kit: a protection barrier to immediate resuscitation at the..., Barcala-Furelos [/bib_ref] In this new scenario, some resuscitation procedures are currently not recommended (eg, inwater resuscitation) [bib_ref] Occupational health recommendations for lifeguards in aquatic emergencies in the Covid-19 era:..., Barcala-Furelos [/bib_ref] and others are not yet certain to be applied (eg, on-boat resuscitation). For this reason and because of the health emergency, scientific societies linked to the prevention and treatment of drowning are calling for progress in researchin order to try to avoid deaths that are collateral to COVID-19.
The initial hypothesis was that on-boat resuscitation on an IRB using PPE is possible and its applicability will be conditioned by two new variables: the level of PPE used and the number of rescuers on board the boat. Moreover, the use of PPE appears to take so much time to dress in special circumstances as aquatic environments.
The main objective of this pilot study has been to test how different types of PPE influence the actual starting time of CPR and its quality. In addition, the fatigue perceived by the rescuers in these new conditions and their ability to use the PPE properly have also been analyzed.
# Methods
## Study design
A comparison study of three PPE methods, using a cross-over design, was carried out to test the time difference in actually beginning on-boat resuscitation and CPR with three different levels of PPE protection [fig_ref] Figure 1: Flow Chart Design [/fig_ref].
## Sample
Fourteen lifeguard volunteers participated in this study. The inclusion criteria were professional lifeguards, updated according to the recommendations of the European Resuscitation Council (Niel, Belgium) Guidelines of Resuscitation (ERC-GR2015) [bib_ref] Resuscitation Council Guidelines for Resuscitation 2015: Section 1. Executive summary, Monsieurs [/bib_ref] and European Resuscitation Council COVID recommendations guidelines (ERC-COVID), 9 who should not present any physical or psychological contraindication to carrying out the study and should authorize their participation by means of written consent. The final sample was 14 rescuers (ten men, four women). The general characteristics were: age 32 (SD = 9) years; weight 72 (SD = 14) kg; and height 173 (SD = 10) cm. This project was approved by the ethics committee of the Faculty of Education and Sport Sciences, University of Vigo, Spain (nº 03-0920).
## Roller refresher
A refresher was carried out before the intervention in order to standardize skills and become familiar with the PPE equipment during CPR with BVM (þHEPA filter). This roller refresher lasted 40 minutes and was organized as follows: Part 1 -Explanation and training in the dressing and use of PPE (20 minutes); Part 2 -CPR training with complete PPE in a team of two rescuers (10 minutes); and Part 3 -CPR training with basic PPE (B-PPE) and plastic blanket (BþPPE) in a team of two rescuers (10 minutes). This training was conducted by a nurse instructor accredited by the Spanish Resuscitation Council (Madrid, Spain; [fig_ref] Figure 2: Phases of Study [/fig_ref].
## Controlled on-board resuscitation (supplementary video online)
Three CPR tests were performed, following the technical recommendations for the ERC-COVID 9 resuscitation, using a sequence in accordance with ERC-GR2015 drowning recommendations. [bib_ref] European Resuscitation Council Guidelines for Resuscitation 2015: Section 4. Cardiac arrest in..., Truhlář [/bib_ref] The sequence consisted of five rescue ventilations (V), followed by cycles of 30 chest compressions (CC) and two Vs, with a duration of two minutes, and the following were compared:
-Test with Basic PPE (B-PPE): Nitrile gloves, FFP mask, and protective glasses; -Test with Full PPE (F-PPE): Nitrile gloves, FFP mask, protective glasses, and waterproof gown; and -Test with Basic PPE þ plastic blanket (BþPPE): Nitrile gloves, FFP mask, protective glasses, and transparent plastic blanket, approximately 250cm long by 150cm wide, according to a previous pilot study. [bib_ref] Plastic blanket drowning kit: a protection barrier to immediate resuscitation at the..., Barcala-Furelos [/bib_ref] In each CPR, an Ambu Mark IV adult BVM (Ambu; Ballerup, Denmark) with an Ambu HEPA filter (Ambu; Ballerup, Denmark) was used on a Laerdal Little Anne QCPR manikin (Laerdal; Stavanger, Norway).
The tests were performed on Broña Beach (Serra de Outes, A Coruña -Spain), GPS positioning: Latitude 42.801747, Longitude -8.929523. In order to be more realistic, each test began having stopped the boat and accelerating at a cruising speed of 20km/hour which was maintained until the end of the test. The IRB model was a Valiant DR-450 (Vila Nova de Cerveira, Portugal), 4.5 meters long and 1.94 meters wide. The weather conditions included a calm sea (0-2 Douglas scale), a light wind between 12 and 19 km/hour (3 Beaufort scale), at an ambient temperature of 22ºC. The weather data were reported by the local weather agency (Meteogalicia; Santiago de Compostela, Spain).
## Variables
Four groups of variables were analyzed: (1) time to start resuscitation; (2) quality of resuscitation; (3) perceived fatigue during resuscitation; and (4) skill in the use of PPE.
Time to Beginning of CPR-The time in seconds (s) was counted from the moment the victim was indicated as being in cardio-respiratory arrest to the start of the first rescue ventilation.
Cardiopulmonary Resuscitation-Three resuscitation variables were analyzed: (1) the quality of the CC in %; (2) the effective V (EV) in %: EV was understood as being when the victim's chest is clearly raised and it provides a positive record in the analysis software; and (3) quality of the CPR (Q-CPR): this is the overall result of CPR estimated by the Laerdal APP CPR instructor software (Laerdal; Stavanger, Norway) installed on an iPhone 7 (Apple Inc.; Cupertino, California USA), connected by Bluetooth to the Little Anne QCPR manikin, programmed according to ERC-GL2015.
Rating of Perceived Exertion-The rating of perceived exertion (RPE) was recorded (measurement of the range 0/10 -sub/ maximal). [bib_ref] A new approach to monitoring exercise training, Foster [/bib_ref] Previously, the lifeguards were trained in the understanding and use of this scale.
Skill in the Use of Waterproof Protection-The skill/correction in dressing the waterproof apron in the F-PPE of each lifeguard and the skill in placing the plastic blanket during the BþPPE by each team of lifeguards was subjectively evaluated. The dichotomous variable considered to be "correct dexterity" is when it provided a complete waterproof barrier between the victim and the lifeguard. If this did not occur, it was considered as "incorrect dexterity" [fig_ref] Figure 3: Dexterity in the Use of Waterproof Apron and Plastic Blanket [/fig_ref].
# Statistical analysis
All statistical analyses were performed with SPSS for Windows, version 22 (IBM Corp.; Armonk, New York USA). The Shapiro-Wilk test was used to evaluate the normality of the data.
The repeated measures ANOVA test with Bonferroni correction was used to compare the parametric variables and the Friedman test was used for non-parametric variables. A significance of P <.05 was established for all analyses on parametric variables and P <.017 on non-parametric variables.
# Results
## Time variables
In the analysis of time to initiation of CPR, it was found that rescuers previously equipped with B-PPE took 17 seconds to initiate CPR. This result was an improvement of 52 seconds compared to [fig_ref] Table 1: Results of the Time, CPR, and RPE Variables [/fig_ref].
## Cpr variables
The CPR was of equally good quality in all three scenarios (B-PPE versus F-PPE versus BþPPE; P >.05). The rescuers obtained values above 79% in all the variables analyzed. There was a nonsignificant trend (P >.05) of a seven percent decrease in the percentage of EV when using F-PPE and an 11% decrease with BþPPE, compared to B-PPE, which obtained the highest value (90%; Table 1; [fig_ref] Figure 4: Visual Chart of Variables [/fig_ref].
## Rpe variable
The RPE was similar in the conditions of all three scenarios (P >.05), with low scores as the values ranged from two to three on a maximum scale of ten [fig_ref] Table 1: Results of the Time, CPR, and RPE Variables [/fig_ref].
## Skill in the use of waterproof protection variable
All teams (100%) were able to use the plastic blanket correctly and to keep it stable throughout the test. However, 43% were not able to correctly don the waterproof apron.
# Discussion
Rescuer protection is essential in any emergency. In addition to the usual risks (usually traumatic), now there is the risk of COVID-19 infection. For this reason, it is suggested that professionals should use isolation devices which prevent contact and virus inhalation if the victim is infected. So far, recommendations for resuscitation have been based on the most common "medical" settings (ie, hospital or ambulance), without yet having evaluated the options for PPE in other less common and less controlled environments (such as lifesaving situations) in which CPR is also performed. Therefore, this study had the novel objective of evaluating three levels of protection during the resuscitation by rescuers on an IRB. In the controlled simulation conditions in a real environment in which the tests were carried out, it was observed that: (1) there was less loss of time at the start of on-boat resuscitation using the BVM and HEPA filter, with a basic level of protection; (2) the use of an extra waterproof barrier such as a plastic blanket seemed faster and easier than a conventional waterproof apron; (3) CPR quality was not affected by the level of protection used; and (4) finally, on-boat CPR with two rescuers did not generate much fatigue amongst trained lifeguards.
Drowning is considered a public health issue by the World Health Organization (WHO; Geneva, Switzerland),and lifeguards are recognized as the first barrier to prevention and intervention. The IRB is commonly used in lifeguarding as it is small, safe, fast, and easy to use, and is common in surveillance and rescue near the coast. [bib_ref] Can surf-lifeguards perform a quality cardiopulmonary resuscitation sailing on a lifeboat? A..., Barcala-Furelos [/bib_ref] The use of IRBs in the event of drowning can gain valuable time in an incident in which every second counts. The systematic review and meta-analysis by Quan, et al 20 found that immersion time is the most influential factor in the prognosis of the victim. Stopping the drowning process quickly 2 and initiating on-boat resuscitation would avoid the time delay involved in the rescue and transfer to land without life support. Another finding by Quan, et al 20 is the favorable outcomes witnessed thanks to the shorter Emergency Medical Service response times. This evidence reinforces the importance of on-boat resuscitation, which is not a common practice but is definitely possible, 7,21 and therefore needs planning and training.
At present, any unknown victim of cardio-respiratory arrest will be considered a potential carrier of SARS-COV-2, and in the case of rescuers, exposure to the risk of contagion may be high since beaches are a place with a large concentration of bathers and rescue techniques inevitably require direct contact. [bib_ref] Occupational health recommendations for lifeguards in aquatic emergencies in the Covid-19 era:..., Barcala-Furelos [/bib_ref] The risk may be reduced depending on the PPE that can be used. [bib_ref] Occupational health recommendations for lifeguards in aquatic emergencies in the Covid-19 era:..., Barcala-Furelos [/bib_ref] From a theoretical perspective, the most complete option of F-PPE could be used on the IRBs; however, it would not be realistic for two reasons:
(1) it is neither viable nor safe to patrol with a waterproof gown while awaiting an incident requiring CPR, since it is a low-probability event (it represents just 0.02% of actions carried out by lifeguards); 22 and (2) the time spent wearing the waterproof gown on a boat, as well as the probability of doing it incorrectly, is a sufficiently important limitation affecting this choice of PPE. Maintaining on-boat resuscitation as a protocol on IRBs involves starting CPR with a basic protection (B-PPE) and ventilating using HEPA-filtered BVMs. However, this procedure does not offer the greatest protection against possible infection and should be assessed by rescue agencies, and the rescuers themselves, to weigh up the risk they are taking considering epidemiological data on local incidence of the virus, the age of the rescuer (usually young) or his/her previous health status (eg, previous pathology, possible immunity due to having previously overcome COVID-19, or other factors), or the type of victim (eg, a child) and the rescue conditions (eg, a short time underwater). An intermediate position could be the use of B-PPE in combination with a plastic blanket (BþPPE) so as to create an insulating barrier between the rescuers and the victim. According to these results, this extra protection allows quick positioning on the patient and does not affect the QCPR. A fundamental criterion for the decision to continue with on-boat resuscitation is to know whether it is possible to perform quality CPR with the PPE. Studies prior to the pandemic have shown good performance by lifeguards or fishermen during on-boat resuscitation, although the QCPR was conditioned by the size of the boat, 6 the waves, 6 the wind, or the speed, 5,23 but until now, neither the PPE variable nor on-boat resuscitation in teams (by two lifeguards) had been introduced. These findings show results in V and CC above 70%, a value arbitrarily assumed in numerous studies as the cut-off point in CPR quality. [bib_ref] ABC of Resuscitation, Perkins [/bib_ref] Similar results were found in a Spanish study on an IRB, 5 with the same maritime conditions and at a very similar speed (10knots/18.52Km/hour) in comparison with the current study (11knots/20Km/hour). The main difference between both studies was the RPE. In the case of a lone rescuer, the perceived effort was five (Heavy/Strong big-major effort) on the RPE scale, [bib_ref] A new approach to monitoring exercise training, Foster [/bib_ref] and when there were two rescuers (ie, in this research), the RPE did not exceed two (a light effort) for the rescuer performing V and three (moderate effort) for the CC rescuer. It seems, therefore, that CPR by two lifeguards has a number of advantages, at least in terms of fatigue, although the crew of an IRB is usually made up of two people (skipper and lifeguard) since a conditioning factor is the limited space. [bib_ref] Circumstances, outcome and quality of cardiopulmonary resuscitation by lifeboat crews, Seesink [/bib_ref] This circumstance could be a limitation for team resuscitation on small IRB models.
## Barcala-furelos © 2021 prehospital and disaster medicine
Correct PPE donning and doffing is not easy and requires specific training, [bib_ref] Occupational health recommendations for lifeguards in aquatic emergencies in the Covid-19 era:..., Barcala-Furelos [/bib_ref] and misuse may lead to a false sense of security.Wind and waves are common circumstances at sea, and tests performed with mild gusts of wind prevented several rescuers from wearing the waterproof gown properly.
This study has a practical and direct impact on lifeguards, regarding how to deal with the most critical situation of drowning (ie, cardio-respiratory arrest). Europe is immersed in the first summer of the COVID-19 Era and this has not prevented the beaches of Mediterranean countries from continuing to be a meeting point for bathers and the scene of potential drownings. Summer will be at the end of 2020 in the Southern Hemisphere, so lifeguards need evidence-based guidance to intervene as safely as possible, in a context in which there are no previous experience and therefore recommendations must be adapted to each specific environment. Preliminary results are offered here in three aspects relevant to on-boat resuscitation: (1) whether it is feasible with current recommendations; (2) how precious time can be saved so as not to delay assistance; and (3) how to protect rescuers to prevent contagion.
# Study limitations
This work presents limitations that should be pointed out. Firstly, it is a pilot study carried out in controlled simulation conditions in favorable weather conditions, with a small, local sample of lifeguards. The same tests with other maritime conditions, human or material resources, could obtain different results. The major limitation is the use of a dummy. In a real victim, the difficulty of resuscitation will be different and more complex.
Three levels of PPE were investigated, based on current knowledge and recommendations for the prevention of COVID-19 during CPR. However, the actual risk is not possible to fully measure with this study.
To the authors' knowledge, this is the first research work that aims to assess the feasibility of on-boat resuscitation during the COVID-19 Era, so there may be other limitations not described and not yet known by the authors.
# Conclusions
The use of PPE during on-board CPR is feasible and does not interfere with quality when performed by trained lifeguards. The use of B-PPE allows for rapid initiation of CPR. The use of PPE which requires wearing a waterproof apron on board is a significant loss of time that delays the start of CPR. The use of a plastic blanket could be a quick and easy alternative to offer extra protection to the lifeguards during on-boat resuscitation on an IRB.
This pilot study could help Lifesavers' Organizations to define their rescue and resuscitation protocols, based on the local situation, pandemic level, experience, training, and available materials.
[fig] Figure 1: Flow Chart Design. Abbreviations: CPR, cardiopulmonary resuscitation; PPE, personal protective equipment; FFP, filtering facepiece; IRB, inflatable rescue boat. Barcala-Furelos © 2021 Prehospital and Disaster Medicine [/fig]
[fig] Figure 2: Phases of Study: On-Shore Roller Refresher Training and On-Boat Resuscitation Test. Abbreviations: PPE, personal protective equipment; B-PPE, basic PPE (gloves, glasses, and FFP mask); F-PPE, full PPE (gloves, glasses, FFP mask, and waterproof coat); BþPPE, basic PPE þ plastic blanket. half-a-minute compared to wearing a waterproof apron on-boat (BþPPE 34 [SD = 6] seconds versus F-PPE 69 [SD = 17] seconds; P = .006; [/fig]
[fig] Figure 3: Dexterity in the Use of Waterproof Apron and Plastic Blanket. [/fig]
[fig] Figure 4: Visual Chart of Variables: Time and CPR. Abbreviations: PPE, personal protective equipment; B-PPE, basic PPE (gloves, glasses, and FFP mask); F-PPE, full PPE (gloves, glasses, FFP mask, and waterproof coat); BþPPE, basic PPE þ plastic blanket; QCPR, quality of cardiopulmonary resuscitation; QCC, quality of chest compressions; EV, effective ventilations. [/fig]
[table] Table 1: Results of the Time, CPR, and RPE Variables [/table]
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Arthrofibrosis of the Ankle
Arthrofibrosis is a common, but often overlooked, condition that imparts significant morbidity following injuries and surgery to the foot and ankle. The most common etiologies are related to soft tissue trauma with subsequent fibrotic and contractile scar tissue formation within the ligaments and capsule of the ankle. This leads to pain, alterations in gait, and ankle dysfunction. Initial treatment often includes extensive physical therapy, however, if severe enough surgical options exist. Although the literature regarding ankle arthrofibrosis is scarce, this review article provides a greater understanding of the pathogenesis of arthrofibrosis and describes the current and future therapeutic options to treat fibrotic joints. Level of Evidence: Level V, expert opinion.
# Introduction
Limited joint motion can have a profound effect on a patient's daily life. Although the etiology of motion loss is multifactorial, arthrofibrosis has more recently been investigated as a key cause. Arthrofibrosis is defined as joint pain and stiffness that does not allow functional range of motion and is commonly due to adhesions, scarring, and contracture of the joint. [bib_ref] Wrist arthrofibrosis, Lee [/bib_ref] [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] Motion-limiting arthrofibrosis has been reported throughout the body, including the knee, shoulder, wrist, and elbow. [bib_ref] Wrist arthrofibrosis, Lee [/bib_ref] [bib_ref] Arthrofibrosis of the knee, Magit [/bib_ref] There is far less literature, however, regarding arthrofibrosis of the ankle.
In the ankle, this condition is mostly commonly due to an inciting trauma. Utsugi et al [bib_ref] Intra-articular fibrous tissue formation following ankle fracture: the significance of arthroscopic debridement..., Utsugi [/bib_ref] reported arthrofibrosis in 73% (24/33) of patients who sustained an ankle fracture, whereas Thomas et al [bib_ref] Chronic pain after ankle fracture: an arthroscopic assessment case series, Thomas [/bib_ref] found it to be present in 40% (20/50) of patients similarly sustaining ankle fractures. Clinically, arthrofibrosis is characterized by pain, diminished range of motion, stiffness, and difficulty with performing daily activities due to impaired gait and abnormal ankle function. Early preventive strategies are critical for minimizing the risk of developing arthrofibrosis, including bracing and splinting the ankle in a neutral position to prevent an equinus contracture. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] [bib_ref] Prevention of permanent arthrofibrosis after anterior cruciate ligament reconstruction alone or combined..., Noyes [/bib_ref] Once fibrosis has developed, conservative and operative management have been shown to improve function. [bib_ref] Treatment of posttraumatic adhesive capsulitis of the ankle: a case series, Cui [/bib_ref] [bib_ref] Evidence-based indications for ankle arthroscopy, Glazebrook [/bib_ref] [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] In part because of the limited current evidence, this entity has rarely been discussed despite the significant impact it can have on a patient's recovery. Therefore, the purpose of this article is to review the current literature on arthrofibrosis of the ankle.
## Etiology
A variety of factors contribute to causing arthrofibrosis. A few common causes are infection, trauma, and surgery. [bib_ref] Stiffness after total knee arthroplasty, Bong [/bib_ref] [bib_ref] Pathological mechanisms and therapeutic outlooks for arthrofibrosis, Usher [/bib_ref] [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] Following the inciting event, fibrous scar tissue may develop in the anterior capsule, posterior capsule, or both. Common surgeries reported to lead to fibrosis of the anterior ankle capsule include open reduction and internal fixation (ORIF) of fractures (73% of ankle fractures), open debridement of anterior osteophytes, and ankle arthroplasty 1% of ankle arthroplasties). [bib_ref] The Scandinavian total ankle replacement: long-term, eleven to fifteen-year, survivorship analysis of..., Brunner [/bib_ref] [bib_ref] Dealing with the stiff ankle: preoperative and late occurrence, Hintermann [/bib_ref] [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] [bib_ref] Current concepts review: Arthroscopic treatment of anterior ankle impingement, Ross [/bib_ref] [bib_ref] Intra-articular fibrous tissue formation following ankle fracture: the significance of arthroscopic debridement..., Utsugi [/bib_ref] Posterior ankle capsular arthrofibrosis may occur following posterior ankle impingement surgery or following posterior approach to the ankle for ORIF. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] However, cause and effect are unclear and initial pathology may predispose to increased fibrosis after these procedures. Prolonged immobilization after trauma or surgery may also play a role in the development of arthrofibrosis, particularly if the ankle is immobilized in equinus. [bib_ref] Cast and splint immobilization: complications, Halanski [/bib_ref] Inflammation, whether caused by trauma, systemic disease, surgery or infection, is implicated as a root cause of arthrofibrosis of all joints. The role that the inflammatory cycle plays is being increasingly explored by the basic science community. [bib_ref] Pathological mechanisms and therapeutic outlooks for arthrofibrosis, Usher [/bib_ref] In particular, the role of cytokines such as interleukin-1 (IL-1) and IL-6, is being investigated since they are frequently upregulated in the setting of infection and lead to cell migration, adhesion, and increased matrix metalloproteinase activity. [bib_ref] Pathological mechanisms and therapeutic outlooks for arthrofibrosis, Usher [/bib_ref] The direct causes of decreased ankle range of motion can be further categorized into either extra-articular or intra-articular pathology. Potential extra-articular pathologies causing ankle stiffness and restricted motion include soft tissue infections, tendon adhesions, or muscle contractures around the ankle. [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] Depending on the muscle or tendon, patients will predominantly have limitations in either plantarflexion or dorsiflexion. Conversely, examples of intra-articular pathology are adhesive capsulitis and intra-articular fractures. Adhesive capsulitis of the ankle is a rarely reported condition that is often preceded by a traumatic injury. [bib_ref] Treatment of posttraumatic adhesive capsulitis of the ankle: a case series, Cui [/bib_ref] [bib_ref] Adhesive capsulitis of the hip and ankle, Griffiths [/bib_ref] [bib_ref] The arthroscopic management of frozen ankle, Lui [/bib_ref] [bib_ref] Adhesive capsulitis of the ankle, Palladino [/bib_ref] The condition typically involves the development of intra-articular adhesions, and therefore limits both ankle plantarflexion and dorsiflexion. [bib_ref] Treatment of posttraumatic adhesive capsulitis of the ankle: a case series, Cui [/bib_ref] [bib_ref] The arthroscopic management of frozen ankle, Lui [/bib_ref] Pathophysiology Following an inciting event, a synovial inflammatory response is activated. There is proliferation of fibroblasts and a significant deposition of extracellular matrix proteins. [bib_ref] Inflammatory cytokines are overexpressed in the subacromial bursa of frozen shoulder, Lho [/bib_ref] [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] [bib_ref] Alpha-smooth muscle actin containing contractile fibroblastic cells in human knee arthrofibrosis tissue...., Unterhauser [/bib_ref] The excess extracellular matrix may then impair blood flow by increasing the distance of the tissue to blood vessels, and thus oxygen delivery to the injured tissue is reduced, resulting in local hypoxia. 14 Deprivation of oxygen triggers the release of inflammatory cytokines, primarily transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). [bib_ref] Pathological mechanisms and therapeutic outlooks for arthrofibrosis, Usher [/bib_ref] These cytokines, in turn, signal a cascade of events resulting in scar tissue formation. In normal healing responses, TGF-beta and PDGF levels decrease over time. However, in patients with arthrofibrosis following injury or infection, these cytokines are dysregulated and continue to be expressed at elevated levels. This overexpression leads to elevated alpha-smooth muscle actin expression in fibroblasts; increased collagen type VI synthesis, which is thought to normally serve as an anchoring element between collagen I/III fibrils and basement membranes; and prolongation of the inflammatory response resulting in fibrous tissue hyperplasia. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] [bib_ref] Arthrofibrosis of the knee, Magit [/bib_ref] [bib_ref] Alpha-smooth muscle actin containing contractile fibroblastic cells in human knee arthrofibrosis tissue...., Unterhauser [/bib_ref] [bib_ref] Immunohistochemical localization of collagen VI in arthrofibrosis, Zeichen [/bib_ref] Additionally, as demonstrated in patients following total knee arthroplasty, reactive oxygen and nitrogen species initiate and sustain the arthrofibrotic response by inducing protein and DNA modifications. [bib_ref] Reactive oxygen and nitrogen species induce protein and DNA modifications driving arthrofibrosis..., Freeman [/bib_ref] Interestingly, other studies have reported an association with human leukocyte antigens (HLAs), suggesting a genetic predisposition to the development of arthrofibrosis. [bib_ref] Screening for arthrofibrosis after anterior cruciate ligament reconstruction: analysis of association with..., Skutek [/bib_ref] Specifically, HLA-Cw*07 was found significantly less often in patients with primary arthrofibrosis following ACL reconstruction than in the general population (P ¼ .022). The opposite effect was seen for HLA-Cw*08, which was found in 17.6% of the study group but only in 3.8% of the control group (P ¼ .045). A significant difference was also seen for HLA-DQB1*06, with 23.5% of the patients possessing this allelic variant as opposed to 48.6% of the control group (P ¼ .048). However, a statistical bias cannot be excluded given the relatively small number of 17 patients reviewed. [bib_ref] Screening for arthrofibrosis after anterior cruciate ligament reconstruction: analysis of association with..., Skutek [/bib_ref] A very recent study attempted to uncover evidence of a genetic predisposition for fibrosis in musculoskeletal tissues. [bib_ref] Human fibrosis: is there evidence for a genetic predisposition in musculoskeletal tissues?, Dagneaux [/bib_ref] They demonstrated that 22% of gene variants were between the lung and the hand, specifically idiopathic pulmonary fibrosis and Dupuytren disease. These genes included ADAM, HLA, CARD, EIF, TGF, WNT, and ZNF genes. Despite these shared genetic variations, the authors concluded that there remains limited information about genetic variants associated with fibrosis in other MSK regions. [bib_ref] Human fibrosis: is there evidence for a genetic predisposition in musculoskeletal tissues?, Dagneaux [/bib_ref] Although bony deformities or arthritic changes may cause bony impingement and altered biomechanics, this review will focus on arthrofibrosis independent of mechanical impingement. These factors should, of course, be carefully evaluated for in any clinical scenario.
## Clinical features and diagnosis
The clinical manifestations of arthrofibrosis may include joint pain, swelling, stiffness, and decreased range motion. First, distinguishing between stiffness and diminished range of motion is important. Although these 2 terms are often used interchangeably, there are distinct differences between them. Stiffness is a symptom reported by the patient. A patient may report joint stiffness even if there is no objective sign of decreased range of motion. Vega et al [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] reported that some patients may refer to the feeling of stiffness as a similar to having a "mass-occupying feeling" in the ankle. In the ankle, stiffness often suggests the presence of impinging scar tissue within the joint. Diminished range of motion is an objective finding defined as a deficit in degrees of motion from the expected normal or in comparison to the contralateral, uninjured side. [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] Potential extra-articular factors, such as muscle contractures, may also contribute to decreased range of motion. Peri-articular causes of diminished range of motion typically involve the capsule and ankle ligaments. [bib_ref] The arthroscopic management of frozen ankle, Lui [/bib_ref] [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] As with any patient encounter, a careful history must be obtained. Patient complaints regarding pain and limitations in activities of daily living such as difficulty navigating stairs or sloped surfaces suggest arthrofibrosis. All patients should be asked about prior trauma or surgeries, as intra-articular fibrous tissue formation occurs commonly after an ankle fracture or dislocation. In 2007, one study graded ankle arthrofibrosis according to the extent of intra-articular fibrous tissue detected by arthroscopy. This was present in 73% of patients who had undergone ankle fracture ORIF. [bib_ref] Intra-articular fibrous tissue formation following ankle fracture: the significance of arthroscopic debridement..., Utsugi [/bib_ref] Other studies demonstrated that arthrofibrosis occurs in more than 20% of patients who underwent total ankle replacement. [bib_ref] The Scandinavian total ankle replacement: long-term, eleven to fifteen-year, survivorship analysis of..., Brunner [/bib_ref] [bib_ref] Dealing with the stiff ankle: preoperative and late occurrence, Hintermann [/bib_ref] [bib_ref] Bone augmentation for revision total ankle arthroplasty with large bone defects, Horisberger [/bib_ref] Providers should also elicit a history of coexistent medical diseases including juvenile arthritis, infectious diseases, and particularly hemophilic arthropathy. Hemophilic arthropathy is a condition most associated with arthrofibrosis and loss of motion in the knee, ankle, and elbow joints. [bib_ref] Management of arthrofibrosis in haemophilic arthropathy, Solimeno [/bib_ref] Patients commonly develop synovial fibrosis and musculotendinous shortening, resulting in joint contracture and diminished motion. [bib_ref] Management of arthrofibrosis in haemophilic arthropathy, Solimeno [/bib_ref] The prevalence of knee, ankle, and elbow joint contractures in patients with severe hemophilia has been reported to be between 50% and 95%, with the most common deformities being knee and elbow flexion contractures as well as ankle equinus. [bib_ref] Joint contractures in the hemophilias, Atkins [/bib_ref] [bib_ref] Management of arthrofibrosis in haemophilic arthropathy, Solimeno [/bib_ref] Following the history, a thorough physical examination should be performed. In the absence of bony deformity or severe arthritic changes, a careful inspection of both lower extremities is performed to identify potential extra-articular causes of joint dysfunction such as tendon adhesions and muscle or fascia contractures. [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] After inspection, palpation of the ankle should be done as patients with arthrofibrosis often present with tenderness to palpation of the joint margins. It is important to observe the patient both standing and walking as they will often present with an altered gait. Abnormal gait, as manifest by shortened stride and early heel lift-off, occurs when patients cannot achieve a minimum of 5 to 10 degrees of dorsiflexion. [bib_ref] Complications of total ankle replacement, Conti [/bib_ref] Lastly, intra-articular injection of a local anesthetic may assist in diagnosis. There is a higher likelihood of extensive ankle arthrofibrosis and structural pathology if only a few milliliters can be injected or if there is dense fibrous tissue in the anterior ankle, which may be felt on needle insertion. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] Imaging studies may be used to diagnose arthrofibrosis and to plan treatment. Plain radiographs and computed tomography (CT) should be obtained to detect other causes of joint motion restriction such as bony incongruity, arthritis, malunion, nonunion, and loose bodies. Dynamic radiographic sequences, or fluoroscopy, can more objectively record the degree of motion loss, particularly in relation to the contralateral side. [bib_ref] Sagittal subtalar and talocrural joint assessment during ambulation with controlled ankle movement..., Mchenry [/bib_ref] One of the more common imaging methods used for diagnosing ankle arthrofibrosis is magnetic resonance (MR) imaging. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] Typical imaging findings consist of capsular and pericapsular thickening (>3 mm) and scarring that is best demonstrated on proton-density MR images [fig_ref] Figure 1: Magnetic resonance images demonstrating [/fig_ref]. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] The signal intensity of the capsular thickening on proton-density-weighted MR images correlates with the stage of arthrofibrosis. Less commonly, ultrasonography can also be used to detect arthrofibrosis. Findings will demonstrate hypoechoic capsular thickening and pericapsular scars. [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] Finally, fluoroscopic-guided arthrography can be used to confirm the diagnosis. Patients with arthrofibrosis typically demonstrate decreases in joint capacity, obliteration of normal joint recesses and high back flow. [bib_ref] Posttraumatic adhesive capsulitis of the ankle: arthrographic diagnosis, Goldman [/bib_ref]
## Treatment options
Early detection of motion loss as well as the underlying etiology of ankle dysfunction, whether it be intra-or extra-articular in origin, is crucial to the management of ankle stiffness. Once defined, management usually follows a stepwise approach starting from conservative therapies to more aggressive treatments.
## Nonoperative management
Conservative treatment begins with physical therapy. Range of motion exercises have been shown to increase strength and motion as one gradually increases intensity. Care should be taken to discourage overly aggressive therapy, in particular passive range of motion, as this can increase the risk of inflammation and fibrosis. [bib_ref] Pathological mechanisms and therapeutic outlooks for arthrofibrosis, Usher [/bib_ref] Another option for the treatment of arthrofibrosis is dynamic splinting with prolonged, passive stretching. The premise of dynamic splinting is to deliver a low load and prolonged duration of passive stretching with measurable dynamic tension to stretch the connective tissue. Furthermore, dynamic splinting involves splint application that allows a patient to achieve 6 to 8 hours of passive stretching each day. When used as home therapy, the combination of dynamic splinting and prolonged passive stretching has demonstrated to be a safe and efficacious treatment for lower extremity joint contractures with minimal risk of adverse events. [bib_ref] Systematic review of contracture reduction in the lower extremity with dynamic splinting, Furia [/bib_ref] A systematic review by Furia et al [bib_ref] Systematic review of contracture reduction in the lower extremity with dynamic splinting, Furia [/bib_ref] reported that mean ankle range of motion was improved between 9 and 31 degrees with the use of dynamic splints. They also found that the degree of improvement was directly and linearly related to the number of hours that stretching was performed. Although the conclusion that dynamic splinting serves as an effective treatment for ankle arthrofibrosis is only based off of one study, the authors believe that these devices can be particularly useful because the effort required to comply with the protocol is relatively low and can be performed without direct supervision.
Adjunctive intra-articular corticosteroid injections may also be used to help alleviate arthrofibrosis, although at a low success rate. One study reported only a 20% long-term response rate. [bib_ref] Treatment of posttraumatic adhesive capsulitis of the ankle: a case series, Cui [/bib_ref] Joint manipulation under anesthesia can be a useful modality but supportive data in the foot and ankle is sparse. [bib_ref] Manipulative therapy for lower extremity conditions: update of a literature review, Brantingham [/bib_ref] [bib_ref] Joint manipulation under anesthesia for arthrofibrosis after hallux valgus surgery, Feuerstein [/bib_ref] Lastly, other adjunctive modalities such as continuous passive motion, whirlpool therapy, and ultrasound stimulation have not been reported in the literature.
## Operative management
If conservative measures fail to improve patient symptoms, surgical treatment is sometimes considered. The procedure depends on the cause of decreased joint motion. Extra-articular issues such as muscle, tendon, or fascial contractures or adhesions may require open or percutaneous release or lengthening procedures. Large osteophytes causing mechanical impingement should be removed as well. The literature is scant with regard to evaluating surgical management of ankle arthrofibrosis and is limited to reports, discussed below, which have evaluated various open and arthroscopic techniques.
Arthroscopy has been advocated as a minimally invasive strategy. The current literature, however, does not robustly support arthroscopic release. The most compelling compilation of objective outcome measurements was by Glazebrook et al [bib_ref] Evidence-based indications for ankle arthroscopy, Glazebrook [/bib_ref] [bib_ref] Operative arthroscopy of the ankle. Three years' experience, Parisien [/bib_ref] Bonnin and Bouysset, however, did not show improvement in 6 patients following arthroscopic debridement for post-traumatic ankle stiffness. [bib_ref] Arthroscopy of the ankle: analysis of results and indications on a series..., Bonnin [/bib_ref] Ankle arthroscopy can be performed both anteriorly and posteriorly depending on the location of scarring and deficit in motion. Anterior arthroscopy allows easy access to the anterior plafond and both the medial and lateral gutters, although access of the posterior ankle can be limited by joint distraction and portal placement. The posterior approach allows easier access to the posterior capsule and tendons. [bib_ref] Arthroscopy and endoscopy of the foot and ankle: indications for new techniques, Lui [/bib_ref] Postoperatively, active and passive range-of-motion exercises should be initiated as soon as possible in order to achieve the best outcomes. [bib_ref] The arthroscopic management of frozen ankle, Lui [/bib_ref] [bib_ref] Ankle arthroscopy: an update, Vega [/bib_ref] Open debridement is another option. Postoperative arthrofibrosis can be treated with open arthrolysis, Achilles tendon lengthening, and/or gastrocnemius recession with good pain relief and increased range of motion reported. [bib_ref] Evaluation and management of the painful total ankle arthroplasty, Hsu [/bib_ref] Similar to arthroscopic treatments, there are few reports on outcomes following open procedures. There is some encouraging literature in the total ankle arthroplasty for open debridement. In Gross and colleagues' retrospective study of surgical treatment of bony and soft-tissue impingement in total ankle replacements, they demonstrated improvement in all pre-to postoperative assessments. [bib_ref] Surgical treatment of bony and soft-tissue impingement in total ankle arthroplasty, Gross [/bib_ref] Of note, there were no significant differences in pain relief, rates of repeat debridements, and functional improvement between the arthroscopic and open debridement groups. AOFAS hindfoot scores improved from 42.3 to 66.1, visual analog scale pain score declined from 7.3 to 3.1, SF-36 increased from 41.4 to 58.2, and SMFA scores decreased from 49.7-30.9. Among the patients who were followed over 1 year postoperatively, 84% remained pain-free at 26.6 months.
Finally, in the foot and ankle, gradual correction with external fixation is often considered for correction of soft tissue contractures and arthrofibrosis. Circular external fixation has been used as a treatment method for many secondary ankle and foot pathologies that result in rigidity. This technique provides for gradual correction of deformity or maintenance of a static position during treatment, often while enabling functional use of the limb. Although external fixation is not recognized as a primary treatment of arthrofibrosis, it has been employed to treat contracture as well as arthrosis that may be present concomitantly. Gradual correction by this method can allow joint realignment and soft tissue lengthening without traditional releases or extensive incisions [fig_ref] Figure 2: Hinged external fixator allowing for gradual stretching and correction of equinus [/fig_ref]. [bib_ref] Correction of rigid equinovarus deformity using a multiplanar external fixator, Cuttica [/bib_ref] [bib_ref] Correction of foot deformities by the Ilizarov method without corrective osteotomies or..., Hosny [/bib_ref] [bib_ref] Correction of complex foot deformities using the Ilizarov external fixator, Kocaoglu [/bib_ref] [bib_ref] Treatment of equinocavovarus deformity in adults with the use of a hinged..., Oganesyan [/bib_ref] Complications commonly include pin tract infection, but this is often preferable to the morbidity of larger, open procedures. [bib_ref] Correction of foot deformities by the Ilizarov method without corrective osteotomies or..., Hosny [/bib_ref] [bib_ref] Correction of complex foot deformities using the Ilizarov external fixator, Kocaoglu [/bib_ref] Ankle joint distraction arthroplasty was introduced in 1978 as an alternative to arthrodesis and arthroplasty.This technique involves temporarily unloading the joint with an external fixator with the goal of improving joint diastasis and range of motion. [bib_ref] Subchondral bone remodeling is related to clinical improvement after joint distraction in..., Intema [/bib_ref] [bib_ref] Clinical benefit of joint distraction in the treatment of ankle osteoarthritis, Marijnissen [/bib_ref] [bib_ref] Intermediate-term follow-up after ankle distraction for treatment of end-stage osteoarthritis, Nguyen [/bib_ref] [bib_ref] Distraction arthroplasty of the ankle-how far can you stretch the indications?, Paley [/bib_ref] [bib_ref] Amendola A. Motion versus fixed distraction of the joint in the treatment..., Saltzman [/bib_ref] [bib_ref] Joint preservation of the osteoarthritic ankle using distraction arthroplasty, Tellisi [/bib_ref] Adjunctive procedures to address soft tissue contracture about the ankle or hindfoot, bony limb malalignment, osteophytosis, nerve compression, and intra-articular pathology can be performed concurrently. [bib_ref] Distraction arthroplasty of the ankle-how far can you stretch the indications?, Paley [/bib_ref] [bib_ref] Amendola A. Motion versus fixed distraction of the joint in the treatment..., Saltzman [/bib_ref] [bib_ref] Joint preservation of the osteoarthritic ankle using distraction arthroplasty, Tellisi [/bib_ref] There is an improved arc of motion after treatment that is more functional, even though the overall range of motion may not change. 48,52,58 Short-and midterm results have been good in patients with severe arthritis, with reduced ankle satisfaction by 8-10 years. [bib_ref] Patient characteristics as predictors of clinical outcome of distraction in treatment of..., Marijnissen [/bib_ref] [bib_ref] Intermediate-term follow-up after ankle distraction for treatment of end-stage osteoarthritis, Nguyen [/bib_ref] [bib_ref] Distraction arthroplasty of the ankle-how far can you stretch the indications?, Paley [/bib_ref] Arthrofibrosis of the ankle and subtalar joints frequently results in asymmetric contracture, resulting in joint incongruity. [bib_ref] Treatment of posttraumatic adhesive capsulitis of the ankle: a case series, Cui [/bib_ref] [bib_ref] Imaging findings in arthrofibrosis of the ankle and foot, Linklater [/bib_ref] [bib_ref] Intra-articular fibrous tissue formation following ankle fracture: the significance of arthroscopic debridement..., Utsugi [/bib_ref] Contractures (eg, ankle equinus) can be safely managed with hinged or multiplanar external fixation and gradual correction. [bib_ref] Correction of foot deformities by the Ilizarov method without corrective osteotomies or..., Hosny [/bib_ref] [bib_ref] Correction of complex foot deformities using the Ilizarov external fixator, Kocaoglu [/bib_ref] [bib_ref] Treatment of equinocavovarus deformity in adults with the use of a hinged..., Oganesyan [/bib_ref] This method uses the concept of distraction histogenesis, whereby soft tissue lengthening occurs in response to gradual stretching. [bib_ref] Correction of foot deformities by the Ilizarov method without corrective osteotomies or..., Hosny [/bib_ref] [bib_ref] Basic science of distraction histogenesis, Tetsworth [/bib_ref] It is a less invasive alternative to traditional methods, and neurovascular (eg, tarsal tunnel decompression) and soft tissue releases may be performed concomitantly. [bib_ref] Correction of complex foot deformities using the Ilizarov external fixator, Kocaoglu [/bib_ref] [bib_ref] Treatment of equinocavovarus deformity in adults with the use of a hinged..., Oganesyan [/bib_ref] Distraction histogenesis is frequently sufficient to treat diagnoses such as equinus contracture, but the addition of osteotomies can allow reorientation of the foot to a more plantigrade position if arthrofibrosis is too severe. [bib_ref] Correction of foot deformities by the Ilizarov method without corrective osteotomies or..., Hosny [/bib_ref] [bib_ref] Correction of complex foot deformities using the Ilizarov external fixator, Kocaoglu [/bib_ref] Prevention Because arthrofibrosis most commonly occurs following trauma or surgery, preventive strategies begin directly after the provoking incident. These tactics include postoperative pain control, early passive and active range of motion exercises, and ongoing communication between therapists and physicians to ensure appropriate rehabilitation. [bib_ref] Clinical benefits of intra-articular anakinra for arthrofibrosis, Brown [/bib_ref] [bib_ref] Passive dorsiflexion flexibility after cast immobilization for ankle fracture, Nightingale [/bib_ref] [bib_ref] Effects of immobilization on plantar-flexion torque, fatigue resistance, and functional ability following..., Shaffer [/bib_ref] The importance of active and passive range-of-motion ankle exercise programs supervised by a physical therapist cannot be overstated. In 2000, Shaffer and colleagues showed that after 8 weeks of cast immobilization after open reduction and internal fixation of ankle fractures, patients experienced significant decreases in muscle performance, functional ability, and fatigue resistance. However, 10 weeks of mobilization with physical therapy can successfully return patients to normal functional performance. [bib_ref] Effects of immobilization on plantar-flexion torque, fatigue resistance, and functional ability following..., Shaffer [/bib_ref] This highlights the need for rigid internal fixation of fractures, which can then allow more rapid return to motion activities to potentially decrease the risk of arthrofibrosis. The authors' protocol following ORIF includes splinting for 2 weeks followed by boot for 4 weeks, then nighttime padded ankle foot orthosis postoperatively. Physical therapy at 6 weeks is prescribed if needed for better range of motion, strengthening, etc.
One preventive strategy that can be overlooked is proper casting and splinting technique. As one molds the cast or splint, the ankle must be held in a neutral position until the plaster or fiberglass hardens to minimize the risk of developing contractures. [bib_ref] Cast and splint immobilization: complications, Halanski [/bib_ref] Maintaining optimal foot position is also paramount during treatment of wounds and for soft tissue coverage procedures. If the splinted ankle is positioned in plantarflexion and immobilized for prolonged periods of time, patients are at increased risk for muscle and joint stiffness. Splint in equinus for short term may be tolerated and resolved with stretching. However, if prolonged immobilization occurs, contractures may become chronic. It is therefore important to limit the duration of immobilization and initiate range of motion exercises early after discontinuation. In more serious cases of contractures, the patient may benefit from physical therapy after the removal of the immobilizing device. Lastly, it is best practice to keep the ankle neutral until the splint/cast hardens and to place the entire foot on a relatively flat surface to ensure the ankle is not immobilized in equinus. The exception is if equinus is desired (ie, after Achilles repair) in which case it should be maintained for as short a period of time as possible. Nevertheless, more studies are needed to develop a better understanding of the prevention of ankle arthrofibrosis.
## Future treatments
Current treatments as described above have limitations. Therefore, the advent of pharmacologic agents that can successfully address excess fibrous tissue from the ankle joint are promising. Two agents with antifibrotic effects have recently emerged and have the potential to effectively treat arthrofibrosis: anakinra and relaxin.
Anakinra is an interleukin-1 antagonist. Interleukin-1 plays an integral role in the inflammatory cascade, promotes pro-fibrotic mediators, and stimulates fibroblast proliferation and chemotaxis. [bib_ref] Regulation of PDGF and its receptors in fibrotic diseases, Bonner [/bib_ref] [bib_ref] Clinical benefits of intra-articular anakinra for arthrofibrosis, Brown [/bib_ref] [bib_ref] Role of cytokines and cytokine therapy in wound healing and fibrotic diseases, Gharaee-Kermani [/bib_ref] In a pilot study, intra-articular injection of anakinra was given to 8 patients with knee arthrofibrosis. A majority of patients (75%) reported improvement in range of motion and pain. [bib_ref] Clinical benefits of intra-articular anakinra for arthrofibrosis, Brown [/bib_ref] Although the cohort of patients was small, anakinra may be a promising agent for ankle or other joint arthrofibrosis.
Relaxin is a versatile endogenous neurohormone. Though traditionally viewed as a pregnancy hormone only secreted by the corpus luteum, it has been subsequently established that relaxin is also produced in the heart, endometrium, mammary gland, placenta, and prostate. [bib_ref] Cardiovascular effects of relaxin: from basic science to clinical therapy, Du [/bib_ref] [bib_ref] Relaxin: a novel agent for the treatment of acute heart failure, Wilson [/bib_ref] Although much of the recent literature has investigated the cardiovascular effects of relaxin in the setting of heart failure, [bib_ref] Relaxin: a novel agent for the treatment of acute heart failure, Wilson [/bib_ref] relaxin possesses multiple other properties, including antifibrotic effects. [bib_ref] Intraarticular injection of relaxin-2 alleviates shoulder arthrofibrosis, Blessing [/bib_ref] [bib_ref] Anti-fibrotic actions of relaxin, Samuel [/bib_ref] Its antifibrotic effect profile may offer a pharmacologic intervention to target arthrofibrosis.
Recently, a rat model of shoulder arthrofibrosis has been produced via prolonged immobilization, and this has been shown to render lasting effects on in vivo kinematics. [bib_ref] Rat model of adhesive capsulitis of the shoulder, Okajima [/bib_ref]
# Conclusion
Arthrofibrosis is a common, but overlooked, condition that imparts significant morbidity following injuries and surgery to the foot and ankle. The most common etiologies are related to soft tissue trauma with subsequent fibrotic and contractile scar tissue formation within the ligaments and capsule of the ankle. This leads to pain, alterations in gait, and ankle dysfunction. The most critical component of treatment is most likely attempting prevention by providing appropriate splinting and physical therapy. However, when arthrofibrosis occurs, there are effective treatments including physical therapy and dynamic splinting. When conservative treatment fails, open or arthroscopic procedures as well as management with external fixation may be effective [fig_ref] Figure 3: Treatment algorithm for ankle arthrofibrosis [/fig_ref]. Although surgery may be beneficial, there are currently few studies on which to guide expectations. The future, however, is hopeful and there may be new treatment options that can be used to treat fibrotic joints without the need for surgical intervention.
## Ethics approval
Ethical approval was not sought for the present study because it was a topical review.
## Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ICMJE forms for all authors are available online.
# Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
## Orcid id
Brian Timothy Velasco, MD, https://orcid.org/0000-0003-3513-4028 Shalin S. Patel, MD, https://orcid.org/0000-0002-7934-7095 David B. Frumberg, MD, https://orcid.org/0000-0001-9926-8960 Christopher P. Miller, MD, https://orcid.org/0000-0001-8259-7629
[fig] Figure 1: Magnetic resonance images demonstrating (A) fibrotic tissue at the anterior ankle joint line (red arrow) and (B) dense fibrotic tissue extending along the capsule of the joint (red arrow). [/fig]
[fig] Figure 2: Hinged external fixator allowing for gradual stretching and correction of equinus. [/fig]
[fig] Figure 3: Treatment algorithm for ankle arthrofibrosis. [/fig]
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Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples
Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severityCoating of Microtube Inner Surface with Halloysite Nanotubes
## 1
. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible .
Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see . A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers . To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay . The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes . This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of >50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.
## Video link
The video component of this article can be found at http://www.jove.com/video/4248/
## Protocol
The following protocol is for the production of a single microtube device.
## Preparation of halloysite nanotube solution
1. Sonicate (10-13 W (rms)) 250 μL halloysite nanotube solution (6.6 wt% in water) 30 sec. Cool solution with cold water and repeat sonication once. Cool again. 2. Draw sonicated solution into a syringe, attach a 0.45 μm syringe filter, and filter solution into clean microfuge tube. Vortex the solution occasionally to maintain homogeneity.
1. Prepare a solution of 10 μg/mL protein G in 1X Dulbecco's phosphate buffered saline (PBS, pH 7.0 -7.2). Draw 50 μL PBS through microtube and then draw 50 μL of the protein-G solution and allow to incubate for 1.5 hr at RT. 2. Prepare a solution containing 5 μg/mL E-selectin-IgG and 50 μg/mL antibody (anti-EpCAM for most cancer samples, anti-PSMA for prostate cancer samples) in PBS. Draw 50 μL of the E-selectin and antibody solution into the microtube and allow to incubate for 2 hr at RT. 3. Nonspecific cellular adhesion is blocked with 5% milk protein. Prepare a solution of 5% (w/v) milk protein in PBS. Draw 50 μL milk protein solution into the microtube and allow to incubate for 1 hr at RT. 4. Draw 50 μL PBS into tube and leave at RT until blood or buffy coat samples are ready for processing. 5. 10 min prior to using the microtube for isolation, draw 50 μL PBS that has been saturated with Ca 2+ ("PBS+") to activate the selectin molecules.
## Preparation of samples for cell isolation
1. Draw or obtain 10 mL blood from patient into heparinized tube. 2. Place 10 mL Ficoll-Paque lymphocyte isolation solution into 50 mL centrifuge tube. Gently layer 10 mL whole blood on top of Ficoll so as not to mix the blood and Ficoll. 3. Centrifuge at 2000x g for 15 min at 4 °C with minimal deceleration. 4. Remove the buffy coat layer and place in new tube. 5. Wash buffy coat with PBS (centrifuge at 230x g for 10 min, discard the supernatant). 6. Gently resuspend cells with 1 mL RBC lysis buffer and incubate for 10 min at RT to lyse erythrocytes. 7. Add 10 mL PBS, mix gently, and centrifuge at 230x g for 10 min. Discard the supernatant and gently resuspend the pellet in 3 to 4 mL PBS+.
## Cell isolation
1. Attach one end of the functionalized microtube to a 5 mL syringe using IDEX adaptors. Accutase. Gently perfuse enough Accutase into the microtube to fill (~50 μL) and allow to incubate at RT for 10 min. 7. Attach a syringe prefilled with 1 mL of growth media (79% RPMI, 20% FBS, 1% penicillin streptomycin) and perfuse into microtube, collecting effluent into tissue culture treated well plate. 8. Culture cells at 37 °C and 5% CO 2 under humidified conditions.
# Representative results
The goal of this technique is to isolate viable cancer cells from the blood of cancer patients. Several methods exist to identify cancer cells in culture; a necessary verification of device success. We have chosen to stain cells in culture with antibodies against epithelial moieties such as EpCAM (epithelial cellular adhesion molecule) or PSMA (prostate specific membrane antigen), in addition to DAPI to identify intact cell nuclei [fig_ref] 2: Insert the syringe onto a syringe pump [/fig_ref]. The number of cancer cells captured using this technique is necessarily a function of the number of CTCs in the starting sample, and patient variability can be high. In processing samples taken from patients diagnosed with stage IV cancers, we routinely capture between 100 and 500 cancer cells per tube of blood, at purities >50%. Immediately following isolation, greater numbers of contaminating leukocytes may be present. However these numbers will be depleted following incubation in culture medium for up to 5 days.
# Discussion
It is often the case that the early steps in the discovery of new cancer therapeutics utilize cancer cell lines, which bear questionable resemblance to primary cancer cells yet remain in use due to their ease of use in the laboratory. Research into the development of new cancer therapies would be expedited if primary human cancer cells were utilized early in novel research. CTC are the most easily accessible type of cancer cell, due to their presence in blood and the ease of a standard blood draw. In addition, circulating tumor cells represent a necessary step in the process of metastasis [bib_ref] Cancer metastasis: Building a framework, Gupta [/bib_ref] , so their relevance to the disease and utility for targeting new drugs is clear. Isolation of CTC from blood in vitro is complicated by their low concentrations: on the order of one per million leukocytes or one per billion erythrocytes [bib_ref] Circulating tumor cells: a window into cancer biology and metastasis, Maheswaran [/bib_ref]. The majority of current methods for detecting CTC in blood, including the only FDA-approved technique CellSearch(Veridex), damage or destroy cells in the detection process, precluding use beyond enumeration. The method described above does not compromise cell viability and thus opens the door for future clinical research on cancer. As the recovery of intact cells is the goal of this technique, it is imperative that cells be handled with care, especially during blood separation steps.
There are a number of tunable parameters in this system that could be altered to achieve improved yield depending on critical characteristics such as cancer type. In the steps described above, we had chosen to use EpCAM as a CTC-specific antibody for all cancer types except when processing prostate cancer samples, wherein anti-PSMA was used. Further substitutions of cancer-specific antibodies can certainly be used to improve performance for individual patients. Furthermore, selectin and antibody concentrations can be altered to enhance capture [bib_ref] Microcontact Printing of P-Selectin Increases the Rate of Neutrophil Recruitment Under Shear..., Lee [/bib_ref].
An essential feature of the device is the incorporation of E-selectin molecules onto the surface. E-selectin is normally expressed on the luminal surface of endothelial cells and function to recruit fast moving leukocytes to sites of inflammation. Flowing leukocytes bind transiently to selectin molecules, resulting in a slower, rolling behavior that facilitates slower and stronger binding of the cell to the endothelium by integrins. Convincing experimental evidence exists that makes the case that CTC can extravasate by a similar mechanism .Thus our device physiologically mimics a venule to biomimetically capture flowing CTC without inflicting cellular injury.
Enhanced device performance can be attributed to the addition of the halloysite nanotube coating to the luminal surface of the device. Previous studies have shown that there are three major components of the nanotube coating that allows for improved functionality. First, the nanotube coating provides increased surface area, allowing for greater protein deposition onto the surface . Second, in performing atomic force microscopy on the nanotube coating we have determined that individual nanotubes protrude from the surface into the flow. This allows selectin molecules to be presented as much as one micron above the surface so that cells can be captured and recruited to the surface earlier in their trajectory through the tube . Finally, the halloysite nanotube coating is able to prevent leukocyte adhesion and spreading on the surface, allowing for a reduced number of leukocytes captured along with the CTC and thus greater subsequent CTC purities . Disclosures M.R.K. is a scientific advisor of CellTraffix, Inc. (Rochester, NY).
[fig] 2: Insert the syringe onto a syringe pump. 3. Submerge the open end of the functionalized microtube into the cell suspension. 4. Process the cell suspension through the microtube at 1 to 4 mL/h. 5. Transfer the open end of the microtube to a tube containing PBS+ and draw 300 μL PBS+ into syringe at 0.016 mL/min to remove unbound and loosely bound cells from the microtube. 6. Place the open end of the microtube into a clean tube. Disconnect the syringe from the microtube and attach a syringe prefilled with [/fig]
[fig] Figure 1: Schematic of functionalized microtube for CTC isolation, showing selectin-mediated rolling followed by antibody-mediated static adhesion. [/fig]
[fig] Figure 3: Representative micrographs from separate donors (A and B) of CTC in culture 5 days subsequent to isolation from a cancer patient blood sample. Cells were fluorescently stained for EpCAM with AlexaFluor 488 (green) and 4',6-diamidino-2-phenylindole (DAPI) to visualize the nucleus (blue). [/fig]
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The SARS‐CoV‐2/COVID‐19 pandemic and challenges in stroke care in India
Stroke care in India has evolved rapidly in the last decade with a focus on stroke awareness, prevention, rapid triage, treatment, and rehabilitation. But acute stroke care and poststroke rehabilitation in the country have limitations owing to the economic constraints and poor access to health care. The SARS-CoV-2/COVID-19 pandemic has made stroke care even more challenging. We outline the unfavorable circumstances in stroke care induced by the pandemic; propose mitigating measures; crisis management; and provide a comparative evaluation of stroke care between India and the United States during the pandemic. There is a need for public health systems in both developed and developing countries to improve awareness, implement proper strategies of triage, acute treatment, well-defined rehabilitation plans, telemedicine services, and virtual check-ins.
# Introduction
Systems of stroke care in India have been evolving at a rapid pace in recent years to meet the stroke burden, with remarkable advances made in stroke awareness, prevention, rapid triage, treatment, and rehabilitation. Yet, acute stroke care and poststroke rehabilitation in the country have limitations owing to the economic constraints and poor access to healthcare, burdening a large section of the population. [bib_ref] Stroke epidemiology and stroke care services in India, Pandian [/bib_ref] It is not surprising that the health impact of the SARS-CoV-2/COVID-19 pandemic sweeping the world has created challenges for stroke a These authors have equal seniority on this study. care in India, which are distinct from those faced by the developed world.
We conducted a survey of stroke experts from 13 established stroke centers in India using a brief questionnaire aimed at capturing the impact of the COVID-19 pandemic and the nationwide lockdown on acute stroke care services. We compare our data with that from a developed country, the United States of America, and attempt to understand and substantiate the observed similarities and differences. In this report here, we, stroke neurologists from high-volume stroke centers in India, identify the weak links in acute stroke care, poststroke rehabilitation, and care of stroke survivors during the COVID-19 outbreak, and suggest remedial measures with a special focus on telemedicine. The SARS-CoV-2 outbreak in India and impact on health care
India is home to 1.37 billion people, nearly one fifth of the world's population.The first case of COVID-19 in India was reported on January 30, 2020 and rapid escalation of cases was noted in mid-March, with confirmed cases crossing 70,756 as of . A nationwide lockdown in India was imposed on March 25, 2020, which brought to a standstill nearly all travel and commercial human activities across the country.The swift and strict restrictive measures adopted have at least temporarily halted the flooding of the health services with COVID-19 cases, though current epidemiological models project the peak of the curve for hospital admissions to be in June or July 2020. [bib_ref] Healthcare impact of covid-19 epidemic in India: a stochastic mathematical model, Chatterjee [/bib_ref] The stroke "chain of survival" and care pathways in India have likely been affected in one way or the other because of the pandemic and lockdown. The shortfall of health insurance coverage and rehabilitative care centers ensures that most of the burden of illness is borne by out-of-pocket payment by patients and families. 5 A large percentage of specialized health services, such as advanced stroke care, is provided by corporate hospitals. [bib_ref] Challenges to healthcare in India-the five A's, Kasthuri [/bib_ref] However, many of these centers are currently unable to extend care for stroke in persons suspected of having COVID-19, as COVID-19 care in India is mostly confined to designated hospitals under the state and central governments. The few exceptions are in regions with a high COVID-19 caseload, where some of the larger private hospitals have been identified for care. Individuals from poorer sections of society and daily-wage workers are the worst affected owing to their already limited access to a healthcare facility. [bib_ref] Challenges to healthcare in India-the five A's, Kasthuri [/bib_ref] The priority has shifted from health to daily sustenance for economically weaker populations. Lower educational levels and traditional cultural beliefs contribute to poor understanding of the need for quarantine and distancing measures. [bib_ref] Gaps in India's preparedness for covid-19 control, Chetterje [/bib_ref] These factors may eventually lead to a rise in COVID-19-related as well as -nonrelated diseases-including stroke-related mortality and morbidity-across the country in the coming days. An additional concern is the high risk of infection among healthcare workers from the shortage of personal protective equipment (PPE), which can lead to a reduced workforce available for stroke services.
## Impact of the sars-cov-2/covid-19 pandemic in acute stroke services in india: survey report
From the data received from apex centers of India [fig_ref] Table 1: Changes in acute stroke care in selected high-volume stroke centers in India... [/fig_ref] through the survey and contacts, it is apparent that the onset of the SARS-CoV-2 pandemic has resulted in significant changes in the number of reported, and treatment of, stroke patients. The questionnaire-based survey was conducted among neurologists in 13 major centers of the country, including eight government/academic institutes and five large private hospitalsand B). The questionnaire consisted of 11 questions related to the changes noted in acute stroke care following the outbreak of the COVID-19 pandemic. An overall reduction of about 61.22% in the reporting of weekly stroke cases across the country has been observed since the lockdown. Intravenous thrombolysis and endovascular procedures were also severely affected, with an overall reduction of 64.76% and 67.21%, respectively, the latter coming to a complete halt in five centers that had regularly provided this service. The drop in the cases occurred equally in both COVID-19-designated and -nondesignated hospitals.
PPE was available for all hospital workers in the triage area only in five among the surveyed centers, and only 58.3% of centers used full PPE during endovascular procedures. Stroke rehabilitation services, especially for outpatient care, were markedly affected in more than 53% of surveyed hospitals because of a lower number of personnel, fear among staff and patients, and poor access.
## A comparison of the impact of covid-19 on stroke care in india and the united states
The adverse impact of the SARS-CoV-2 pandemic on stroke care showed several similarities and differences between India, a developing country, and the United States. There are reports in the United States of a significant decrease in all acute stroke cases presenting to the emergency rooms at stroke centers, 7 similar to reports in India. The magnitude of this decrease has been estimated in surveys to be around 30-40% in the United States, about two thirds of the reduction seen in the Indian survey. The larger magnitude of decrease in India is deeply troubling, as India has a higher incidence of stroke compared with the United States. [bib_ref] Incidence & prevalence of stroke in India: a systematic review, Kamalakannan [/bib_ref] There is an approximately 50% decrease in all reperfusion therapies in the United States, including thrombolysis and thrombectomies, whereas these numbers are 64.76% for thrombolysis and 67.21% for thrombec-tomy in India. Though the survey included both COVID-19-designated and -nondesignated hospitals, the possibility of triaging of stroke patients to nonstroke centers was likely to lead to poor outcomes. There is a clear similarity in both countries, however, in that neither country's public health system made proactive efforts to raise awareness among the public to not ignore life-threatening emergencies such as stroke during the pandemic, or efforts to deal with the developing atmosphere of fear of going to hospitals. This is likely because public health systems all over the world, regardless of country income levels, were completely unprepared for handling the pandemic and its impacts on other health conditions. This is one of the key lessons for all countries in developing future pandemic preparedness policies and protocols.
## Challenges and solutions for stroke care in india during the pandemic
## Acute stroke care
The extreme measures adopted to contain the spread of SARS-CoV-2 have resulted in a dramatic fall in the number of stroke patients reaching hospitals in India. A similar change in the pattern of stroke patients was noted in Italy, with more severe patients reaching hospitals and at longer times after onset. [bib_ref] Acute stroke management pathway during Coronavirus-19 pandemic, Baracchini [/bib_ref] This may be mostly owing to patients with minor stroke and transient ischemic attack not seeking care because of unavailability of transport, fear of contracting infection, and financial liabilities. The lockdown has also created difficulty in timely consultation with caregivers, leading minor stroke cases to remain undiagnosed. India lacks emergency medical services; studies have shown that from 1.5% to less than 10% of patients reach hospitals by ambulance. [bib_ref] Ischemic stroke profile, risk factors, and outcomes in India: the Indo-US collaborative..., Sylaja [/bib_ref] [bib_ref] Factors delaying admission to a hospital-based stroke unit in India, Pandian [/bib_ref] The onset-to-door and treatment time is longer that before COVID-19 [bib_ref] Stroke-related education to emergency department staff: an acute stroke care quality improvement..., Puri [/bib_ref] and it is likely to increase further, as observed in many severely affected countries, thereby compromising acute care. [bib_ref] Acute stroke management pathway during Coronavirus-19 pandemic, Baracchini [/bib_ref] Stroke patients from remote areas of the country are at greater risk of not reaching hospitals because of the suspension of public transport. [bib_ref] Factors delaying admission to a hospital-based stroke unit in India, Pandian [/bib_ref] [bib_ref] Factors delaying hospital arrival of patients with acute stroke, Ashraf [/bib_ref] Acute stroke care in individuals suspected of having COVID-19 is of special concern. According to the Ministry of Health and Family Welfare (MoHFW) Government of India guidelines, these cases are supposed to go to designated COVIDcare government hospitals-several of them are in smaller districts and are state hospitals. Many of the COVID-19-designated hospitals either may not have facilities for acute stroke care or are not stroke ready. The consequent disruption of hyperacute stroke treatment requiring mechanical thrombectomy is particularly acute because resources are particularly strained: the bulk of endovascular therapy in India is provided by corporate hospitals, 1 which are currently excluded from caring for stroke patients suspected of having COVID-19. Those patients who reach a healthcare facility having no stroke expertise are undertreated and need referral to higher-level centers, often after a considerable delay with unavoidable complications. 14 As noted in many severely affected countries, the onset-todoor and door-to-treatment times are prolonged in the SARS-CoV-2 epidemic, compromising acute care. [bib_ref] Acute stroke management pathway during Coronavirus-19 pandemic, Baracchini [/bib_ref] The reluctance of patients to report to hospitals is rooted in fears of acquiring SARS-CoV-2 infection and economic implications/consequences of hospital admission. Awareness campaigns to reeducate the public on the high morbidity and mortality of noncommunicable diseases (NCDs), including stroke, and the high risk of a second wave of mortality due to these illnesses, are essential. In virtually all stroke cases, however, the need to get timely care in hospitals supersedes the risk of possible exposure to SARS-CoV-2. Even when the "core" government machinery is directed toward the control of the pandemic, operating special ambulances and other emergency services could be a game-changer for acute NCD needs.
Stroke management guidelines would need to be followed with concessions made for changes in accessibility. Acute therapy may see an inevitable shift from larger centers to smaller, peripheral hospitals and from endovascular to more intravenous thrombolysis during the COVID-19 crisis. Moreover, especially in the case of suspected situations of hyperacute strokes, optimal use of telemedicine facilities would help ensure the services of stroke specialists even in peripherally located COVID-19designated centers. Patients who present later with symptoms could be referred to designated stroke centers with facilities for isolation and concurrent COVID-19 care. The recent consensus statement of the Indian Stroke Association suggests organizing hospital stroke pathways during the ongoing pandemic. [bib_ref] Consensus statement-suggested recommendations for acute stroke management during the covid-19 pandemic: expert..., Bhatia [/bib_ref] The risk of infectious exposure to clinical teams can be mitigated by adopting the Protected Code Stroke guidelines.Defined and designated areas for all stroke patients and screening by an emergency room physician for suspected COVID-19 symptoms, with full protective gear (full-sleeved gown, surgical mask, eye protection (face shield and/or goggles), gloves, and head cover), have been proposed. [bib_ref] Acute stroke care in the coronavirus disease 2019 pandemic, Dafer [/bib_ref] Since testing currently does not include a point-of-care device, universal precautions to healthcare workers become even more crucial.
## Poststroke rehabilitation
Recovery from a disabling stroke greatly depends on early and easy access to tailored, comprehensive rehabilitative measures, including physical, speech, and occupational therapies, especially in the initial 3-6 months poststroke. [bib_ref] Early rehabilitation after stroke: a narrative review, Coleman [/bib_ref] India has few rehabilitation centers, and most of the patients with mild-to-moderate strokes receive postacute rehabilitation at home or through outpatient clinics of large hospitals. [bib_ref] Rehabilitation needs of stroke survivors after discharge from hospital in India, Kamalakannan [/bib_ref] The stroke recovery curves are likely to turn suboptimal, as patients will have even less access and availability to comprehensive rehabilitation facilities poststroke. The sudden interruption in rehabilitation may lead to other acute difficulties, including worsening of ambulation that patients may assume to be stroke recurrence, causing panic.
During the pandemic, hospitals are forced to complete treatment/evaluation and discharge patients early to minimize the risk of infection, which shortens or eliminates in-hospital rehabilitation services. Shifting of resources toward the care of COVID-19 patients has also resulted in fewer dedicated centers for assisting stroke patients. The ability of patients to avail the services of experienced therapists at home is also impaired by the lack of transport services and mandatory cessation of all nonemergency services during the lockdown. Trained speech and occupational therapists, who were scarce in normal circumstances, have become almost unavailable with the continuing cutback in travel. Here again, relatively weaker sections of communities are affected disproportionately, with subsidized government facilities being centered around COVID-19 care.
Developing a caregiver-driven stroke rehabilitation program has been attractive in India to address the scarcity of rehabilitation centers and trained therapists. Implementing such programs will be crucial in (and perhaps after) COVID-19 in order to avoid preventable disabilities. As one example, video-and app-based programs developed in the local language can facilitate home therapy. [bib_ref] Stroke coach: a pilot study of a personal digital coaching program for..., Kamoen [/bib_ref] Care of stroke survivors and risk factor control Stroke review clinics across the country have been suspended to prioritize the care of COVID-19 cases. Curb of public transport and fear of SARS-CoV-2 infection prevent timely reviews even in areas where centers are active. Consequently, stroke survivors are facing difficulties in receiving follow-up care, which may lead to a surge in new cerebrovascular and cardiovascular events.
Poor accessibility to testing facilities for risk factor monitoring has rendered risk factor control challenging in the remote areas of the country. India has a higher burden of rheumatic heart diseases compared with developed countries; afflicted patients require vitamin K antagonists for anticoagulation and secondary stroke prevention. [bib_ref] Ischemic stroke profile, risk factors, and outcomes in India: the Indo-US collaborative..., Sylaja [/bib_ref] [bib_ref] Predictors of ischemic stroke in rheumatic heart disease, Gupta [/bib_ref] Hence, the proportion of anticoagulated patients using warfarin, acenocoumarol, or related drugs is likely to be comparable to those using nonvitamin K-antagonist or -anticoagulation. [bib_ref] Ischemic stroke profile, risk factors, and outcomes in India: the Indo-US collaborative..., Sylaja [/bib_ref] [bib_ref] Predictors of ischemic stroke in rheumatic heart disease, Gupta [/bib_ref] Patients generally have poor knowledge of the optimal use of anticoagulation and monitoring, which is likely to worsen further with decreased interaction with physicians. [bib_ref] Knowledge regarding oral anticoagulation therapy among patients with stroke and those at..., Alphonsa [/bib_ref] With both laboratory and hospital access hampered, adverse events related to both poor physical therapy maintenance and suboptimal vascular risk-factor control are likely to surface. Mental health issues have also come more to the fore, with depression and anxiety associated with separation from loved ones and fear for well-being, lack of money, and inability to work because of COVID-19 implications and consequences. With India under lockdown, the availability of essential medications is also hampered. There are increased chances of stroke recurrence if patients fail to take their medications due to unavailability, limitations in venturing out to buy medications, and inability to reach public hospitals for prescriptions and refilling from free shops. The high demand for medicines, in turn, increases prices, making it difficult for some patients to attain essential medicines. This makes patients highly susceptible to availing medicines from unauthorized sources or even receiving incorrect medications.
Teleconsultation facilities are being set up in tertiary care facilities to ensure that patients receive timely medical advice and, more importantly, motivation and counseling to endure the crisis. Such stroke teleconsultation facilities should use the opportunity to reinforce the need to also continue preventive measures for NCDs and to see that timely treatment of life-threatening diseases like myocardial infarction and stroke is not compromised.Government drug suppliers in India and defense personnel are reaching out to ensure that available drugs reach the difficult-to-access locations in the country,whereas political action is required to ensure the resumption of drug manufacturing. In many parts of the country, local administrations have established counseling centers to tackle depression and stress. Interestingly, popular media are attributing the decrease in reporting of acute myocardial infarction and strokes to an actual reduction in incidence of both related to the improvement of certain risk factors: reduced consumption of fast food, more sleep, decreased environmental pollution, and lower work-related stress are touted as possible positive influencers. 27
## Stroke care during covid 19: shifting to telemedicine
Major developments in the field of telemedicine have taken place at a rapid pace during the COVID-19 crisis. [bib_ref] The coronavirus disease 2019 crisis as catalyst for telemedicine for chronic neurological..., Bloem [/bib_ref] Telemedicine, by virtue of minimizing travel and reducing physical contact, provides a pragmatic advantage by avoiding in-person consultation of patients while providing access to both primary and specialist care. Electronic consultations (e-consults) thereby greatly assist in delivery of outpatient health care without compromising the benefits of specialty expertise, which can be readily made available to many patients. [bib_ref] Utility, appropriateness, and content of electronic consultations across medical subspecialties: a cohort..., Ahmed [/bib_ref] Virtual consultations have further clear advantages. Concerns of patients with COVID-19 can be addressed and triaged, and individuals in quarantine or recently discharged to home can be provided care. For non-COVID-19 issues, telemedicine can provide care without the risk of exposure, which is a major concern for the old and those with chronic conditions. This could have a net effect of reduced exposure to patients and clinicians while limiting the demands of emergency departments. [bib_ref] Rapidly converting to "virtual practices": outpatient care in the era of covid-19, Mehrotra [/bib_ref] One of the major concerns about telemedicine, especially in the West, has been to ensure privacy the Health Insurance Portability and Accountability Act of 1996 (HIPAA). In the United States, HIPAA noncompliant platforms have generally not been allowed. However, the U.S. Department of Health and Human Services recently waived potential penalties for HIPAA violations and declared that it would not enforce compliance rules during the COVID-19.Policymakers in India have also opened the route of telemedicine by a fast track issuing of telemedicine guidelines.These guidelines provide authorization to use various telemedicine platforms by registered medical practitioners and hospitals. It, however, prohibits any technology platforms based on artificial intelligence or machine learning to counsel or prescribe medicines to patients.
Stroke management using telestroke platforms can be utilized in India to provide high-quality care for triaging patients, as well as the entire stroke care pathway, even in resource-poor settings. [bib_ref] Telestroke in resource-poor developing country model, Sharma [/bib_ref] As the number of COVID-19 patients continues to increase, there may be a greater resource crunch in the routine outpatient care of stroke patients, which may be avoided to a significant extent if telemedicine platforms could be integrated early on into health systems within the framework of the guidelines of the MoHFW, Government of India.
# Conclusions
The COVID-19 pandemic has made stroke care even more challenging. Various facets of stroke treatment need reorganization to provide optimized services during such crises. There is a need for public health systems in both developed and developing countries to improve stroke awareness and to implement proper strategies of triage, acute treatment, well-defined rehabilitation plans, teleservices, and virtual check-ins. This will help maintain the continuum of care for stroke care and reduce morbidity and mortality.
[fig] Figure 1: (A) Map of India showing the location of apex medical centers where the questionnaire-based survey was conducted (Map of India adapted from Bhuvan, ISRO). (B) Graph representing the percentage reduction of total reporting of stroke cases post-COVID-19, along with percentage reduction in IV thrombolysis and thrombectomies in apex medical centers across India. [/fig]
[table] Table 1: Changes in acute stroke care in selected high-volume stroke centers in India during the COVID-19 outbreak ER, emergency room; IV, intravenous; NA, not applicable; PPE, personal protective equipment. [/table]
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Longitudinal analysis of primary and secondary factors related to fatigue in multiple sclerosis
# Introduction
Fatigue is a poorly understood symptom in multiple sclerosis (MS), despite its high frequency and influence on the quality of life. Studies suggest a multifactorial model, separating between primary MS-related and secondary factors. Primary factors entail disease activity and immune activation, in particular the influence of endocrines and proinflammatory cytokines, as well as alternations to brain areas suggestive to be involved in the perception of fatigue. Secondary mechanisms involve comorbidities such as additional autoimmune disease and psychiatric disorders. Multiple studies found poor sleep quality to be linked with higher fatigue levels and additionally associated with increased depression scores. Indeed, major depressive disorder (MDD) is one of the most common fatigue-influencing comorbidities in patients with MS. MDD is not only highly prevalent in MS patients but is also associated with immune system alterations and hyperactivity of the hypothalamic-pituitary-adrenal axis with subsequently altered inflammatory neuroendocrine factors and is thus directly linked to primary diseases mechanisms in MS.
Our goal was to describe the evolution of fatigue in MS compared with a healthy population, especially in regard to primary and secondary factors associated with fatigue.
# Patients and methods
For this retrospective analysis, we selected patients and controls from ongoing observational cohort studies (Berlin-CIS-COHORT, NCT01371071, and VIMS, EA1/182/10). Inclusion criteria were a diagnosis of CIS or RRMS according to the 2010 McDonald criteria. Of 170 screened patients, 133 patients were included (CIS N = 100, RRMS N = 33, median follow-up time (time between first and last visit, inter-quartile range (IQR)): 2.40 years (1.45, 3.81)) and 30 healthy controls (HCs) matched for age (p = 0.784) and sex (p = 0.072).HCs had no relevant comorbidities. Inclusion criteria were a baseline diagnosis of CIS or early RRMS according to the McDonald criteria (revised version 2010)with less than 3 years since disease onset, and a minimum age of 18. Exclusion criterium was a missing FSS questionnaire. The cohort studies were approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent.
Disability was assessed using the expanded disability status scale (EDSS). were measured with Beck's depression inventory (BDI-II, cut-off: 13) and the Pittsburgh Sleep Quality Index (PSQI, cut-off: 6), respectively.
Whole-brain lesion volume was obtained using the lesion segmentation toolbox with manual correction in ITK-SNAP using MPRAGE and FLAIR images of a 3 T MRI scanner (MAGNETOM Trio Tim Siemens, Erlangen, Germany).
Group comparisons were performed with χ 2 (test statistic: χ 2 ) or Wilcoxon test (test statistic: W). Correlations were analyzed using parametric or Spearman's correlation. All tests were performed using R 3.6.0. P values < 0.05 were considered significant.
# Results
We found no differences in fatigue distribution between patients and HCs at baseline (HC vs. patients: χ 2 = 3.135, p = 0.209).
During follow-up, 88 patients had a consistent fatigue status (fatigued = 10 [8%], non-fatigued = 78 [59%]). 45 (34%) patients changed fatigue status (inconsistent fatigue (IF)): Twelve patients lost their fatigue status at follow-up (9%), whereas 8 patients developed fatigue at follow-up (6%). The remaining patients showed only a minor change of scores and switched from or to borderline fatigued, black arrows). The majority (13 [81.25%]) of HCs had a consistent fatigue status, with only one (6.25%) HC categorization changing from borderline to non-fatigued at follow-up. Fewer patients (N = 78; 59%) were consistently categorized as not fatigued than HCs (N = 13; 81%). More patients (N = 45; 34%) than HCs (N = 1; 6.25%) had an unstable fatigue status over time (p = 0.012, χ 2 = 6.309).
We then analyzed primary and secondary factors in patients (n = 30) categorized as consistently high fatigued (CF, n = 10,or IF (n = 20,, green and red arrows). Regarding primary factors, 7 (23%) patients had an EDSS increase (≥ 1) at follow-up (4 CF, 3 IF). At baseline, EDSS was higher in fatigued than non-fatigued patients (p = 0.021). Changes in EDSS and in the fatigue group were associated (p = 0.042). In contrast, lesion volume did not correlate with fatigue at baseline (R = 0.680, p = 0.502) or follow-up (R = 0.847, p = 0.406) and was not associated with consistency of fatigue status ([W] 699.0, p = 0.774).
Investigating secondary mechanisms, the majority of IF (18 [90%]) and CF (9 [90%]) patients showed comorbidities. 8 IF (40%) and 6 CF (60%) had comorbid autoimmune disorders. Thyroid disorders were observed in 1 CF (10%) with Hashimoto-thyroiditis, 4 IF (20%) with hypothyroidism (2 IF (10%) with Hashimoto-thyroiditis, 1 IF (5%) after thyroidectomy due to focal thyroid autonomy, 1 IF (5%) not further classified) and 1 IF (5%) with an elevated Thyroid-Stimulating-Hormone level (not further classified). 9 IF (45%) and 8 CF (80%) showed psychiatric comorbidities: Depressive symptoms were present in 8 IF (40%) and
# Discussion
We observed a high prevalence and persistence of fatigue without a general alteration pattern in early MS compared with HCs. In our study at disease onset, only 15% of MS/CIS patients reported fatigue. This is in contrast to two previous studies from one center, which reported frequencies of up to 45%. Concerning primary fatigue, disability and disability worsening were associated with fatigue at onset and development of fatigue, whereas MRI T2-weighted lesion load did not contribute. Most relevant in our study were secondary factors of fatigue, namely concomitant autoimmune and mood disorders as well as poor sleep. In contrast, primary mechanisms hardly are associated with a higher prevalence of fatigue. Thus, MS patients appear to be either prone to fatigue or stay stable not fatigued after their first event. Disease progression as the most important primary mechanism appears to play only a minor role since fatigue did not increase over time and was not associated with disease activity. However, increasing disability (e.g. newly developed sensory disorder or spasticity) might lead to perceived mobility difficulties, which could be felt as weakness or be interpreted as fatigue. As many patients are stable, not fatigued even though EDSS increases, a direct link between disability and fatigue is unlikely. Studies supporting this multifactorial highly interactive disease model have further found psychiatric comorbidities, such as mood and anxiety disorders, to be associated with higher EDSS scores leading to the assumption that also these comorbidities confound the correlation of EDSS and fatigue.
Limitations of this study include the lack of data prior to the MS diagnosis and the shorter follow-up in HC. The low prevalence of fatigue may limit our sample's representativeness.
To conclude, secondary factors have to be taken into account when assessing and managing fatigue in MS. These comorbidities may open an important avenue of interventions in patients suffering from fatigue and should thus be evaluated before considering treatment options for primary fatigue. In contrast, primary factors of fatigue were less important in our sample of patients at disease onset.
Author contributions JS: data collection, data analysis, writing the manuscript. FCO: data acquisition, supervision of data analysis, writing the manuscript. GC: data acquisition, lesion quantification, data analysis. JBS/AUB: study concept, design, study coordination, data collection, supervision of data analysis, writing the manuscript. All authors revised the manuscript for intellectual content and read and approved the final version. |
Predictors of successful percutaneous transvenous mitral commissurotomy using the Bonhoeffer Multi-Track system in patients with moderate to severe mitral stenosis: Can we see beyond the Wilkins score?
Objective: To know the predictors of a successful outcome of percutaneous transvenous mitral commissurotomy (PTMC) other than described in the Wilkins scoring system. Methods: Two hundred fifty-eight consecutive patients were enrolled for this observational study in a tertiary care heart center of Pakistan who had a Wilkins score of ≤8. Patients with more than mild mitral regurgitation (MR) or having a clot in the left atrium were excluded. The Bonhoeffer multi-track system was used as a default technique. Successful PTMC was defined as achieving a mitral valve area (MVA) of ≥1.5 cm 2 with no more than mild MR. Results: Out of 258 PTMC procedures, 197 were successful. The Bonhoeffer multi-track system was used in ~94% cases. Among unsuccessful procedures, 41 patients did not achieve the required valve area, and 21 patients developed more than mild MR, including those 8 patients who did not achieve the required valve area and had more than mild MR. Bigger mean annulus size (33.5±2.6 versus 32.8±2.1 mm; p=0.02) and preprocedure MVA (0.93±0.1 versus 0.87±0.1 cm 2 ; p=0.002) had a significant effect on successful PTMC. Lower mean preprocedure systolic right ventricular pressure on echo (65.4±19.4 versus 75.3±18 mm Hg; p=0.000) and on cath (74±21.5 versus 81.5±24.6 mm Hg; p=0.002), lower grade of left ventricular dysfunction (p=0.04), and tricuspid regurgitation on echo (p=0.003) also had positive effects on the outcome. Conclusion: Bigger preprocedure mitral valve annulus size and mitral valve area, and better left and right ventricular hemodynamics are correlated with successful PTMC. (Anatol J Cardiol 2015; 15: 373-9)
# Introduction
Rheumatic mitral stenosis (MS) is one of the common valvular heart diseases in southeast Asia. Percutaneous transvenous mitral commissurotomy (PTMC) is an established alternative to surgical mitral commissurotomy and is often a preferred method of treatment in patients with symptomatic moderate to severe mitral stenosis and with suitable anatomy [bib_ref] Systematic comparison of the effectiveness of percutaneous mitral balloon valvotomy with surgical..., Hu [/bib_ref] [bib_ref] Balloon mitral valvuloplasty: immediate and short term haemodynamic and clinical outcome, Rahman [/bib_ref]. The Wilkins scoring system is an established method of predicting the outcome of PTMC [bib_ref] Prediction of successful outcome in 130 patients undergoing percutaneous balloon mitral valvotomy, Abascal [/bib_ref]. However, there are studies that have questioned the accuracy and validity of this scoring system as a predictor of outcomes and that have suggested a more refined and comprehensive assessment in light of recent data [bib_ref] Validation of a new score for the assessment of mitral stenosis using..., Anwar [/bib_ref] [bib_ref] Ten Cate FJ. New scores for the assessment of mitral stensosis using..., Soliman [/bib_ref] [bib_ref] Assessment of mitral valve commissural morphology by transoesophageal echocardiography predicts outcome after..., Babu [/bib_ref]. Moreover, The Wilkins scoring system is mainly based on the structural changes of the mitral valve apparatus and is lacking in demographic, technical, and other echocardiographic features. Secondly, in the majority of cases (17 out of 22), a single-balloon technique was used by Wilkins et al. [bib_ref] Percutaneous balloon dilatation of the mitral valve: an analysis of echocardiographic variables..., Wilkins [/bib_ref]. Therefore, despite the common incidence in our part of the world, we did not know if there were other predictors of outcome besides that described by In particular, if we are using a double-balloon (Bonhoeffer multi-track system) technique, then it is more important to know the other predictors of the outcome in our setup [bib_ref] Percutaneous mitral valve dilatation with the multi-track system, Bonhoeffer [/bib_ref].
This study was designed to determine the predictors of a successful outcome of PTMC in patients with symptomatic moderate to severe mitral stenosis by Bonhoeffer multi-track system [bib_ref] Percutaneous mitral valve dilatation with the multi-track system, Bonhoeffer [/bib_ref].
# Methods
## Study design and patients
This observational study was conducted at a tertiary care cardiovascular center of the National Institute of Cardiovascular Diseases, Karachi, Pakistan, during the years 2009 to 2011. A total of 258 consecutive patients presenting with moderate to severe mitral stenosis and with a Wilkins score of ≤8 were included in study. Patients with more than mild mitral regurgitation (MR) and/or a clot in the left atrium (LA) and/or a Wilkins score of >8 were excluded from the study. Patients with concomitant significant valvular disease besides tricuspid regurgitation (TR) were also excluded from the study.
The double balloon (Bonhoeffer multi-track system) was used as the default technique. The single-balloon technique was used as an alternative when the double-balloon technique was not feasible.
## Procedure
A right femoral approach was used for catheterization; 6 F and 8 F sheaths were introduced into the femoral artery and vein, respectively. Right heart pressure hemodynamics were checked and recorded with a balloon angiographic catheter. Similarly, left heart pressures were recorded with a 6 F pigtail catheter, and then it was left in the ascending aorta for continuous pressure monitoring and for an anatomical landmark. An 8 F Mullins sheath was introduced into the femoral vein, and the interatrial septum was punctured with a Brockenbrough needle. A Mullins sheath was then introduced into the LA with the dilator. Once the position in the LA was confirmed with the help of a small injection of contrast material, the dilator and Brockenbrough needle were removed. Simultaneous pressure of the LA and LV was recorded with the Mullins in the LA and a pigtail catheter in the LV to obtain the gradient across the mitral valve. A flowdirected end-hole balloon catheter was introduced into the Mullins sheath, and the mitral valve was crossed, followed by the aortic valve, and then it was kept just distal to the arch of aorta. A 0.035" wire with a 6-cm floppy J-tip was then introduced into the balloon catheter and positioned preferably just distal to the arch of aorta to get better support and stability. The balloon catheter, along with the Mullins sheath, was removed, and the skin and interatrial septum were dilated with a 14 F dilator. The balloons of the multi-track system were prepared with contrast, and the first multi-track balloon was introduced and positioned across the mitral valve, and then, the second balloon was advanced over the wire and lined up with the first one. The balloons were then inflated simultaneously under fluoroscopic vision once, twice, or more until the disappearance of the socalled waist (which is formed around the inflated balloon by tight mitral stenosis). The balloons were removed, and pressures of the right and left heart were recorded with the help of multi-track angiographic and pigtail catheters. After the achievement of an acceptable transmitral gradient, the system was removed, and hemostasis was secured.
## Data collection and definitions
The study was approved by the hospital research ethics committee. Written informed consent was taken by all eligible patients, and information was collected in the form of a detailed questionnaire, included the following: patient's demographics; prior history of surgery, commissurotomy, or thromboembolic event; electrocardiographic findings; echocardiographic findings before and after the procedure; and hemodynamic and catheterization data.
Transthoracic echocardiography was performed just after and 24-48 hours after the procedure, and all findings were recorded. The same experienced echocardiographer performed all measurements using the parasternal short and long axis and four-chamber views.
Prospectively collected procedure-related complications included: death, pericardial tamponade, thromboembolism, and moderately severe MR. Procedure-related death was defined as in-hospital death that was directly related to PTMC. MR was graded as follows: mild, moderate, moderately severe, and severe. More than mild MR was defined as a central jet of >4 cm 2 or occupying >20% of the LA area on color Doppler echocardiography. Successful PTMC was defined as achieving a post-procedural mitral valve area (MVA) of ≥1.5 cm 2 with no more than mild MR.
# Statistical analysis
Data were entered and analyzed using SPSS, version 17 (SPSS Inc., Chicago, IL, USA). Mean±SD was computed for all quantitative variables. Frequency and percentages were calculated for all categorical variables. Independent sample t-test was applied to compare means of various quantitative variables, like age, weight, and annulus size, etc, whereas chi-square test was applied for the association between relative frequencies of qualitative variables. Binary logistic regression was performed to determine the significant predictive factors for a successful PTMC outcome. A p value of <0.05 was considered significant.
# Results
Out of 258 PTMC procedures, 197 (76.3%) were successful and 61 (23.6%) were unsuccessful. Among unsuccessful procedures, 41 (15.8%) patients did not achieve the required valve area, and 21 (8.1%) patients developed more than mild MR, including those 8 (3.1%) patients who did not achieve the required valve area and had more than mild MR. Tamponade developed in 5 patients (1.9%). One patient died, and two procedures were abandoned. shows the baseline demographic and preprocedural echo and cath features of the successful and unsuccessful procedures of PTMC. A trend of successful PTMC was observed toward taller and heavier patients, but the difference was not significant among the two groups (p=0.05). However, a significant difference was observed between annulus size and preprocedural MVA among the successful and unsuccessful groups.
Similarly, a significant difference in RV pressure was observed among the two groups, and high RV pressure was correlated with unsuccessful procedures. Moreover, moderate to severe TR was frequently observed among unsuccessful procedures (p=0.003). LV dysfunction was also frequently found with unsuccessful procedures (p=0.003). [fig_ref] Table 2: Technical and postprocedural catheterization and echocardiographic features of patients who underwent PTMC [/fig_ref] shows the technical features and postprocedural cath and echo findings of successful and unsuccessful procedures. The PTMC wire was most commonly placed in the ascending aorta and least commonly placed in the LV apex, but this placement did not show any significant difference over the success of the procedure. Regarding balloon sizes, a 14x16-mm balloon set was most successful, although the 14x14-mm balloon set was used most frequently. Similarly, two or more balloon inflations correlated more frequently with successful out-
[formula] Successful Unsuccessful P n=197 (%) n=61 (%) value [/formula]
Age, years (±SD) [bib_ref] Impaired left ventricular systolic function in mitral stenosis, Shikano [/bib_ref] comes. Right ventricular (RV) systolic pressure and mean pressure gradient (MPG) across the mitral valve on cath was significantly high in unsuccessful procedures as compared to successful procedures just after the procedure. Similarly, MVA, MPG, and RV pressure were significantly raised on echocardiography in unsuccessful procedures when performed on Day 1 of the procedure. [fig_ref] Table 3: Univariate analysis of demographic, echocardiographic, and catheterization features of patients who underwent... [/fig_ref] shows the categorical analysis of the PTMC procedure in different groups. The findings strengthened the observation mentioned in . MVA of <0.9 cm 2 and mitral annulus size of ≤33 mm predicted a greater chance of an unsuccessful procedure. Similarly, the higher the RV pressure, the greater the chance was of predicting an unsuccessful procedure. [fig_ref] Table 4: Factors predicting successful outcome of PTMC on multivariate analysis Figure 1 [/fig_ref] shows the multivariate analysis for factors predicting a successful outcome of PTMC. The variables age, gender, annulus size, and preprocedure echocardiographic and catheterization features were included in the model to evaluate if they were significant predictive factors for a successful PTMC outcome. It was found that annulus size, preprocedure MVA, and RV pressure on echo and cath were individually significant predictors, but the multivariate logistic regression showed that only MVA (p=0.030) and RV pressure (p=0.040) were significant. depicts the same relation of higher RV pressure with an unfavorable outcome of the PTMC procedure. Discussion PTMC is considered the mainstay of treatment in patients with MS [bib_ref] Percutaneous balloon valvotomy for patients with severe mitral stenosis, Palacios [/bib_ref] [bib_ref] Indications, complications and short-term clinical outcome of percutaneous transvenous mitral commissurotomy, Nobuyoshi [/bib_ref] [bib_ref] Percutaneous transarterial balloon dilatation of the mitral valve: five year experience, Babic [/bib_ref]. It is a time-tested, safe, and effective treatment [bib_ref] Percutaneous transarterial balloon dilatation of the mitral valve: five year experience, Babic [/bib_ref] [bib_ref] Results of percutaneous mitral commissurotomy in 200 patients, Vahanian [/bib_ref] [bib_ref] Long-term results of percutaneous mitral valvuloplasty with the Inoue balloon catheter, Chen [/bib_ref]. Despite the careful selection of patients using Wilkins score and selecting patients with a Wilkins score of <8, PTMC carries a considerable failure rate [bib_ref] Comparison of immediate and long-term results of mitral balloon valvotomy with the..., Leon [/bib_ref] [bib_ref] Which patients benefit from percutaneous mitral balloon valvuloplasty? Prevalvuloplasty and postvalvuloplsty variables..., Palacios [/bib_ref]. This led us to search for other predictors of successful PTMC.
Bonhoeffer et al. [bib_ref] Percutaneous mitral valve dilatation with the multi-track system, Bonhoeffer [/bib_ref] have described the use of a multitrack system, which is actually a refinement of the double-balloon technique. This technique is one of the two main techniques that are currently used. The other one is the Inoue balloon technique, which was first described in 1984, and is claimed to be the most commonly used technique [bib_ref] Clinical application of intravenous mitral commissurotomy by a new balloon catheter, Inoue [/bib_ref]. However, in our institute, the Bonhoeffer (Multi-track) double-balloon technique is used in the majority of cases. To the best of our knowledge, this is the largest dataset showing the experience of the Bonheoffer technique for PTMC procedures in our part of the world. Secondly, this is the first study showing the predictors of the outcome in PTMC procedures other than Wilkins score.
Wilkins score concentrates on mitral valve apparatus and does not consider patient demographics and technical and echocardiographic features, including other systems, like pulmonary artery hypertension [bib_ref] Prediction of successful outcome in 130 patients undergoing percutaneous balloon mitral valvotomy, Abascal [/bib_ref]. According to our findings, there are several other features that may help us in predicting the outcome of the procedure. However, these findings need discussion.
Among demographic features, one important finding is the tendency of a successful PTMC in patients with increased BSA. It is an established fact that the annular dimensions of the mitral valve increase correspondingly with body surface area [bib_ref] The study of mitral valve annular dimension in relation to the body..., Rajila Rajendran [/bib_ref]. Although some other mitral valve structures, like interpapillary muscle and papillary muscle annular distance, have the same correlation, MV annular area shows the highest correlation with BSA [bib_ref] The study of mitral valve annular dimension in relation to the body..., Rajila Rajendran [/bib_ref] [bib_ref] Age and body surface area dependency of mitral valve and papillary apparatus..., Sonne [/bib_ref]. Hence, the mitral valve shows a steady rise in its diameter with a rise in body surface area [bib_ref] The study of mitral valve annular dimension in relation to the body..., Rajila Rajendran [/bib_ref]. This is probably the reason for the tendency of our operator to use a more aggressive approach-i.e., use of a bigger balloon and multiple inflations-in patients with increased BSA to get the optimum result; hence, they got better results.
Among echocardiographic features, we observed in our data that patients with a smaller annular size and valve area showed less favorable results [fig_ref] Table 3: Univariate analysis of demographic, echocardiographic, and catheterization features of patients who underwent... [/fig_ref]. Mitral annular calcification and valvular size have been reported recently as factors predicting suboptimal outcomes [bib_ref] Mitral annular calcification predicts immediate results of percutaneous transvenous mitral commissurotomy, Salarifar [/bib_ref] [bib_ref] Acute results of mitral balloon valvuloplasty, Korkmaz [/bib_ref]. It is a known fact that MVA in rheumatic heart disease (RHD) reduces by 0.1 mm 2 /year. Therefore, a smaller MVA and annular size reflects more chronic disease and, understandably, more complex histopathological changes in mitral valve apparatus, including subendothelial collagen fibrosis and calcinations [bib_ref] Heart valves in rheumatic heart disease in the light of scanning electron..., Krymskii [/bib_ref]. This apparently requires a bigger balloon with multiple dilations to get the favorable outcome. In contrast, our operators used a less aggressive approach, and (in general) they used an under-sized balloon in patients with a smaller annular size as a bid to prevent MR. Had they used a bigger balloon, would it have made the outcome better? It was not in our scope to answer this question, and study did not have sufficient power to settle this issue. However, we observed in our study that a bigger set of balloons is more successful than a smaller set of balloons [fig_ref] Table 2: Technical and postprocedural catheterization and echocardiographic features of patients who underwent PTMC [/fig_ref].
Similarly, increased pulmonary artery and right heart pressures are also a reflection of chronic disease and poor medical management [bib_ref] Relation between pulmonary artery pressure and mitral stenosis severity in patients undergoing..., Otto [/bib_ref]. This is understandable in our setting, in which patients from far-flung rural areas do not have access to better medical facilities, and they are left untreated for a longer period of time until they become symptomatic [bib_ref] Cardiac valvular lesions in patients with rheumatic heart disease, Shaikh [/bib_ref] [bib_ref] Status of rheumatic heart disease in rural Pakistan, Rizvi [/bib_ref]. Usually, they present with extensive disease, and obviously, their outcome is not as good as in those patients with better right heart hemodynamics. This is the fact that we observed and documented in our data. In fact, we observed that pre-procedural RV pressure on echocardiography and right heart catheterization had a strong relation with the outcome of the procedure [fig_ref] Table 3: Univariate analysis of demographic, echocardiographic, and catheterization features of patients who underwent... [/fig_ref]. The higher the RV pressure was, the poorer the outcome was in our study .
Left ventricular dysfunction is another echocardiographic finding that predicted a less favorable outcome in our study . Although it lost its significance in the multivariate analysis, it can be said that it may have an impact on outcomes, keeping in mind the fact that LV dysfunction is not an uncommon association with mitral stenosis [bib_ref] Impaired left ventricular systolic function in mitral stenosis, Shikano [/bib_ref]. In addition to that, the infarct shows a severe form of disease and further complicates the disease with atrial fibrillation and thrombus formation, which is, again, more commonly seen with mitral stenosis associated with LV dysfunction [bib_ref] Impaired left ventricular systolic function in mitral stenosis, Shikano [/bib_ref] [bib_ref] Severe mitral stenosis with atrial fibrillation -a harbinger of thromboembolism, Farman [/bib_ref]. Hence, with a worse LV geometry and deformed subvalvular apparatus, the outcome of PTMC understandably could not be as favorable as observed with normal LV function.
One surprising technical finding that we observed was the negative relation of the wire positioning on the outcome of the procedure. Ideally, the PTMC wire should be parked in the arch of aorta. It is believed that this position gives better support and alignment to the balloon and makes the procedure easier and quicker. Equivalent results, irrespective of the place of the wire positioning, may be related to our experienced operators in high-volume center, leading to better balloon control.
It may be argued that the overall success rate of the PTMC procedures, ~77% in our study, is not up to the mark. Although the success rates of PTMC procedures in other landmark studies are comparable [bib_ref] Comparison of immediate and long-term results of mitral balloon valvotomy with the..., Leon [/bib_ref] [bib_ref] Which patients benefit from percutaneous mitral balloon valvuloplasty? Prevalvuloplasty and postvalvuloplsty variables..., Palacios [/bib_ref] , it should be noted that the postprocedural mean MVA of unsuccessful procedures was 1.4 cm 2 [fig_ref] Table 2: Technical and postprocedural catheterization and echocardiographic features of patients who underwent PTMC [/fig_ref] , whereas it was significantly smaller (0.8 cm 2 ) before the procedure as compared to successful procedures (0.9 cm 2 ). Therefore, though technically speaking, these procedures were unsuccessful, practically most of these patients showed remarkable clinical improvement. It is probably due to the achievement of more than 50% of the valve area from the baseline value in most of these so-called unsuccessful proce-dures. Secondly, in ~6% of cases, our operators used the singleballoon technique. These were the high-risk and/or pregnant patients in whom the operator wanted to make the procedure simpler and quicker. Hence, they used a single balloon in these patients, despite knowing the suboptimal outcome of this technique [bib_ref] Percutaneous catheter commissurotomy in rheumatic mitral stenosis, Lock [/bib_ref] [bib_ref] Balloon angioplasty for congenital and rheumatic mitral stenosis, Kveselis [/bib_ref]. Therefore, not surprisingly, the single-balloon technique was found inferior to the double-balloon technique in our study, and it further validates what is already reported in the literature [bib_ref] Percutaneous catheter commissurotomy in rheumatic mitral stenosis, Lock [/bib_ref] [bib_ref] Balloon angioplasty for congenital and rheumatic mitral stenosis, Kveselis [/bib_ref].
What are the implications of our findings? Our study suggests that there is a need to look for other demographic, echo, and technique-related predictors of a successful PTMC rather than just sticking to only mitral valve apparatus-related predictors. It also highlights the need for a modified Wilkins score that includes other parameters, like BSA, pulmonary artery pressure, and LV dysfunction.
# Study limitations
As the selection of balloon size was absolutely at the discretion of the operators, they did not use any uniform criteria to select the balloon size. Some of our operators used BSA, and others used annular size for the selection of the balloon set. Therefore, the results may not be standardized. Secondly, echocardiography was done just after the procedure, and it was repeated 24-48 hours after the procedure. Most of our patients belonged to far-flung rural areas, and they were followed up in their local hospital. Therefore, we could not record their followup echo findings and did not know if there was any long-term impact on the results.
# Conclusion
Besides Wilkins score, increased body surface area, bigger preprocedure mitral valve annulus size and mitral valve area, and better left and right ventricular hemodynamics are significantly correlated with successful PTMC. Our study urges the need for percutaneous intervention before the worsening of annulus size and pulmonary artery pressure, along with a suitable mitral valve apparatus. It further suggests the development of a modified Wilkins score incorporating other predictors of successful PTMC.
[table] Table 2: Technical and postprocedural catheterization and echocardiographic features of patients who underwent PTMC [/table]
[table] Table 3: Univariate analysis of demographic, echocardiographic, and catheterization features of patients who underwent PTMC [/table]
[table] Table 4: Factors predicting successful outcome of PTMC on multivariate analysis Figure 1. Trend of successful PTMC outcome with preprocedure right ventricular pressure. Note that success rate decreases as RVP increases [/table]
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Breast awareness mobile apps for health education and promotion for breast cancer
# Introduction
Breast cancer (BC) is the most common cancer globally [bib_ref] Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for..., Sung [/bib_ref]. It accounts for about 30% of female cancers and a mortalityto-incidence ratio of 15% as women's first oncological cause of death [bib_ref] Cancer statistics, Siegel [/bib_ref]. Similarly, BC was the most often diagnosed malignancy among Malaysian women from 2007 to 2011, accounting for roughly 32.1% of all cases. According to the National Cancer Institute, the 5-years survival rate for BC in Malaysia is around 87.5% for stage I, 80.7% for stage II, 59.7% for stage III, and 23.3% for stage IV from 2007 to 2016 [bib_ref] National cancer registry department national cancer institute ministry of health. In: Malaysian..., Myscan [/bib_ref].
Screening for BC is available in Malaysia, and diagnosing the disease sooner and treating it in its early stages is feasible. Many measures have been launched in Malaysia to prevent BC mortality and morbidity by raising awareness through campaigns and screening programmes offered by government institutions. BC awareness month, often known as "Pink October, " has aided in raising awareness among women. However, the number of women who regularly get screened is far from satisfactory. A lack of knowledge of the various cancer screening methods, cultural attitudes, and a lack of encouragement by family members and doctors are the major reasons for the poor response to screening. Despite being an upper-middle-income nation with a robust healthcare system and effective socioeconomic initiatives, Malaysia's cancer survival rates are lower than the global average. The rate is attributable to several factors, including poor cancer awareness and screening rates, delays in seeking medical help, delays in detection and diagnosis, and insufficient access to highquality care. These barriers are particularly obvious for people who live in rural areas because cancer centers are typically located near major cities [bib_ref] Cancer in my community: addressing increasing cancer cases in Malaysia, Mastura [/bib_ref]. Therefore, Malaysia's awareness program for BC and health promotion and education needs to be reinforced.
As the use of mobile phones grows, so does the need for mobile phone apps. These applications might be used for various purposes, including social engagement, education, entertainment, and personal health. Mobile applications have positively impacted health-related behaviors and clinical health outcomes. Application users were more satisfied with using mobile health applications to manage their health than users of conventional care [bib_ref] 6. Nasution A, Yusuf A, Keng SL, Rasudin NSP, Iskandar YH, Ab..., Han [/bib_ref]. A mobile app for the women population at risk in Malaysia was developed as a new tool for BC's health education and promotion. This study has shown its usability (6). However, we do not know whether this mobile app can improve users' knowledge, attitudes, and behavioral changes on BC. Therefore, this study aimed to assess the efficacy of the BrAware mobile app in improving the knowledge of risk factors of BC, warning signs awareness and confidence level for BSE among women aged 18 years and older who are the population at risk for the disease in northeast peninsular Malaysia.
# Methods
## Research design and study participants
A quasi-experimental pre and post-test research design were conducted among women in Kelantan, Malaysia, to measure the efficacy of a mobile application (BrAware) in increasing the knowledge of BC risk factors, awareness of warning signs, and confidence in breast self-examination (BSE). The state of Kelantan is largely rural, and its culture is quite distinct from other Malaysian states (7). The inclusion criteria were women aged 18 years and above who owned a smartphone and had never been diagnosed with BC. No age range was specified because the risk of BC increases with age. In addition, participants were excluded if their self-reported mobile app literacy was low and the phone did not run appropriately after downloading the mobile application. Low mobile app literacy is the inability to find, use, understand and evaluate the apps [bib_ref] Content, usability, and utilization of plain language in breast cancer mobile phone..., Ginossar [/bib_ref]. A non-probability sampling method using social media such as Facebook and WhatsApp group were used to recruit public women living in Kelantan. The sampling technique was considered because it was more cost-effective and time-effective than probability sampling, and it was also impossible to do a probability sampling (9).
## Instrument
The questionnaire consisted of two sections: sociodemographic and BC awareness. Sociodemographic variables included age, occupation, monthly household income, ethnicity, race, highest education level, BC family history, trained BSE, and period to seek medical help if there was a change in the breast). The Breast Cancer Awareness Measure (B-CAM) and B-CAM-M (M for Malay language) were validated in the United Kingdom (10), and Malaysia (11) were adapted. The content and response format were modified to make it more culturally relevant for the local women. Of the seven domains of the B-CAM, three domains were included: Knowledge of risk factors, awareness of warning signs, and BSE. Nineteen items were adapted for BC awareness (9 items on knowledge of risk factors, eight items on awareness of warning signs and two items on confidence in BSE). A 5-point Likert scale was used: strongly agree, agree, neutral, disagree, and strongly disagree. The total score for each domain was summated into a percentage score. The mean percentage score of BC awareness before and after using the BrAware App was computed and compared. Findings showed that the BC awareness Cronbach alphas coefficient was 0.89, indicating good internal consistency [bib_ref] The use of cronbach's alpha when developing and reporting research instruments in..., Taber [/bib_ref]. The instruments were pilot tested on ten women similar to the samples but not included in the final sample. No changes were made to the instruments.
. /fpubh. .
## Procedure
Using the Malay language, data was collected online using the Google Forms platform between October 1, 2021, and December 1, 2021. This study applied various strategies to maintain social distance and observe the Movement Control Order. These include relying on the researchers' professional and personal networks to publicize and disseminate the advertisement via posters and social media like Facebook and WhatsApp and sharing the poster through email. An introduction statement and instructions for completing the pre and post-test online and a link for participants to download the BrAware App are included in the information. Participants were expected to become familiar with the BrAware App. The researchers would remind the subjects to complete a post-test 2 months after the pre-test date through Whats App text message or phone call. Instructions for completing the questionnaire online were also supplied by clicking the "Continue" button. Participants were given a choice to respond to the survey using the "Yes/No" option to confirm their willingness to participate. The participant was instructed to finish the online questionnaire after receiving confirmation to continue "Yes, " whilst "No" indicates a refusal to participate in the survey. To avoid duplicated or exaggerated data, participants were limited to one response. The survey took ∼10-15 min to complete. For the post-test, the same instrument was employed. Therefore, the data set only included participants who completed both the pre-test and post-test.
## Braware app
The mobile app, BrAware, from the abbreviation of Breast Awareness, was developed based on the Android platform by the researchers (6). The app was user-friendly, constructed with simple point form sentences, including a share button, infographic images or video, dual language capabilities (English and Malay language), a Google Map navigator and a reminder function. BrAware App content includes information about breast anatomy, BC, risk factors of BC, treatment modalities, BSE, screening examination, doctor examination, survival rate, finding support group, hotline number, screening reminders and myths and facts based on Malaysian cultural beliefs.
# Statistical analysis
Collected data were coded and analyzed using IBM Statistical Package for Social Sciences (SPSS) (version 26, IBM Corp., Armonk, NY). Descriptive data were used to describe the characteristics of sociodemographic variables. The test score is presented as means ± standard deviation (SD) and analyzed by a paired t-test. P < 0.05 is considered statistically significant.
# Results
A total of 41 women completed the online survey questionnaire. Sociodemographic characteristics are shown in . The mean age was 39.71 (SD = 8.80). The ethnic distribution of participants was 95.1% Malay while the remainder, 4.8%, was non-Malay. More than half (51.2%) of the participants obtain a secondary education. Most of the participants (78%) were married women. The mean household income of participants was 5,582.93 (SD = 8,158.21), and 9.8% had a BC family history, whilst 24.4% were not trained in BSE. The mean period to seek medical help if found a change in the breast was 6.29 (SD = 10.72).
Results of the paired t-test show that the mean knowledge score of BC warning signs differs before using BrAware (mean 70.62, SD 11.74) and after using BrAware (mean 79.83, SD 10.15) at the <0.001 level of significance. On average, knowledge of BC warning signs was about 9.21 points higher after using BrAware. However, for BSE knowledge, the mean score before and after using BrAware was increased to 9.75 (p = 0.007). In addition, the mean knowledge of risk factors for BC changed before (mean 65.79, SD 14.63) and after (mean 77.07, SD 16.57) using BrAware at the 0.005 level of significance. Therefore, this implies that it makes sense to conclude that the intervention might be responsible for improving knowledge of BC risk factors, awareness of warning signs, and confidence in breast self-examination (BSE) among the participants .
# Discussion
The pre and post-tests show an improvement in knowledge of BC among the women population after using the BrAware app. As Nasution et al. (6) mentioned, it is best to approach their knowledge and awareness to change human behavior and promote early BC detection in the community (6). Therefore, the BrAware app can be considered feasible in a real clinical context to promote behavioral changes in the lifestyles of women in performing BSE and screening. The plausible explanation was the regular practice of correct skills while using the app may increase the users' memorisation [bib_ref] Effects of smartphone application education combined with hands-on practice in breast self-examination..., Kang [/bib_ref]. Most of the participants who had secondary-level education would easily understand simple point-form sentences in the app. This study highlights the importance of mobile apps based on how people's health and well-being can be improved via monitoring [bib_ref] A mobile system to improve quality of life via energy balance in..., Lozano-Lozano [/bib_ref]. This result matches those observed in a study in Iran that implemented the smartphone app, which improved BSE practice and even reported abnormal findings among participants by mass palpation or visually inspected nipple retraction [bib_ref] The effect of a smartphone application on women's performance and health beliefs..., Shakery [/bib_ref]. According to the Health Belief Model (HBM), an individual's opinion that she is vulnerable to BC, the severity of BC, and the benefits, as well as a barrier to preventative action such as doing the BSE and screening at the hospital, all influence health-seeking behavior toward BC prevention (6). In addition, to our knowledge, no BC awareness support apps are targeting Malaysian women.
A combination of dual language strategies of knowledge dissemination will help facilitate the knowledge transfer to the user as most apps do not provide educational content in the local language. The Malay language is used widely in ASEAN as the official language of Indonesia, Brunei, Singapore and Malaysia, and a lingua franca spoken by communities in southern Thailand, southern Philippines, and parts of Myanmar and Cambodia (17). Therefore, the BrAware app can be a user-friendly tool for the Malaysian community and ASEAN countries on BC. This app provides credible information on breast anatomy, BC, risk factors of BC, treatment modalities, BSE, screening examination, doctor examination, survival rate, finding support group, hotline number, screening reminders and myths and facts based on Malaysian cultural beliefs. The credibility of the BrAware app content is supported by the source of reference info on each page.
Meanwhile, another study promotes the app's credibility by enabling direct communication with the therapist [bib_ref] The effect of a smartphone application on women's performance and health beliefs..., Shakery [/bib_ref]. Furthermore, the reminder feature notifications as cues in the app improve user engagement and BSE routine in a time duration set according to the user's menstrual cycle. Many studies have supported mobile apps in improving the user's or patient's knowledge and awareness of the diseases (6). A study in China suggested that using a mobile app could improve the user's experience, especially on the accessibility to health information, leading to positive health outcomes [bib_ref] Effectiveness of the colorapp mobile app for health education and promotion for..., Lu [/bib_ref]. Another time-intervention effect study involving 50 years and older group population in Kedah, Malaysia, showed a significantly improved overall knowledge among participants in the intervention group compared with the control group about colorectal cancer (20). Therefore, this study shows that the BrAware app can be fully used to deliver health education and promotion to intended users and enhance the effect of education to change people's behavior.
In the present advancement in communication and digital technology, it can be concluded that the BrAware app is a way forward for health promotion and education, particularly in preventing and early detection of BC. However, there are potential limitations of this study that should be noted. First, the results may not be generalisable to all women in Malaysia as only one state was selected. Therefore, the participants need to be expanded to urbanized women in Malaysia. Also, since this study evaluated the outcomes after 2 months, it is not certain what the knowledge of BC awareness retention is and how long it will be retained. Therefore, we will need to evaluate the app's effectiveness over the long term in the future.
# Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
# Ethics statement
The studies involving human participants were reviewed and approved by Human Research Ethics Committee (HREC) of Universiti Sains Malaysia (USM/JEPeM/18080380). 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
AY, YP, IA, and SL contributed to the conception and design of the study. AY and AN implemented the methods and execution of the study. AY performed the statistical analysis. AY, AN, and SL wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.
# Funding
This work was supported by the USM short-term grant (STG/304.PPSK.6315141).
[table] TABLE Socio -: demographic characteristics of participants (n = ). [/table]
[table] TABLE Breast cancer: awareness (n = ). [/table]
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Micturition in the toilet compared with bedpan in laboring Nulliparas: a randomized controlled trial
Background: Bladder overdistension in labor may lead to prolonged postpartum urinary retention. We hypothesized that nulliparas mobilizing to toilet is more likely to achieve satisfactory micturition.Methods:One hundred sixteen (58 in each arm) term nulliparas in labor with filled bladders were randomized to mobilizing to the toilet or using bedpan to micturate. Primary outcome was satisfactory micturition defined as ultrasound derived post-void bladder volume < 150 ml. Following unsatisfactory micturition, participants crossover to the opposed intervention. Participants were catheterized if after crossover, residual bladder volume was ≥250 ml.Results: Satisfactory micturition rates were 55/58 (95%) vs. 43/58 (74%) RR 1.28 95%CI 1.08-1.51 NNT b 4.8 95%CI 3.0-12.4 P = 0.008, failure to micturate 1/58 (2%) vs. 8/58 (14%) RR 0.13 95%CI 0.02-0.97 NNT b 8.3 95%CI 4.6-38.7 P = 0.047. After cross over following unsatisfactory bladder voiding, satisfactory micturition rates were 0/3 (0%) vs 13/15 (87%) P = 0.024, bladder catheterization rates were 3/58 (5%) vs. 2/58 (4%) RR 95%CI 1.5 (0.26-8.65) P = 0.648, maternal satisfaction with allocated intervention 55/58 (95%) vs. 9/58 (16%) RR 95%CI 6.1 (3.3-11.2) NNT b 95%CI 1.3 (1.1-1.5) P < 0.0001 and preference for mobilizing to the toilet if micturition was needed again during labor 55/58 (95%) vs. 53/58 (92%) for mobilizing to the toilet compared to bedpan use arms respectively. Labor and neonatal outcomes were similar.Conclusion: Satisfactory micturition was more frequently achieved with mobilization to the toilet than bedpan use. Women in both arms overwhelmingly prefer to mobilize to the toilet to urinate.
birth expected within 2 hours of pushing. Nulliparity and prolonged labor particularly in the second stage are well-established risk factors for postpartum voiding dysfunction [bib_ref] The risk factors of postpartum urinary retention after vaginal delivery: a systematic..., Li [/bib_ref] [bib_ref] Postpartum urinary retention: absolute risk prediction model, Barba [/bib_ref].
Nulliparas due to their tendency for labor dystocia, often need oxytocin augmentation [bib_ref] Labor dystocia in nulliparous women, Lefevre [/bib_ref] with concomitant continuous cardiotocography, intravenous hydration [bib_ref] Intravenous fluid rate for reduction of cesarean delivery rate in nulliparous women:..., Ehsanipoor [/bib_ref] and neuraxial analgesia as their labor pain is perceived to be more intense [bib_ref] The pain of labour, Labor [/bib_ref] ; hence are often connected to multiple infusion and monitoring devices limiting mobility during labor. Care providers maybe wary of interrupting monitoring and treatment, and of inadvertent birth into the toilet when mobilizing laboring women to micturate in the toilet. In our busy publicly funded university hospital, bedpan use or even catheterization is increasingly seen as time and labor-saving for intrapartum bladder care. The UK National Institute of Clinical Excellence (NICE) guidance recommends recording frequency of passing urine during first stage of labor without further elaboration on bladder voiding management.
Women's toileting behavior related to micturition can be defined as voluntary actions related to the physiological event of emptying the bladder comprising specific attributes of voiding place, voiding time, voiding position and voiding style influenced also by the physical and social environments [bib_ref] Women's toileting behaviour related to urinary elimination: concept analysis, Wang [/bib_ref]. For these reasons, it is postulated that micturition in the toilet is more likely to be satisfactorily performed compared to with bedpan use for nulliparas at high risk of in-labor voiding dysfunction. We sought to evaluate this hypothesis in a randomized trial.
# Methods
This trial was approved by the Medical Ethics Committee of University Malaya Medical Centre (reference number: 2019330-7272 on 04/07/2019) and registered with ISRCTN (https:// doi. org/ 10. 1186/ ISRCT N1778 7339 on 17/07/2019). The trial was performed in accordance with International Conference on Harmonization -Guidelines for Good Clinical Practice (ICH-GCP) and Declaration of Helsinki. Informed consents were obtained from all participants.
We sought in our labor ward, nulliparas (no prior delivery beyond 20 weeks gestation) in labor (at least three contractions in 10 minutes, cervical dilatation within the last 2 hours of 4 to 8 cm and ruptured membrane but without evidence of second stage) who had the urge to micturate or a palpable bladder and ultrasound scan bladder volume > 300 ml, aged > 18 years old with a singleton live fetus in cephalic presentation for trial participation. An adult functional bladder can comfortably hold between 300 and 400 ml [bib_ref] A healthy bladder: a consensus statement, Lukacz [/bib_ref]. Wall pressure of 5-15 mmHg creates a sensation of bladder fullness while 30 mmHg and beyond is painful [bib_ref] Neural sensing of organ volume, Umans [/bib_ref]. For this trial a filled bladder that warrants voiding is defined as the urge to micturate or a palpable bladder and ultrasound bladder volume > 300 ml [bib_ref] Intrapartum ultrasound estimation of total bladder volume, Gyampoh [/bib_ref]. Women with known bladder dysfunction, recurrent antenatal urinary tract infections, lower segment uterine fibroid, on neuraxial analgesia and prior bedpan or urine catheter use during labor were excluded. Eligible women were approached, verbally informed and provided with the participant information sheet by the investigator (WKC). Written informed consents were obtained from all participants. Relevant demographic and clinical data were recorded in the Case Report Form.
Pre and postvoid ultrasound for bladder volume was performed (solely by investigator WKC) using a 3.5 MHz convex transducer with the participant in semirecumbent position. Bladder measurements were performed in two planes (transverse and sagittal images) in between contractions. For transverse image, the transducer is positioned horizontally along the long axis of the bladder, and adjusted until the maximum transverse section is found and the maximum transverse width measured (horizontal diameter). For the sagittal image, the transducer is rotated 90-degree into the sagittal plane, position adjusted until the maximum sagittal section is obtained and sagittal depth and height were then measured. Sonographic measurements were recorded for sagittal height, sagittal depth and transverse width of the bladder (i.e., h, d and w). Using the prolate ellipsoid method [bib_ref] Measurements of urinary bladder volume: comparison of five ultrasound calculation methods in..., Dicuio [/bib_ref] , volume = 0.52 x h x d x w [bib_ref] Can we replace the catheter when evaluating urinary residuals?, Araklitis [/bib_ref].
Prior to randomization women were asked on their preferred method to urinate using bedpan, mobilize to toilet, catheterization or no preference. Participants were randomized to micturition by mobilizing to toilet or using bedpan by opening the lowest numbered, sealed and opaque envelope that remained. The randomization sequence was computer generated in blocks of 4 or 8 using random. org by investigator (PCT) who was not involved with recruitment.
Participants randomized to mobilizing to toilet were disconnected from medical devices and can choose to go to the toilet with a companion (birth partner or the midwife) or by themselves with the door closed, for up to 10 min to micturate on a standard sitting toilet. The voided urine was collected into a disposable plastic bag that was hold firmly under the toilet lid. The voided urine volume was measured after decanting to a measuring jug. Participants in the bedpan arm was provided with the bedpan and be in any position that was comfortable to her (lying or sitting on the bed) for up to 10 min to micturate. The voided volume was measured after decanting to a measuring jug.
Primary outcome of satisfactory micturition is defined as residual urine volume < 150 ml by ultrasound. A residual bladder volume ≥ 150 ml typically defines covert postpartum urinary retention [bib_ref] Delivery-related risk factors for covert postpartum urinary retention after vaginal delivery, Mulder [/bib_ref] [bib_ref] Postpartum voiding dysfunction: identifying the risk factors, Buchanan [/bib_ref]. Participants who were not able to micturate, or if their residual urine volume ≥ 150 ml, were asked to cross-over to the opposite intervention for another attempt. If after attempting both methods and residual urine volume was > 250 ml, a standard "in and out" bladder catheterization was offered and performed under an aseptic technique.
Immediately after completion of their micturition attempt, participants provided a 5-grade Likert's scale response of their satisfaction with their allocated intervention and stated their preferred bladder voiding method to "if you have to pass urine again during labor, what would you prefer?". Labor and neonatal outcomes were retrieved from participants' charts. Prespecified secondary outcomes were in-out catheterization, duration of the second stage of labor, mode of delivery, estimated delivery blood loss and maternal satisfaction after allocated intervention prior to any crossover.
Participants, investigator (WKC) who performed the ultrasound and care providers were not masked to the interventions as it was impractical due to its obvious nature.
As there was no direct pilot data available, sample size calculation was based on in-out catheterization rates of 19% vs 54% in recovery room among postoperative patients (early mobilization vs bedpan) [bib_ref] The number of in-out catheterisations is reduced by mobilising the postoperative patient..., Hansen [/bib_ref] as surrogate outcome to represent unsatisfactory micturition of this study. We applied a more conservative difference of 25% vs 50%, alpha 0.05, power 80%, 1:1 randomization and Chi-square test, 58 participants were needed in each arm (N = 116) using the PS: Power and Sample Size Calculation [bib_ref] Power and sample size calculations. A review and computer program, Dupont [/bib_ref].
Data were entered into SPSS (Version 23, IBM, SPSS Statistics). Student t test was applied for means with normally distributed continuous data, Mann-Whitney U test for non-normally distributed data or ordinal data, Chi-square test for categorical data across trial arms and paired t test for continuous data within trial arms. Twosided P values were reported and p < 0.05 was regarded as significant. Analysis was based on intention-to-treat. [fig_ref] Figure 1: Recruitment flow chart into a randomized trial of mobilization to the toilet... [/fig_ref] depicts the recruitment flow: 156 potentially eligible women were approached, 17 declined participation, and 23 women refused to be randomized, wanting mobilization to the toilet. Recruitment stopped on reaching target sample size of 116 (58 randomized to each arm). [fig_ref] Table 1: Characteristics of trial participants randomized to mobilization to the toilet compared with... [/fig_ref] shows trial participants' characteristics: prevoid bladder volume, contraction frequency, cervical dilatation and bladder voiding preferences in particular were similar across trial arms. [fig_ref] Table 2: Primary outcome and other bladder voiding outcomes after randomization to mobilization to... [/fig_ref] reports the primary outcome and other micturition related outcomes. Primary outcome of satisfactory micturition was 55/58 (95%) vs. 43/58 (74%) RR 95%CI 1.28 (1.08-1.51) NNT b 95%CI 4.8 (3.0-12.4) P = 0.008 and prespecified secondary outcome bladder catheterization 3/58 (5%) vs. 2/58 (4%) RR 95%CI 1.5 (0.26-8.65) P = 0.648 for toilet vs. bedpan respectively. Other (post hoc) significant secondary outcomes of failure to micturate (0 ml voided) were 1/58 (2%) vs. 8/58 (14%) RR 95%CI 0.13 (0.02-0.97) NNT b 95%CI 8.3 (4.6-38.7) P = 0.047, and after cross-over, successful micturition was 0/3 (0%) vs. 13/15 (87%) P = 0.024 for toilet vs. bedpan respectively. [fig_ref] Table 3: Secondary outcome after randomization to mobilization to the toilet compared with bedpan... [/fig_ref] shows maternal and neonatal secondary outcomes. Prespecified secondary outcomes of maternal satisfaction with allocated intervention was 55/58 (95%) vs. 9/58 (16%) RR 95%CI 6.1 (3.3-11.2) NNT b 95%CI 1.3 (1.1-1.5) P < 0.0001, heavily favored mobilization to the toilet arm, but duration of the second stage of labor, mode of delivery and estimated delivery blood loss were not different. When asked to state a preference if they need to micturate in labor again, 55/58 (95%) mobilizing arm vs. 53/58 (92%) bedpan arm P = 0.554 responded overwhelmingly and uniformly in favor of mobilization to the toilet in both trial arms. Neonatal outcomes were similar.
# Results
# Post-hoc analysis
We sought to evaluate a) difference in ultrasound derived mean voided volume (calculated from pre and post ultrasound volumes) and the measured actual voided volume using paired t test for the entire trial population and separately for within trial arms and; b) the accuracy of ultrasound (US) over measured actual voided volume with the use of the ratio of ultrasound derived voided volume to measured actual voided volume (Supplementary [fig_ref] Table 1: Characteristics of trial participants randomized to mobilization to the toilet compared with... [/fig_ref]. US derived voided volume vs. measured volume were, mean ± standard deviation 364 ± 178 vs. 356 ± 185 ml P = 0.151 (entire trial population), 406 ± 138 vs. 390 ± 143 ml P = 0.058 (mobilize to toilet) and 322 ± 202 vs. 321 ± 215 ml P = 0.946 (bedpan); no significant difference between ultrasound derived to measured voided volumes in all three analyses indicating that ultrasound derived parameters are accurate and closely comparable to measured volumes. With accuracy defined as concordance within 20% range of ultrasound to measured actual voided volume in participants who managed to void (any volume, excluding complete overt retention), the accuracy rate by ultrasound 42/56 (75%) vs. 37/48 (77%) RR 95%CI 0.89 (0.36-2.20) P = 0.805 for toilet and bedpan arms respectively with no indication of operator bias across trial arms.
# Discussion
Nulliparas in labor randomized to mobilizing to the toilet were more likely to micturate satisfactorily (NNT b 4.8) and less likely to have overt urinary retention (NNT b 8.3). Amongst women who had overt urinary retention or complete failure to void with their allocated intervention, 13/15 (87%) who cross-over to the toilet after failure with the bedpan managed to micturate satisfactorily whilst no participant 0/3 (0%) who cross-over to the bedpan after failure in the toilet managed to micturate satisfactorily. However, after cross-over was permitted, the need for catheterization was low (toilet 5% vs. bedpan 4%) and not significantly different across trial arms based on original intentionto-treat analysis. A PubMed search (https:// pubmed. ncbi. nlm. nih. gov/) was performed on April 6 2021 using search terms "toilet bedpan labor trial" retrieved only a solitary trial report [bib_ref] Walking reduces the post-void residual volume in parturients with epidural analgesia for..., Weiniger [/bib_ref]. In that report, parturients with epidural analgesia for labor who managed the walk to the toilet, more managed to void, fewer required urinary bladder catheterization in labor and their postvoid residual volume was significantly lower. However, 59% of the walk to toilet arm were unable to walk and actually voided in a bedpan [bib_ref] Walking reduces the post-void residual volume in parturients with epidural analgesia for..., Weiniger [/bib_ref]. These findings [bib_ref] Walking reduces the post-void residual volume in parturients with epidural analgesia for..., Weiniger [/bib_ref] concur with our findings of a better micturition performance in the privacy of the toilet amongst nulliparas in labor without neuraxial analgesia. Participants in mobilization to the toilet arm were more likely to report satisfaction (NNT b 1.3) and 95% would prefer to mobilize to the toilet to urinate if they needed to do so again. In women randomized to bedpan, although 74% achieved satisfactory micturition with bedpan, 91% would still prefer to mobilize to toilet to micturate, which suggested that the preference for the toilet was not motivated by a bad micturition performance with bedpan, but a positive personal choice and a near universal one at that. The bedpan is a subject of everyday nursing practice but little research was found concerning this issue; its use has been described by patients as uncomfortable, embarrassing [bib_ref] Frequency of using the bedpan in acute care, Saxer [/bib_ref] [bib_ref] Patient experience with bedpans in acute care: a cross-sectional study, Gattinger [/bib_ref] and creating a feeling of dependency [bib_ref] Patient experience with bedpans in acute care: a cross-sectional study, Gattinger [/bib_ref] which might have contributed to our satisfaction and preference findings in the disfavor of the bedpan.
Our primary outcome was predicated on ultrasound derived post-void residual volume which was calculated using the prolate ellipsoid method [bib_ref] Measurements of urinary bladder volume: comparison of five ultrasound calculation methods in..., Dicuio [/bib_ref] We used the proportionality constant of 0.52 in our volume formula as it correlated best [bib_ref] Measurements of urinary bladder volume: comparison of five ultrasound calculation methods in..., Dicuio [/bib_ref] [bib_ref] Can we replace the catheter when evaluating urinary residuals?, Araklitis [/bib_ref] with measured volumes. Our post hoc analyses also showed that our ultrasound derived voided volumes were very similar to the measured actual voided volumes. There was also no indication of an operator bias in ultrasound measurements across trial arms (Supplementary [fig_ref] Table 1: Characteristics of trial participants randomized to mobilization to the toilet compared with... [/fig_ref].
The overall urinary catheterization rate in our trial was low at about 5% and similar across trial arms due to an almost 90% cross-over success rate after unsatisfactory micturition with the bedpan, whereas after unsatisfactory micturition in the toilet, cross-over success rate was 0%. This finding reinforced our primary finding of the toilet being the better set-up for micturition. Our data indicated that a two-step approach of bedpan and if unsatisfactory, switch-over to toilet compared to mobilization to the toilet might still be viable but cumbersome option with the catheterization rate as the endpoint. Use of the bedpan can be convenient but the privacy of the toilet appeared the most suitable for micturition. With regard to strength, this is an original and powered study to evaluate the micturition performance in toilet or by bedpan of nulliparas in labor without neuraxial analgesia with positive finding on the primary and a number of supportive secondary outcomes in favor of the toilet environment. The study was powered and data replete with no drop-out. A single investigator was involved, eliminating inter-operator bias of ultrasound assessment. The investigator had 6 years of experience after certified training in obstetrics and gynecologic ultrasound. We believed our findings to be generalizable.
With regards to limitations, this study was not blinded to the investigator-sonographer. This could be important as our primary outcome was ultrasound derived post-void residual urine volume and there could be investigator bias in image capture and measurements. Post hoc analyses of our data on ultrasound accuracy did not suggest operator bias across trial arms. There is continuing debate about the accuracy of ultrasound estimation of urine volume particularly during labor compared to measured catheterized volumes and 2-dimensional vs. 3-dimensional bladder volumes using ultrasound [bib_ref] Intrapartum ultrasound estimation of total bladder volume, Gyampoh [/bib_ref] [bib_ref] Can we replace the catheter when evaluating urinary residuals?, Araklitis [/bib_ref] [bib_ref] Validity of bladder volume measurement by ultrasound in women postpartum, Jensen [/bib_ref] [bib_ref] Prospective 3D ultrasonographic evaluation of immediate postpartum urine retention volume in 100..., Demaria [/bib_ref]. In the bedpan arm, we allowed choice of lying down or sat up, and as posture could plausibly affect micturition performance, these choices could cause confounding. It is possible that women who are in the most advance stages of labor or very distressed are less likely to be recruited which may introduce selection bias.
# Conclusions
In conclusion, nulliparas in labor are more likely to have satisfactory micturition and far higher maternal satisfaction when mobilized to the toilet compared to bedpan use. However, with cross-over to go to the toilet if bedpan use is unsatisfactory, urinary catheterization rates are similar indicating a two-step approach with initial bedpan may still be viable. As a woman-centered quality of care issue, mobilizing to the toilet in labor to micturate is overwhelmingly preferred over the alternatives of the bedpan or catheterization.
[fig] Figure 1: Recruitment flow chart into a randomized trial of mobilization to the toilet compared with bedpan use to micturate in nulliparas with a [/fig]
[table] Table 1: Characteristics of trial participants randomized to mobilization to the toilet compared with bedpan use to micturate in nulliparas with a filled bladder during labor Data expressed as mean ± standard deviation or number (%). Analyses by Student t test for comparison of means for continuous data and Chi-Square test categorical datasets. 2-sided analyses a other ethnicities: 4 Indonesian, 4 Myanmar national, 2 Sabah native, 2 Filipino, 1 Yemeni [/table]
[table] Table 2: Primary outcome and other bladder voiding outcomes after randomization to mobilization to the toilet compared with bedpan use to micturate in nulliparas with a filled bladder during labor [/table]
[table] Table 3: Secondary outcome after randomization to mobilization to the toilet compared with bedpan use to micturate in nulliparas with a filled bladder during labor [/table]
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Development of a system for the automated identification of herbarium specimens with high accuracy
Herbarium specimens are dried plants mounted onto paper. They are used by a limited number of researchers, such as plant taxonomists, as a source of information on morphology and distribution. Recently, digitised herbarium specimens have begun to be used in comprehensive research to address broader issues. However, some specimens have been misidentified, and if used, there is a risk of drawing incorrect conclusions. In this study, we successfully developed a system for identifying taxon names with high accuracy using an image recognition system. We developed a system with an accuracy of 96.4% using 500,554 specimen images of 2171 plant taxa (2064 species, 9 subspecies, 88 varieties, and 10 forms in 192 families) that grow in Japan. We clarified where the artificial intelligence is looking to make decisions, and which taxa is being misidentified. As the system can be applied to digitalised images worldwide, it is useful for selecting and correcting misidentified herbarium specimens.Herbarium specimens were first collected about 500 years ago 1 , and approximately 380 million specimens are stored in approximately 3000 museums globally 2 . Herbarium specimens have long been used by a limited subset of researchers, such as plant taxonomists, as a reference for scientific names or voucher specimens, or as a source of information on morphology and distribution. However, research in the field of museomics 3 , in which DNA, proteins, metabolites, radioisotopes 4 , and heavy metals 5 are extracted from specimens, has recently become prevalent. As the digitisation of label data (taxon name, collection location, collection date, images, etc.) related to plant specimens has progressed, data have been accumulated in international databases such as Global Biodiversity Information Facility (GBIF, https:// www. gbif. org/) and Integrated Digitized Biocollections (iDigBio, https:// www. idigb io. org/). Specimen images have become big data, and can be used freely by anybody to study, for example, the effects of climate change by examining the flowering season of certain plant species 6 . They are also beginning to be used in comprehensive research to address imminent social issues related to conservation and food security 7-9 . However, there are problems that must be resolved for the future development of these studies. One of the major problems is that the data contain misidentified specimens[10][11][12]reported that at least 58% of the 4500 specimens of African gingers had a wrong name prior to a recent taxonomic study. It is difficult for non-taxonomists to notice misidentified specimens and their presence is likely to result in analyses using incorrect data. In studies that deal with big data, the amount of labour required to check for misidentifications is enormous. Misidentified specimens need to be found and corrected quickly to ensure the value of collections as a data set, but taxonomists are unevenly distributed and too few to re-examine whole specimens OPEN
www.nature.com/scientificreports/ for their identification. There is an urgent need to develop an artificial intelligence (AI)-based plant identification system with high accuracy. Florian Schroff et al. [bib_ref] FaceNet: A unified embedding for face recognition and clustering, Schroff [/bib_ref] developed a system that can judge a human face. It distinguishes 8 million people from about 200 million images, and its accuracy is 99.63% [bib_ref] FaceNet: A unified embedding for face recognition and clustering, Schroff [/bib_ref]. The automated identification of plant species from images of a leaf [bib_ref] Deep learning for plant identification using vein morphological patterns, Grinblat [/bib_ref] [bib_ref] How deep learning extracts and learns leaf features for plant classification, Lee [/bib_ref] or seedling [bib_ref] Plant species classification using deep convolutional neural network, Dyrmann [/bib_ref] [bib_ref] Plant seedlings classification using deep learning, Ashqar [/bib_ref] [bib_ref] Improving weeds identification with a repository of agricultural pre-trained deep neural networks, Espejo-Garcia [/bib_ref] is a research field with a rich recent literature, mostly concerning agriculture [bib_ref] Deep learning in agriculture: A survey, Kamilaris [/bib_ref]. The LifeCLEF 2020 Plant Identification Challenge was conducted using field images of plants in addition to specimens and showed that such images can also be used for classification [bib_ref] Overview of LifeCLEF Plant Identification task 2019: Diving into data deficient tropical..., Goëau [/bib_ref]. Recently, various researches using deep learning technology to determine species names from specimen images have been actively conducted [bib_ref] Going deeper in the automated identification of herbarium specimens, Carranza-Rojas [/bib_ref] [bib_ref] Automated Identification of Herbarium Specimens at Different Taxonomic Levels, Carranza-Rojas [/bib_ref]. In 2017, Carranza-Rojas et al. [bib_ref] Going deeper in the automated identification of herbarium specimens, Carranza-Rojas [/bib_ref] constructed a semi-automatic identification system using 113,205 images of 1000 species obtained from the iDigBio portal. GoogLeNet (InceptionV1) was used for the analysis, and the accuracy was 70.3%. In 2018, 253,733 images of 1191 species obtained from the iDigBio portal were analysed using GoogLeNet, and the accuracy was 63.0% [bib_ref] Automated Identification of Herbarium Specimens at Different Taxonomic Levels, Carranza-Rojas [/bib_ref]. In 2019, the Herbarium Challenge was held using 46,469 images of 683 melastome species (Melastomataceae) provided by the New York Botanical Garden, and the winning team used SeResNext-50, SeResNext-101, and ResNet-152, with an accuracy of 89.8% 2 .
In this study, we investigated the optimum number of specimens per taxa required for improving the accuracy. We also investigated whether the accuracy would improve if specimens without leaves or those with large or many holes in the leaves were excluded. In addition, we investigated which taxa were mistaken for other taxa and what part of the image was focused on when making the identification.
# Results
Improvement of data sets and identification accuracy. The targets of this study were taxa growing in Japan. In addition to images (about 290,000) digitised by the authors using a scanner [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] [bib_ref] Establishment of high-speed digitization method of herbarium specimen and construction of maintenance-free..., Moriguchi [/bib_ref] or camera [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] [bib_ref] Simple but long-lasting: A specimen imaging method applicable for small-and medium-sized herbaria, Takano [/bib_ref] , approximately 260,000 specimen images were downloaded from the database. Finally, 546,184 images of 3,114 taxa (2871 species, 25 subspecies, 181 varieties, 37 formas) in 219 families were obtained. Images of specimens were collected from across Japan. The number of specimens varied depending upon taxa; 838 (27%) taxa had ≤ 50 specimens, 742 (24%) had ≤ 40 specimens, 539 (17%) had ≤ 30 specimens, and 108 (3%) had ≤ 20 specimens.
Using the collected images, a plant taxa identification system was developed. For the experiments, Inception-ResNet-v2 was used, as it is the one of the most accurate function in pre-trained deep neural network [bib_ref] Improving weeds identification with a repository of agricultural pre-trained deep neural networks, Espejo-Garcia [/bib_ref]. The results of the first experiment showed an accuracy of 92.3%; Supplementary Data 1). There were 319 taxa with average macro f-scores ≤ 0.6, calculated as 2 × (precision × recall)/(precision + recall), and the average number of images used in these experiments was 48 per taxa. To exclude these taxa from the analysis target in the second experiment, we decided to use only ≥ 50 images per taxa. In the case of an image containing multiple individuals or shoots in one sheet [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] , the individual of plants or shoots were cut out to increase the number of images [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref]. The second experiment was conducted using 534,778 images from 2,191 taxa (2,084 species, 9 subspecies, 88 varieties, and 10 formas), and the accuracy of the results increased to 93.9%; Supplementary Data 2).
In the second set of experiments, 11,950 images were incorrectly identified. Among them, there were 767 (6.4%) specimens that had only twigs without leaves and flowers/fruits [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] and/or had large or many holes in the leaves [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] , and they were clearly misidentified. Such images were discarded, and the third set of experiments was conducted using 500,554 specimens from 2171 taxa. The accuracy in this experiment was 96.2%; Supplementary Data 3). In the system developed in this study, the most probable taxa were extracted as the Top-1 and the Top-5. The correct answer rate of the Top-1 was 96.2%, while the correct answer rate within the Top-5 was 99.4%. In this experiment, the AI misidentified 5,195 images. We investigated whether the AI had actually misidentified them, or whether this was caused by the AI correctly identifying a sample that was previously misidentified. We re-identified 181 specimens in the Herbarium of University Archives and Collections, Fukushima University (FKSE). As a result, at least 34 (19%) of the 181 specimens had been previously misidentified. We constructed the system nine times. In the preliminary experiment, the system was constructed six times by changing the combination of training data and test data. All these results were analysed together. We selected specimens that were misidentified six times or more by the AI and re-identified them. At least 32 (28%) of 113 specimens had been previously misidentified. Subsequently, an identification system was developed focussing only on pteridophytes (353 taxa). The number of specimen images per pteridophyte taxon was higher than that of flowering plant taxa, averaging 578 per taxon (230 in the third experiment, which excluded damaged and misidentified specimens). While the number of taxa decreased to about one-sixth, the number of specimens per taxon doubled. The accuracy of the results was 98.4%; Supplementary . The relationship between the number of images and the average macro f-score was investigated in the analysis performed on 2171 taxa (the third experiment) and the analysis only on pteridophytes; the larger the number of images used in the analysis, the higher the average macro f-score [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref].
Inception-ResNet v2 analysis method used in this study was compared with Inception-ResNet v2_base, Inception v3, and VGG16 using the images included in the third experiment. The method used in this study (Inception-ResNet v2) was found to be the most accurate. The classification accuracy of Inception-ResNet v2, Inception v3, and VGG16 showed the same tendency as classification by ImageNet, and the accuracy of Inception-ResNet v2 was the highest. The proposed method adds two 4096-dimensional, fully connected layers after the average pooling of Inception-ResNet v2. Unlike Inception-ResNet v2_base, the class of target image can be predicted from a vector with more dimensions than the number of classes; thus, the prediction accuracy is improved compared to Inception-ResNet v2 (Supplementary Data 6-8). The influence of collection method on identification. When collecting herbarium specimens, a collector may take several samples from the same individual plant. Even if these specimens were mounted onto different sheets, they were collected from the same plant on the same day. Therefore, these specimens may be visually much more similar than those of other samples collected from another plant of the same species, in another region, at another period of the year. Thus, the evaluation may be biased by these specimens.
Of the 2171 taxa used in this experiment, 1902 taxa (87.6%) contained samples collected on the same day. there is a possibility that one individual was divided and treated as multiple specimens. The average f-score of the 82 taxa that comprised 10% of all samples collected on the same day and at the same location was 0.8992, which was lower than the average (0.958). Some of the samples used in this experiment may have been sampled from the same place on the same day, but the effect of these samples on identification accuracy was not observed.
The influence of labels, colour bars, scales, stamps, etc. in the sample image on identification. For the Herbarium Challenge 2019 data set, the labels on herbarium sheets were removed to prevent the AI from using the plant name and other information written on the label 2 . The image input size of the training www.nature.com/scientificreports/ data used in this study was 299 × 299 pixels. This size was used in ImageNet 25 . It is difficult for even humans to read the written characters in these images. The effect of labels on identification was investigated using the following method. A set of 5000 correctly identified sample images were randomly selected, and the images were processed so that only the label of the sample images remained. Identification was then performed. The probability of obtaining the correct answer by chance was 0.05% (2.3 images/5000 images). Only three images were correctly identified, giving a correct answer rate of 0.06%. In addition to the labels, some sample images contained colour bars, scales, and stamps [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref]. To investigate the effect of these factors on identification, they were deleted from the images [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] and a fourth set of experiment was conducted to investigate their effect on the identification accuracy. The accuracy was only slightly lower when using the images with the colour bars, scales, and stamps removed, Supplementary Data 4). From these results, it was clarified that the presence of a label, colour bar, scale, or stamp in the image does not significantly affect the accuracy of identification.
## Does ai make the same misidentifications as humans?
We investigated whether the AI could correctly identify plants that are frequently misidentified by collectors and collection managers (hereinafter referred to as experts). First, taxa that were frequently misidentified by experts were selected according to the records of identification history of the specimens in FKSE. Of the taxa stored in the FKSE that had 50 or more specimens, 17 taxa with a misidentification rate (number of misidentified or previously misidentified specimens/number of specimens) ≥ 15% were classified as 'frequently misidentified taxa' [fig_ref] Table 2: List of 17 taxa that are frequently misidentified in the records of... [/fig_ref]. The average number of images used per taxon in the third set of experiment was 230, while the average number of images used per taxon of the 17 taxa was 328. The average macro f-score of the 2171 taxa was 0.962, while the average value of the 17 taxa was 0.890. Experts often misidentified Platanthera tipuloides (L.f.) Lindl. as Platanthera minor (Miq.) Rchb.f. In addition, Lespedeza homoloba Nakai was frequently misidentified as Lespedeza cyrtobotrya Miq. or Lespedeza bicolor Turcz. We investigated whether the AI also misidentified them. It was found that, for all taxa, the AI made the same mistakes as the experts [fig_ref] Table 2: List of 17 taxa that are frequently misidentified in the records of... [/fig_ref].
We investigated whether the AI and experts tend to make the same misidentification. Although bluegrasses (Poa spp., Poaceae) are morphologically similar to each other, the AI rarely misidentified Poa nipponica Koidz. (Poaceae) as Poa trivialis L., Poa annua L., and Poa pratensis L. subsp. pratensis, but often as Corydalis pallida (Thunb.) Pers. var. tenuis Yatabe (Papaveraceae) and as Pilea hamaoi Makino (Urticaceae). Experts do not misidentify C. pallida var. tenuis, Pilea hamaoi, and Poa nipponica because they are very different in shape. Therefore, we did not understand why the AI confused these species.
To clarify what kind of taxa are selected for the Top-2 by AI when AI identifies taxa successfully, we selected the 1022 images that the AI identified correctly. The taxa that the AI identified as the Top-2 were checked. Floral characters are important for species diagnosis, but those of willow species are small, less than 10 mm long, and each specimen contains either female or male flowers. The shape of leaves can be useful for taxon recognition, but willow leaves usually start to emerge after anthesis. All the willow specimens were used as training data without separating the males and females, and the image input size was 299 × 299 pixels, which is too small for the small floral organs to be recognized. A crosstabulation table was created for the recall and precision values of 17 willow taxa, and it was found that willow taxa were often misidentified within the same genus. The Gradient-weighted Class Activation Mapping (Grad-CAM) results (an analysis method that displays the most important parts in different colours during AI identification 27 ) showed that the AI displayed a tendency to use the inflorescences and infructescence, and some of the branches to which they were attached for identification, and then use entire leaves for identification after using the infructescence.
Is AI misidentifying taxa of the same genus? Species misidentified by experts are mostly within the same genus. Therefore, we investigated whether the taxa misidentified by the AI belonged to the same genus. We examined the genera of the Top-5 taxa for 137,483 images that were correctly identified by the AI. All five of the Top-5 taxa were from the same genus (including the Top-1 correctly identified taxon name) in 4.6% (6293) of the cases, four in 9.5% of cases, three in 17.1% of cases, and two in 27.5% of cases. , which are in different families. We investigated whether the AI misidentified these species in the same way, and the same misidentifications were found .
## Identification of parts where ai is important for identification. easy-to-identify pteridophytes
include Thelypteris acuminata (Houtt.) C.V. Morton with its long terminal leaflet and Polystichum tripteron (Kunze) C. Presl with its long, cross-shaped basal pinnae. The average macro f-score of T. acuminata was 0.993, and that of P. tripteron was 0.998. Looking at the Grad-CAM analysis results, we found that the characteristic parts for each species were captured . For T. acuminata 352 of the 648 images (54.3%) focused on the www.nature.com/scientificreports/ particular part of the image [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref]. Subsequently, from each image two images were created-one in which the non-focal parts were cut outand the other in which the focal parts were removed. These images were then analysed. The accuracy rate for the images containing only the non-focal parts decreased to 72.4%, while the correct answer rate for the images that included only the focal part was 54.0%. Although the area of the focal part was small and the area of the non-focal parts was large, the accuracy rate of images containing only the focal parts was low. This was the opposite of what was expected. From these results, it was expected that AI would first look at the whole and narrow down, and then look at specific parts to narrow down further. After processing an image that contained two individuals in one specimen [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref] to produce an image containing only one individual, the accuracy was 82%. We prepared an image in which the sample image was halved vertically and horizontally and further divided it into four both vertically and horizontally . When tested, the accuracy decreased to 54%.
Publication of the system. [fig_ref] Figure 6: A system that automatically identified the taxa name from an image of... [/fig_ref]. www.nature.com/scientificreports/ misidentified specimens by AI revealed that at least 19% were misidentified. From this, it was found that in order to further improve the identification accuracy, it is necessary to improve the quality of the training data rather than improving the method. Plants vary in size and shape depending on the stage and environment in which they grow, and some have flowers and fruits. Furthermore, different colours and morphologies are shown, depending on the method used to make the specimenswww.nature.com/scientificreports/ the latter case, the AI often misidentified these taxa as other taxa in the same genus. In other words, they were misidentified as taxa with similar morphologies. To solve this problem, it is necessary to use more accurately identified specimens for taxa with low average macro f-scores. The effects of specimen labels, stamps, rulers, colour bars, etc. contained in the specimens on the identification were investigated and it became clear that the accuracy of the identification did not increase even if these were removed.
## Similarities and differences between ai and human identification methods.
From previous studies, it was not clear what the AI was using to make its decisions. In this study, the Grad-CAM analysis revealed the important areas in a specimen image used for AI identification. As the accuracy decreased if parts of the image were removed and only a section of the plant was used for identification, or when the image was divided and the identification made using a reduced area , the AI appeared to first observe the whole plant and then add specific characteristic parts. The identification method of the AI may be similar to that performed by experts.
It was also not apparent in previous studies which taxon is mistaken for which taxon. In this study, we created a cross-table, Supplement Data 1-8) and investigated this information. As a result, it became clear that the AI made mistakes in the taxa of the same genus. Furthermore, it became clear that taxa of different genera and families that are similar in their morphology were also mistaken as in the case of experts.
In the willow genus (Salix spp.), it was found that the identification method is different between AI and experts because the floral parts that experts are paying attention to are too small for the AI. If the part required for identification is small, it was thought that identification would be possible by preparing an enlarged image of the part and training it. In the case of willows, identification was possible at a certain level without using such a small part, so it was considered that the accuracy of identification could be improved by increasing the number of specimens to be trained.
## Utilization of the system created in this study.
In Japan, the number of plant taxonomists who are able to classify plant taxa accurately is declining, and this trend is expected to continue. While the number of people who can correctly identify taxa is decreasing, the need for environmental investigation is increasing owing to active human activities and environmental change. Thus, it is necessary to develop technology that can help non-experts to correctly identify taxa. The identification system developed in this study is a good candidate. By constructing a system multiple times by changing the combination of training data and test data, it is possible to select particular specimens in which AI makes a mistake in identification multiple times. Since about 28% of the specimens selected in this way were misidentified and then the correct specimen data was registered in GBIF, it can be said that our system is a good one for selecting the misidentified specimens and correcting the data. Herbaria and databases are full of misidentified specimens [bib_ref] An examination of confidence in open data of specimens: Cuscuta australis (Convolvulaceae), Fujii [/bib_ref] [bib_ref] On natural history collections, digitized and not: A response to Ferro and..., Sikes [/bib_ref] [bib_ref] Widespread mistaken identity in tropical plant collections, Goodwin [/bib_ref]. The method developed in this study is considered to be effective for the correction of such specimens and the reduction of erroneous data due to misidentified specimens in the database.
# Methods
Digitisation of specimens and collection of digitised specimen images. Specimens in the FKSE, Tottori Prefectural Museum, Rikuzentakata City Museum, Kagoshima University Museum, Shimane Nature Museum of Mt. Sanbe, and Shimane University Faculty of Life and Environment Sciences were digitised using a scanner (EPSON DS-50000G, ES-7000HS, or ES-10000G). The method has been described previously [bib_ref] Establishment of high-speed digitization method of herbarium specimen and construction of maintenance-free..., Moriguchi [/bib_ref]. Specimens from the Museum of Nature and Human Activities, Hyogo were digitised using a camera (SONY α6500 Samyang AF 35 mm F2.8 FE, ISO 100), as described previously [bib_ref] Simple but long-lasting: A specimen imaging method applicable for small-and medium-sized herbaria, Takano [/bib_ref]. Digitalised images of TNS, College of Life Science, National Taiwan University, and Flora of Tokyo specimens were downloaded from the website (the URL is shown in [fig_ref] Figure 1: Specimen images taken with a scanner [/fig_ref]. The TNS specimens were digitised using a camera, while the College of Life Science, National Taiwan University, and Flora of Tokyo specimens were digitised using a scanner. The images were downsized to 299 × 299 pixels for input size in this study.
Deep learning model. It has been clarified that deep learning, which was used in this study, is more accurate than non-deep methods [bib_ref] Automated plant species identification: Trends and future directions, Wäldchen [/bib_ref]. A convolutional neural network is a neural network model mainly consisting of convolutional, pooling, and fully connected layers. The convolutional layer has a weight parameter called a filter. The input image is converted into a feature map by applying the filter. The pooling layer extracts representative values from a specific region and reduces the spatial size. In the fully connected layer, all the nodes in the layer are connected to each other, and each edge has an independent weight. Different convolutional neural network models can be designed depending on the composition of the layers. In recent years, models such as VGG 32 , Inception 33 , and ResNet 34 have been confirmed to be highly accurate. Inception is composed of inception blocks that integrate the results of multiple convolutional and pooling processes within a single layer. ResNet has a shortcut connection that prevents gradient loss. Inception-ResNet-v2 consists of an inception block with an added shortcut connection, and has been shown to possess high classification accuracy [bib_ref] Inception-v4, Inception-ResNet and the impact of residual connections on learning, Szegedy [/bib_ref]. The performance of this model was evaluated using the ImageNet dataset with 1000 different classes, and the Top-5 accuracy was approximately 95%. In this study, we used Inception-ResNet-v2 with two additional fully connected layers, with 4096 nodes each, after average pooling, to perform classification on a dataset with a large number of classes. The output of the first fully connected layer was normalised using Batch Normalisation. In Inception-ResNet-v2, the number of nodes after average pooling is 1792, so if the number of classes exceeds 1792, the probability of belonging to each class is predicted using fewer nodes than the number of classes. By Scientific Reports | (2022) 12:8066 | https://doi.org/10.1038/s41598-022-11450-y www.nature.com/scientificreports/ adding a fully connected layer, it becomes possible to predict the probability of belonging to each class using a larger number of nodes.
Evaluation. To evaluate our experiments, we examined the accuracy and f-score of taxa classification. Accuracy is defined as the rate of correct answers among all test data. Top-1 accuracy is considered correct when the class ranked first in the prediction results is the correct answer. Top-5 accuracy is considered correct when the top five classes in the prediction results contain the correct answer. The f-score is defined as a harmonic mean of precision (P i ) and recall (R i ).
In these formulae, for a class (i), a i is the number of positive answers to positive samples; c i is the number of negative answers to positive samples; and b i is the number of positive answers to negative samples. The f-score of each class (f i ) is defined as a harmonic mean of precision and recall, and the whole f-score is the macro average and weighted average.
## Method of removing stamps, colour bars, and scales from images.
In-house software was developed to remove stamps, colour bars, and scales with a priori knowledge of their shapes and colours.
## Data availability
Some of the data used in this study can be downloaded from the database. The processed and other data for which we have the copyright are available upon reasonable request emailed to the corresponding author. However, data for which we do not have the copyright are unavailable.
[fig] Figure 1: Specimen images taken with a scanner (a1) and with a camera (a2). (b1) Multiple individuals in one specimen image and (b2) image divided into two. (c) Specimen images showing only branches and leaves that have fallen. (d) Specimen image with many holes in a leaf eaten by insects. (e1) Specimen image with a scale, stamp, and colour bar and (e2) the image after the scale, stamp, and colour bar were removed. Scientific Reports | (2022) 12:8066 | https://doi.org/10.1038/s41598-022-11450-y www.nature.com/scientificreports/ [/fig]
[fig] Figure 2: (a) List of herbarium-stored specimens used in this study. (b) Locations of specimen archives. Scientific Reports | (2022) 12:8066 | https://doi.org/10.1038/s41598-022-11450-y [/fig]
[fig] Figure 3: (a) A cross-tabulation table was created for the recall and precision values of 17 taxa of Salix L in the third experiment. (1) S. integra Thunb., (2) S. futura Seemen, (3) S. pierotii Miq., (4) S. udensis Trautv. et C.A.Mey., (5) S. miyabeana Seemen subsp. gymnolepis (H.Lév. et Vaniot) H.Ohashi et Yonek., (6) S. vulpina Andersson subsp. vulpina, (7) S. dolichostyla Seemen subsp. serissifolia (Kimura) H.Ohashi et H.Nakai, (8) S. vulpina Andersson subsp. alopochroa (Kimura) H.Ohashi et Yonek., (9) S. eriocataphylla Kimura, (10) S. japonica Thunb., (11) S. dolichostyla Seemen subsp. dolichostyla, (12) S. eriocarpa Franch. et Sav., (13) S. triandra L. subsp. nipponica (Franch. et Sav.) A.K.Skvortsov, (14) S. gracilistyla Miq., (15) S. caprea L., (16) S. chaenomeloides Kimura, (17) S. sieboldiana Blume var. sieboldiana, (18) others. (b) Results of Grad-CAM analysis of Salix integra and Salix futura. Red indicates the more important parts while blue represents less important parts. Scientific Reports | (2022) 12:8066 | https://doi.org/10.1038/s41598-022-11450-y [/fig]
[fig] Figure 4, Figure 5: (a) Astilbe thunbergii (Siebold & Zucc.) Miq. var. thunbergii, (b) Astilbe odontophylla Miq., (c) Machilus thunbergii Siebold & Zucc., and (d) Lithocarpus edulis (Makino) Nakai. Species (a) and (b) and species (c) and (d) are different plants with similar morphologies. These species are often misidentified by experts and were misidentified by AI in this study. improve identification accuracy? In this study, it was clarified whether the method of select-Results of Grad-CAM analysis of (a) Thelypteris acuminata (Houtt.) C.V. Morton and (b) Polystichum tripteron (Kunze) C. Presl. (c) In the Grad-CAM analysis, only part of the specimen was considered important (red). (c2) Images in which the focal part was cut out and (c3) images in which only the non-focal part was cut out were created and used for Grad-CAM analysis. (d1) Multiple individuals in one specimen image. The image was divided into two images (d2, 3) and used for Grad-CAM analysis. (e) The sample image was halved vertically and horizontally, and further divided it into four both vertically and horizontally. Scientific Reports | (2022) 12:8066 | https://doi.org/10.1038/s41598-022-11450-y [/fig]
[fig] Figure 6: A system that automatically identified the taxa name from an image of a plant (http:// tayou sei. life. shima ne-u. ac. jp/ ai/ index_ all. php). The identification system for pteridophytes is located at http:// tayou sei. life. shima ne-u. ac. jp/ ai/ index_ Pteri dophy tes. php. Drag and drop an image of the plant for which you want to identify the taxa name, or select files and click the send button. Plant taxa from Top-1 to Top-5 are displayed as candidates, and the accuracy is also displayed.Scientific Reports | (2022) 12:8066 | https://doi.org/10.1038/s41598-022-11450-y [/fig]
[table] Table 1: (a) List of experiments and results. (b) List of methods and results. These experiments were performed with the specimens used for the third experiment (excluding broken and misidentified specimens). [/table]
[table] Table 2: List of 17 taxa that are frequently misidentified in the records of identification history of the specimens in the Herbarium of University Archives and Collections, Fukushima University (FKSE). CAM, we identified the parts of the image that are important for AI identification. First, we investigated the Grad-CAM analysis results and selected 287 images that were identified by focussing on a [/table]
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Asymptomatic intrathyroidal pyriform sinus fistula mimicking thyroid cancer
Rationale: There have been many reports of non-thyroidal lesions which can be mistaken for thyroidal lesions on ultrasound (US) examination. However, it is not known that pyriform sinus fistula (PSF) can manifest as an incidental thyroid nodule and cause serious complication on fine-needle aspiration (FNA).Patient concerns: We present a 34-year-old man with PSF incidentally detected on US. US examination showed hypoechoic nodule with several bright echogenic spots at the uppermost part of left thyroid gland. With the suspicion of thyroid cancer, although there would have been some morphologic changes between the 2 US examinations, FNA was performed.Diagnoses: Cytologic specimen revealed some clusters of ciliated columnar cells mixed with inflammatory and lymphoid cells. On computed tomography (CT) before FNA, there were tiny air bubbles within the thyroid nodule. Laryngoscopy revealed fistula originating from the pyriform sinus.Interventions: After FNA, he had to undergo tracheostomy and removal of abscess due to infectious complication.Outcomes: The deep neck abscesses and infections were controlled after the treatment. At 1 year after FNA, successful chemocauterization with 40% trichloracetic acid solution was performed for PSF found on laryngoscopy.Lessons: PSF can manifest as an incidental thyroid nodule mimicking thyroid cancer. Special care should be taken when FNA is planned for the nodule with air foci and morphologic changeability at the uppermost part of left thyroid gland.Abbreviations: FNA = fine-needle aspiration, PSF = pyriform sinus fistula, US = ultrasound.
# Introduction
Ultrasonography (US) plays a key role in the diagnosis and management of thyroid nodules and US guided fine-needle aspiration (FNA) is primarily recommended for the further evaluation of thyroid nodules. [bib_ref] American Thyroid Association Management guidelines for adult patients with thyroid nodules and..., Haugen [/bib_ref] Along with increase of use of high resolution US, there have been many reports of various nonthyroidal lesions including Killian-Jamieson diverticulum and paraesophageal air cyst, which can be mistaken for thyroidal lesions on US. [bib_ref] Characteristics of Killian-Jamieson diverticula mimicking a thyroid nodule, Kim [/bib_ref] [bib_ref] Paratracheal air cysts: sonographic findings in two cases, Kim [/bib_ref] [bib_ref] Thyroid ultrasonography: pitfalls and techniques, Choi [/bib_ref] In most literature, pyriform sinus fistula (PSF) has been presented with the complication cases of acute suppurative thyroiditis and recurrent deep neck infection and emphasized as an important infection source, especially in children. [bib_ref] Neck infection associated with pyriform sinus fistula: imaging findings, Park [/bib_ref] [bib_ref] Diagnosis and management of pyriform sinus fistula: experience in 48 cases, Sheng [/bib_ref] Herein, we report the case of asymptomatic intrathyroidal PSF mimicking thyroid cancer on US, which entrapped into FNA that could cause serious infectious complication.
## Case report
This study was approved by the institutional review board of Seoul National University Hospital, and the requirement for informed consent was waived due to its retrospective nature.
A 34-year-old man was referred for an incidental left thyroid nodule. The results of thyroid function tests and complete blood count were within normal ranges. The US demonstrated a 1.1 cm nodule at the uppermost part of left thyroid gland. The nodule showed mild hypoechogenicity, irregular shape, nonparallel orientation, ill-defined margin, and many bright echogenic spots without acoustic shadowing on US [fig_ref] Figure 1: A longitudinal US image [/fig_ref]. [bib_ref] Ultrasonography diagnosis and imaging-based management of thyroid nodules: revised Korean Society of..., Shin [/bib_ref] Between the 2 US exams with 7 months interval, there were some unrecognized changes in the shape and number of internal bright echogenic foci of the nodule [fig_ref] Figure 1: A longitudinal US image [/fig_ref]. In addition, there was 1.2 cm lowattenuating nodule with air bubble at the uppermost part of left thyroid gland on chest CT [fig_ref] Figure 1: A longitudinal US image [/fig_ref]. Nevertheless, the FNA operator could not notice these findings and FNA was performed under the impression of thyroid cancer. The cytologic specimen revealed many ciliated columnar cells in the background of inflammatory and lymphoid cells. Unfortunately symptoms of neck pain, swelling, fever, and dyspnea developed sequentially during 3 days after FNA and aggravated in spite of oral antibiotics. When he visited emergency room at 3 days after FNA, the white blood cell count was elevated (17,400 /mL) and computed tomographic (CT) scan revealed abscess involving left upper thyroid gland, adjacent perithyroid tissue and retropharyngeal space along with diffuse swelling of hypopharynx and supraglottic larynx [fig_ref] Figure 2: Three days after the US-guided fine needle aspiration, CT images depict an... [/fig_ref]. Because of the rapid progression of respiratory distress, he had to undergo tracheostomy and the subsequent incision and drainage of abscess. At 1 year after FNA, successful chemocauterization with 40% trichloracetic acid solution was performed for PSF found on laryngoscopy [fig_ref] Figure 3: A laryngoscopy obtained 1 year later depicts a fistulous opening [/fig_ref].
# Discussion
Although bacterial infection rarely occurs in thyroid gland due to its rich blood supply and lymphatics and thick fibrous capsule, when PSF exists, the fistula tract can act as pathway for the infection propagation to the thyroid gland. [bib_ref] Neck infection associated with pyriform sinus fistula: imaging findings, Park [/bib_ref] As most PSF cases manifest as an infection, it is difficult to interfere with asymptomatic PSF. We firstly report that asymptomatic PSF can manifest as a thyroid nodule and undergo FNA on the clinical scenario. [bib_ref] Neck infection associated with pyriform sinus fistula: imaging findings, Park [/bib_ref] [bib_ref] Diagnosis and management of pyriform sinus fistula: experience in 48 cases, Sheng [/bib_ref] Although the embryologic origin is controversial, PSF appears to be a remnant originates from the failure of the third or fourth branchial pouches to atrophy and perish in utero, which results in the sinus tract that lie in close proximity to, or inside, the thyroid gland. [bib_ref] Ten years of experience with third and fourth branchial remnants, Liberman [/bib_ref] Until now, there were some reports of intrathyroidal branchial cleft or cleft-like cyst that manifested mainly as cystic lesions. [bib_ref] Intrathyroidal branchial cleft-like cyst in chronic thyroiditis, Haba [/bib_ref] However, they were different from our case in the fact that they were lined by squamous epithelium, and they were not usually located in uppermost part of left thyroid gland. In addition, the connection to the pyriform sinus was not reported in previous studies. Under the name of intrathyroidal branchial cleft sinus, Kim et al [bib_ref] Imaging feature of intrathyroidal branchial cleft sinus, Kim [/bib_ref] reported a similar case of this study, which presented the thyroid nodule with the suspicious malignant features on US but FNA could be avoided because the lesion changed morphologically during FNA procedure and subsequent CT showed air tract between pyriform sinus and the thyroid lesion. Followings can be the clues for intrathyroidal PSF. First, the lesions are located in the uppermost part of the thyroid gland. The predominance of left-sided location up to 93% in PSF may be related to the embryologic asymmetry in the persistence of a patent thymopharyngeal duct. [bib_ref] Neck infection associated with pyriform sinus fistula: imaging findings, Park [/bib_ref] [bib_ref] Diagnosis and management of pyriform sinus fistula: experience in 48 cases, Sheng [/bib_ref] Uppermost location within the thyroid gland can be explained by anatomic proximity to pyriform sinus. Second, air bubbles from pyriform sinus seem to make the internal bright echogenic foci on US. Because internal echogenic spots from air bubbles can be confused with microcalcifications, they may be the important US features in which intrathyroidal PSF can be misregarded as thyroid cancer. Air foci depicted on US or CT seem to be an important clue for the diagnosis of various intrathyroidal structure which communicates with laryngo-pharyngo-esophageal structures. [bib_ref] Characteristics of Killian-Jamieson diverticula mimicking a thyroid nodule, Kim [/bib_ref] [bib_ref] Paratracheal air cysts: sonographic findings in two cases, Kim [/bib_ref] [bib_ref] Thyroid ultrasonography: pitfalls and techniques, Choi [/bib_ref] In the previous series of PSF complicated by neck infections, foci containing air were noted as bubbles or tracts on 9 out of 17 cases. [bib_ref] Neck infection associated with pyriform sinus fistula: imaging findings, Park [/bib_ref] Third, the morphologic changeability can be noted between US scans or even during US scan. It can be explained by movability of the extended pharyngoesophageal structure and internal air bubbles. Among the various non-thyroidal lesions mimicking thyroid nodules, Killian-Jamieson diverticulum showed similar features in the aspects of left sided predominant location, foci containing air, and morphologic changeability. [bib_ref] Characteristics of Killian-Jamieson diverticula mimicking a thyroid nodule, Kim [/bib_ref] However, uppermost location of left lobe without connection to esophagus can be an essential clue for differentiating intrathyroidal PSF from Killian-Jamieson diverticulum.
# Conclusion
PSF can manifests as an incidental thyroid nodule with high suspicious malignant features on US. Because FNA for this lesion can cause a disaster, special care should be taken in the case of the thyroid nodule with air foci and morphologic changeability, in the uppermost part of left thyroid gland.
[fig] Figure 3: A laryngoscopy obtained 1 year later depicts a fistulous opening (white arrow) at the left pyriform sinus. [/fig]
[fig] Figure 1: A longitudinal US image (A) shows a nodule (arrowheads) with mild hypoechogenicity, irregular shape, nonparallel orientation, ill-defined margin, and bright echogenic spots without acoustic shadowing in the uppermost part of left thyroid gland. On longitudinal US images (B) obtained 7 months later, there are some changes in the shape of the nodule (arrowheads) and in the number of its internal bright echogenic foci. On chest CT scan (C), there is a 0.2 cm air bubble (white arrow) within a 1.2 cm low-attenuating nodule at the uppermost part of left thyroid gland. CT = computed tomography, US = ultrasound. [/fig]
[fig] Figure 2: Three days after the US-guided fine needle aspiration, CT images depict an abscess (arrowheads) involving left upper thyroid gland, adjacent perithyroid tissue, and retropharyngeal space along with diffuse swelling of the hypopharynx and supraglottic larynx. CT = computed tomography, US = ultrasound. [/fig]
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Dissecting Functions of the Conserved Oligomeric Golgi Tethering Complex Using a Cell-Free Assay
Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting.Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.
Vesicle transport is responsible for protein sorting within the eukaryotic secretory pathway. It can be divided into four principal stages: formation of the vesicle (budding); movement of the vesicle to the target membrane (transport); loose attachment of the vesicle to the target (tethering) and merger of the vesicle and target membranes (fusion). Of these stages, budding and fusion are the best characterized. In both cases, detailed mechanistic characterization has been greatly facilitated by the development of cell-free reconstitutions in the form of budding [bib_ref] Functional reconstitution of COPI coat assembly and disassembly using chemically defined components, Reinhard [/bib_ref] and fusion [bib_ref] Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity, Brandhorst [/bib_ref] assays. Conversely, the lack of cell-free assays for other steps in the transport pathway has caused our mechanistic understanding of these processes to lag behind. In particular, we know very little about the molecular details of vesicle tethering. Earlier efforts for reconstituting the whole of vesicle transport (5-7) used an enzymatic reaction in the lumen of the target organelle to indicate successful cargo delivery. This assay has the disadvantage of monitoring a step far downstream of vesicle tethering. The large number of steps between the actual tethering process and the assay readout means that subtle changes in tethering efficiency, which have to be monitored for a detailed mechanistic description of the process, will likely go unnoticed in this assay. Indeed, only one study has used it for the mechanistic dissection of the vesicle targeting process in close to 30 years [bib_ref] Sequential involvement of p115, SNAREs, and Rab proteins in intra-Golgi protein transport, Gmachl [/bib_ref]. More recently developed assays using fluorescent labelling of membranes do not reconstitute vesicle transport per se, but rather the transport and fusion of whole organelles [bib_ref] Reconstitution of Rab-and SNARE-dependent membrane fusion by synthetic endosomes, Ohya [/bib_ref] , such as vacuoles or endosomes, and therefore do not recapitulate the sorting aspect of vesicle-based transport.
The posttranslational modification of secreted proteins is compartmentalized into several cisternae at the Golgi [bib_ref] Early and late functions associated with the Golgi apparatus reside in distinct..., Dunphy [/bib_ref] , and the enzymes performing the modifications use different retrograde transport vesicles to be sorted into their appropriate locations [bib_ref] Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport, Harris [/bib_ref]. The Golgi apparatus is therefore ideally suited for reconstituting vesicle transport and especially vesicle-based sorting [bib_ref] Reconstitution of the transport of protein between successive compartments of the Golgi..., Balch [/bib_ref]. Isolated Golgi membranes retain their transport activity [bib_ref] Reconstitution of the transport of protein between successive compartments of the Golgi..., Balch [/bib_ref] , and can be used in conjunction with transport vesicles to reconstitute the retrograde vesicular sorting of resident Golgi enzymes [bib_ref] Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport, Love [/bib_ref]. The transport of these retrograde vesicles depends on several transport factors, with the conserved oligomeric Golgi (COG) complex playing a central role [bib_ref] Characterization of a mammalian Golgi-localized protein complex, COG, that is required for..., Ungar [/bib_ref] [bib_ref] Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells, Zolov [/bib_ref]. COG is a hetero-octamer that combinatorially interacts with several Rab-type small GTPases and coiled coil tethers of the golgin family to accomplish vesicle tethering [bib_ref] Molecular insights into vesicle tethering at the Golgi by the Conserved Oligomeric..., Miller [/bib_ref]. It is therefore not surprising that COG defects cause aberrant glycosylation [bib_ref] Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in..., Kingsley [/bib_ref] because of the missorting of glycosylation enzymes [bib_ref] The COG and COPI complexes interact to control the abundance of GEARs:..., Oka [/bib_ref].
A large proportion of the inherited glycosylation defects in humans are classed as congenital disorders of glycosylation (CDGs). CDGs are rare because they are very difficult to diagnose based on clinical symptoms alone, yet the cases diagnosed so far have arisen from defects in over 30 different genes, including genes involved in Golgi organization and trafficking [bib_ref] Golgi glycosylation and human inherited diseases, Freeze [/bib_ref] [bib_ref] Congenital disorders of glycosylation (CDG): it's (nearly) all in it!, Jaeken [/bib_ref]. Because of a lack of suitable cellular assays, the defective gene in novel CDG cases is difficult to pin down, especially when Golgi organization rather than a specific glycosylation reaction is affected. The first described CDG patients with COG defects were siblings with a Cog7 defect [bib_ref] Mutation of the COG complex subunit gene COG7 causes a lethal congenital..., Wu [/bib_ref] , and since then CDG-causing mutations in six of the eight COG subunits (COGs 1-8) have been described [bib_ref] Deficiencies in subunits of the Conserved Oligomeric Golgi (COG) complex define a..., Zeevaert [/bib_ref]. The most severe of these are the aforementioned Cog7 and a Cog6 mutation [bib_ref] Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric..., Luebbehusen [/bib_ref] , both of which result in death within the first few months after birth. Most of the patient-derived mutations fall into the Cog5-8 part of the complex, also known as lobe B. Lobe B has been implicated in mediating the vesicular sorting of trans-Golgi enzymes [bib_ref] Differential effects of lobe A and lobe B of the Conserved Oligomeric..., Peanne [/bib_ref] [bib_ref] COG complex specifically regulates the maintenance of Golgi glycosylation machinery, Pokrovskaya [/bib_ref] , likely by its functional interactions with the tethering and fusion machinery in this Golgi region [bib_ref] COG complexes forms spatial landmarks for distinct SNARE complexes, Willett [/bib_ref]. Lobe A (Cog1-4), in contrast, plays a role in Golgi organization and cis-Golgi sorting [bib_ref] Differential effects of lobe A and lobe B of the Conserved Oligomeric..., Peanne [/bib_ref] [bib_ref] COG complexes forms spatial landmarks for distinct SNARE complexes, Willett [/bib_ref] , yet its potential contribution to trans-Golgi vesicular sorting has not been elucidated.
Here, we use the recycling of the late-Golgi enzyme β-1,4-galactosyltransferase I (GalT) [bib_ref] Mapping the distribution of Golgi enzymes involved in the construction of complex..., Rabouille [/bib_ref] as the basis for the cell-free reconstitution of vesicle transport. Our system can be used to dissect the role of COG's different parts in trans-Golgi vesicle tethering. The data imply that lobe A is inhibitory for lobe B-mediated late-Golgi vesicle targeting. Moreover, the assay shows sensitivity to a novel patientderived mutation opening up the possibility of its use for the cellular confirmation of CDG mutations that are defective in Golgi-enzyme trafficking.
# Results
## Development of the cell-free assay
While cisternal maturation carries secretory cargo forward through the Golgi stack, vesicle trafficking is required for the recycling of glycosylation enzymes to their steady-state locations [bib_ref] A cisternal maturation mechanism can explain the asymmetry of the Golgi stack, Glick [/bib_ref]. Thus, vesicles carrying a specific enzyme are targeted to the cisterna housing that enzyme. We used this property to design a cell-free reconstitution of vesicle transport that could be used to investigate the details of vesicle tethering. The enzyme GalT was fluorescently labelled to monitor the locations of both its vesicular carrier and target compartment in vitro [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref]. Two stable HEK293 cell lines were generated, expressing fulllength GalT tagged with either eCFP or eYFP. Confocal microscopy [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref] and data not shown) was used to confirm that the C-terminal fluorescent tags did not perturb the late-Golgi localization of GalT. While tagged GalT colocalized well with the trans-Golgi marker TGN46, it consistently localized right next to the cis-Golgi marker GM130 [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref]. This shows that fluorescently tagged GalT localizes at the trans-Golgi as does the endogenous protein [bib_ref] Mapping the distribution of Golgi enzymes involved in the construction of complex..., Rabouille [/bib_ref]. Golgi membranes isolated by gentle cell disruption followed by sucrose gradient floatation (5) yielded 0.5-1.5 μm large membrane particles [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref]. Concurrently, vesicles isolated by Optiprep gradient floatation [bib_ref] Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport, Love [/bib_ref] A) Schematic view of the assay in which Golgi and vesicle fractions are isolated from HEK293 cells stably expressing CFP-tagged (magenta) or YFP-tagged (green) GalT. Following their coincubation under assay conditions, colocalization is determined using fluorescence microscopy. B) Confocal fluorescent micrographs of stable GalT-CFP (green)-expressing HEK293 cells immunostained for TGN46 (left, red) or GM130 (right, red). Blue in the merge image shows DAPI staining, scale bar is 2 μm. C and D) Negatively stained electron micrographs of Golgi (C) and vesicle (D) fractions. Scale bars are 500 nm (C) and 200 nm (D). E) Immunoblot against Sec22b of equivalent Golgi and vesicle amounts. The two lanes on the right contain 2.5-fold more membrane material than the left ones. F) Typical IMAGEJ-based data analysis workflow after image acquisition. Following noise reduction and background subtraction, binary images were overlaid and the particles containing at least one colocalizing pixel exported to be counted. For details, see Materials and Methods. Scale bar, 10 μm.
1D), as expected. Isolated Golgi fractions contained 2.20 ± 0.51 x 10 7 , whereas vesicle fractions contained 2.11 ± 0.25 x 10 8 (n = 4 for both) fluorescent particles per microgram of protein. The relative amount of the Golgivesicle marker Sec22b [bib_ref] Quantitative proteomics analysis of the secretory pathway, Gilchrist [/bib_ref] was significantly enriched in vesicles compared with Golgi membranes, demonstrating that the isolated vesicles were not simply small Golgi fragments [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref].
In a typical 50-μL assay, Golgi and vesicle membranes containing approximately 7 and 0.7 μg protein, respectively, were used to ensure a good number of fluorescent particles during imaging. Membranes were mixed with cytosol and an energy regeneration system (5) before incubation at 37 - C for 40 min. Control reactions were kept on ice, a temperature shown to inhibit membrane transport at a stage preceding vesicle tethering [bib_ref] SNARE function is not involved in early endosome docking, Geumann [/bib_ref]. An aliquot of each sample was then imaged using an emCCD camera, and the images processed to remove noise and overlay the two different fluorescent channels [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref]. Counting of the colocalized Golgi and vesicle particles gives a measure of transport activity in the assay. For each assay, 10-15 different fields of view were imaged per sample, which usually resulted in well over 1000 counted particles for both Golgi and vesicles. At 0 - C the colocalizing particles could make up between 0.5 and 2% of the total, whereas at 37 - C these values ranged from 2 to about 7%. Activity is expressed as the ratio of the 37 - C and 0 - C results, and yields 3.16 ± 0.33 [standard error of the mean (SEM) for n = 8] for a full assay. This shows that physiological incubation in the presence of energy and cytosol resulted in a threefold increase of Golgi-vesicle colocalization when compared with the identical mix incubated on ice.
## Physiological relevance of the cell-free reconstitution
We next performed a series of control experiments to test whether the assay reconstitutes physiological membrane transport. When only the protease inhibitor Pefabloc was present during the assay, activity was unaffected, whereas incubation with the non-specific protease proteinase K for 30 min prior to inhibitor protection reduced activity to background levels [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref]. This implies that transport in the assay depends on one or more intact proteins. It has been shown previously that energy is necessary for in vitro vesicle transport within the Golgi (13). When energy was omitted from our assay, activity fell to background levels [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref]. Similarly, vesicle transport has been shown to require cytosolic proteins such as NEM-sensitive factor (NSF) and α-SNAP, particularly when the membranes are stripped of essential components by treatments such as high-salt washes [bib_ref] Purification of three related peripheral membrane proteins needed for vesicular transport, Clary [/bib_ref]. We found that activity became cytosol dependent when a concentrated vesicle stock was diluted at least 450-fold into the assay mix, with increasing amounts of cytosol leading to a proportional increase in activity [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref]. Salt washing of vesicle membranes also caused cytosol dependence, although in this case the total activity was lower (data not shown).
Membrane transport is critically dependent on the divalent cations Mg 2+ and Ca 2+ . Mg 2+ is needed for the activity of nucleotide-binding proteins such as Rab GTPases (31), whereas a local increase in Ca 2+ concentration is essential for the fusion of intra-Golgi transport vesicles [bib_ref] Regulation of intra-Golgi membrane transport by calcium, Porat [/bib_ref]. The generic chelator ethylenediaminetetraacetic acid (EDTA) potently inhibited activity [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref] , showing that at least one cation is essential for activity in this assay. In contrast, the chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N ,Ntetraacetic acid (BAPTA), which sequesters Ca 2+ but not Mg 2+ , did not significantly affect activity [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref]. This is in line with the need for Mg 2+ during Rab function at the tethering stage, and implies that the assay may indeed reconstitute tethering rather than fusion, because the latter would need Ca 2+ . To further investigate this point, the effect of RabGDI on activity was tested. This protein inhibits Rab GTPases by sequestering them in the cytosol. RabGDI was able to suppress activity in a dose-dependent manner [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref] , further implicating the sensitivity of the assay to the vesicle tethering step.
## Dissection of tethering functions using the new assay
Given that the assay satisfied several criteria for the physiological reconstitution of vesicle tethering, we wondered whether it could be used to assess the function of tethering factors. The COG complex was chosen for these investigations, because it is needed for all retrograde vesicle tethering reactions at the Golgi [bib_ref] Molecular insights into vesicle tethering at the Golgi by the Conserved Oligomeric..., Miller [/bib_ref]. Fibroblasts derived from Cog6-and Cog7-deficient patients who have defects in terminal glycosylation reactions including galactosylation [bib_ref] Mutation of the COG complex subunit gene COG7 causes a lethal congenital..., Wu [/bib_ref] [bib_ref] Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric..., Luebbehusen [/bib_ref] were first used. When cytosol isolated from Cog6-and Cog7-deficient fibroblasts was compared to cytosol from healthy human fibroblasts, transport activity was reduced to background levels [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref]. The activity could be restored by addition of a highly enriched COG fraction purified from bovine brain to the Cog6-deficient cytosol [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref] , third and fourth bars). Thus, the assay can indeed assess COG function, and is therefore suitable for the ex vivo analysis of COG-dependent CDG defects. To expand the potential usefulness of the assay, a hitherto uncharacterized CDG case, CDG-X, was also analysed. Serum from this patient displayed an abnormal mass spectrometric transferrin profile, including both sialylation and galactosylation defects, which was consistent with a possible defect in Golgi trafficking. Yet, no mutation in COG was found in this patient (H.H. Freeze, unpublished data). The cytosol derived from this patient lacked transport activity [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref] , last bar), demonstrating that our assay is also sensitive to a CDG-derived defect that is independent of COG. The experiments using Cog6-and Cog7-deficient cytosol suggest that lobe B of the COG complex is necessary for the tethering of GalT-containing vesicles. The two lobes have been suggested to play differential roles in the targeting of early-and late-Golgi retrograde transport vesicles, with lobe A playing a more prominent role at the early Golgi and lobe B playing a more prominent role at the late Golgi [bib_ref] Differential effects of lobe A and lobe B of the Conserved Oligomeric..., Peanne [/bib_ref] [bib_ref] COG complex specifically regulates the maintenance of Golgi glycosylation machinery, Pokrovskaya [/bib_ref] [bib_ref] COG complexes forms spatial landmarks for distinct SNARE complexes, Willett [/bib_ref]. Lobe A mutants were therefore also tested for their effect on assay activity using the Chinese hamster ovary (CHO) cell lines ldlB, which is Cog1 deficient,
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Traffic 2014; 15: 12-21
Cell-Free Vesicle Transport Assay Escherichia coli cytosol was used in the control without cytosol. SEM calculated for no cytosol, COG6 and COG7 n = 4, CDG-X n = 3 and complemented COG6 and human fibroblast n = 2. See [fig_ref] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker [/fig_ref] , Supporting Information for the individual assays used for calculating the ratios for the COG7-deficient cytosol as an example. B) Transport activity in the presence of cytosol isolated from COG mutant CHO cell lines. ldlB cells (horizontal stripes) are COG1 deficient, whereas ldlC cells (slanted stripes) are COG2 deficient. All details as in 'A', activities are shown in ratio to WT CHO cytosol. Error bars represent SEM for n = 4. Although E. coli cytosol was used as a protein mix in 'A' to suppress non-specific membrane aggregation, we found that omission of this did not cause a difference, and therefore conducted some of the experiments in 'B' without this additive.
and ldlC, which is Cog2 deficient [bib_ref] Characterization of a mammalian Golgi-localized protein complex, COG, that is required for..., Ungar [/bib_ref]. Although the phenotypes of these cells are nearly identical [bib_ref] Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in..., Kingsley [/bib_ref] , ldlBand ldlC-derived cytosols behaved differently in the assay. ldlC cytosol supported assay activity consistently above the level of wild type (WT), whereas ldlB cytosol failed to support transport [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref]. The primary difference between ldlB and ldlC cytosol is the ratio of lobe B and the bulk of lobe A. In the ldlC cytosol lobe B is in excess, as lobe A is absent, whereas in ldlB cytosol the Cog2-4 trimer of lobe A is in excess [bib_ref] Genetic analysis of the subunit organization and function of the COG complex:..., Oka [/bib_ref]. Thus, it is tempting to speculate that this novel assay could uncover a potential new inhibitory role for lobe A in trans-Golgi transport that could not be detected in previous in vivo studies.
# Discussion
This study describes the development of a new cellfree assay reconstituting intra-Golgi transport. By several criteria, our assay reconstitutes a physiological process. First, as protease treatment eliminates activity, it is protein-dependent. This finding argues that activity does not arise from simple membrane aggregation. Activity also depends on physiological temperatures, as would be expected from a process requiring membrane fluidity, such as membrane fusion or budding, but not necessarily from membrane tethering. Although synthetic liposomes containing SNARE proteins are seen to associate at 0 - C (34), endosomes tether to each other only at physiological temperatures [bib_ref] SNARE function is not involved in early endosome docking, Geumann [/bib_ref]. A further requirement for both endosomal transport and intra-Golgi transport in vitro is an energy source [bib_ref] Coupled ER to Golgi transport reconstituted with purified cytosolic proteins, Barlowe [/bib_ref] [bib_ref] SNARE function is not involved in early endosome docking, Geumann [/bib_ref] [bib_ref] Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport, Love [/bib_ref] , which is likewise required for activity in our assay. In addition, the universal need for Rab GTPases in vesicle transport could also be recapitulated by inhibition of the activity through treatment with RabGDI.
The classic reconstitution of intra-Golgi transport relies on the enzymatic processing potential of the organelle by using the incorporation of radiolabelled sugar into a model glycoprotein (5). The new assay described here provides several advantages. First, while an enzymatic readout depends on a number of factors including the efficiency of the enzymatic reaction and the completion of other glycosylation reactions that are a prerequisite for label incorporation [bib_ref] Compartmental organization of the Golgi stack, Dunphy [/bib_ref] , the new assay provides a more explicit readout of tethering without a coupled enzymatic step. Successful tethering results in colocalization of the differently coloured markers allowing direct visualization. Therefore, this assay should be more useful in elucidating molecular mechanistic details of tethering, as we have started to do with the COG complex. Second, the fluorescent readout informs solely on the specific transport step involving the labelled glycosylation enzyme -in this case Traffic 2014; 15: 12-21
GalT. Conversely, the classical assay is similarly sensitive to the perturbation of all SNAREs throughout the Golgi stack without preference for a given intra-Golgi vesicle transport step [bib_ref] Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi..., Volchuk [/bib_ref]. It should be straightforward in the future to generate fluorescently marked enzymes residing in medial and cis-Golgi compartments to study the different vesicle targeting requirements throughout the Golgi stack.
The usefulness of the assay is highlighted by its ability to start dissecting functions of the two lobes of COG that so far could not be directly tested. COG has been shown to stimulate the classical Golgi-transport assay, and antibodies against the lobe B subunit Cog5 inhibited the same [bib_ref] Purification and characterization of a novel 13S hetero-oligomeric protein complex that stimulates..., Walter [/bib_ref]. Because of the exquisite sensitivity to a specific trans-Golgi transport step, the fluorescence-based assay described here is capable of further dissecting COG's involvement in intra-Golgi retrograde transport. Cytosol containing reduced levels of lobe B, derived either from mutant CHO cells or from patients, cannot sustain transport activity when lobe A levels are unchanged [bib_ref] Mutation of the COG complex subunit gene COG7 causes a lethal congenital..., Wu [/bib_ref] [bib_ref] Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric..., Luebbehusen [/bib_ref] [bib_ref] Genetic analysis of the subunit organization and function of the COG complex:..., Oka [/bib_ref]. However, the same amount of lobe B in ldlC cells is perfectly competent for transport, albeit its level is still only 10% of the WT steady-state level, when lobe A is eliminated [bib_ref] Genetic analysis of the subunit organization and function of the COG complex:..., Oka [/bib_ref]. The property of lobe B to promote transport by itself at the trans-Golgi is consistent with previous in vivo reports of lobe B function [bib_ref] Differential effects of lobe A and lobe B of the Conserved Oligomeric..., Peanne [/bib_ref] [bib_ref] COG complexes forms spatial landmarks for distinct SNARE complexes, Willett [/bib_ref]. Yet, a possible inhibitory effect of lobe A is a novel suggestion for COG function, which will have to be further tested in the future.
The sensitivity of the assay to trans-Golgi transport, which relies on the choice of GalT as a marker, is essential for the shown functional dissection of COG. At the same time this activity opens up new possibilities for the characterization of novel CDG cases. COG-dependent CDGs can be identified by a characteristic delay of the brefeldin A-induced relocalization of enzymes to the endoplasmic reticulum [bib_ref] COG-7-deficient human fibroblasts exhibit altered recycling of Golgi proteins, Steet [/bib_ref] [bib_ref] Deficiencies in subunits of the Conserved Oligomeric Golgi (COG) complex define a..., Zeevaert [/bib_ref] [bib_ref] Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase..., Kornak [/bib_ref]. This is likely because similarly pleiotropic glycan aberrations earlier in the glycosylation pathway would not survive embryonic development. For this reason an assay that is a sensitive measure of vesicle transport at the trans-Golgi will be useful for the cellular characterization of many new CDG cases involving Golgi-organization defects, as we have started to do with the described CDG-X case. This is another advantage compared with the classical Golgi-transport assay, which used GlcNAc incorporation as the readout (5), and thus a glycosylation step that is too early for the successful investigation of most trafficking-related CDGs. For some of these CDGs though, a sialyltransferase-based assay, which can be easily constructed based on our GalTbased template, may be even more appropriate. Having a new assay opens up the possibilities of complementation and mechanistic studies that will help better understand these diseases.
# Materials and methods
# Materials
All chemicals were from Sigma and cell culture reagents from Invitrogen, unless otherwise stated. Affinity-purified rabbit α-Sec22b (used at 1:1000) was a kind gift from J. Hay (U. Montana). All procedures except for the appropriate assay incubations were performed at 4 - C or on ice, unless otherwise stated. Borosilicate microscope slides and cover slips were from Menzel Gläser, and were cleaned before use by soaking in several changes of 3% decon90 (Decon Laboratories Limited) for a day followed by thorough rinsing in water and drying in a 60 - C oven. Five-microlitre dry volume of 5-μm silica beads (Bangs Laboratories) was suspended in 500 μL of 150 mM KCl, 10 mM HEPES, pH 7.2, by vigorous vortexing and 30-second agitation in a sonicating water bath. Beads were used at a density to give one to two beads per field of view in the assay.
## Plasmids
Full-length GalT cDNA (kind gift from M. Fukuda) was cloned into a pCR3.1+ vector modified to contain an HA or myc tag followed by a seven-amino acid linker and an eCFP or eYFP tag, all downstream of the GalT gene.
## Cell culture
HEK293 cells and human-derived fibroblasts were cultured in DMEM supplemented with 10% FBS and 2 mM GlutaMAX-I. HEK293 cell clones stably expressing GalT-XFP constructs were selected in complete media in the presence of 0.8 mg/mL Geneticin. WT and mutant CHO cells were cultured in Ham's F-12 Nutrient Mixture supplemented with 5% FBS and 2 mM GlutaMAX-I.
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## Cell-free vesicle transport assay
## Isolation of membranes and cytosol
Golgi membranes were isolated as described (5) Recombinant histidine-tagged RabGDI was purified using Ni-NTA followed by MonoQ ion-exchange chromatography as described [bib_ref] Expression and purification of recombinant His6-tagged guanine nucleotide dissociation inhibitor and formation..., Peter [/bib_ref].
The purified protein was concentrated to 10 mg/mL, dialysed into 7.5 mM HEPES, 75 mM KCl, 0.5 mM DTT, pH 7.2, and filtered before use in the assay.
Partial purification of the COG complex through ammonium sulphate precipitation and butyl sepharose chromatography as described (14) was followed by TSK4000SW (Toso Haas) gel filtration chromatography (in cytosol buffer) that results in a highly enriched COG fraction [bib_ref] Characterization of a mammalian Golgi-localized protein complex, COG, that is required for..., Ungar [/bib_ref]. Enriched COG was concentrated to 1.4 mg/mL and filtered before use in the assay. This fraction is only active for about 10 days at 4 - C.
## Immunofluorescence and confocal microscopy
Cells grown on cover slips coated with poly-D-lysine (BD Biosciences) were fixed with paraformaldehyde, permeabilized and stained with α-TGN46 polyclonal antibody (Millipore, 1:600), α-GM130 monoclonal antibody (BD Biosciences, 1:500) followed by Alexa-568-conjugated goat α-rabbit (for TGN46) or α-mouse (for GM130) antibody (Life Technologies, 1:400) and DAPI . Cover slips were mounted using Aqua-Poly/Mount (Polysciences) and imaged using an inverted Zeiss confocal microscope with a 63× objective.
## Electron microscopy
Four-microlitre diluted membrane particles were placed onto a copper grid (square 200 mesh, Agar Scientific) and left for 5 min. Golgi membranes were briefly fixed with glutaraldehyde prior to dilution.
The grid was rinsed with water and then negatively stained for 5 min with 1% uranyl acetate. Stained grids were viewed in a Tecnai G2 12 BioTWIN transmission electron microscope (FEI) at 120 kV. To generate the assay mix, very small amounts of occasionally viscous stock solutions had to be handled, resulting in substantial variation in background activity. The effect of this variation could be minimized by splitting an assay after mixing to generate the 0 - C and 37 - C samples, and reporting the activity as a ratio of the 37 - C and 0 - C activities.
## Cell-free assay
Images were collected using an Evolve 512 emCCD camera (Photometrics) attached to a Zeiss Axiovert 200M fully motorized inverted microscope (Carl Zeiss) with a Zeiss Plan-Apochromat 63×/1.40 Oil DIC objective and an eCFP/eYFP filter set (Chroma Technology). Illumination was provided by an X-Cite 120Q light source (Lumen Dynamics Group). The adjustable iris of the X-Cite was opened 75%. The Traffic 2014; 15: 12-21 analogue-to-digital gain was 3 and image quality was 16-bit for all images. Before capturing an image the microscope was focussed onto the vesicles using a 100-millisecond exposure time with an EM gain of 200 and 2 × 2 binning. Vesicles were then imaged using a 5-second exposure, EM gain of 23, while Golgi with an exposure of 3 seconds and EM gain of 6, both without binning. EM gain parameters had to be slightly altered as the illumination source slightly decayed over a period of months. At least 10 fields of view were imaged for each sample.
# Data analysis
Image processing and analysis was performed using IMAGEJ. Following their conversion to 8-bit, noise in the images was reduced using the 'subtract background' routine with a rolling ball radius of 2.0 pixels and a sliding paraboloid. The threshold was then set to 'triangle dark' for Golgi images and the same for vesicle images except that the upper threshold was multiplied by 1.4. Binary images were generated from the selections, and the 'lookup table' for the binary image changed to 'red' for Golgi and 'green' for vesicles. After converting the Golgi (red) and vesicle (green) binary images to RGB they were merged, and the number of yellow (colocalizing) as well as all Golgi and vesicle particles counted. Using Excel, the number of colocalized particles was expressed as a percentage of the total number of particles (Golgi + vesicles + colocalized), which is a measure of activity in the sample. The activity of each sample measured at 37 - C was normalized to the activity of the same sample at 0 - C. For more complex series of data using cytosols from mutant cells the normalized activities are presented as a ratio of the mutant and WT activities (see [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref]. Error bars were calculated as standard error of the mean for averaged experiments carried out on separate occasions.
# Statistical analysis
For pairwise comparison of data [fig_ref] Figure 2: Testing the assay under physiological conditions [/fig_ref] a one-tailed Student's t-test (unpaired, unequal variance) was performed with the hypothesis that non-standard assay mixtures will reduce activity.
For data presented in [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref] a one-way ANOVA was used either with a Dunnett post hoc test (for [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref] using WT fibroblasts as the control or with a Tukey-Kramer post hoc test [fig_ref] Figure 3: Functional distinction between lobe A and lobe B of the COG complex [/fig_ref]. Significant differences are indicated as *p < 0.05 and **p < 0.01.
[fig] Figure 1: Setup of a cellfree assay using fluorescently tagged GalT as a marker. [/fig]
[fig] Figure 2: Testing the assay under physiological conditions. A) Assays in the presence of membranes, cytosol and energy were performed (grey bar) in the presence of the protease inhibitor Pefabloc (dotted bar) and in the presence of Pefabloc added following a 30-min incubation on ice with Proteinase K (striped bar). Error bars show SEM for n = 2. B) Assays performed in the presence of membranes and cytosol with (grey bar) or without (dotted bar) energy source. Error bars show SEM for n = 3. C) Assay activity in the presence of increasing amount of cytosol. Error bars show SD for n = 2. Note that standard assays used 57 μg cytosol to allow the addition of other factors without increasing osmolarity beyond 310 mOsm. D and E) Activity in the presence of the chelators EDTA (D, n = 6) or BAPTA (E, n = 5). Error bars represent SEM. F) Assay activity in the presence of increasing amounts of RabGDI. Full assay mix was preincubated with the indicated amount of RabGDI for 20 min on ice before addition of the energy regeneration system and normal assay incubation. Error bars represent SEM for n = 3 (0 and 25 μg RabGDI) or n = 2 (10 μg RabGDI). Note that the value of 1.0 is the activity observed in the ice-control sample. Statistical significance was determined as described in the Materials and Methods section, *p < 0.05; ns, not significant. [/fig]
[fig] Figure 3: Functional distinction between lobe A and lobe B of the COG complex. A) Transport activity with cytosol from fibroblasts of COG-deficient patients as well as a novel CDG-X patient with an unknown mutation. Because of differences in cytosol and membrane concentrations between different experiments the bars show the average of the ratio between the given cytosol's activity and WT cytosol activity. The value of 1.0 therefore represents WT activity in this graph. [/fig]
[fig] A: total of 3.8 μL of Golgi membranes, 0.8 μL vesicle membranes (diluted sevenfold with KHM in the case of the more concentrated stock) and 57 μg cytosol were mixed in a final volume of 50 μL containing 150 U/mL creatine phosphokinase, 1 mM GTP, 0.5 mM TP, 20 mM creatine phosphate, 30 mM HEPES, 30 mM KCl, 66 mM sucrose, 2 mM MgCl 2 and 0.3 mM DTT, pH 7.4. Combined with the sucrose derived from the Golgi membrane aliquot the components in the assay mix gave an osmolarity close to 310 mOsm, which is the osmolarity of tissue culture media. fter thorough mixing, 25 μL was incubated for 40 min at 37 • C, while the remaining 25 μL was kept on ice. Both samples were kept in the dark. Reactions were stopped by placing samples on ice, and following the addition of a 1-μL suspension of 5-μm beads, 3 μL of each sample was mounted using 22-mm square cover slips and then fully sealed with clear nail varnish. Slides were stored at 4 • C until imaging. [/fig]
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Efficacy and Safety of Immune Checkpoint Inhibitors in Patients with Cancer and Hepatitis B or C: A Systematic Review and Meta-Analysis
Background. Immune checkpoint inhibitors (ICIs) have changed the situation of tumor therapy in recent years. However, for security reasons, those special populations are often excluded from clinical trials, such as infected hepatitis B or hepatitis C patients. ICIs are systematically reviewed and meta-analyzed for the frst time in patients infected with hepatitis B or C in this paper. Methods. Te relevant studies were searched in PubMed, EMBASE, Cochrane Library, and Web of Science until October 2022. Trials and observational studies meeting the inclusion criteria were included. Te outcomes included the efectiveness of ICIs in patients with HBC/HCV (ORR, DCR, mOS, and mPFS), the incidence of adverse reactions, high-grade adverse reactions, and abnormal liver enzymes. At the same time, these indexes were compared with those of uninfected patients. Results. A total of 2,625 patients were enrolled, involving 1,179 patients with hepatitis (HBV or HCV). We found that ICIs showed higher ORR (25.80% vs. 18.10%) and DCR (66.22% vs. 58.74%) in patients with hepatitis B/C than those without infection. In terms of survival time, patients with hepatitis virus infection showed longer mOS (15.44 m vs. 13.30 m) but shorter mPFS (4.94 m vs. 5.01 m) than uninfected patients. As for safety data, patients with hepatitis showed a lower incidence of all-grade irAEs (68.02% vs. 70.43%) than uninfected patients, while that of 3-4 irAEs (21.27% vs. 21.79%) was similar in the two groups. However, hepatic dysfunction was more common and serious in hepatitis patients. Four HBVr and no HCVr were observed. Conclusion. According to this metaanalysis, ICIs are efective and safe for patients with hepatitis B or C, but basic liver enzymes have to be evaluated before treatment to avoid liver adverse events.
# Introduction
Over the past few years, the immune checkpoint inhibitors (ICIs) that target programmed cell death receptor-1 (PD-1), programmed cell death-ligand 1 (PDL1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have shown encouraging efcacy in a variety of tumor types such as lung cancer, melanoma, and liver cancer [bib_ref] Immune checkpoint inhibitors in cancer immunotherapy, Himmel [/bib_ref]. Although ICIs have presented a revolutionary alternative therapeutic approach for patients with tumor, its adverse reactions also pose a threat to them.
Hepatitis C virus (HCV) and hepatitis B virus (HBV) are the main causes of chronic liver disease worldwide and are the leading causes of liver cancer and overall mortality globally [bib_ref] EASL recommendations on treatment of hepatitis C: fnal update of the series, Pawlotsky [/bib_ref] [bib_ref] Hepatitis B virus: advances in prevention, diagnosis, and therapy, Nguyen [/bib_ref]. Te total infection prevalence of HBV worldwide has risen to 3.9%, which means that at least 292 million people sufer from HBV [bib_ref] Hepatitis B virus: advances in prevention, diagnosis, and therapy, Nguyen [/bib_ref]. As for HCV, about 71 million people worldwide are chronically infected with the hepatitis C virus [bib_ref] EASL recommendations on treatment of hepatitis C: fnal update of the series, Pawlotsky [/bib_ref]. Regardless of the fact that there are not a small number of people with hepatitis, these patients are often excluded from ICIs clinical trials for the associated theoretical risk of hyperimmune response or causing hepatitis B/C reactivation. Taking into account this concern, there is limited evidence of the safety and efcacy of ICIs in patients with viral hepatitis.
Te Checkmate 040 study showed that in patients with advanced hepatocellular carcinoma, the disease control and response rates of hepatitis-infected and uninfected patients were similar and hepatitis patients did not show a higher tendency of adverse reactions [bib_ref] Safety and efcacy of anti-PD-1 therapy for metastatic melanoma and non-small-cell lung..., Kothapalli [/bib_ref]. A retrospective cohort study found that 5.3% of patients had HBV reactivation, which is concerning and cannot be ignored [bib_ref] Hepatitis B virus reactivation in cancer patients with positive Hepatitis B surface..., Zhang [/bib_ref].
Terefore, it is crucial to conduct a comprehensive evaluation of ICIs' efcacy and safety in patients with hepatitis B/C for clinical decision-making. Previously published studies are usually retrospective and observational, with only a small number of intervention studies, so we conduct a meta-analysis to combine their results to obtain convincing evidence.
# Methods
## Search strategy. te preferred reporting items for
Systematic Reviews and Meta-Analyses (PRISMA) statement was followed during this meta-analysis, and it was registered in the International Prospective Register of Systematic Reviews (PROSPERO) with the number CRD42022341247.
## Study selection.
Searches were conducted in PubMed, EMBASE, the Cochrane Library, and Web of Science until October 2022, for studies pertaining to ICIs in tumor patients with hepatitis B or C. Te search terms utilized keywords and Medical Subject Headings (MeSH) terms to defne conditions such as ICIs, hepatitis B, and hepatitis C. Specifc retrieval strategies are presented in Appendix.
Literature screening was carried out by two authors (HJD and CXX) independently by reading the title, abstract, and full text to select the studies eligible for inclusion that met the following inclusion criteria: (1) the type is intervention trial (randomized or nonrandomized and controlled or noncontrolled) or observational study (cohort studies, case-control studies, or case series with more than 5 hepatitis, and prospective or retrospective), (2) participants were treated with ICIs, either alone or in combination with other treatments, (3) the study reported the efcacy of ICIs in tumor patients with hepatitis B or C, with or without safety outcomes, and (4) the study was published in English.
If an update of the same population data was given, the latest literature would be selected. If there was any disagreement during the literature screening, it would be decided after a full discussion with the third researcher (HJC).
## Data extraction.
Two authors (HJD and CXX) extracted the data independently. Incongruities would be resolved by discussions with the third author (HJC). Following are the characteristics of the extracted data in the included studies: authors, year of publication, country, study types, carcinoma, type of hepatitis, ICIs, ICIs types, number of patients, mean age, efective outcomes (mOS, mPFS, ORR, DCR), and security outcome (incidence of adverse reactions). All data were recorded in the table.
## Assessment of study quality and publication bias.
Tree tools were used to adjust for diferent types of studies in this meta-analysis. Te Cochrane risk-of-bias tool (RoB 2.0) was used for randomized controlled trials, while Risk Of Bias In Non-randomised Studies of Interventions (ROBINS I) was used for nonrandomized intervention studies. Evaluation of observational studies was done using Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). In addition, potential publication bias was assessed by Begg's test.
## Data
Analysis. All statistical analyses were conducted using STATA software (version 14.10). A value of P < 0.05 was considered statistically signifcant. Meta-analysis of rates was carried out with Freeman−Tukey double arcsine transformation (metaprop command, ftt option). In order to assess statistical heterogeneity, the I 2 statistic was used. Subsequently, considering the heterogeneity of research design and study types, the meta-analysis was mainly based on a random-efect model. Te subgroup analysis was based primarily on the location of the tumor.
# Results
## Study selection.
As a result of the retrieval strategy, we identifed 1840 records, which were subsequently reduced to 1211 after removing duplicate records. Using the title and abstract to assess eligibility, 1012 studies were excluded. Reading 199 studies in their entirety, 24 articles [bib_ref] Safety and efcacy of anti-PD-1 therapy for metastatic melanoma and non-small-cell lung..., Kothapalli [/bib_ref] were deemed to be eligible for inclusion, including 7 prospective studies and 17 retrospective studies. Finally, 2,625 patients were enrolled, involving 1,179 patients with hepatitis (HBV or HCV). In [fig_ref] Figure 1: Flow diagram of search and selection [/fig_ref] , one can see a detailed description of the retrieval process. [fig_ref] Table 1: Te pooled incidence rate of AST and ALT abnormalities [/fig_ref]. Te inclusion criteria of hepatitis patients in each study are detailed in . Among all the studies included, the efcacy of ICIs in patients with HBV or HCV was evaluated and safety was evaluated in 13 studies. Among 1,179 patients with hepatitis, 11 studies included patients with HBV, 2 studies included patients with HCV, and 11 studies included patients with HBV or HCV. In ICIs, the vast majority was anti-PD-1 or anti-PD-L1 [bib_ref] Nivolumab in patients with metastatic renal cell carcinoma and chronic hepatitis C..., Tsimafeyeu [/bib_ref] , followed by anti-CTLA-4 (4). 1 study was anti-PD-1 combined with anti-CTLA-4, and 1 study did not specify the type of ICIs. Te categories of tumors included the following: liver (15), lung [bib_ref] Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative,..., El-Khoueiry [/bib_ref] , melanoma (4), kidney (3), stomach (2), colorectum (1), biliary (1), esophagus (1), head and neck tumor (1), glioblastoma (1), and urothelium (1).
## Study characteristics
## Clinical efcacy response
## Orr.
A total of 21 studies reported ORR data , involving 815 uninfected patients and 1,061 hepatitis patients. Tis meta-analysis showed [fig_ref] Figure 2: Continued [/fig_ref] [fig_ref] Figure 1: Flow diagram of search and selection [/fig_ref]. If one study included both liver tumors and other tumors, it would be classifed into other tumors group in the subgroup analysis, which was also the same in other analysis.. . Te pooled DCR of uninfected patients and HCV and HBV patients was 58.74% (95% CI: 46.89%-70.13%), 67.83% (95% CI: 52.01%-82.14%), and 65.79% (95% CI: 58.86%-72.43%). After the merger of 658 patients with hepatitis, the pooled value was 66.22% (95% CI: 60.02%-72.20%). Te forest plot is given in [fig_ref] Figure 3: Continued43% [/fig_ref]. According to the subgroup analysis based on the categories of tumors among hepatitis patients, the pooled value of the liver group was 67.67% (95% CI: 62.30%-72.82%), which was higher than other tumors group (64.10%, 95% CI: 49.96%-77.28%) (Supplementary [fig_ref] Figure 1: Flow diagram of search and selection [/fig_ref].
## Dcr. fourteen studies included dcr parameters
## Mpfs.
Median progression-free survival (mPFS) was published by 11 studies [bib_ref] Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative,..., El-Khoueiry [/bib_ref] [bib_ref] P1.04-010 CheckMate 870: an open-label safety study of nivolumab in previously treated..., Lu [/bib_ref] [bib_ref] Clinical outcomes and prognosis factors of nivolumab plus chemotherapy or multitarget tyrosine..., Chen [/bib_ref] [bib_ref] Efcacy and safety of atezolizumab and bevacizumab in the real-world treatment of..., Himmelsbach [/bib_ref] [bib_ref] Safety and efcacy of immune checkpoint inhibitors in patients with non-small cell..., Pertejo-Fernandez [/bib_ref] [bib_ref] Nivolumab in patients with metastatic renal cell carcinoma and chronic hepatitis C..., Tsimafeyeu [/bib_ref] [bib_ref] Safety and efcacy of anti-PD-1 inhibitors in Chinese patients with advanced lung..., Xu [/bib_ref] [bib_ref] Association of hepatitis B virus infection status with outcomes of non-small cell..., Zhang [/bib_ref] [bib_ref] Hepatitis B virus infection does not afect the clinical outcome of anti-programmed..., Zhong [/bib_ref] [bib_ref] Pembrolizumab (PEM) plus granulocyte macrophage colony stimulating factor (GM-CSF) in advanced biliary..., Kelley [/bib_ref] [bib_ref] Comparison of efectiveness and safety of camrelizumab between HBV-related and non-B, non-C..., Liu [/bib_ref]. Te pooled mPFS [fig_ref] Figure 4: Forest plots depicting pooled mPFS of [/fig_ref] [fig_ref] Figure 1: Flow diagram of search and selection [/fig_ref].
## Mos.
Median overall survival (mOS) was published by 12 studies . According to the meta-analysis, the pooled mOS was 13.
## Adverse events.
Te incidence of immune-related adverse events (irAEs) in hepatitis patients and uninfected patients was further combined. A total of 13 studies reported irAEs . irAEs were classifed into all grades and grades 3-4. Te highest pooled value of all-grade irAEs was HCV patients (71.53%, 95% CI: 49.66%-89.58%), followed by uninfected patients (70.43%, 95% CI: 51.84%-86.31%), HBV patients (68.33%, 95% CI: 54.78%-80.55%), and hepatitis patients (68.02%, 95% CI: 57.37%-77.87%). Forest plots are presented in . Te pooled value of the incidence of grade 3-4 irAEs was, respectively, 21.79% (95% CI: 10.48%-35.43%), 32.93% (95% CI: 23.05%-43.53%), 15.18% (95% CI: 7.74%-24.31%), and 21.27% (95% CI: 13.89%-29.61%) in uninfected patients and those who had HCV, HBV, and hepatitis [fig_ref] Figure 7: Forest plots depicting the pooled value of grade 3-4 irAEs of [/fig_ref].
Among hepatitis patients, the pooled incidence of allgrade irAEs in the liver group (76.67%, 95% CI: 64.06%-87.45%) was higher than in other tumors group (53.34%, [fig_ref] Figure 2: Continued [/fig_ref]. We further analyzed glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST). Patients with HCV show a higher pooled incidence rate of elevated liver enzymes, both in all-grades and grade 3-4 irAEs. Detailed data are provided in [fig_ref] Table 1: Te pooled incidence rate of AST and ALT abnormalities [/fig_ref]
## Subgroup
Analysis. In addition to the location of the tumor, we carried out a subgroup analysis of ICI types. Te results suggested that the combination of ICIs showed higher ORR and DCR. At the same time, the incidence of irAEs for patients receiving combined therapy of anti-PD-1 and anti-CTLA-4 drugs was higher than that of a single drug (81.27% vs. 59.95%) and the incidence of high-grade irAEs was also higher (34.04% vs. 14.89%). Detailed results are provided in [fig_ref] Figure 8: Te [/fig_ref]. Te forest plots are shown in Supplementary [fig_ref] Figure 4: Forest plots depicting pooled mPFS of [/fig_ref].
## Quality assessment and publication bias.
We evaluated 2 randomized studies with ROB 2.0, 5 nonrandomized intervention studies with ROBINS-I, and 17 observational studies with STROBE. Only one study showed high risk, while others showed low to medium risk . Tere was a publication bias in ORR and grade 3-4 irAEs calculated by Begg's test and funnel plots, and others did not show publication bias [fig_ref] Figure 9: Begg's funnel plots [/fig_ref]. Sensitivity analysis indicated that the results were stable, and the detailed results are reported in [fig_ref] Figure 5: Forest plots depicting pooled mOS of [/fig_ref]
# Discussion
Tere has been no systematic review or meta-analysis that evaluated ICIs' efcacy and safety in patients with hepatitis B or C based on the available studies. In our study, we found that ICIs showed higher ORR (25.80% vs. [bib_ref] Abstract 3230: safety and efcacy of immune checkpoint inhibitors (ICIs) in patients..., Shah [/bib_ref] Journal of Oncology conventional T cells. CTLA-4 is homologous to T-cell costimulatory protein CD28 and shares two ligands, namely, CD80 and CD86. Te interaction between ligands and CTLA-4 is helpful to inhibit T-cell response [bib_ref] CTLA-4: a moving target in immunotherapy, Rowshanravan [/bib_ref]. ICIs can restore T-cell function by blocking the binding of PD-1 or CTLA-4 to ligands, thus achieving the purpose of tumor therapy. However, in patients with hepatitis B/C, this situation becomes complicated.
## Journal of oncology
Tere has been evidence that PD-1 was signifcantly overexpressed on total and HCV-specifc CD8 cytotoxic T lymphocytes (CTLs) in the liver and peripheral blood of patients with persistent HCV infection [bib_ref] Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virusspecifc CD8+..., Golden-Mason [/bib_ref]. Similarly, HBV-specifc T cells in the peripheral blood of patients with chronic HBV infection also express high levels of PD-1 [bib_ref] Characterization of hepatitis B virus (HBV)-specifc T-cell dysfunction in chronic HBV infection, Boni [/bib_ref]. In addition to PD-1, the upregulation of CTLA-4 on virus-specifc T cells from chronic HBV and HCV was likewise repeatedly observed [bib_ref] Programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)..., Cho [/bib_ref]. Under the action of negative costimulatory molecules such as CTLA-4 and PD-1, specifc T-cell dysfunction occurs in patients with persistent hepatitis B or C infection. Terefore, while blocking this process to inhibit tumors, ICIs may also reverse T-cell depletion and play an antiviral efect, which has been observed in some clinical studies. In addition, Han found that serum-soluble PD-L1 (sPD-L1) levels in patients with HBV-related hepatocellular carcinoma (HCC) was markedly increased, which was positively correlated with the expression of PD-L1 in tumor tissues [bib_ref] Pre-treatment serum levels of soluble programmed cell death-ligand 1 predict prognosis in..., Han [/bib_ref]. Te upregulation of PD-L1 was also observed in patients with HCV infection [bib_ref] Te PD-1/PD-L1 Axis and virus infections: a delicate balance, Schönrich [/bib_ref]. In view of the fact that the expression level of PD-L1 in tumor tissues has become a biomarker for predicting the efcacy of immunotherapy [bib_ref] Immune checkpoint inhibitors: recent progress and potential biomarkers, Darvin [/bib_ref] , it can be speculated that the high ORR and DCR of ICIs in patients with HCV/HBV are related to the high expression of PD-L1.
However, ICIs may also weaken the ability of T cells to inhibit viral hepatitis, resulting in HBV/HCV reactivation [bib_ref] Ipilimumab administration for advanced melanoma in patients with pre-existing Hepatitis B or..., Ravi [/bib_ref]. Te incidence of HBV reactivation (HBVr) and HCV reactivation (HCVr) induced by immunotherapy is not clear. Among the 878 hepatitis patients we included, 4 HBVr and no HCVr were observed [bib_ref] Correlation of HBV DNA and hepatitis B surface antigen levels with tumor..., Pan [/bib_ref] [bib_ref] Association of hepatitis B virus infection status with outcomes of non-small cell..., Zhang [/bib_ref] [bib_ref] Interaction between baseline HBV loads and the prognosis of patients with HCC..., Hu [/bib_ref] , so we thought that the probability of hepatitis reactivation caused by ICIs was relatively low. In addition to HBVr and HCVr, immunemediated hepatotoxicity constitutes one of the reasons why researchers exclude clinical trials because hepatitis patients are often accompanied by baseline damage of liver function. Immune-mediated hepatic dysfunction was found to be more common and severe in patients with hepatitis than in patients without infection, according to our study. In HBV patients, this is more prevalent than in HCV patients. Te reason for this might be that patients with HCV have a higher risk of sufering from liver damage (micronodular cirrhosis, lymphoid aggregates, damage to the bile ducts, etc.) than those with HBV [bib_ref] Histopathological study of chronic hepatitis B and C: a comparison of two..., Rozario [/bib_ref]. Meanwhile, in the subgroup analysis, the incidence of immune-mediated hepatic dysfunction was higher in those given the combination of anti-PD-1 and anti-CTLA-4, both in all grades and high grades (except the incidence of ALT increases in all grades).
Tere are certain limitations that originated from the fnite number of studies present in this meta-analysis. Te literature included in this study is dominated by observational studies. Terefore, the interpretation still needs to be further verifed by larger samples and randomized clinical trials. In addition, the majority of patients in related studies were diagnosed with liver cancer, possibly because HBV/ HCV and liver cancer are closely related, which may lead to a certain bias. In addition, due to the lack of analysis of viral hepatitis stages in many studies, there was no subgroup analysis of this factor.
# Conclusion
According to this meta-analysis, ICIs in patients with hepatitis B or C are efective and safe, but the baseline of liver enzyme should be evaluated before use, especially when multiple ICIs are used in combination. Besides, the infectious disease physician should be invited to evaluate and follow up the patients.
## Data availability
Detailed information about the original contributions to the study is included within the article/Supplementary Materials. Further data are available from the corresponding author upon request.
## Conflicts of interest
Te authors declare no conficts of interest.
# Supplementary materials
Te following supporting information can be obtained from Supplementary Materials: Appendix: search strategy; [fig_ref] Table 1: Te pooled incidence rate of AST and ALT abnormalities [/fig_ref] : basic characteristics of the included studies; : immune-related adverse reactions of the included studies; : HCV/HBV inclusion criteria of the included studies; : risk of bias assessment; [fig_ref] Figure 1: Flow diagram of search and selection [/fig_ref] : subgroup analysis of ORR, DCR, mPFS, and mOS; [fig_ref] Figure 2: Continued [/fig_ref] : subgroup analysis of all-grade irAEs and grade 3-4 irAEs; [fig_ref] Figure 3: Continued43% [/fig_ref] : the pooled incidence rate of AST, ALT abnormalities; [fig_ref] Figure 4: Forest plots depicting pooled mPFS of [/fig_ref] : the subgroup analysis of ICIs; [fig_ref] Figure 5: Forest plots depicting pooled mOS of [/fig_ref] : the sensitivity analysis.
[fig] 30: months (95% CI: 8.24-18.36), 18.29 months (95% CI: −0.61-37.18), 12.90 months (95% CI: 9.85-15.96), and 15.44 months (95% CI: 8.86-22.01) for patients who were uninfected and those who had HCV, HBV, and hepatitis (Figure 5). Te pooled mOS of liver tumors was 12.94 months (95% CI: 11.15-14.74), which is shorter than that of the other tumors group (19.37 m, 95% CI: 8.37-30.38) (Supplementary Figure 1). We also calculated the mean of mOS, with values 14.17 m (uninfected), 19.94 m (HCV), 14.67 m (HBV), and 17.17 m (hepatitis), respectively. [/fig]
[fig] Figure 1: Flow diagram of search and selection. [/fig]
[fig] Figure 2: Forest plots depicting pooled ORR of (a) uninfected patients and patients with HCV and HBV and (b) patients with hepatitis. [/fig]
[fig] Figure 3: Forest plots depicting pooled DCR of (a) uninfected patients and patients with HCV and HBV and (b) patients with hepatitis. [/fig]
[fig] Figure 4: Forest plots depicting pooled mPFS of (a) uninfected patients and patients with HCV and HBV and (b) patients with hepatitis. [/fig]
[fig] Figure 5: Forest plots depicting pooled mOS of (a) uninfected patients and patients with HCV and HBV and (b) patients with hepatitis. [/fig]
[fig] Figure 6 0: Forest plots depicting the pooled value of all-grade irAEs of (a) uninfected patients and patients with HCV and HBV and (b) patients with hepatitis. Iˆ2 = 77.0744%, p = 0.0000) Overall (Iˆ2 = 77.5423%, p = 0.0000); Heterogeneity between group: p = [/fig]
[fig] Figure 7: Forest plots depicting the pooled value of grade 3-4 irAEs of (a) uninfected patients and patients with HCV and HBV and (b) patients with hepatitis. [/fig]
[fig] Figure 8: Te [/fig]
[fig] Figure 9: Begg's funnel plots. (a) ORR. (b) DCR. (c) mOS. (d) mPFS. (e) All-grade irAEs. (f ) Grade 3-4 irAEs. [/fig]
[table] Table 1: Te pooled incidence rate of AST and ALT abnormalities. [/table]
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Propofol Modulates Early Memory Consolidation in Humans
Maintenance of memory across time is crucial for adaptive behavior. Current theories posit that the underlying consolidation process depends on stabilization of synapses and reorganization of interactions between hippocampus and neocortex. However, the temporal properties of hippocampal-neocortical network reconfiguration during consolidation are still a matter of debate. Translational research on this issue is challenged by the paucity of techniques to transiently interfere with memory in the healthy human brain. Here, we report a neuropharmacological approach with the GABA A ergic anesthetic propofol and a memory task sensitive to hippocampal dysfunction. Patients undergoing minor surgery learned word lists before injection of an anesthetic dose of propofol. Results show that administration of the drug shortly after learning (;13 min) impairs recall after awakening but spares recognition. By contrast, later administration (;105 min) has no effect. These findings suggest significant changes in memory networks very early after learning that are decisive for later recall. Propofol general anesthesia provides an experimental tool to modulate the first steps of hippocampus-mediated memory consolidation in humans.Consolidation of memories depends both on mechanisms at the synaptic and the systems level. How and when these mechanisms interact is currently unclear. Here, we have used the anesthetic drug propofol to create a transient pharmacological "lesion" of the neural substrates of memory consolidation in humans undergoing minor surgery. Our results show that there is a brief time window after learning where hippocampus-dependent memories are susceptible to GABAergic modulation with propofol. Later recall appears to depend significantly on integrity of these first steps of memory formation. We infer that there is significant rearrangement of memory networks during the first hours after learning. Propofol general anesthesia provides an experimental approach to interfere with early memory consolidation in humans.
# Introduction
A defining feature of memory is the creation of cerebral representations that bridge temporal gaps between experience and behavior. It has been known since the 19th century that memory is not a static mental image of the past but rather a dynamic and re-constructive process that alters memory traces and involves distinct neural substrates as time proceeds . The mechanisms that stabilize memories have been termed memory consolidation. A key clinical finding that has shaped the currently prevailing view on the neural substrates underlying consolidation is that some patients with lesions affecting the hippocampus show a temporally graded amnesia with relative sparing of remote memories, i.e., memories that were acquired months to years before hippocampal damage. The "standard model" posits that consolidation involves a re-distribution of memories between hippocampus and neocortical networks with a decreasing role of the hippocampus with increasing memory delays. However, the time scales addressed in patient studies of memory consolidation are not easy to reconcile with results from more recent imaging studies, showing that interactions between hippocampus and neocortex during the seconds and minutes that follow memory encoding are predictive of later recall. To account for the wide range of memory delays, it has been suggested that consolidation should be seen as a family of processes on multiple time scales that transform, stabilize and update memory traces according to contextual demands. Drawing largely from results from experimental studies in animals, it has been proposed that processes on a synaptic level at a time scale of up to some hours may provide iterative subroutines for consolidation on a systems level at much longer time scales.
It has proven difficult to provide complimentary experimental data for humans. There are virtually no studies that link clinical investigations in humans with hippocampal dysfunction and synaptic accounts of memory consolidation. An ideal patient model for the investigation of memory consolidation would consist of a transient brain lesion that acts selectively on a distinct phase of memory consolidation. However, most brain lesions are permanent and thus simultaneously affect encoding, consolidation and retrieval. Moreover, the hippocampus and surrounding structures are not accessible for current transcranial brain stimulation techniques. Modulation of long-term potentiation (LTP) by direct microstimulation of the human entorhinal cortex during memory tasks is a promising tool in this respect but limited to patients undergoing evaluation for epilepsy surgery .
Here, we have taken a new neuro-pharmacological approach on human memory consolidation. We tested whether general anesthesia with the anesthetic propofol (2,6-diisopropylphenol) interferes with memory consolidation when applied shortly after learning and whether these effects are time dependent. Propofol is a shortacting anesthetic drug that is broadly used for sedation during invasive diagnostic and surgical procedures and for sedation in intensive care units. Propofol is both an agonist on GABA A receptors and a partial antagonist on NMDA receptors. Studies in rat hippocampal slices suggest that these properties account for reduction of LTP and affect synaptic consolidation. Systemic administration of propofol immediately after learning of a location in a water maze has moreover been shown to affect consolidation of spatial memory in rats.
Since ethical constraints limit experiments with anesthetic doses of propofol in healthy volunteers, we investigated patients undergoing minor ophthalmic surgery receiving propofol as a centrally acting drug during a short general anesthesia. Subjects performed a verbal learning and memory task that has previously proven to be sensitive to hippocampal dysfunction. Verbal material was learned preoperatively at two different time points and tested postoperatively both for recall and recognition.
# Materials and methods
## Participants
We included subjects between 18 and 60 years of age without any history of neuropsychiatric disorders, hearing disorders or substance abuse. Four groups with a total of 96 subjects were tested (4 Â 24 age-matched and sexmatched subjects; 49 females;. Two groups received general anesthesia with propofol for strabismus surgery at two different timepoints after learning ("early injection" and "late injection," respectively; . In the early injection group, we aimed to act on early steps of memory consolidation immediately following learning. We thus kept the delay between end of learning and injection of propofol as short as possible. In the late injection group, it was aimed to act on a later phase of memory consolidation, i.e., clearly beyond the effects of propofol on maintenance of LTP in rat hippocampal slicesand longer than the expected duration of surgery/anesthesia in the early injection group (;60 min). We thus aimed at a delay of ;90 min between end of learning and injection of propofol. A third group consisted of healthy controls without any surgical procedure (control, no anesthesia; . A fourth group consisted of subjects undergoing local anesthesia for minor surgical procedures (control, local anesthesia; . This group was included to control for any presurgical arousal effects on our task. All subjects spoke German fluently. Subjects undergoing surgery were recruited during preparatory visits in the outpatient departments of the Charité -Universitätsmedizin Berlin at least 3 d before surgery. Control subjects were recruited with advertisements via the intranet of the Charité -Universitätsmedizin Berlin. All procedures reported in this manuscript were approved by the ethics committee of the Charité -Universitätsmedizin Berlin. All subjects gave written informed consent before participation.
## Behavioral testing
Subjects were informed that they should perform a memory task before surgery and that they would receive a short additional testing after awakening from anesthesia. Subjects Values are medians and interquartile ranges; n.a., not applicable. . Task and experimental conditions. First row, Early injection condition. Second row, Late injection condition. Third row, Control condition. Fourth row, Local anesthesia condition. In all conditions, subjects learned a list of semantically unrelated and emotionally neutral words. In the early injection condition, subjects received general anesthesia with propofol ;13 min following learning and were tested for recall and recognition about 3 h after learning. In the late injection condition, subjects received general anesthesia ;105 min after learning and were tested ;4.5 h after learning. In the control condition, subjects received no anesthesia and were tested 3 h after learning. In the local anesthesia condition, subjects received local anesthesia and were tested 3 h after learning.
were not informed about the precise structure and purpose of the task and were not informed about the necessity to maintain to-be-remembered items across anesthesia. Subjects were tested with the Verbaler Lern-und Merkfähigkeitstest (VLMT;, a German version of the widely used Auditory Verbal Learning Test (AVLT;. None of the subjects was familiar with the task. In the learning phase of this test, the examiner read a list of 15 semantically unrelated and emotionally neutral words (e.g., "drum," "coffee," "river") to the subject at a rate of one word every 2 s. After each presentation, the subject was requested to recall as many words as possible and to report all recalled words orally to the examiner. This list was presented five times to the subject and was each time recalled. After the fifth recall, a distractor list with 15 other words was presented and recalled. Afterwards, the original word list had to be recalled again. Learning took ;15-20 min. Depending on the condition, subjects then received propofol general anesthesia, local anesthesia or were free to fill the delay until testing with intermediate activities. At testing, subjects were requested to recall the original word list and to report all recalled words orally to the examiner. Then, a recognition test was given. The examiner read a list which consisted of the 15 original words, the 15 words of the distractor list and 15 new words in pseudorandom order. For each word, the subject was requested to respond with "yes" or "no" whether the word had been part of the original word list.
## Procedure
In the early injection group, subjects learned the word lists in a preparatory room adjacent to the operating theater, while being in a supine position. The time between end of learning and induction of anesthesia [median (Mdn) 13.0 min, interquartile range (IQR) 10-17] was filled with small talk with the examiner and explanatory remarks of the anesthesiologist. Then, subjects were preoxygenated with a face mask and received a bolus of propofol for induction of anesthesia (Mdn 200 mg, IQR 200-215) followed by a continuous infusion of propofol for maintenance of anesthesia (Mdn 6 mg/kg/h, IQR 6-6) and remifentanil for analgesia (Mdn 0.2 mg/kg/min, IQR 0.15-0.2). After loss of consciousness, the airway was managed with a laryngeal mask and subjects were mechanically ventilated. During anesthesia, subjects underwent surgery for ocular misalignment with recession, plication or resection of eye muscles according to established surgical standards. Anesthesia was continued for about 1 h (Mdn 58 min, IQR 53-65). After surgery, patients remained in a supine position until the end of testing. Apart from occasional communication with nurses and physicians, the postsurgical period was free of any specific activities. Postsurgical pain was treated with ibuprofen and paracetamol. Testing for delayed recall and recognition was conducted about 2 h after recovery from anesthesia (Mdn 113.5 min, IQR 106.5-128) and about 3 h after learning .
In the late injection group, subjects learned the word lists in a room on the ward, while being in a supine position. The time between end of learning and induction of anesthesia was filled with periods of rest, small talk with nurses and the examiner and explanatory remarks of the anesthesiologist (Mdn 105 min, IQR 95.25-115; U = 0, p , 0.001 difference with early injection). Subjects maintained a supine position during the entire delay between learning and anesthesia. Subjects underwent the same surgical procedure and received a comparable dose of propofol (bolus: Mdn 200 mg, IQR 155-237.5; maintenance: Mdn 6.0 mg/kg/h, IQR 6-6.75; U = 272, p = 0.723 and U = 235, p = 0.108 difference with early injection group) and remifentanil as the early injection group (Mdn 0.2 mg/kg/min, IQR 0.2-0.2; U = 218.5, p = 0.093 difference with early injection group). Duration of anesthesia and postanesthesia recovery was like in the early injection group (Mdn 56 min, IQR 46.25-64.75; Mdn 113 min, IQR 108.5-116.75; U = 251, p = 0.445 and U = 250, p = 0.433 difference with late injection group). General postsurgical management was like in the early injection group.
In the control, local anesthesia group, subjects learned the word lists in a preparatory room adjacent to the operating theater, while being in a supine position. The minutes between end of learning and local anesthesia (,10 min) were filled with small talk with the examiner and explanatory remarks of the surgeon. Depending on the surgical procedure, subjects then received local injections of lidocaine close to the region of surgery. Memory was tested after a 3-h delay (Mdn 180 min, IQR 150-180). General postsurgical management was like in the two propofol groups.
In the control, no anesthesia group, subjects learned word lists in a seated position in a room on the ward. After learning, subjects were free to walk in the hospital, but were requested to return after ;170 min. Testing was performed about 3 h after end of learning (Mdn 180 min, IQR 180-180).
## Experimental design and statistical analyses
All data obtained in this study are openly available at the Open Science Framework (osf) at https://osf.io/ 3x95n/. Data were analyzed by using IBM SPSS, version 25. Performance was described as percent correct responses in each subject. For initial learning, we analyzed the number of correctly recalled items from the original word list after presentation of the distractor list. For delayed recall, we analyzed the number of correctly recalled items from the original word list after the delay. For delayed recognition, we analyzed the number of correctly recognized items (hits) minus the number of erroneously recognized items (false alarms), thus yielding a "corrected recognition" value for each subject. In order to analyze possible subtle impairments in source memory, we further separately analyzed false alarms to items from the distractor list and false alarms to new items. Group averages are given as Mdn and IQR. Since accuracy in behavioral tests is rarely normally distributed and since Kolmogorov-Smirnov testing showed that the assumption of a normal distribution had to be rejected (p , 0.05 for at least one subject group in learning, recall, and recognition conditions), non-parametric statistical testing was used for statistical analysis. Kruskal-Wallis ANOVA was used for analysis of group differences and two-tailed Mann-Whitney tests were used for post hoc comparisons between groups. Spearman rank order correlation was used for correlation analysis. Significance was accepted at a p , 0.05 level.
# Results
After five repetitions of the original word list and presentation of the distractor list, all four groups showed similar retention of original word lists, with no significant differences between groups (x 2 (3) = 6.204, p = 0.102;. This suggests that presurgical arousal did not significantly affect initial learning of verbal stimuli. However, after the memory delay, significant group differences were found for recall of word lists (x 2 (3) = 19.459, p , 0.001;. Compared with control, no anesthesia and control, local anesthesia subjects, late injection patients showed unimpaired performance with no significant differences in recall of word lists (late injection, Mdn 93.3%, IQR 86.7-93.3; control, no anesthesia, Mdn 86.7%, IQR 81.7-93.3; control, local anesthesia, Mdn 86.7%, IQR 80.0-93.3; U = 261.0, p = 0.565 and U = 241.5, p = 0.332, respectively;. Any hangover effects of general anesthesia or the surgical procedure on recall are thus unlikely. By contrast, early injection patients showed a significant decrease in recall of word lists compared with control, no anesthesia subjects and late injection patients (early injection, Mdn 66.7%, IQR 60-85; U = 123.0, p = 0.001 difference with control, no anesthesia; U = 107.5, p , 0.001 difference with late injection;. Importantly, recall in early injection patients was also significantly different from the control, local anesthesia group (U = 129, p = 0.001 difference;. This result and the almost identical performance in both control conditions (U = 274, p = 0.767 difference;suggest that presurgical arousal or some other direct reaction to the surgical procedure did not significantly affect initial consolidation of word lists. Similar to previous observations of a differential susceptibility of delayed recall and recognition of word list learning to hippocampal dysfunction, corrected recognition of word lists did not differ significantly between groups, although a statistical trend might have been present (x 2 (3) = 7.363, p = 0.061). Comparison of corrected recognition scores shows that this trend was mainly driven by a slightly lower performance of the control, local anesthesia group rather than by subtle performance deficits in the propofol groups (late injection, Mdn 93.3%, IQR 86.7-100.0; early injection, Mdn 90%, IQR 81.7-93.3; control, no anesthesia, Mdn 93.3%, IQR 86.7-93.3; control, local anesthesia, Mdn 86.7%, IQR 80-93;. Moreover, when hit rates and false alarms were analyzed separately, no significant differences were found between groups (hit rate, x 2 (3) = 4.733, p = 0.192; false alarms to items from the distractor list, x 2 (3) = 1.626, p = 0.653; false alarms to new items, x 2 (3) = 2.926, p = 0.403).
In order to analyze the selectivity of the recall-recognition dissociation in early injection patients, we next compared difference between recall and corrected recognition in all four groups ("D-R-R"). As expected, there was a significant difference of D-R-R between groups (early injection, Mdn 20%, IQR 6.67-33.33; late injection Mdn 0%, IQR À5.0-6.67; control, no anesthesia, Mdn 0%, IQR À5.0-13.33; control, local anesthesia Mdn 0%, IQR 0-0; x 2 (3) = 25.111, p , 0.001 difference between groups). Post hoc testing further showed that there was a significant difference of D-R-R between early injection and all other three groups but not between the other three groups (early injection vs all other groups, U 146, p 0.003; all other comparisons, U ! 221, p ! 0.157). This analysis shows that the difference between recall and corrected recognition is selective for the early injection group.
Because of the clinical setting, subjects both in the early injection and late injection condition showed some variability in time between end of learning and injection of propofol (ranges: early injection, 6-21 min; late injection, 72-140 min). To more precisely infer on a possible time window for propofol effects on word list consolidation, we tested whether recall performance showed a relationship with time to injection in both groups. However, we found no significant correlation between these variables when calculated separately for both groups (early injection, r = 0.286; p = 0.175; late injection, r = À0.007; p = 0.975), thus suggesting that susceptibility of word list consolidation to propofol general anesthesia ends at some time point between 21 and 72 min following learning.
# Discussion
The findings of our study show that propofol general anesthesia interferes with declarative memory in a task that is commonly used to assess integrity of the human hippocampus. The amnesic effect of propofol general anesthesia is critically time dependent and appears to be limited to a brief time window following learning. We infer that propofol general anesthesia modulates presumably hippocampus-dependent initial steps of memory consolidation.
Since its approval at the end of the eighties of the last century, propofol has become a dominant anesthetic agent for induction and maintenance of general anesthesia, ambulatory surgical procedures and sedation in intensive care patients. Propofol has a rapid onset and is quickly eliminated. With infusions of a duration of 1 h, the contextsensitive half-time of propofol is ,10 min. Clinically, this accounts for rapid recovery times compared with other anesthetics (10-30 min). Apart from its clinical applications, propofol has increasingly been used as a recreational drug. Since soybean oil is used as a solubilizer, propofol has a milk-like appearance and has thus been nicknamed the "milk of amnesia". Although this sobriquet implies some interference of the drug with memory processes, there are surprisingly few experimental investigations of propofol effects on the neural substrates underlying memory formation.
LTP and long-term depression (LTD) of synaptic transmission are thought to represent key mechanisms underlying transformation of labile representations of perceptual input into longer-lasting memories. Recordings of EPSPs from the CA1 region of the rat hippocampus have shown that an injection of propofol transiently (,60 min) inhibits field EPSPs in CA1 and affects maintenance of LTP, if given after LTP induction. Additional experiments on rat hippocampal slices showed that propofol can also inhibit induction of LTP and that this effect can be blocked by agents that block GABA A receptors, but not by agents that block NMDA receptors. GABA A receptors are densely expressed in the hippocampus and the deep layers of the cortex where they are pivotal for learning and memory, with some isoforms being particularly important for memory formation. Pharmacological modulation of GABA A receptors has moreover been shown to affect memory consolidation-related sharp wave-ripple complexes in hippocampal networks. For example, at clinical concentrations, the anesthetic thiopental affects the incidence, rhythmicity and synchrony of sharp waves and the quantity of ripple oscillations in the CA1 region of hippocampal slices. These effects appear to be mediated by distinct subunits of GABA A receptors. In particular, a5GABA A receptors appear to reduce hippocampal excitability and may inhibit memory formation. Accordingly, stimulation of a5GABA A receptors with therapeutic concentrations of diazepam has been shown to reduce the number, duration and power of ripple oscillations and to produces a partial temporal dissociation between ripples and sharp waves. Application of high concentrations of diazepam can also reduce the frequency of sharp waves. Computational modeling of the effects of various GABAergic drugs suggests that changes in power and duration of ripple oscillations reflects altered dynamics of interneuron networks in the CA1 region of the hippocampus. Correspondingly, when propofol is systemically administered to rats immediately after learning of a location in a water maze, memory retention 24 h following learning is impaired in a dose-dependent way.
While these findings suggest that propofol should act on consolidation of human memory too, a transfer of these results on clinical settings has not been successful so far. It has been controversial whether it is possible to induce deficits in preoperatively learned material by subsequent administration of anesthetic agents. Early experiments showed that sedative doses of propofol, i.e., doses that leave subjects able to communicate and breathe spontaneously, may affect memory of visual and verbal material, when stimuli are learned and tested during a continuous infusion of the drug, with effects being largely independent of the level of sedation. Subsequent experiments with eventrelated potential recordings (ERPs) from subjects performing a continuous picture recognition task during propofol infusion showed a selective drug effect on pictures that were tested after 27 s, but not after 6 s. ERP amplitudes during recognition decreased in parallel. More recently, functional magnetic resonance imaging (fMRI) during encoding of emotional pictures and continuous propofol infusion showed suppression of hippocampal responses that correlated with the degree of memory impairment for the stimuli. While these studies make a strong point for modulation of memory-related neural activity in the human hippocampus by sedative doses of propofol, their focus was on revealing the mechanisms and the prevention of surgeryinduced posttraumatic stress disorder.
Thus, it is difficult to disentangle the relative contributions of encoding, consolidation and retrieval to the antegrade amnesia induced in these experiments.
A critical prerequisite for studies of the time course of consolidation with anesthetic agents like propofol is the induction of retrograde memory effects, i.e., effects on material that is learned before infusion of the drug and tested after discontinuation. So far, there has been no convincing evidence for anesthetic-induced retrograde amnesia. A previous study on patients with depression however successfully used electroconvulsive therapy (ECT) in deep anesthesia as an intervention to study reconsolidation of emotionally negative stories learned one week before treatment. Recall of these stories was impaired, when memory of the story was cued immediately before ECT and tested 24 h afterward. By applying the same behavioral paradigm to patients receiving sedation for endoscopy, a recent study showed that propofol at sedative doses may induce similar, albeit slightly weaker, effects on reconsolidation of emotional story contents.
Our results add significantly to previous work by showing that propofol general anesthesia can indeed exert retrograde amnesia for emotionally neutral declarative tobe-remembered items. Normal performance in the late injection condition of our study suggests that the amnesic effect of propofol general anesthesia may extend up to ;30-60 min before injection. This new finding suggests that propofol general anesthesia acts on postencoding processes that are decisive for initial consolidation and later recall. Electrophysiological signatures of early memory formation have been found in direct recordings of ERPs from the hippocampus of patients undergoing evaluation for epilepsy surgery. ERPs recorded during learning of word lists separated subsequently recalled from unrecalled words. Studies with fMRI have further shown that interactions between hippocampus and neocortex during the minutes that follow encoding of visual associative stimuli are predictive of later recall. Similarly, activity in hippocampus and caudate nucleus following stimulus offset can predict memory of audiovisual episodes (Ben-Yakov and. Despite the heterogeneity of approaches, these and related studies therefore provide evidence for a pivotal role of the hippocampus for the very first steps of declarative memory consolidation.
It must be conceded that clinical propofol anesthesia is always administered in the context of invasive procedures, mostly in combination with intravenous opioid analgesia. Whether this might have contributed to the deficits in the early injection condition of our study remains elusive. A recent review concluded that opioid signaling is not required for, but can sometimes act to constrain, hippocampus-dependent memory. Likewise, it is possible that arousal before a surgical procedure may influence memory consolidation. We deem this factor not to be decisive, at least for the task in our study, as surgery in local anesthesia did not produce a memory impairment.
We are therefore confident that the effects on early consolidation observed here are mainly attributable to pharmacological actions of propofol.
One reason why previous pharmacological studies did not reveal the same retrograde effects observed here may be lower serum concentrations of propofol in experiments with sedative doses of propofol in cooperative and spontaneously breathing normal subjects. In a study on reconsolidation of emotional story contents, retrograde propofol effects on reactivated memory of stories before propofol sedation were observed when subjects were tested 24 h after anesthesia, but not when tested after a delay of up to 106 min (Galarza. Compared with this study, the anesthetic doses applied to our patients are significantly higher. At least in animal experiments, propofol effects on LTP are critically dose dependent. fMRI studies on pain processing at different propofol concentrations have moreover shown that connectivity changes within cerebral large-scale networks are critically dose dependent. A further point may be a differential sensitivity of the mnemonic representations across tasks to GABA A ergic drugs and to altered neuronal activity in distinct brain regions. The task used here has proven to be a reliable marker of hippocampal integrity, particularly for its recall component. Thus, although propofol general anesthesia is likely to act on a wide network of brain regions, the pattern of results is most consistent with modulation of hippocampal neural activity. Predominant effects on recall in our experiments and the abovementioned reconsolidation studyfurther show that application of propofol after memory encoding does not lead to an unselective impairment but rather tends to affect some memory domains more than others, presumably sparing less hippocampusdependent routes of memory consolidation.
Which level of consolidation has been modulated in our experiment? The time window identified in our study is suggestive of propofol actions on synaptic memory consolidation. Systems and synaptic levels of memory consolidation have traditionally been considered separately and with distinct experimental approaches. It is only recently that the interaction between these two levels has been discussed within a common conceptual framework. Current models of synaptic consolidation propose mechanisms by which synaptic plasticity impacts on memory-guided behavior at various timescales, including the short delays addressed here. Complimentary data from humans have been scarce so far. While it is of course not possible to infer from our behavioral results on modulation of synaptic and/or systems levels of memory consolidation, combination of the neuropharmacological approach of our study with imaging techniques may provide a way to link synaptic and systems consolidation in humans.
# Conclusion
The results of our study show that propofol general anesthesia may create a transient pharmacological "lesion" of the neural substrates supporting early memory consolidation. The lack of effect beyond this time window further suggests rapid subsequent reconfiguration of hippocampus-dependent memory networks. While our approach is spatially not selective, it nevertheless circumvents restrictions of traditional patient-based approaches and makes the initial steps of memory consolidation accessible to experimental modulation, without affecting encoding or memory retrieval. Importantly, it allows for the study of memory consolidation in human subjects with brains that are unaltered by neuropsychiatric disorders or brain surgery. Combination of propofol general anesthesia with subsequent functional imaging of memory replay in the hippocampus may ultimately reveal how transient modulation of GABAergic neurotransmission affects mechanisms of memory consolidation in humans. |
Stimuli-responsive controlled-release system using quadruplex DNA-capped silica nanocontainers
[fig] Figure S2, Figure S3, Figure S4, Figure S5, Figure S6 3, Figure S7: X-ray diffraction pattern of MSN. Thermogravimetric analysis of the samples a) MSN-NH2, b) MSN-COOH, c) MSN-DNA. TGA showed ca. 2.2% more weight loss for MSN-DNA compared with MSN-COOH, indicating that the grafting density of DNA is about 3.49 µmol/g SiO2. The result agreed well with the absorption results obtained after Release profiles of rhodamine B dye from (a) MSN-DNA and (Release profiles of rhodamine B dye from unfunctionalized MCM-The fluorescence profiles of 2 µM of rhodamine B at pH 5.0 and 8.0. Structures of CPM and the thiol derivative of CPM. [/fig]
[fig] Figure S8, Figure S9: (A) Fluorescence images of CPM-loaded MSN-cDNA: (a-b) without thiol at pH 5.0 and 8.0, (c-d) with thiol at pH 5.0 and 8.0. The control experiments indicated that the involvement of i-motif DNA was critical for the function of nanoreactor. (B) Fluorescence images of CPM-loaded MSN-cDNA before (a) and after (b) centrifugation. The result suggested that the thiol indeed got into the MSN. Fluorescence spectra of CPM-loaded unfunctionalized MSN after reaction with thiol in citrate buffer at pH 5.0 (a) and pH 8.0 (b). [/fig]
[fig] Figure S10: Fluorescence spectra of thiol-derivatized CPM in citrate buffer at pH 5.0 (a) and pH 8.0 (b). [/fig]
|
Emotional Intelligence as a Protective Factor against Victimization in School Bullying
Previous research has identified the main predictors of being a victim of school bullying. This study focused on the phenomenon of school bullying and its relationship with self-perceived emotional intelligence. The main aim was to analyze the mediating effect of emotional attention, clarity, and repair in relation to school victimization. The sample was made up of 822 primary school pupils from 10 public schools. Data were collected through self-reports, exploring the profile of victims of school bullying, and the dimensions of self-perceived emotional intelligence (PEI). A multivariate analysis and multinomial regression showed a relationship between the two variables; the probability of being a victim of school bullying was 5.14 times higher among pupils with low clarity, 2.72 times higher among pupils with low repair, and 2.62 times higher among pupils with excessive attention. The results demonstrated that the better their emotional regulation and understanding, the less likely pupils are to be victims of school bullying. This confirmed that adequate emotional attention and excellent emotional clarity and repair are protective factors against victimization.
# Introduction
Bullying is acknowledged around the world as a serious problem in educational settings during childhood and adolescence . Due to its high prevalence, bullying is an important topic of study. According to national and international epidemiological studies, 2-12% of schoolchildren worldwide are victims of bullying, while in Spain, the figure is 9.3%.
This phenomenon is defined as a form of abuse perpetrated by peers repeatedly over timeand is characterized by a constant abuse of power, as bullies engage in deliberate acts to harm victims who find it hard to defend themselves.
School bullying gives rise to two profiles: an aggression profile and a victimization profile. The aggression profile is determined by students building an aggressive and generally violent relational dynamic toward those they see as weak, while the victims are usually the target of hostile attacks without any incitement, systematically suffering the situation with a great degree of emotional intensity that can lead to severe states of anxiety or depression and social isolation. A perverse relationship of extended domination and submission emerges between the two profiles, leading to processes of victimization. Victimization is defined as the experience of being targeted with physical, verbal, and psychological abuse by peers in the school environment on a recurrent basis.
Adequate interaction with peers in a school setting is usually a source of emotional support, but pupils who find themselves caught up in a process of victimization perceive this setting to be unsafe. Victimization can have serious consequences on children's development, both at the time and in the longer term, causing personal, social, and emotional problems that can lead
## The present study
In the context of these considerations, this study focused on the phenomenon of school bullying and its relationship with self-perceived emotional intelligence. Drawing on previous research linking a lack of emotional intelligence with bullying, the main aim of the study was to analyze the mediating effect of the dimensions of emotional intelligence-attention, clarity, and repair-in relation to school victimization. As factors of protection against victimization, we expect to feel emotions and express feelings without hypervigilance (adequate emotional attention), to know and understand our emotions, to be able to distinguish one from another, to understand how they evolve and to integrate them into our thinking (excellent emotional clarity), and to regulate and control positive and negative emotions, moderating or intensifying them at our convenience (excellent emotional repair).
# Materials and methods
## Participants
The participants were among 20,000 pupils enrolled in years 5 and 6 at primary schools in the autonomous community of Extremadura, Spain, during the 2018-2019 school year, considering a sampling error of 3.5% and a confidence level of 96%. The total sample was made up of 822 primary school pupils from 10 public and semi-private schools, selected using intentional or convenience sampling; 45% of the sample of primary school pupils were in year 5, and 55% were in year 6. The age range was 10-12 years (M = 10.58; SD = 0.663); 46% were female and 54% male.
## Instruments
## School coexistence questionnaire (scq), spanish ombudsman, 2006
The SCQis a self-administered questionnaire that analyzes the incidence of school bullying from three perspectives: the observer, the aggressor, and the victim. Each scale includes 13 harassing behaviors classified into six modalities of bullying. The victim scale in the questionnaire requires participants to indicate whether they have experienced any of 13 different bullying scenarios since the school year began on a Likert-type scale with four response options ranging from 1 ("never") to ("4 always"). The scenarios are: being ignored, being excluded from participating, being insulted, being called offensive or humiliating nicknames, being spoken badly of, having belongings hidden, having belongings broken, having belongings stolen, being hit, being threatened, being sexually harassed, being forced to do things they do not want to do using threats, and being threatened with weapons. Based on the direct scores from the victim scale, three categories or levels were obtained using a frequency criterion: no victimization (M ≤ 14), occasional victimization (range from M > 14 to M ≤ 17), and victimization (M > 17).
## Trait meta-mood scale-24 (tmms-24)
To evaluate self-perceived emotional intelligence, the Spanish version of the Trait Meta-Mood Scale-24 (TMMS-24) questionnaire was used. The questionnaire features 24 items, with a Likert-type scale offering five response options (1 = strongly disagree, 5 = strongly agree). It assesses three different dimensions (eight items per dimension): attention (ability to have and express feelings adequately), clarity (understanding emotional states), and repair (adequate emotional regulation).
The authors of the questionnaire classified the scores for the different dimensions in three categories: low, adequate, and excessive or excellent, with different cut-off points for boys and girls. In the emotional attention dimension, the cut-off points are: low attention (boys ≤ 21, girls ≤ 24), adequate attention (boys ≥ 22 to ≤32, girls ≥ 25 to ≤35), and excessive attention (boys ≥ 33, girls ≥ 36). In the emotional clarity dimension, the cut-off points are: low clarity (boys ≤ 25, girls ≤ 23), adequate clarity (boys ≥ 26 to ≤35, girls ≥ 24 to ≤34), and excellent clarity (boys ≥ 36, girls ≥ 35). In the emotional repair dimension they are: low repair (boys ≤ 23, girls ≤ 23), adequate repair (boys ≥ 24 to ≤35, girls ≥ 24 to ≤34), and excellent repair (boys ≥ 36, girls ≥ 35). High scores in clarity and repair indicate a good emotional intelligence in these dimensions, but this is not the case for emotional attention, as high scores can indicate an excessive vigilance of emotions and a need to improve this aspect.
## Procedure
To carry out this research, the first step was to contact headteachers and teachers from the selected schools to explain the study objectives and to request their collaboration and permission. A consent form was then sent to parents and guardians, and the pupils who were given permission to participate were included in the study. The data collection process involved administering questionnaires by class group during school hours. The questionnaire took approximately 25 min to complete, in a quiet atmosphere to avoid pupils sharing opinions and contaminating the data. The ethical guidelines set out by the American Psychological Association were followed, ensuring the anonymity of the responses, the confidentiality of the data, and the exclusive use of this data for research purposes. The study was approved by the ethics committee of the University of Extremadura, Spain.
# Data analysis
Reliability analyses (Cronbach's alpha and McDonald's omega) and confirmatory analyses of the instruments were carried out, followed by a multivariate analysis of the mean scores in the different dimensions of emotional intelligence (attention, clarity, and repair), based on the degree of victimization (no victimization, occasional victimization, and victimization). Finally, as the authors of the TMMS-24 established three categories (low, adequate, excessive/excellent) for the different dimensions of emotional intelligence, and the victim scale includes three degrees of victimization based on frequency, it was necessary to conduct a multinomial logistic regression to establish the degree of association between the study variables, calculating the odds ratios and the respective 95% confidence intervals. Statistical analysis was carried out using the SPSS 21.0 (IBM Corp, New York, NY, USA, 2012) package for PC and JASP Free.
# Results
## Reliability and validity analysis of the instruments used in the study
The victim scale exhibited good internal consistency, with Cronbach's alpha (α = 0.84) and McDonald's omega (Ω = 0.856). With respect to the TMMS-24, the reliability of each component was: attention (α = 0.79; Ω = 0.79); clarity (α = 0.71; Ω = 0.70); repair (α = 0.80; Ω = 0.81). To determine whether the models found in the original validation study of the questionnaires properly adjusted to our data, we used GFIs (goodness-of-fit indexes). The adjustment indexes were close to the desirable values, showing evidence of reliability and validity. Although the authors of the TMMS-24 did not validate the instrument with subjects under 12 years old, the tests confirmed reliability and validity for children up to 11.
## Analysis of the tmms-24 factors based on degree of victimization and gender
A correlation analysis of the variables in the study was carried out.shows the directly significant correlations between the victim and emotional attention variables and inverse significant correlations with clarity and emotional repair variables. All three factors of the TMMS-24 were directly significantly correlated. Multivariate comparison of the mean scores in the attention, clarity, and emotional repair dimensions of the TMMS-24 was carried out based on the degree of victimization (no victimization, occasional victimization, and victimization), gender, and the interaction between the two variables.
The Univariate contrasts in the degree of victimization showed significant differences in emotional clarity (F(2, 814) = 5.853. p = 0.003, η = 0.014) and emotional repair (F(2, 814) = 10.895, p < 0.001. η = 0.026). No significant differences were found in emotional attention (F(2, 814) = 1.647. p = 0.193. η = 0.004). Bonferroni pairwise comparisons showed that: (1) participants in the no victimization category scored significantly higher in emotional clarity than those in the occasional victimization (p = 0.017) and victimization (p = 0.001) categories; (2) participants in the no victimization category scored significantly higher in emotional repair than those in the occasional victimization (p = 0.003) and victimization (p < 0.001) categories, while those in the occasional victimization category scored higher than those in the victimization category (p = 0.027).
## Emotional intelligence and victimization: multinomial logistic regression analysis and odds ratios (ors)
A multinomial logistic regression analysis was performed to determine which TMMS-24 variables predict victimization in school bullying. The analysis used the TMMS-24 dimensions of attention, clarity, and repair, grouped into three categories as predictor variables, and the degree of victimization, grouped into no victimization, occasional victimization, and victimization, as the dependent variable. The gender of the participants was included in the model as a control variable.
The multinomial regression analysis showed satisfactory fit (χ 2 = 83.304, p < 0.001; χ 2 Pearson = 135.069, p = 0.097; R Nagelkerke = 0.161), allowing 58% of the cases to be classified correctly.
Examining the results of the model with the no victimization reference category in detail, the estimates of the parameters showed that excessive attention (Wald = 6.514, p = 0.001), adequate attention (Wald = 11.990, p = 0.001), low clarity (Wald = 14.733, p < 0.001), adequate clarity (Wald = 11.685, p = 0.001), and low repair (Wald = 7.045, p = 0.008) were significantly and directly associated with victimization, while adequate attention (Wald = 9.284, p = 0.001), low attention (Wald = 17.003, p < 0.001), and adequate clarity (Wald = 7.746, p = 0.005) were significantly and directly associated with occasional victimization. Meanwhile, being a boy (Wald = 5.656, p = 0.017) was directly associated with victimization. The OR estimates for the model showed that the probability of being a victim of school bullying was 2.62 times higher among pupils with excessive attention, 2.44 times higher among pupils with adequate attention, 5.14 times higher among pupils with low clarity, 3.47 times higher among pupils with adequate clarity, and 2.72 times higher among pupils with low repair. The probability of being an occasional victim was 1.81 times higher among pupils with adequate attention, 3.43 times higher among those with low clarity, and 1.93 times higher among those with adequate clarity. A pattern emerged, showing victims of school bullying characterized by excessive attention, low clarity, and low repair.
Finally, the predictive model estimating the parameters for the occasional victimization reference category, which allowed a comparison of the victim and occasional victim groups, showed that low repair (Wald = 7.781, p = 0.005) was significantly and directly associated with victimization, meaning that probability of being a victim of school bullying was 2.55 times higher among pupils with low repair. Boys were more likely to be involved in processes of victimization (Wald = 0.757 p < 0.001).
# Discussion
To fulfill the study objectives, the reliability and validity of the instruments were evaluated. The factorial models found in the original studies of the instrumentsdisplayed satisfactory fit with our data. The indices were close to the optimal desired values, showing sufficient reliability and validity.
The aim of this study was to investigate the relationship between emotional intelligence and victimization in school bullying. The results of the multivariate analysis confirmed the existence of an association between the two variables: pupils who struggled to understand and regulate their emotional states were more likely to be involved in school bullying. Other studies have indicated significant differences between those involved and those not involved in bullying, with those involved displaying lower emotional repair. Victims in particular are characterized by low clarity and repair.
A detailed analysis of the results confirmed the relevance of self-perceived emotional intelligence as a predictor of victimization. Typically, the lower the excessive attention and the greater the clarity and repair among pupils, the less likely they were to be victims of school bullying. Intense attention to emotions and poor abilities to understand and regulate them made children more likely to fall victim to school bullying, confirming the lack of social efficacy among victims. These results support existing evidence that the clarity and repair dimensions are clearly linkedand that intense attention is associated with low clarity: the more focused the mind is on a problem, the less it is able to understand emotions and to seek effective solutions, with a negative impact on the ability to resolve conflicts with others. Victims of school bullying have a more negative social and emotional perception of themselves.
Other studies have also revealed associations between emotional intelligence and interpersonal relationships. Low emotional intelligence scores have been positively correlated with low scores in empathy, self-control in social situations, social skills, cooperation with peers, and affective relationships with others, while low levels of clarity and emotional repair have been found to be associated with greater social anxiety, lower empathy, and lower interpersonal satisfaction.
Our findings raised an interesting question: Why does clarity have stronger predictive power than emotional repair or attention? The results showed that the probability of being a victim of school bullying was 5.14 times higher among pupils with low clarity, 2.72 times higher among pupils with low repair, and 2.62 times higher among pupils with excessive attention. A limited ability to understand emotions makes it more difficult to interpret others' intentions, potentially giving rise to errors in assessing certain behaviors. This goes some way to explaining why low clarity had the greatest predictive power.
Finally, our results showed that being a boy is directly associated with victimization, i.e., boys are more likely to be involved in processes of victimization than girls. More boys than girls said they had been victims of verbal aggression, hitting, threats, and blackmail. Our findings echo those of other studies, which indicate that boys are more frequently involved in bullying as victims.
Some studies have shown that women are more sensitive than men. Generally, women are also more expressive than men. Some authorshave demonstrated that female gender are more perceptive, show greater empathy, and are better at recognizing others' emotions, which explains their more positive social interactions and lesser involvement in school bullying.
## Limitations and future research
This study has several important limitations, including the use of self-reports as a data collection method for evaluating emotional intelligence. Pupils may have underestimated or exaggerated their perceptions of their emotional competence when completing the self-reports. Other tools for measuring emotional skills are available, which evaluate participants' ability to complete a task. When assessing school bullying, it is important to include other informants besides those directly involved. We believe that teachers are particularly well placed to analyze victimization behaviors, as they can observe and evaluate them on a daily basis in the classroom. Although the authors of the TMMS-24 did not validate the instrument with children aged under 12, the tests carried out in this study confirmed its reliability and validity for use with children aged up to 11. Other limitations include the cross-sectional design, which makes it difficult to establish further inferences about the relationship between the study variables. Finally, cultural considerations mean that caution should be exercised when extrapolating the results to other countries, especially those outside the Western world.
# Conclusions
This study has made a valuable contribution to understanding the links between emotional intelligence and school bullying. The findings showed that the lower their excessive attention and the higher their emotional regulation and understanding, the less likely pupils are to be victims of school bullying. This confirms that adequate emotional attention and excellent emotional clarity and repair are protective factors against victimization.
Bullying at school is a topic of great social concern and the source of significant financial costs to the public health system. With the relevance of emotional intelligence skills as a protective factor, we recommend that emotional education is included in any actions taken to prevent or reduce school bullying. Schools are ideal settings for encouraging the development of emotional skills to allow pupils to manage their feelings and emotions, as well as those of others, and to respond appropriately to bullying. As school gives children the opportunity to interact with their peers, it is the ideal setting to develop emotional abilities in a practical way by group dynamics, role-playing, communication, stimulation games, and cooperative learning. To introduce emotional education in school, a movement has arisen demanding the inclusion of social and emotional aspects as a solution to some of the most urgent problems in the education system. Emotional intelligence development programs entitled "Social and Emotional Aspects of Learning" have been implemented. They train basic abilities directly related to EI, such as emotional perception, emotional understanding, or emotional regulation, and broader and superior order aspects such as personality, self-esteem, perseverance, assertiveness, and optimism. Funding: This research were funded by Junta de Extremadura, grant number IB16090.
## Conflicts of interest:
The authors declare no conflict of interest. |
Coil Now, Pipe Later: Two-stage Treatment for Acute Intracranial Aneurysm Rupture
# Introduction
Acutely ruptured intracranial aneurysms can pose a treatment challenge in certain clinical situations. Currently, the main two modalities of treatment are endovascular embolization and surgical clipping [bib_ref] Treatment of ruptured complex and large/giant ruptured cerebral aneurysms by acute coiling..., Brinjikji [/bib_ref] [bib_ref] Ultra-early versus delayed coil treatment for ruptured, Luo [/bib_ref] [bib_ref] Risk of hemorrhagic complication associated with ventriculoperitoneal shunt placement in aneurysmal subarachnoid..., Mahaney [/bib_ref]. Endovascular treatment carries the benefit of accessing ruptured aneurysms that are surgically difficult to navigate and have been shown to have a better outcome in patients with subarachnoid hemorrhage (SAH) versus surgical clipping [bib_ref] Treatment of ruptured complex and large/giant ruptured cerebral aneurysms by acute coiling..., Brinjikji [/bib_ref]. However, endovascular coiling of morphologically complex or large aneurysms is also associated with low rates of angiographic occlusion and high rates of recurrence [bib_ref] Pipeline for uncoilable or failed aneurysms: results from a multicenter clinical trial, Becske [/bib_ref]. The Pipeline Embolization Device (PED) (Medtronic, Irvine, CA), a flow-diverting stent (FDS), has gained momentum in recent years for the treatment of these complex intracranial aneurysms. Yet, its use in the acute ruptured aneurysm setting is limited by the need for dual antiplatelet therapy and the concomitant risk of hemorrhagic complications [bib_ref] Treatment of ruptured complex and large/giant ruptured cerebral aneurysms by acute coiling..., Brinjikji [/bib_ref] [bib_ref] Pipeline for uncoilable or failed aneurysms: results from a multicenter clinical trial, Becske [/bib_ref] [bib_ref] Pipeline embolization device in aneurysmal subarachnoid hemorrhage, Cruz [/bib_ref]. Here we present an interesting and unique case of an acutely ruptured irregular intracranial aneurysm that was partially coiled in the acute stage and later underwent definitive treatment with flow diversion.
## Case presentation
A 72-year-old female with a past medical history of hypertension presented to our emergency room with a three-day history of worsening headache complicated by new onset weakness, dizziness and lethargy. She responded easily to verbal commands but was oriented to name only with minimal verbal response. Emergent computerized tomography (CT) of the head showed aneurysmal subarachnoid hemorrhage and developing hydrocephalus [fig_ref] FIGURE 1: Non-contrast head computed tomography shows diffuse subarachnoid hemorrhage [/fig_ref]. Two long coils were successfully placed within the body and dome of the two largest lobules but attempts at placing the third coil were unsuccessful as it repeatedly herniated into the parent vessel [fig_ref] FIGURE 4: Fluoroscopic lateral projection image after placement of the flow diversion device [/fig_ref]. The decision was made to defer complete embolization until optimal timing of flow diversion therapy. Two weeks later, after the status of the ventriculostomy catheter had been codified, the patient successfully underwent left PCommA pipeline flex embolization device treatment of the aneurysm and was started on aspirin and clopidogrel antiplatelet therapy [fig_ref] FIGURE 5: Fluoroscopic lateral projection image after placement of the flow diversion device [/fig_ref]. Follow-up angiogram six months later showed successful occlusion of the aneurysm with no residual aneurysm filling identified [fig_ref] FIGURE 6: Delayed lateral projection angiographic image from a six-month follow-up study reveals a... [/fig_ref].
# Discussion
The PED uses a flexible low-porosity endoluminal sleeve that allows for direct reconstruction of the affected parent artery, reducing the hemodynamic communication between the aneurysm and its parent artery [bib_ref] Pipeline for uncoilable or failed aneurysms: results from a multicenter clinical trial, Becske [/bib_ref] [bib_ref] Pipeline embolization device in aneurysmal subarachnoid hemorrhage, Cruz [/bib_ref]. Over time, this FDS promotes thrombosis within the aneurysm sac, acts as a scaffold for neointima formation, and effectively leads to long-term reconstruction of the parent artery [bib_ref] Pipeline for uncoilable or failed aneurysms: results from a multicenter clinical trial, Becske [/bib_ref]. The need for dual antiplatelet therapy complicates PED use in the acute setting due to the potential risk of spontaneous hemorrhage or necessity of further invasive procedures, such as ventriculostomy placement [bib_ref] Treatment of ruptured intracranial aneurysms with the pipeline embolization device, Chalouhi [/bib_ref] [bib_ref] International retrospective study of the pipeline embolization device: a multicenter aneurysm treatment..., Kallmes [/bib_ref]. Mahaney, et al. described an increased risk of symptomatic intracranial hemorrhage in patients on dual antiplatelet therapy who underwent ventriculoperitoneal shunt placement. Four patients (33%) on dual antiplatelet therapy had a hemorrhagic complication versus zero patients (0%) not on antiplatelet therapy [bib_ref] Risk of hemorrhagic complication associated with ventriculoperitoneal shunt placement in aneurysmal subarachnoid..., Mahaney [/bib_ref]. Few studies have described the novel use of a PED after patients with complex ruptured aneurysms undergo initial staged endovascular coiling in the acute phase where dual antiplatelet therapy is largely contraindicated such as in our patient. In a study by Brinjikji, et al., 31 patients underwent endovascular coiling in the acute phase, 29 of which had experienced aneurysmal rupture, with the intention to possibly undergo flow diversion in the subacute phase. After coiling, 45.2% of their patients developed symptomatic hydrocephalus, subsequently undergoing ventricular drain placement. Additionally, 16.1% of patients had vasospasm that required endovascular intervention, further outlining the potential benefit of avoiding dual antiplatelet use in the acute phase. Given the complexity of the aneurysms, 100% of their patients had incomplete or near-complete occlusion of the aneurysmal sac at the time of flow diversion treatment. Of the 27 patients that underwent subsequent flow diversion treatment, 15 (55.6%) patients had complete occlusion and seven (25.9%) had nearly complete or stable-incomplete occlusions on last imaging studies [bib_ref] Treatment of ruptured complex and large/giant ruptured cerebral aneurysms by acute coiling..., Brinjikji [/bib_ref]. In another study, Chalouhi, et al. demonstrated similar success in complex, unruptured aneurysms with 80% of patients achieving complete occlusion, 20% of whom underwent staged coiling followed by PED treatment [bib_ref] Treatment of ruptured intracranial aneurysms with the pipeline embolization device, Chalouhi [/bib_ref]. Furthermore, the Pipeline for Uncoilable or Failed Aneurysms, a multicenter, prospective single-arm trial, demonstrated PED use in 108 anatomically complicated aneurysms. The mean aneurysm size in this study was 18.2 mm with 20.4% being larger than 25 mm in maximum distension. The study reported technical success in 99.1% of patients who underwent PED placement with major ipsilateral stroke or neurologic death reported in 5.6% of patients [bib_ref] Pipeline for uncoilable or failed aneurysms: results from a multicenter clinical trial, Becske [/bib_ref]. Our case resonates with the studies outlined above to demonstrate how an anatomically complex ruptured intracranial aneurysm may initially be treated with staged endovascular coiling in the acute phase until dual antiplatelet therapy use and FDS treatment is no longer contraindicated. This paradigm allows the aneurysm rupture site to be secured in the short term and the patient to undergo potentially invasive procedures in the acute phase that may not have been possible while on dual antiplatelet therapy.
# Conclusions
Acute intracranial aneurysm rupture is a potentially life-threatening condition requiring prompt treatment. Many treatment modalities are available, each with its own benefits and risks. In the case presented here, a combination of endovascular coiling with subsequent antiplatelet therapy and FDS treatment was utilized to ensure optimal treatment of her anatomically complex intracranial aneurysm. Physicians should be readily prepared to recognize and treat intracranial aneurysm rupture while accounting for the unique anatomical variations of each patient in order to avoid potentially dangerous outcomes.
# Additional information disclosures
Human subjects: Consent was obtained 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 1: Non-contrast head computed tomography shows diffuse subarachnoid hemorrhage (arrow) and hydrocephalus (double arrow).CT angiography revealed a large dysplastic left posterior communicating artery (PCommA) aneurysm with irregular contour, measuring up to 1 cm(Figure 2). [/fig]
[fig] FIGURE 2 of 9 FIGURE 3: Axial computed tomography angiogram shows the wide neck multilobulated left posterior communicating artery region aneurysm (arrow). The patient underwent right frontal ventriculostomy placement and was later taken for endovascular embolization of the rupture left PCommA aneurysm. Cerebral angiography confirmed a large, irregular aneurysm with three separate lobules (Figure 3). 2017 Haider et al. Cureus 9(11): e1876. DOI 10.7759/cureus.1876 3 Lateral projection image from a left internal carotid artery angiogram reveals the multilobulated posterior communicating artery region aneurysm along the left posterior carotid wall (arrow)."Left" indicates the patient's left side. [/fig]
[fig] FIGURE 4: Fluoroscopic lateral projection image after placement of the flow diversion device (double arrow). The coils have a stable configuration within the aneurysm sac (arrow). "Left" indicates the patient's left side. [/fig]
[fig] FIGURE 5: Fluoroscopic lateral projection image after placement of the flow diversion device (double arrow). The coils have a stable configuration within the aneurysm sac (arrow). "Left" indicates the patient's left side. [/fig]
[fig] FIGURE 6: Delayed lateral projection angiographic image from a six-month follow-up study reveals a stable configuration of the coils in the aneurysm sac (arrow). The previously noted area of persistent aneurysm filling at the base of the aneurysm is no longer visible (double arrow). This is consistent with complete aneurysm occlusion. [/fig]
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Androgen Mediated Regulation of Endoplasmic Reticulum-Associated Degradation and its Effects on Prostate Cancer
The endoplasmic reticulum (ER) comprises thirty percent of the newly translated proteins in eukaryotic cells. The quality control mechanism within the ER distinguishes between properly and improperly folded proteins and ensures that unwanted proteins are retained in the ER and subsequently degraded through ER-associated degradation (ERAD). Besides cleaning of misfolded proteins ERAD is also important for physiological processes by regulating the abundance of normal proteins of the ER. Thus it is important to unreveal the regulation patterns of ERAD. Here, we describe that ERAD pathway is regulated by androgen, where its inhibitor SVIP was downregulated, all other ERAD genes were upregulated. Consistently, androgen treatment increased the degradation rate of ERAD substrates. Using several independent techniques, we showed that this regulation is through androgen receptor transactivation. ERAD genes found to be upregulated in prostate cancer tissues and silencing expression of Hrd1, SVIP, and gp78 reduced the in vitro migration and malignant transformation of LNCaP cells. Our data suggests that expression levels of ERAD components are regulated by androgens, that promotes ERAD proteolytic activity, which is positively related with prostate tumorigenesis.Prostate cancer is the second leading cause of cancer mortality and the most prevalent cancer among males with an estimation of more than 3.3 million men in the United States 1,2 . Androgen and the androgen receptor (AR), which is a transcription factor of the nuclear steroid receptor family, play a critical role in any stage of normal or neoplastic growth of the prostate. After androgen binding, AR dissociates from heat shock proteins and forms a homodimer. Dimerized AR then acts as a ligand-dependent transcription factor and binds to the androgen response elements (AREs) of androgen-regulated target genes. As a transcription factor, androgen-bound AR recruits RNA polymerase II and a basal transcriptional complex for the transcription of AR target genes 3 . Since androgen target genes are the mediators of several diverse metabolic processes 4 , it is crucial to specifically identify these androgen-responsive genes. Besides normal prostate growth and pathologies, androgen signaling is also critical for female physiology and other male characteristics, such as muscle mass, strength, bone mineral density and neuronal remodeling 5 . There are several diseases that have been associated with androgen signaling besides prostate cancer such as breast cancer, diabetes, metabolic syndrome, cardiovascular diseases and Alzheimer's disease 5-7 . Therefore, it is important to delineate the biochemical processes that are altered by androgen action.In addition to their regulation by hormones, prostate cancer cells are also known to be highly secretory. The Endoplasmic Reticulum (ER) is the organelle responsible for the synthesis and maturation of proteins that are destined for the secretory pathways. There is a sophisticated protein quality control mechanism called the ER-associated degradation (ERAD) that eliminates misfolded or unassembled polypeptides and ensures that only fully maturated proteins reach their sites of function. ERAD is also essential for physiological processes by regulating the abundance of normal proteins of the ER, such as monooxygenase cytochrome p450; cholesterol metabolism regulatory proteins 3-hydroxy-3-methylglutaryl-CoA reductase, insulin-induced gene-1 and apolipoprotein B; neurodegenerative disease proteins superoxide dismutase-1 and ataxin-3; and the metastasis suppressor KAI1/CD82 8-12 . Considering its critical role on the regulation of cellular homeostasis, it is not surprising that aberrant ERAD is involved in the pathogenesis of many diseases, such as cancer, cystic fibrosis, neurodegenerative diseases, and diabetes 13 .Understanding the regulation of ERAD is one of the main questions of cellular proteostasis. Some of ERAD factors, namely Hrd1, Hrd3 and Derl1 are reported to be induced upon activation of unfolded protein response (UPR) in yeast14,15. Ubiquitination of ERAD components also regulates ERAD. For example, autoubiquitination of Hrd1p is required for retrotranslocation in yeast 16 . For mechanism still not clear, deubiquitination enzymes (DUBs) can also act as positive regulators in ERAD 17 . There are two additional specific regulatory patterns for gp78-mediated ERAD. The first mechanism is to control the level of gp78 by Hrd1, which targets gp78 for ubiquitination and proteasomal degradation18,19. The second mechanism is via the endogenous ERAD inhibitor, namely SVIP, which inhibits gp78-mediated ERAD by competing with p97/VCP and Derlin1 20 .There is very limited information on ERAD and androgen signaling pathways in prostate cancer cells to date. In 2009, Romanuik et al. identified SVIP as one of the novel androgen-responsive genes by sequencing of LongSAGE libraries 21 . Since the previously characterized ERAD inhibitor SVIP found to be negatively regulated by androgen treatment in LNCaP cells, we were prompted to test regulation of ERAD pathway by androgen.In this study, we showed that ERAD is an androgen-regulated process where both the mRNA and protein levels of ERAD components are regulated with the treatment of the synthetic androgen, R1881. We found that while the level of SVIP, the endogenous ERAD inhibitor, is decreased, all other tested ERAD proteins are increased by the R1881 treatment. This pattern is present in androgen sensitive prostate cancer cells, namely LNCaP and 22RV1, but not in androgen insensitive prostate cancer cells, PC3 and DU145. In addition, we showed that anti-androgen bicalutamide efficiently antagonizes the androgenic induction of ERAD proteins in these cells. Moreover, by using a chemical IRE1α inhibitor we found that regulation of ERAD by androgen is partially or fully independent of UPR. Consistent with androgen-mediated regulation of ERAD genes, R1881 treatment increases ERAD proteolytic activity since the degradation rate of two ERAD substrates, CD3δ and KAI1. Finally, the effect of Hrd1, gp78, and SVIP was evaluated on the cell proliferation rate, wound healing, migration and malignant transformation of LNCaP cells using the RNAi approach, and our data suggests that ERAD may be involved in in vitro migration and malignant transformation in LNCaP cells.
# Results
## Differential expression of erad proteins in prostate cancer cell lines.
To determine the role of ERAD components in prostate tumorigenesis, we first examined their protein expression levels by immunoblotting (IB) in 6 prostate epithelial cell lines. For this aim, two non-tumorigenic prostate cell lines: normal prostate epithelial cell line (RWPE1) and benign prostatic hyperplasia epithelial cell line (BPH1) were utilized as controls. As tumorigenic cell lines, two androgen-sensitive prostate cancer cell lines (LNCaP and 22RV1) and two androgen-insensitive prostate cancer cell lines (DU145 and PC3) were included. Among all the tested ERAD components, two ubiquitin ligases, Hrd1 and gp78, and glycan binding lectin, OS9, were expressed significantly higher in the hyperplastic (BPH1) and androgen sensitive cells (LNCaP and 22RV1); whereas the ERAD inhibitor SVIP was expressed only in the LNCaP and 22RV1 cells [fig_ref] Figure 1: Expression of ERAD components in different prostate cell lines [/fig_ref]. Almost all of the tested ERAD components except p97/ VCP were either not expressed or expressed in very low levels in the normal prostate epithelial cell line, RWPE1 [fig_ref] Figure 1: Expression of ERAD components in different prostate cell lines [/fig_ref]. In summary, our data indicates that ERAD component levels are all high in androgen-sensitive LNCaP and 22RV1 cells.
## Regulation of erad components by androgen.
Since the prostate cancer cell lines with intact androgen receptor showed higher expression levels of all tested ERAD component proteins and ERAD inhibitor of SVIP was reported as one of the novel androgen-responsive genes by sequencing of LongSAGE libraries [bib_ref] LNCaP Atlas: gene expression associated with in vivo progression to castration-recurrent prostate..., Romanuik [/bib_ref] , we hypothesized that the ERAD pathway might be regulated by androgen. LNCaP cells were treated with increasing concentrations of R1881 (0.1-10 nM) for 24 h and processed for protein expression analyses. R1881 treatment caused a dose dependent increase of E3 ubiquitin ligases, Hrd1 and gp78; retrotranslocation complex members p97/VCP, Ufd1, Npl4 and Derlin1; E3/E4 ubiquitin ligase Ufd2a and glycan binding lectin, OS9. The increase of the expression level of these proteins was in parallel with the dose dependent induction of AR and the endogenous AR target, PSA . Interestingly, the level of the ERAD inhibitor SVIP was decreased dose dependently, while the levels of all other ERAD components were increased. Together, this data suggests that the expression of ERAD components is regulated by androgen in a dose-dependent manner, in other words androgen treatment downregulates ERAD inhibitor SVIP levels but upregulates other ERAD genes.
A time-course study was performed in LNCaP cells by using different treatment lengths (2-24 h) with 10 nM R1881. Once again the expression levels of almost all the ERAD components showed significant increase in a time-dependent manner, whereas only ERAD inhibitor SVIP level was decreased . Together, our data indicate that ERAD is regulated by in vitro androgenic stimulation in a time-and dose-dependent manner.
In order to see whether the effect of androgen on ERAD components is on the protein or mRNA level, we treated LNCaP cells with 10 nM R1881 for 24 h and ERAD genes were tested for their altered mRNA expression levels using RT-qPCR. All the ERAD genes except Ufd2a, showed statistically significant alterations (p < 0.05 for gp78, SVIP, p97/VCP, Ufd1 and p < 0.005 for Hrd1, Derlin1, Npl4 and OS9) on mRNA levels . Consistent with protein expression results, R1881 treatment decreased the SVIP mRNA level, whereas increased the mRNA expression of other ERAD genes. In this assay system, PSA was used as a positive control and its mRNA expression was increased 10-fold with 10 nM R1881 treatment . Interestingly, our data showed that mRNA level of AR was decreased by R1881 treatment , while its protein level was augmented . In fact, our results are consistent with a previous publication by Yeap et al. [bib_ref] Differential posttranscriptional regulation of androgen receptor gene expression by androgen in prostate..., Yeap [/bib_ref] , which reports that androgen downregulates AR mRNA transcription, while increases the AR protein expression due to the stabilization of the ligand receptor complex after ligand binding [bib_ref] Differential posttranscriptional regulation of androgen receptor gene expression by androgen in prostate..., Yeap [/bib_ref]. To summarize, our data suggests that androgen regulates ERAD component levels in both the protein and mRNA level . To further analyze whether the androgen-mediated regulation of ERAD is at the gene transcriptional or translational level we pretreated LNCaP cells either with the RNA synthesis inhibitor actinomycin D (1 μ g/ml) or the protein synthesis inhibitor cycloheximide (1 μ g/ml) and then added R1881 to the medium [bib_ref] Androgen stimulates matrix metalloproteinase-2 expression in human prostate cancer, Liao [/bib_ref]. Both of these inhibitors significantly abolished R1881 induced expression of Hrd1, gp78, Derlin1, p97/VCP, Npl4, Ufd2 and OS9, where well known AR-target PSA was used as the positive control .
In order to see whether the regulation of ERAD by androgen is only limited to metastasis derived androgen-sensitive LNCaP cells, we performed a similar dose-response study with the non-metastasis derived androgen-sensitive 22RV1 cell line and found that all ERAD components were similarly regulated by androgen in both LNCaP and 22RV1 cells . This data suggests that regulation of ERAD by androgen is not limited to LNCaP cells since SVIP levels were downregulated, while all other ERAD component expressions were upregulated both in LNCaP and 22RV1. Consistently, ERAD was not regulated by androgen in two AR-negative prostate cancer cell lines, PC3 and DU145 .
Androgens show their biological effects via the intracellular AR, which is a ligand-activated transcription factor and pretreating cells with androgen antagonists abolishes the androgen action. Among several antiandrogen agents, bicalutamide (Casodex) is known to act as a pure androgen receptor antagonist in LNCaP cells [bib_ref] Androgen stimulates matrix metalloproteinase-2 expression in human prostate cancer, Liao [/bib_ref]. To examine whether androgen mediated regulation of ERAD is mediated via AR transactivation, we pretreated LNCaP cells with the effective dose of bicalutamide (10 μ M) for 1 h that was followed by the R1881 treatment. PSA has been used a positive control. Pretreatment of bicalutamide ablated R1881 induced induction of ERAD proteins, such as Hrd1, gp78, Derlin1, p97/VCP, Ufd1, Npl4, Ufd2, OS9. When bicalutamide was present alone in culture, SVIP expression was upregulated, while the downregulation of SVIP level following R1881 treatment was significantly blocked in the presence of bicalutamide. This data suggests that the regulation of ERAD pathway by androgen is specifically mediated via the AR [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref].
The levels of Hrd1, Hrd3 and Derlin1 are reported to be enhanced upon activation of UPR in yeast 14 and a recently published paper described androgen-mediated induction of IRE1α branch and inhibition of PERK signaling of UPR [bib_ref] Divergent androgen regulation of unfolded protein response pathways drives prostate cancer, Sheng [/bib_ref]. Thus we wanted to test whether IRE1α induction is responsible for androgen-mediated induction of ERAD by utilizing a chemical IRE1α inhibitor, 4μ 8c. R1881 mediated upregulation of ERAD genes such as gp78 was observed both in 4μ 8c-treated cells [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] , lane 1 versus 4) and in the cells with intact IRE1α pathway [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] , lane 2 versus 3). In this assay system we also checked the success of IRE1α inhibition by detecting the expression level of XBP1s, the downstream effector of IRE1α branch. While R1881 treatment caused an upregulation [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] , lane 2 versus 3), 4μ 8c treatment caused a downregulation on XBP1s expression level [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] , lane 1 versus 2). Furthermore, co-treatment of R1881 with 4μ 8c did not increase the XBP1s expression compared to the cells treated with only 4μ 8c [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] , lane 1 versus 4), confirming that unlike ERAD genes, the upregulation of XBP1s expression by R1881 is solely dependent on IRE1α activity [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref]. Interestingly, 4μ 8c treatment caused significant decrease on the basal expression levels on ERAD component levels [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] , lane 1 versus 2), but not on BIP and Ire1α levels. Together our results suggest that regulation of ERAD by R1881 is mediated via AR and is partially or fully independent on the androgen-mediated induction of IRE1α branch of UPR [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref].
## Figure 2. erad is regulated by r1881 in a dose-and time-dependent manner. (a) androgen-starved
LNCaP cells were treated with R1881 at indicated doses for 24 hour and the levels of ERAD components; AR and PSA were analyzed by immunoblotting using antibodies raised against them. (B) Androgen-starved LNCaP cells were treated with 10 nM R1881 for indicated times and expression levels were analyzed as in 2A.
(C) Androgen-starved LNCaP cells were treated with 10 nM R1881 and the mRNA levels of the indicated genes were investigated using quantitative PCR (QPCR). Controls were treated with vehicle and set to 1. Data represent the mean of two independent biological replicates in triplicates and error bars represent SE. p-values were calculated with respect to vehicle-treated cells by two-tailed equal variance Student's t-test. (*p < 0.005, # p < 0.05) (D) Androgen-starved LNCaP cells were pretreated first with 1 μ g/ml cycloheximide or actinomycin for 1 h followed by R1881. 24 h after R1881 treatment, cells were lysed and the expression levels of the proteins of interest were tested using immunoblotting as in 2A.
Scientific RepoRts | 7:40719 | DOI: 10.1038/srep40719
Androgen treatment increases ERAD activity. In this study we showed that androgen treatment decreases the expression level of the ERAD inhibitor SVIP, while increases the expression levels of all other tested ERAD components; indicating a general induction of the ERAD pathway. Thus, we hypothesized that AR signaling produces homeostatic adjustments in the ERAD activity. To test this hypothesis, we transfected LNCaP cells with a well-known ERAD substrate, CD3δ 25 , and determined its degradation rate using cyclohexamide chase analysis. As seen in [fig_ref] Figure 5: ERAD activity is enhanced by R1881 [/fig_ref] , the degradation rate of CD3δ increased significantly with R1881 treatment in LNCaP cells. We also checked the degradation rate of another ERAD substrate, a transmembrane metastasis suppressor,
## Figure 3. regulation of erad by androgen is present in androgen-sensitive cell lines but not in androgeninsensitive cells. (a) androgen sensitive cells (b)
Androgen-insensitive cells were treated with R1881 at indicated doses for 24 hour and the level of ERAD components and PSA were analyzed by immunoblotting as in .
Scientific RepoRts | 7:40719 | DOI: 10.1038/srep40719 KAI1 11 . Since the endogenous KAI1 in LNCaP cells was not detectable, we also overexpressed KAI1 by using a plasmid coding for the KAI1 gene for ectopic expression [bib_ref] Frequent downregulation of the KAI1(CD82) metastasis suppressor protein in human cancer cell..., White [/bib_ref] [bib_ref] Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes, Kim [/bib_ref]. As for CD3δ , the degradation rate of KAI1 significantly increased with R1881 treatment [fig_ref] Figure 5: ERAD activity is enhanced by R1881 [/fig_ref]. Our data strongly suggests that androgen treatment downregulates ERAD inhibitor SVIP and upregulates all other ERAD genes, which in turn enhances ERAD proteolytic activity.
ERAD components are upregulated in prostate cancer tissues and induce prostate cancer cell proliferation and oncogenicity. We checked the expression level of some ERAD genes in prostate tissue samples by using a Prostate Cancer Tissue Array containing 9 normal and 39 prostate cancer tissues. Our data revealed that gp78, Hrd1, SVIP and AR showed increased expression (p < 0.05 for AR and p < 0.005 for gp78, Hrd1, SVIP) in prostate cancer tissues [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref]. As expected prostate cancer patient samples had diverse levels of ERAD component mRNA detected by RT-qPCR. It is noteworthy to mention 51% of prostate tumors (20 of 39) and 25% of tumors (10 of 39) had 5 fold higher gp78 mRNA expression and Hrd1 mRNA expression, respectively, compared to normal prostate tissue controls (n = 9) .
To further investigate the potential role of ERAD in prostate tumorigenesis, we transiently silenced Hrd1, gp78 or SVIP expression in LNCaP cells [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref] and analyzed the cell proliferation rates of cells by measuring the impedance-based signals every 30 min for 60 h using a real time cell analyzer system. Our data showed that silencing Hrd1 or gp78 expression caused significant reduction (p < 0.005 for Hrd1, p < 0.05 for gp78) in cell growth rate of LNCaP cells [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref]. We obtained similar results when LNCaP cells were treated with 10 nM R1881, where cells with silenced Hrd1 or gp78 expression had slower proliferation rate (p < 0.005 for Hrd1, gp78). On the other hand, there was no change detected in the proliferation rate of SVIP silenced cells. In order to test the role of ERAD components on the motility of LNCaP cells, an in vitro wound healing model was carried out using IBIDI linear exclusion systems which prevents cell growth in a predefined, standardized region. After removal of the insert, cells were monitored for their motility. Our data indicates that silencing of gp78 and Hrd1 resulted in a decrease in the rate of wound closure (p < 0.005 for Hrd1, p < 0.05 for gp78) [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref].
Next, we analyzed the effect of selected ERAD genes on the migration of LNCaP cells using the Boyden Chamber assay to assess their serum-stimulated chemokinesis. Our data indicates that silencing Hrd1, gp78 or SVIP expression decreased the serum stimulated-migration ability of LNCaP cells [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref]. Lastly, soft agar assay was performed to examine the effect of Hrd1, gp78 or SVIP on anchorage independent growth, which is a hallmark of malignant transformation. Instead of using the classical soft agar assay, which involves manual counting of colonies, we used a 96-well fluorescence cell transformation assay. This assay system also has a relatively shorter incubation time (around 6 days), which makes it possible to work with cells that are transiently transfected with siRNAs. As seen in [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref] , silencing of Hrd1, gp78 or SVIP caused a significant decrease on both the size and number of colonies, as well as a decrease in the measured fluorescence intensity [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref]. In conclusion, our data suggests that silencing of Hrd1 and gp78 affect the proliferation rate, whereas Hrd1, gp78 and SVIP have role in malignant transformation of prostate cancer cells.
# Discussion
ERAD is the most effective, rapid and direct means to remove misfolded proteins. Besides degrading these potentially toxic misfolded proteins, ERAD also regulates the level of some properly folded proteins, such as HMG-CoA reductase; rate-limiting enzyme of cholesterol biosynthesis; apolipoprotein B, assembly factor of cholesterol-containing liposomes, and KAI1, tumor metastasis suppressor [bib_ref] Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterolregulated ubiquitination..., Song [/bib_ref] [bib_ref] Overexpression of the tumor autocrine motility factor receptor Gp78, a ubiquitin protein..., Liang [/bib_ref] [bib_ref] The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation, Tsai [/bib_ref]. Considering its critical role in the regulation of cellular homeostasis, it is believed that any aberration on ERAD has significant effects on cell physiology. To date, there are almost 70 ERAD substrates linked to a variety of human diseases including cancer, neurodegenerative diseases and diabetes [bib_ref] The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in..., Guerriero [/bib_ref]. Therefore, there is an ongoing extensive research on the ERAD substrates, their association with diseases, elucidation of the steps of ERAD mechanism and its regulation. Despite all of these studies, little is known about the intracellular regulation of mammalian ERAD. This study, to the best of our knowledge, is the first study to characterize the androgen-mediated regulation of the ERAD pathway.
As a multistep process, more than 50 proteins are involved in ERAD [bib_ref] Cleaning up in the endoplasmic reticulum: ubiquitin in charge, Christianson [/bib_ref] [bib_ref] The mammalian endoplasmic reticulum-associated degradation system, Olzmann [/bib_ref]. In this study, we examined ERAD through several selected genes including OS9, which functions in substrate recognition and targeting; gp78 and . (C) The proliferation rates of cells silenced as indicated were determined with real time cell growth assay of three biological and six technical replicates. Cells were treated with vehicle and R1881 in left and right, respectively. (D) Wound healing assay was performed using LNCaP cells seeded on 35 mm dishes with high culture-insert coating (IBIDI). The closure of the gap created by the removal of insert was monitored for three days. Representative images are shown. The analysis of wound closure% was determined using the ImageJ software. Two independent biological and three technical repeats per experiment were used. p-values were calculated with respect to control siRNA transfected cells by two-tailed equal variance Student's t-test (*p < 0.005, # p < 0.05). (E) Boyden chamber assay was done using 24-well transwell chamber as explained in the Material and Methods section. The migrated LNCaP cells on the lower surface of the membrane were fixed and stained with Giemsa. Representative images are shown. Migration was quantified by counting stained cells. The mean percentage of migrated cells compared to control groups were given using the data obtained from two independent biological replicates in triplicates (*p < 0.005). (F) The colonogenic assay of LNCaP cells was performed as explained in Material and Methods. Representative microimages are shown. Quantification of colony formation of cells was performed with CyQuant GR dye using a fluorometer (*p < 0.005). Hrd1, E3 ubiquitin ligases; Ufd2a functioning as E3/E4 ubiquitin ligase; Derlin1, p97/VCP, Ufd1 and Npl4 as components of the retrotranslocation complex and SVIP, which is the first identified endogen ERAD inhibitor. In 2009, SVIP was reported as one of the novel androgen-responsive genes by sequencing of LongSAGE libraries [bib_ref] LNCaP Atlas: gene expression associated with in vivo progression to castration-recurrent prostate..., Romanuik [/bib_ref]. Considering that SVIP is an ERAD inhibitor and found to be negatively regulated by R1881 treatment in LNCaP cells [bib_ref] LNCaP Atlas: gene expression associated with in vivo progression to castration-recurrent prostate..., Romanuik [/bib_ref] , we tested the regulation of the ERAD pathway using LNCaP prostate cancer cell line using the synthetic androgen, R1881. Besides the proliferation pattern of LNCaP, its expression in differentiated secretory pathway and the control of some cellular pathways, such as lipid synthesis and accumulation also remains to be androgen responsive [bib_ref] Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the..., Swinnen [/bib_ref] [bib_ref] Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic..., Swinnen [/bib_ref].
## Relative expression levels
In this study, we observed that the expression levels of ERAD components are highest in androgen-responsive prostate cancer cells among other tested prostate cells [fig_ref] Figure 1: Expression of ERAD components in different prostate cell lines [/fig_ref]. The expression of some ERAD components was also present in androgen insensitive prostate cancer cells and hyperplastic prostate cells. However, almost no ERAD components, except p97/VCP, were detected in normal prostatic epithelial cells [fig_ref] Figure 1: Expression of ERAD components in different prostate cell lines [/fig_ref]. Our data strongly suggests that all tested ERAD components, except SVIP, were upregulated by R1881 treatment in a dose-and time-dependent manner . However, SVIP, the endogen inhibitor of ERAD, was downregulated by androgen treatment . The regulation of ERAD by androgen is not limited to LNCaP cells, but also to another androgen responsive cell line, 22RV1, which has a similar androgen-mediated ERAD regulation pattern . The effect of androgen observed on the ERAD components levels was mediated through the AR, since bicalutamide (androgen antagonist) pretreatment reduced the effect of R1881 on ERAD protein levels [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref]. Since the ERAD inhibitor protein SVIP was downregulated while all other tested ERAD components were upregulated, we hypothesized that ERAD activity should be augmented in the R1881-treated LNCaP cells. Indeed, our cycloheximide chase assay results suggest that the ERAD substrates were degraded faster in R1881-treated cells when compared to control cells [fig_ref] Figure 5: ERAD activity is enhanced by R1881 [/fig_ref].
In a prostate cancer tissue panel of patients we found the mRNA expression levels of Hrd1, gp78, and SVIP are upregulated in prostate cancer [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref]. Thus, we also examined the role of Hrd1, gp78, and SVIP on prostate tumorigenesis. The silencing expression of ubiquitin ligases, Hrd1 and gp78 (but not SVIP), decreased the proliferation rate of LNCaP cells both with and without R1881 treatment [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref]. Surprisingly, silencing of all tested ERAD components very drastically inhibited in vitro transwell migration and colony formation in LNCaP cells [fig_ref] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis [/fig_ref] , suggesting that both positive (Hrd1, gp78) and negative (SVIP) regulators have similar role in malignant transformation of LNCaP cells. This might be due to multiple functions that have been reported for SVIP. Besides being an ERAD inhibitor, SVIP has been characterized as a regulator of autophagy pathway 32 and as p97/VCP independent myelin protein component in neurons [bib_ref] Structure and expression of a novel compact myelin protein -small VCPinteracting protein..., Wu [/bib_ref]. More extensive work on the mechanisms of tumor invasion and metastasis needs to be performed in the future including in vivo tumor growth assays.
While we were working on this manuscript, a report identifying a divergent androgen regulation of UPR in prostate cancer cells was published [bib_ref] Divergent androgen regulation of unfolded protein response pathways drives prostate cancer, Sheng [/bib_ref]. In this paper, it was suggested that androgens activate the inositol requiring enzyme 1α (IRE1α ) branch, but inhibit the protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling in prostate cancer cells. In accordance with these findings, we also found that the IRE1α branch was activated and XBP1s expression level were significantly increased by R1881 treatment in LNCaP cells [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref]. Our data in [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref] indicates gp78, Hrd1, OS9, Derlin1, Ufd1 expression levels were also upregulated by R1881 in cells that IRE1α activity was inhibited by using 4μ 8c [fig_ref] Figure 1: Expression of ERAD components in different prostate cell lines [/fig_ref]. Therefore, upregulation of ERAD by R1881 is partially or fully independent of androgen-mediated UPR induction. However, it is interesting that treatment with only 4μ 8c caused decrease on the expression of especially gp78, Os9, Ufd1 and Derlin1, which might be the reason that 4μ 8c treated cells have lower degree of upregulation by R1881 than the response obtained in cells with intact IRE1α activity [fig_ref] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on... [/fig_ref].
In an effort to find putative binding sites for AR (ARE) we examined the human genomic sequences of ERAD genes tested in this study using in silico MatInspector bioinformatic tool (Genomatix Software, Munich, Germany, http://www.genomatix.de). A restrictive threshold of 0.85 and V$GREF matrix were used for prediction of putative AREs [bib_ref] Androgen receptor binding sites identified by a GREF_GATA model, Masuda [/bib_ref]. This scanning results in the identification of three putative ARE sites for Hrd1, one for gp78, six for p97/VCP gene, seven for Ufd1, one for SVIP, six for OS-9, four for Ufd2a and six for Npl4 (Supplemental . Further tests are required to analyze whether those putative AREs are really functioning. This in silico screening results together supports our experimental results suggesting that ERAD is regulated with androgen action via androgen receptor.
Besides its effect on prostate, androgens play several roles in different tissues such as androgen-mediated augmentation of the axonal regeneration after peripheral nerve injury [bib_ref] Testosterone regulation of the regenerative properties of injured rat sciatic motor neurons, Kujawa [/bib_ref]. Androgens might also act directly in the AR-containing cell populations in the nerve to enhance axonal growth and myelination [bib_ref] Androgen receptor messenger RNA and protein in adult rat sciatic nerve: implications..., Jordan [/bib_ref]. Recently, SVIP was identified as a novel compact myelin protein in the sciatic nerve, independent of its interaction with p97/VCP suggesting another role of SVIP in the central and peripheral nervous systems, in addition to being an ERAD inhibitor [bib_ref] Structure and expression of a novel compact myelin protein -small VCPinteracting protein..., Wu [/bib_ref]. Sciatic nerves from adult male and females rats were previously reported to contain both the AR mRNA and protein. In addition, endoneurial fibroblasts have implications for site of androgen actions and the AR might mediate the effects of androgens in the neuromuscular systems [bib_ref] Androgen receptor messenger RNA and protein in adult rat sciatic nerve: implications..., Jordan [/bib_ref]. The expression of Glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22), two proteins that play a crucial role in the structure of peripheral myelin, were shown to be modulated by androgens in the sciatic nerve in adult male rats 37 . Moreover, the age-related reduction of Po and myelin basic protein expression was associated with myelin degeneration, which was partially reversed by steroid derivatives [bib_ref] Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration..., Leonelli [/bib_ref]. It is noteworthy to mention that similar to Po and myelin protein 22; SVIP, the novel myelin protein is also found to be regulated by androgen in this study. Therefore, the androgen regulation of ERAD genes might be of great importance in other systems and pathologies besides prostate cancer.
In conclusion, our findings suggest that protein and mRNA expression levels of ERAD components are regulated by androgens, that promotes ERAD proteolytic activity, which is positively related with prostate tumorigenesis.
Scientific RepoRts | 7:40719 | DOI: 10.1038/srep40719
# Materials and methods
Materials. All cell culture grade reagents including media, fetal bovine serum (FBS), and growth factors were obtained from either Life Technologies or LONZA. Polyclonal antibodies against Hrd1 (147773), gp78 (9590), Npl4 (13489), Derlin1 (8897), CHOP (2895), PSA (5365), AR (5153), PERK (3192), IRE1a (3294), XBP1-s (12782) were purchased from Cell Signaling Technology. Mouse monoclonal antibodies against p97/ VCP (612182), Ufd1 (611642) and Ufd2a (611966) were obtained from BD Transduction Laboratories. Anti-BIP (G90043), anti-actin (A5316), anti-HA (H9658) and anti-myc (M4439) antibodies were from Sigma Aldrich; anti-KAI1 (sc17752) from SantaCruz; anti-OS-9 (ab19853) from Abcam and HRP-conjugated anti-mouse or anti-rabbit IgG was purchased from Pierce. Polyclonal anti-SVIP antibody was described previously [bib_ref] Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation, Ballar [/bib_ref].
Actinomycin D, Tunicamycin, Cycloheximide were purchased from Calbiochem and Bicalutamide from Sigma Aldrich.
Cell culture and treatments. Human prostate cell lines RWPE-1 (normal prostate epithelial cell), 22RV1 (prostate adenocarcinoma), PC3 (prostate adenocarcinoma, bone metastatic site), DU145 (prostate adenocarcinoma, brain metastatic site) and LNCaP (prostate adenocarcinoma, lymph node metastatic site) were obtained from American Type Culture Collection (ATCC, USA), while BPH-1 (benign prostatic hyperplasia epithelial cell line) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). The DU145 and PC3 cell lines were cultured and routinely passaged in DMEM/F12 media containing 10% FBS, while LNCaP and 22RV1 cell lines were propagated in RPMI 1640 containing 10% FBS. RWPE-1 was cultured in Keratinocyte Serum-Free Medium supplemented with 5 ng/ml EGF, 0.05 mg/ml bovine pituitary extract, and 1% Pen-Strep antibiotics cocktail. BPH-1 was propagated in RPMI 1640 containing 20% heat inactivated FBS, 20 ng/ml testosterone, 5 μ g/ml transferrin, 5 ng/ml sodium selenite and 5 μ g/ml insulin.
All the compounds were prepared as a 1000-fold concentrated stock in the solvent, DMSO or ethanol, thus final concentration of solvent did not exceed 0.1%.
All the hormone treatments and RNAi applications were performed in LNCaP cells that are below passage 15.
In order to remove steroids and growth factors during hormone treatment, LNCaP cells were grown in starvation medium containing 2% and 0.5% CT-FBS (Charcoal treated-FBS) for 2 days and 1 day, respectively. Cells were then exposed to R1881 as indicated in each experiment. 22RV1 cells were also treated with hormone following the same protocol used for LNCaP cells.
In indicated experiments, after serum starvation, cells were first pretreated either with 10 μ M bicalutamide, 1 μ g/ml actinomycin or 1 μ g/ml cycloheximide for 1 h and then treated with 10 nM R1881 synthetic androgen.
In the cycloheximide chase assay, cells were treated with either with 10 nM R1881 or ethanol as control. After 18 h and 21 h of R1881 treatment, 25 μ g/ml CHX was added into 6 h and 3 h samples, respectively. Samples were harvested 24 hours after the initiation of the R1881 treatment.
Transfections were performed either with Lipofectamin-2000 (Invitrogen) or X-tremeGENE HP (Roche) following instructions of manufacturer.
Protein preparation and Immunoblotting (IB). Cell lysates were prepared by homogenizing cultured cells in RIPA buffer (1xPBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0). After removal of insoluble materials by centrifugation at 14.000 rpm for 10 min at 4 °C, protein concentrations were determined using BCA protein assay kit (Thermo Scientific). Typically, 40 μ g of total cellular protein were used for immunoblotting. Samples were denatured in 6x Laemmli buffer at 95 °C for 5 min and were separated on either handcast polyacrylamide gels or gradient precast ready gels (BioRAD). Gels were transferred onto PVDF membranes (Millipore). Following classical immunoblotting steps (blocking, incubating with primary and secondary antibodies), proteins were visualized using enhanced chemiluminescence (BioRAD) by Fusion FX7 (Vilber Lourmat). Densitometric analyses of protein bands were performed using ImageJ software (http://imagej.nih. gov/ij/).
## Total rna isolation and expression analysis by quantitative rt-pcr.
The total RNA was isolated using Total RNA Isolation Kit (Norgen) following the manufacturer's instructions. RNA concentration and purity were determined by Nanovette (Beckman Coulter). cDNAs were synthesized using the ProtoScript II First Strand cDNA Synthesis Kit (NEB) using 1 μ g of total RNA and oligo dT primers according to the manufacturer's instructions. The gene expression analysis, quantitative RT-PCR was performed using The SYBR Green I Mastermix (Roche) and LightCycler480 thermocycler (Roche). Specific primers were designed against ERAD genes, PSA and AR, and all primer sequences are listed in Supplemental . Twenty-microliter reactions were performed with 300 nM of primer pairs. Fold change for the transcripts were normalized to the housekeeping gene TBP1 (TATA-Box Binding Protein1, general RNA polymerase transcription factor, M5564) [bib_ref] Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion..., Michel [/bib_ref]. The following PCR conditions were used: denaturation at 95 °C for 10 min, followed by 45 cycles of: 10 s at 95 °C, 10 s at 60 °C, and 15 s at 72 °C. For relative quantification, reaction efficiency incorporated Δ Δ Cq formula was used. Two independent biological replicates with three technical replicates per experiment were used for each PCR. For patient samples, Origene TissueScan Prostate Cancer Tissue Array III (HPRT503) containing 46 tissues covering 39 prostate cancer tissues (18 Stage 2, 19 stage3, 2 Stage4) and 9 normal tissues was used in (3 technical replicas). siRNAs, Plasmids and Transfection. Silencer ® Negative Control siRNA #1, gp78 (siRNA ID: 110862, sense sequence: CGUAUGUCUAUUACACAGA), SVIP (sense sequence: GACAAAAAGAGGCUGCAUC), Hrd1 (siRNA ID: 124188, sense sequence: CCGUUUUUCGGGAUGACUU) were ordered from Ambion [bib_ref] Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation, Ballar [/bib_ref] [bib_ref] Identification of ERAD components essential for dislocation of the null Hong Kong..., Zhong [/bib_ref].
pCI-CD3δ -HA has been previously described [bib_ref] The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic..., Ballar [/bib_ref]. pCMV6-KAI1-myc is obtained from Origene. Proliferation, colony formation and migration assays. Proliferation rate of LNCaP cells was monitored using real-time cellular analysis system (xCELLigence, ACEA) measuring impedance-based signals. 7500 cells/well were seeded into 96-well E-plate (ACEA) and cell proliferation was monitored every 30 min for 60 h. Data was expressed as "cell index", which is defined as "impedance of the well with cells" minus "the background impedance". Normalization was done at 12 hours, where LNCaP cells were attached and regained their morphology. Three independent biological and eight technical repeats per experiment were used. For the wound healing assay, 35 mm dishes with high culture-insert coating (IBIDI) consisting of two reservoirs was utilized. After confluent monolayers of LNCaP cells were established on dishes, the insert was gently removed creating a gap of 500 μ m. The closure of the gap was monitored for three days and images were taken using Olympus CKX41 microscope. The analysis of wound closure % was determined by using the ImageJ software (http://imagej.nih.gov/ij/). Three independent biological and two technical repeats per experiment were used.
Boyden chamber assay was performed to assess the migration rate by using a 24-well transwell chamber that includes a porous polycarbonate membrane with 8-μ m pore size (Corning). Serum-starved LNCaP cells (10000 cells in medium with 0.5%CT-FBS) were seeded onto the Transwell filters (upper chamber). To stimulate cell migration through the membranes, 20% FBS was added to the lower chamber as a chemoattractant. The cells were kept at 37 °C in a CO 2 incubator for 48 hours. The migrated LNCaP cells on the lower surface of the membrane were fixed with methanol and stained with 0.2% crystal violet solution (Sigma Aldrich). Migration was quantified by counting stained cells and the results were expressed as the mean percentage of migrated cells compared to control groups.
Soft agar colony formation assay was performed with CytoSelect cell transformation assay (Cell Biolab, Inc.) following manufacturer's instructions. Equal volumes of 2xRPMI-1640 with 20% FBS and 1.2% agar solution were mixed and transferred onto wells in a 96-well plate. Cell suspensions prepared in 25 μ l were mixed with 25 μl of 2xRPMI-1640 with 20% FBS and 25 μ l of 1.2% agar, and then placed on the solidified bottom agar layer. After the addition of 100 μ l of 2xRPMI-1640 containing 10% FBS to each well, the plates were incubated for 6 days under conventional cell culture conditions. The medium was changed every 2-3 days. The images of colonies were taken using Olympus CKX40 microscope. Colonies were lysed and quantified with CyQuant GR dye using a fluorometer equipped with a 485/520 nm filter set (Varioscan, Thermo Scientific).
Statistics. Data are presented as means ± standard deviation (SD). The statistical significance of differences between groups was assessed by by two-tailed equal variance Student's t-test using GraphPad Prism software. Values of p < 0.05 were considered significant.
[fig] Figure 1: Expression of ERAD components in different prostate cell lines. The expression levels of ERAD components, AR and PSA levels in prostate cell lines were determined by immunoblotting. Actin was used as the loading control in all immunblotting analyses in this study. Scientific RepoRts | 7:40719 | DOI: 10.1038/srep40719 [/fig]
[fig] Figure 4: Effect of androgen antagonist and inhibition of IRE1α branch of UPR on the androgen regulation of ERAD. Androgen-starved LNCaP cells were pretreated first with (A) 10 μ M bicalutamide (B) 1 μM 4μ 8c for 1 h and then with R1881. 24 h after R1881 treatment, cells were lysed and the expression levels of the proteins of interest were tested using immunoblotting as in 2A. (C) Schematic representation of androgen mediated regulation mechanism of ERAD. [/fig]
[fig] Figure 5: ERAD activity is enhanced by R1881. LNCaP cells were transfected with (A) CD3δ and (B) KAI1 on 100 mm dishes. Six hours later, cells were splitted onto 6 well dishes. Next day, cells were first androgen-starved and then treated with R1881. Cycloheximide was added into indicated samples 18 h and 21 h after R1881 treatment and cells were harvested 24 hours after R1881 addition. The level of CD3δ and KAI1 was detected by immunoblotting against their tags HA and myc, respectively and quantified by normalizing samples to actin levels. The degradation rates of substrates (right) were analyzed using three independent experiments.Scientific RepoRts | 7:40719 | DOI: 10.1038/srep40719 [/fig]
[fig] Figure 6: The role of gp78, Hrd1 and SVIP in prostate cancer tumorigenesis. (A) Relative expression of some ERAD genes and AR in prostate cancer tissues (n = 39) compared to normal prostate tissue (n = 9) detected by RT-qPCR. Experiment was performed in 3 technical replicates. B-actin gene is used as reference gene. p-values were calculated with respect to vehicle-treated cells by two-tailed equal variance Student's t-test (*p < 0.005, # p < 0.05). (B) LNCaP cells were transfected with siRNA for the indicated genes. Silencing the expression of indicated proteins were analyzed with immunoblotting as in [/fig]
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Detection of genes mutations in cerebrospinal fluid circulating tumor DNA from neoplastic meningitis patients using next generation sequencing
Background: This study profiled the somatic genes mutations and the copy number variations (CNVs) in cerebrospinal fluid (CSF)-circulating tumor DNA (ctDNA) from patients with neoplastic meningitis (NM).Methods: A total of 62 CSF ctDNA samples were collected from 58 NM patients for the next generation sequencing. The data were bioinformatically analyzed by (Database for Annotation, Visualization and Integrated Discovery) DAVID software. Results: The most common mutated gene was TP53 (54/62; 87.10%), followed by EGFR (44/62; 70.97%), PTEN (39/62; 62.90%), CDKN2A (32/62; 51.61%), APC (27/62: 43.55%), TET2 (27/62; 43.55%), GNAQ (18/62; 29.03%), NOTCH1 (17/62; 27.42%), VHL (17/62; 27.42%), FLT3 (16/62; 25.81%), PTCH1 (15/62; 24.19%), BRCA2 (13/62; 20.97%), KDR (10/62; 16.13%), KIT (9/62; 14.52%), MLH1 (9/62; 14.52%), ATM (8/62; 12.90%), CBL (8/62; 12.90%), and DNMT3A (7/62; 11.29%). The mutated genes were enriched in the PI3K-Akt signaling pathway by the KEGG pathway analysis. Furthermore, the CNVs of these genes were also identified in these 62 samples. The mutated genes in CSF samples receiving intrathecal chemotherapy and systemic therapy were enriched in the ERK1/2 signaling pathway.Conclusions: This study identified genes mutations in all CSF ctDNA samples, indicating that these mutated genes may be acted as a kind of biomarker for diagnosis of NM, and these mutated genes may affect meningeal metastasis through PI3K-Akt signaling pathway.
# Background
Neoplastic meningitis (NM) refers to the dissemination of malignant cells to the leptomeninges, and is associated with very poor survival of patients. The primary cancers are mostly lung and breast cancers or brain tumors, such as medulloblastoma. In clinic, early NM detection and timely treatment could significantly impact the outcome of patients. However, the present diagnosis is primarily based on the clinical signs and symptoms plus cerebrospinal fluid (CSF) cytology and neuroimaging [bib_ref] Meningeal carcinomatosis in solid tumors, Martins [/bib_ref]. Furthermore, although the detection of tumor cells in the CSF is the key to make NM diagnosis, the CSF cytology may not be reliable due to insufficient sensitivity and specificity. Thus, the detection of cell-free circulating tumor DNA (ctDNA) could be another diagnostic strategy [bib_ref] Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma..., Murtaza [/bib_ref] [bib_ref] Analysis of circulating tumor DNA to monitor metastatic breast Cancer, Dawson [/bib_ref]. This can detect cancer-associated genes and gene alterations in the plasma or CSF to monitor the tumor progression and/or treatment responses [bib_ref] Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma..., Murtaza [/bib_ref] [bib_ref] Analysis of circulating tumor DNA to monitor metastatic breast Cancer, Dawson [/bib_ref]. In patients with brain tumor, the plasma ctDNA analysis has revealed either its absence, or extremely low levels [bib_ref] Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain..., Mattos-Arruda [/bib_ref]. Fortunately, CSF ctDNA has been well demonstrated to be present and even abundant in brain tumor patients [bib_ref] Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain..., Mattos-Arruda [/bib_ref] [bib_ref] Detection and quantification of mutations in the plasma of patients with colorectal..., Li [/bib_ref]. In order to better understand characterization of ctDNA in CSF of NM patients, the detection of mutated genes in the CSF could help medical oncologists identify the primary tumor and make effective treatment options for patients. Therefore, the present study aimed to investigate the gene mutations in the CSF ctDNA samples obtained from NM patients using the cutting-edge technique of next generation sequencing (NGS). This approach can help to characterize NM genetic profiles and profile of gene mutations, which can thereby be potentially applied for molecularly targeted therapy. Towards this end, a recent study [bib_ref] Unique Genetic Profiles from Cerebrospinal Fluid Cell-free DNA in Leptomeningeal Metastases of..., Li [/bib_ref] genetically profiled the CSF ctDNA obtained from NM patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). Therefore, characterization of gene mutations in CSF ctDNA samples can provide valuable clinical guidance for precision medicine.
# Methods
## Study population and samples
In the present study, a total of 58 patients with NM, who received lumbar puncture for the CSF cytology examination between October 2014 and June 2018 in the Department of Neurology, The Second Hospital of Hebei Medical University (Hebei, China), were enrolled. The diagnosis of these 58 NM patients was entirely according to the clinical signs and symptoms, positive CSF cytology, and/or neuroimaging findings, such as contrast-enhanced brain magnetic resonance imaging (MRI) or computed tomography (CT), and the results from the CSF ctDNA. The NM clinical signs and symptoms included headache, nausea, vomiting, convulsion, lower back pain, cranial nerve paralysis, paresthesia, gait disturbances, vertigo and defects. The positive CSF cytology was defined by the distinctive pattern of the neoplastic cell morphology. That is, cells that had an irregular size and shape, and contained large and polymorphic nuclei with a lobulated state and malformed buds. The chromatin size increased with the basophilic coarse particles, and the mitotic activity was enhanced with aberrant mitosis. The nuclear membrane was usually thickened with a saw-tooth-shaped and wear edge. In addition, the positive neuroimaging revealed the presence of leptomeningeal enhancement. The clinical data are presented in [fig_ref] Table 1: Characteristics of 58 NM patients [/fig_ref].
The present study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (Hebei, China), and a written informed consent was obtained from each patient or their legal surrogates.
## Csf cytology examination
About 0.3 ml CSF was centrifuged at 750 r/min for 4 min (Therm-4, Shandon Cytospin, US). After naturally drying on the slide, the deposit was dyed with May-Grünwald-Giemsa liquid (Yucai, Beijing, China) and Alcian blue staining (Yucai, Beijing, China), according to the manufacturer's protocol, and observed under a light microscope (OLYMPUS-BX41, Japan). The determination of the positive result was as follows: cells that had an irregular size and shape, and contained large and polymorphic nuclei with a lobulated state and malformed buds. The chromatin size increased with the basophilic coarse particles, and the mitotic activity was enhanced with aberrant mitosis. The nuclear membrane was usually thickened with a saw-tooth-shaped and wear edge. Analysis was done using a cell medical image analysis system (MCDS-20, Chongqing, China).
Next-generation sequencing CSF samples and processing
The CSF samples were collected from each patient and placed into EDTA tubes, according to our hospital routine protocols. Then, these were centrifuged for five minutes at 1000 g. The pellet was stored at − 20°C, while the supernatant was centrifuged at 10, 000 g for an additional 30 min, according to a previous study [bib_ref] Evaluating Cancer of the central nervous system through next-generation sequencing of cerebrospinal..., Pentsova [/bib_ref]. The supernatant was transferred into pre-labeled cryotubes and stored at − 80°C. Next, the ctDNA was extracted from at least 5 mL of the CSF supernatant using a QIAamp Circulating Nucleic Acid kit (QIAGEN), according kit instructions, and the ctDNA was quantified using a Qubit2.1 Fluorometer and Qubit dsDNA HS Assay kit (Life Technologies, Carlsbad, CA, USA).
Preparation of the ctDNA library and the next generation DNA sequencing
The ctDNA samples were subjected to preparation of the Ion Proton library and DNA sequencing, according to the methodologies from previous studies [bib_ref] Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer..., Cai [/bib_ref] [bib_ref] Frequent KIT mutations in human gastrointestinal stromal tumors, Xu [/bib_ref] [bib_ref] PIK3CA and TP53 gene mutations in human breast cancer tumors frequently detected..., Bai [/bib_ref]. Briefly, for each sample, an adapter-ligated library was generated using the Ion AmpliSeq Library Kit 2.0 (Life Technologies). That is, the pooled amplicons made from 10 to 20 ng of ctDNA samples were endrepaired and ligated to Ion Adapters X and P1, and purified using AMPure beads (Beckman Coulter, Brea, USA) to obtain the adapter-ligated products, followed by nick-translation and PCR-amplification for a total of five cycles. Then, the products were subjected to analysis using the Agilent 2100 Bioanalyzer and Agilent Bioanalyzer DNA High-Sensitivity LabChip (Agilent Technologies) to determine the concentration and size of the library, and sample emulsion PCR and emulsion breaking using the Ion (Life Technologies) was used for the sequencing reactions. Then, the SV-OCP143-ctDNA panel (San Valley Biotech Inc., Beijing, China) was used to detect the somatic mutations of 143 cancer-related genes. Since the CSF ctDNA samples contained short DNA fragments, the amplicons in the panel were specially designed for the efficient amplification of ctDNA, and the total read counts were more than 25 million to ensure an average base coverage depth over 10,000 folds. In addition, strict quality control criteria were used to ensure that the average uniformity of the base coverage is no less than 95.5% for the reliability of the DNA sequencing and mutation detection.
## Processing and analysis of dna sequencing data
The raw DNA sequencing data were processed and analyzed using the Ion Proton platform-specific pipeline (Torrent Suite v5.0) with a specific plug-in (Variant Caller v5.0), which included the readouts of the raw DNA sequences, the trimming of the adapter sequences, and the filtering and removal of poor signal sequences according to previous studies [bib_ref] Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer..., Cai [/bib_ref] [bib_ref] Frequent KIT mutations in human gastrointestinal stromal tumors, Xu [/bib_ref] [bib_ref] PIK3CA and TP53 gene mutations in human breast cancer tumors frequently detected..., Bai [/bib_ref]. These three filtering steps were applied to eliminate the erroneous base calling, and the final variant calling was generated. That is, the first step evaluated the DNA sequences using the following criteria: the average total coverage depth is > 10,000, each variant coverage is > 10, the variant frequency for each sample is > 0.1%, and the P-value is < 0.01. The second step was to eliminate ant potential DNA strand-specific errors after visual examination of the gene mutations using the Integrative Genomics Viewer (IGV; http//www.broadinstitute.org/igv) or Samtools (http://samtools.sourceforge.net) software. For the third step, the total amplicon read counts from the Coverage Analysis Plugin were utilized to identify the copy number variants (CNVs). Then, the read counts per amplicon of each sample was normalized to the total number of reads for a given sample, and divided by normalized counts from a composite normal male genomic DNA sample, which yielded a copy number ratio after correcting for GC content. The gene-level copy number was estimated through the determination of the coverage-weighted mean of the GC-corrected per-probe ratio, which was corrected with the expected error, according to the probe-to-probe variance [bib_ref] Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data, Grasso [/bib_ref]. Afterwards, genes with a copy number of < 1 or > 4 were regarded as loss or gain, respectively.
## Egfr mutations in lung cancer tissues
Information of EGFR mutations in lung cancer tissues were obtained from patient's medical record. Lung cancer tissues of most patients were sequenced before this study by various methods.
## Functional annotation and pathway enrichment analysis
Next, the mutated genes were bioinformatically analyzed using the gene ontology (GO) terms [bib_ref] Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources, Huang Da [/bib_ref] and the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway enrichment analysis with the Database for Annotation, Visualization, and Integrated Discovery (DAVID; v.6.8, https://david.ncifcrf.gov/tools.jsp). Then, the data were clasisified as the functional annotation and the KEGG pathway enrichment. A P-value of < 0.05 was set to be statistically significant.
# Statistical analysis
The statistical significance in gene mutation frequency between the two groups was analyzed using Fisher exact test. Pearson correlation analysis was used for correlation analysis. Nonparametric test was used for two independent samples that did not meet the normal distribution. Statistical analyses were performed using SPSS version 19, and a two tailed P-value of < 0.05 was considered statistically significant.
# Results
## Patient characteristics
Clinical characteristics are shown in [fig_ref] Table 1: Characteristics of 58 NM patients [/fig_ref]. For NM patients with lung cancer, the majority of these patients had lung adenocarcinoma (27/42, 64.3%), while of 55 patients had known primary malignancies and 10 patients (18.2%) had NM as the first clinical manifestation. These patients were followed up for two month or longer.
A total of 62 CSF samples were collected from these 58 NM patients, in which three CSF samples were collected from a single patient, while two CSF samples were collected from other two patients at distinct time points. Furthermore, among the 62 CSF samples, 30 CSF samples were collected from 28 patients who received intrathecal chemotherapy and systemic radiotherapy, chemotherapy, and/or molecule-targeted therapy, 11 CSF samples were obtained from 11 patients who received intrathecal chemotherapy, and 12 CSF samples were obtained from 12 patients who received systemic therapy. The remaining nine CSF samples were collected from nine patients who did not receive any anticancer therapy.
Cancer-associated genes mutations in the 62 CSF specimens, regardless of the origin of the primary cancer and the mutated genes functional enrichment analysis
The 62 CSF samples were all positive for ctDNA and mutations of cancer-associated genes. Specifically, 68 (47.6%) of the 143 cancer-associated genes analyzed in the present study had mutations in at least one NM CSF sample, and 62 (100%) of the NM CSF samples carried at least one mutated gene. The most commonly mutated gene was TP53 (54/62, 87.10%), followed by EGFR (44/ [fig_ref] Figure 1: Profiling of genes mutations in the CSF samples obtained from NM patients,... [/fig_ref]. Furthermore, GO (Gene Ontology) annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were used to explore the potential functions of these high frequency mutated genes, and it was found that these high frequency mutated genes were rich in the PI3K-Akt signaling pathway [fig_ref] Figure 2: The GO analysis [/fig_ref].
Furthermore, the variant allele frequency was divided into five groups: ≥50, 30-50%, 10-30%, 1-10% and 0.2-1%, respectively. Then, the variant allele frequency was associated with the detectable tumor cells in the CSF samples, and it was found that the frequency of variant allele frequency (≥1%) was higher in the group with detectable tumor cells than that in the group without detectable tumor cells (P < 0.001, [fig_ref] Figure 3: The association of variant allele frequency with detectable tumor cells in the... [/fig_ref].
## Copy number variations (cnvs)
Data on the CNVs in these CSF ctDNA samples were also obtained, and it was found that high CNVs occurred in 22 of 58 NM patients. Among these 22 patients, the primary tumors were 15 lung cancers (13 lung adenocarcinoma, one squamous cell carcinoma and one unspecified), four gastric cancers, and one each of breast cancer, parotid carcinoma, and unknown primary cancer. The deletion of the CDKN2A copy number was the most frequent CNV that occurred in seven CSF ctDNA samples from six non-small cell lung cancers (6/22, 27.3%). In the increase in CDK4 copy number that occurred in five lung adenocarcinomas, four of these exhibited an increase in MDM2 copy number. In addition, an increase in MDM2 copy number was also detected from another lung adenocarcinoma patient. Two CSF ctDNA samples had a gain of ERBB2 (HER2) copy number from a parotid carcinoma patient, while an increase in CD44 copy number was identified in three patients, in which each patient has breast cancer, gastric cancer and unknown cancer, respectively. In addition, an increased EGFR copy number occurred in three lung adenocarcinoma patients. Other CNVs of tumor-associated genes were detected in five patients (six positive CSF ctDNA samples) with decreased AR copy numbers, five patients had decreased CD274 copy numbers, three patients each has a decreased PDCD1LG2 copy number, two patients each has an increase in FGFR2, CCNE1, or NKX2-1 copy numbers, respectively, and one patient had increased TIAF1, GAS6, or IL6 copy numbers, or reduced CSNK2A1, JAK2, MED12, or SMAD4 copy numbers.
## Association of gene mutations with intrathecal chemotherapy and systemic therapy
The data on these unique mutated genes were summarized for each group of patients, followed by a geneannotation enrichment analysis. It was found that the ERK1/2 pathway was mostly enriched by the GO analysis in patients who received both intrathecal chemotherapy and systemic therapy [fig_ref] Figure 4: The GO analysis [/fig_ref] and [fig_ref] Table 2: Unique mutated genes in each treatment group [/fig_ref].
The association of CSF ctDNA concentration with Karnofsky performance status (KPS) score, gene mutation and CSF tumor cells
The CSF ctDNA concentration was not statistically associated with the KPS scores (r = − 0.038, P = 0.768; [fig_ref] Figure 5: The association of CSF ctDNA concentration with the Karnofsky performance status [/fig_ref]. Furthermore, the number of gene mutations was not associated with the KPS score (r = 0.192, P = 0.135; [fig_ref] Figure 5: The association of CSF ctDNA concentration with the Karnofsky performance status [/fig_ref]. However, it was found that the CSF ctDNA concentration was associated with tumor cells in the CSF, when compared to that without circulating tumor cells (Z = -2.883, P = 0.004; [fig_ref] Figure 5: The association of CSF ctDNA concentration with the Karnofsky performance status [/fig_ref].
Cancer-associated genes mutations in the 45 CSF samples obtained from 42 NM patients with lung cancer A total of 45 CSF samples were collected from 42 NM patients with lung cancer, in which three CSF samples were collected from a single patient at distinct time [fig_ref] Table 3: The number of mutation-bearing CSF samples [/fig_ref].
## Enriched genes and gene pathways in nm patients with lung cancer
Mutated genes in the 42 NM patients with lung cancer were analyzed by GO annotation and KEGG pathway analyses. The top three GO terms were negative regulation of cell proliferation, peptidyl-tyrosine phosphorylation and positive regulation of ERK1 and ERK2 cascade. KEGG pathway analysis found these genes were associated with various biological processes, which included the general signaling pathways underlying the progression of cancer (P = 5.21 × 10 − 30 ; q = 5.05 × 10 − 28 ), chronic myeloid leukemia (P = 7.01 × 10 − 20 ; q = 3.40 × 10 − 18 ), endometrial cancer (P = 3.82 × 10 − 19 ; q = 1.24 × 10 − 17 ), bladder cancer (P = 6.39 × 10 − 19 ; q = 1.55 × 10 − 17 ), melanoma (P = 1.78 × 10 − 18 ; q = 3.44 × 10 − 17 ), glioma (P = 1.62 × 10 − 17 ; q = 2.61 × 10 − 16 ), prostate cancer (P = 8.66 × 10 − 17 ; q = 1.20 × 10 − 15 ), and non-small cell lung cancer (P = 1.59 × 10 − 15 ; q = 1.93 × 10 − 14 ), while the related signaling pathways were the ErbB signaling (P = 4.92 × 10 − 10 ; q = 3.67 × 10 − 9 ), VEGF signaling (P = 6.00 × 10 − 7 ; q = 3.06 × 10 − 6 ), MAPK signaling (P = 1.33 × 10 − 6 ; q = 5.88 × 10 − 6 ), p53 signaling (P = 4.14 × 10 − 6 ; q = 1.61 × 10 − 5 ), and m-TOR signaling (P = 1.00 × 10 − 5 ; q = 3.33 × 10 − 5 ) pathways [fig_ref] Figure 6: The data on the GO analysis [/fig_ref]. Furthermore, the KEGG pathway analysis revealed that EGFR, TP53, CDKN2A, CDK4, BRAF, NRAS, HRAS, JAK3, KRAS, MAP2K1, MAP2K2, PIK3CA and RB1 were strongly associated with non-small cell lung cancer.
A number of gene mutations were statistically more common in the present cohort of NM, when compared to the lung cancer noted in the COSMIC database, and these genes were also further analyzed by GO annotation and KEGG pathway analyses .
## The association of egfr mutations between lung cancer tissues and nm csf samples
Next, EGFR mutations were associated between lung cancer tissues and the NM CSF samples available in 10 patients [fig_ref] Table 4: EGFR activating mutations in primary lung cancer and NM CSF samples CSF... [/fig_ref]. Specifically, CSF samples were collected from N033, N063, N077, N1088, N156, N331, N355 and N1286 during the TKI therapy, while N079 and N090 before the TKI. It was found that there were roughly the same EGFR mutations between lung adenocarcinoma tissues and CSF of nine patients, except for N1088, in which the EGFR mutation was undetectable in the CSF sample.
## A representative case
In the present cohort, there was a lung adenocarcinoma patient who underwent surgical lung cancer resection, and tumor tissues had an EGFR 19Del mutation detected by NGS. Thus, the patient orally received 125 mg of icotinib three times a day for six months and thereafter. However, the patient had a headache during the icotinib therapy for the primary tumor. The head contrast enhanced MRI showed the linear and strip abnormal enhancement of the cerebellar sulcus [fig_ref] Figure 8: A representative case [/fig_ref] after the patient's cancer spread into the leptomeninges, and the CSF cytology examination showed tumor cells in the CSF [fig_ref] Figure 8: A representative case [/fig_ref]. Furthermore, the CSF sample revealed EGFR 19Del and T790M mutations in the CSF ctDNA by NGS technology. Given such a situation, the patient was given 80 mg of AZD9291 once a day to replace the icotinib for 18 months, and the patients overall health condition improved. Furthermore, a complete response was confirmed by the contrast-enhanced brain MRI [fig_ref] Figure 8: A representative case [/fig_ref] , CSF cytology [fig_ref] Figure 8: A representative case [/fig_ref] , and undetectable EGFR mutations in the CSF samples. The patient has been alive for nearly 3 years since the diagnosis of NM.
# Discussion
In the present study, NM patients were enrolled for the detection of CSF ctDNA, gene mutations and copy number variations. Furthermore, the present cohort of NM patients revealed that the large majority of primary cancers was lung adenocarcinoma, and 10 patients had NM as the first clinical manifestation, although seven of these 10 patients were clarified for their primary tumor. Afterwards, 62 CSF samples were acquired from 58 NM patients, and all samples contained detectable ctDNA, indicating that detection of CSF ctDNA is a sensitive biomarker for NM patients, since the ctDNA may not originate from benign tumors and non-neoplastic conditions, according to previous studies [bib_ref] Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain..., Mattos-Arruda [/bib_ref] [bib_ref] Detection and quantification of mutations in the plasma of patients with colorectal..., Li [/bib_ref]. Indeed, a previous study of ctDNA in 640 patients with different cancers [bib_ref] Detection of circulating tumor DNA in earlyand late-stage human malignancies, Bettegowda [/bib_ref] revealed that plasma ctDNA can be detected in at least 75% of patients vs. less than 50% patients with brain tumors, such as glioma, suggesting that CSF ctDNA can be an alternative source of samples for brain tumor diagnosis, since the present data detected positive CSF ctDNA in all 62 CSF specimens. However, it may also be observed that all patients in the present study had NM, and tumor cells in NM can disseminate over the leptomeningeal surface, followed by neoplastic cell shedding into the CSF. Thus, it needs to be further determined whether the CSF could be used to detect early stage brain tumors. However, it is true that the CSF can be a best source to detect ctDNA in NM patients. Previous studies have also reported that all 26 patients [bib_ref] Unique Genetic Profiles from Cerebrospinal Fluid Cell-free DNA in Leptomeningeal Metastases of..., Li [/bib_ref] and three patients [bib_ref] Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain..., Mattos-Arruda [/bib_ref] had positive ctDNA in the CSF samples, and the present study further supports these previous studies. In addition, the present study further demonstrated that CSF ctDNA is a useful resource to analyze gene mutations, which can help medical oncologists identify primary tumors that can cause NM. It was found that the mutations of cancer-associated genes occurred in all 62 CSF ctDNA samples, with the highest frequency on TP53 (54/62, 87. samples; (4) the PI3K-Akt and ERK1/2 signaling pathways are the most altered signaling pathways for these mutated genes; (5) novel gene mutations are induced by intrathecal chemotherapy and systemic therapy in NM patients; (6) lung cancer (especially lung adenocarcinoma) is the major primary tumor in the present cohort of NM patients. Future studies would investigate the usefulness of the CSF and ctDNA for the early detection of NM patients, and target these mutated genes for the therapy of NM patients or even patients with these primary tumors. Indeed, the PI3K-Akt signaling pathway, including but is not limited to TP53, EGFR, PTEN, KIT and KDR, could be crucial or at least partially crucial in mediating primary cancer for meningeal metastasis. In particular, numerous isoforms and/or spliced variants of PI3Ks participate in the regulation of various cell processes, such as cell cycle progression, cell polarization, migration, survival and metabolism, as well as tumor angiogenesis [bib_ref] PI3K and cancer: lessons, challenges and opportunities, Fruman [/bib_ref]. Furthermore, Akt is amenable to the vast majority of PI3K-mediated responses [bib_ref] PI3K at the crossroads of tumor angiogenesis signaling pathways, Soler [/bib_ref] , and the alterations of Akt upstream regulators, elevated Akt expression, and/or Akt activation all result in the promotion of tumor metastasis. For example, activated PI3K-Akt signaling could stimulate the translocation of α-actinin-4 from the nucleus to the cytoplasm and plasma membrane, which in turn induce changes in cell morphology and motility [bib_ref] A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners, Ding [/bib_ref]. In human carcinogenesis, the PI3K-Akt signaling pathway inhibited the expression of tumor suppressor gene E-cadherin, which led to tumor cell epithelial mesenchymal transition and metastasis [bib_ref] Cyclin D1, E-cadherin and beta-catenin expression in FIGO stage IA cervical squamous..., Koay [/bib_ref] [bib_ref] Roles of STAT3 and ZEB1 proteins in E-cadherin Down-regulation and human colorectal..., Xiong [/bib_ref] [bib_ref] E-cadherin inhibits tumor cell growth by suppressing PI3K|[sol]|Akt signaling via |[beta]|-catenin-Egr1-mediated PTEN..., Lau [/bib_ref]. Previous studies have revealed that the PI3K-Akt signaling pathway plays a crucial role in the progression and metastasis of lung cancer [bib_ref] PI3K/AKT inhibition induces compensatory activation of the MET/STAT3 pathway in non-small cell..., Bian [/bib_ref] , ovarian cancer, nasopharyngeal carcinoma [bib_ref] LZTS2 inhibits PI3K/AKT activation and radioresistance in nasopharyngeal carcinoma by interacting with..., Xu [/bib_ref] , prostate cancer [bib_ref] The PI3K/AKT pathway in the pathogenesis of prostate cancer, Chen [/bib_ref] , colorectal cancer [bib_ref] The PI3K/AKT Signaling Pathway: associations of miRNAs with dysregulated gene expression in..., Slattery [/bib_ref] , and gastric cancer [bib_ref] Nectin-4 promotes gastric cancer progression via the PI3K/AKT signaling pathway, Zhang [/bib_ref]. The present study further supports and confirms the important role of the PI3K-Akt signaling pathway in NM patients, which is novel, and to date, there has been no report in the literature. Hence, further studies are needed to verify the importance of this signaling pathway in NM. Furthermore, in the present study, ERK1/2 signaling was found to be enriched in NM patients after intrathecal chemotherapy and systemic therapy, indicating that the change in the ERK1/2 signaling could be associated with treatment resistance. Indeed, the inhibition of the ERK1/2 signaling pathway was caused by the Dioscorea bulbifera-induced apoptosis in human colorectal carcinoma cells [bib_ref] Dioscorea bulbifera induced apoptosis through inhibition of ERK 1/2 and activation of..., Ahmad [/bib_ref]. Conversely, the GABAergic signaling facilitated breast cancer metastasis through promotion of the ERK1/2-dependent phosphorylation [bib_ref] GABAergic signaling facilitates breast cancer metastasis by promoting ERK1/2-dependent phosphorylation, Zhang [/bib_ref]. This speculation needs to be further studied to determine whether this is associated with treatment resistance to both intrathecal chemotherapy and systemic therapy.
In addition, the present study also identified the CNVs of different genes in the CSF ctDNA samples, and the most affected ones were CDKN2A, CDK4 and MDM2. As it is known, the deletion of tumor suppressor CDKN2A was associated with melanoma and pancreatic neuroendocrine tumors metastasis, and the reduced survival rate of patients [bib_ref] Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation, Zeng [/bib_ref] [bib_ref] Loss of chromatin remodeling proteins and/or CDKN2A associates with metastasis of pancreatic..., Roy [/bib_ref]. Indeed, during the DNA replication in cells, gene amplification could generally create a risk for gene overexpression, which could be involved in cancer initiation and progression [bib_ref] Amplification of CDK4 and MDM2 in malignant melanoma, Muthusamy [/bib_ref]. Furthermore, a previous study revealed that CDK4 and MDM2 mutations occurred in melanomas and liposarcoma, while ERBB2 mutations occurred in breast cancer, EGFR mutations occurred in astrocytoma, and MYCN mutations occurred in neuroblastoma [bib_ref] Amplification of CDK4 and MDM2 in malignant melanoma, Muthusamy [/bib_ref]. Altered CD44 expression was associated with the aggressive clinicopathological characteristics of various human cancers [bib_ref] CD44, a therapeutic target for metastasising tumours, Orian-Rousseau [/bib_ref]. In addition, the present study revealed the EGFR and TP53 mutations in the CSF samples of NM patients with lung cancer, in which the COSMIC database also confirmed that these were the most frequently mutated genes in the CSF of NM patients, when compared to primary lung cancer. Furthermore, a higher mutational rate was found from EGFR [fig_ref] Table 1: Characteristics of 58 NM patients [/fig_ref] and BCL9 genes in NM with lung cancer, when compared to lung cancer patients without NM. A previous study revealed that EGFR mutations predisposed to the leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer, and the present study further supports this notion. In spite of the relatively small sample size, the rate of the above mutated genes was much higher than what was reported in patients with lung adenocarcinoma from the COSMIC database, implicating that these genes mutations and the alteration of the signaling pathways are involved in and have become risk factors for NM.
After associating these EGFR activating mutations between primary lung cancer and NM, the present data shows that the EGFR activating mutations in the CSF samples were roughly consistent with those of primary lung cancer. For example, the T790M in two CSF samples were sequentially collected during the TKI therapy. Lastly, the representative patient in the present study provided a unique showcase. The genetic profiling of the CSF ctDNA clearly reflected the dynamic changes in these identified driver genes and treatment responses. That is, this patient was detected to have the EGFR 19Del mutation in the CSF sample, and thereby received icotinib. Thereafter, the CSF sample revealed the EGFR 19Del and T790M mutations. Hence, the treatment was switched to AZD9291, which is a third generation EGFR TKI agent. A high response rate was exhibited by patients with tumors harboring the EGFR T790M mutation, as well as a high capacity to penetrate into the CSF by crossing the blood-brain barrier [bib_ref] AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in..., Cross [/bib_ref] [bib_ref] Next-generation epidermal growth factor receptor tyrosine kinase inhibitors in epidermal growth factor..., Tan [/bib_ref]. The NGS allowed the physician to modify treatment option, in order to help the patient archive complete remission. As it is known, the EGFR T790M mutation mediates the acquired resistance to EGFR TKI [bib_ref] AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer, Jänne [/bib_ref] [bib_ref] Rociletinib in EGFR-mutated non-small-cell lung cancer, Sequist [/bib_ref] , and a previous study reported that AZD9291 was designated as a powerful agent capable of overcoming the acquired EGFR T790M resistance mutation [bib_ref] Epidermal growth factor receptor tyrosine kinase inhibitors for non-smallcell lung Cancer patients..., Liao [/bib_ref]. The data on the GO analysis (a) and KEGG pathway analysis (b) of significant mutated genes in the CSF obtained from NM patients with lung cancer compared to mutated genes of lung cancer in COSMIC database However, the present study does have some limitations. For example, the cohort has a relatively small number of patients, and the different treatment options could not be separated to analyze the association of gene mutation, and the changes in gene copy number with treatment responses in these patients. Thus, future studies with a larger sample size from multiple institutions could help to solve these issues. Though we have found that mutated genes detected in CSF are enriched in the PI3K-Akt and ERK1/2 signaling pathways, it is hard for us to explain the detected mutations are gain of function mutations or loss of function mutations in such pathways. Thus, we will perform the functional analysis in the following studies. Due to the target amplicon sequencing, we could not address the whole genome alterations and yield the comprehensive landscape of NM. So there are any other area we will focus on to make more depth analyses in the future studies.
# Conclusions
This study identified gene mutations in all CSF ctDNA samples, indicating that these mutated genes may be acted as a kind of biomarker for diagnosis of NM, and these mutated genes may affect meningeal metastasis through PI3K-Akt signaling pathway.
[fig] Figure 5a: or the number of gene mutations (r = − 0.195, P = 0.129; [/fig]
[fig] Figure 1: Profiling of genes mutations in the CSF samples obtained from NM patients, regardless of the primary cancer origin points, while two CSF samples were collected from the other patient at distinct time points. In the subgroup analysis, it was found that CSF ctDNA was detected in all 45 CSF samples obtained from 42 lung cancer patients with NM, and gene mutations were also detected in all patients. Specifically, EGFR mutations occurred in 39 of 45 CSF samples (86.67%), followed by TP53 (38/ 45, 84.44%), PTEN (27/45, 60.00%), TET2 (18/45, 40.00%), APC (17/45, 37.78%), CDKN2A (14/45, 31.11%), GNAQ (14/45, 31.11%), and NOTCH1 (11/45, 24.44%). A number of gene mutations previously reported with lung cancer were identified in CSF with NM, while EGFR, TP53, PTEN, TET2, APC, CDKN2A, GNAQ, NOTCH1, FLT3, VHL, BRCA2, PTCH1, CBL, MLH1, BRAF, NRAS, TSC2, CSF1R, KIT, MAP2K1, MSH2, TSC1, HRAS, IFITM1 and BCL9 mutations were [/fig]
[fig] Figure 2: The GO analysis (a) and KEGG pathway analysis (b) of mutated genes in the CSF obtained from NM patients regardless of the primary cancer origin. BP, biological process; CC, cellular components; MF, molecular function statistically more common in the present cohort of NM, when compared to the lung cancer noted in the COS-MIC database (https://cancer.sanger.ac.uk) ( [/fig]
[fig] Figure 3: The association of variant allele frequency with detectable tumor cells in the CSF. MF, mutation frequency [/fig]
[fig] Figure 4: The GO analysis (a) and KEGG pathway analysis (b) of mutated genes in the CSF obtained from NM patients receiving both intrathecal chemotherapy and systemic therapy. BP, biological process; CC, cellular components; MF, molecular function. [/fig]
[fig] Figure 5: The association of CSF ctDNA concentration with the Karnofsky performance status (KPS) scores, the number of gene mutations and the presence of CSF tumor cells. (A) CSF ctDNA concentration vs. KPS (r = − 0.038, P = 0.768). (B) CSF ctDNA concentration vs. the number of gene mutations (r = − 0.195, P = 0.129). (C) The number of gene mutations vs. KPS scores (r = 0.192, P = 0.135). (D) CSF ctDNA concentration vs. the presence of detectable circulating tumor cells in the CSF (Z = -2.883, P = 0.004) [/fig]
[fig] Figure 6: The data on the GO analysis (a) and KEGG pathway analysis (b) of mutated genes in the CSF obtained from NM patients with lung cancer Zhao et al. BMC Cancer (2020) 20:690 [/fig]
[fig] Figure 8: A representative case. (a) The head contrast enhanced MRI. This showed the linear and strip abnormal enhancement of the cerebellar sulcus (red arrow) after the patient had a headache during the icotinib therapy for the primary tumor. (b) The May-Gruwald-Giemsa staining of the CSF sample. The data showed the tumor cells in the CSF (× 1000). (c) The head contrast enhanced MRI. This showed the dramatic improvement and complete response. (d) The May-Gruwald-Giemsa staining. The data showed fewer tumor cells (× 1000), when compared to that in B [/fig]
[table] Table 1: Characteristics of 58 NM patients [/table]
[table] Table 2: Unique mutated genes in each treatment group [/table]
[table] Table 3: The number of mutation-bearing CSF samples (total number of investigated CSF samples: 45) per cancer-associated gene (total number of investigated genes: 143) [/table]
[table] Table 4: EGFR activating mutations in primary lung cancer and NM CSF samples CSF sample No. [/table]
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Rate and Predisposing Factors for Sacroiliac Joint Radiographic Progression After a Two‐Year Follow‐up Period in Recent‐Onset Spondyloarthritis
Objective. To evaluate the rate of radiographic structural progression in the sacroiliac (SI) joints in patients with radiographic or nonradiographic axial spondyloarthritis (SpA), and to determine factors predisposing to such progression, over 2 years.Methods. Patients with recent-onset axial SpA (from the Devenir des Spondyloarthropathies Indiffer erenci ees R ecentes cohort) were assigned a radio-graphic SI joint score according to the modified New York criteria. Demographic characteristics, smoking status, HLA-B27 positivity, inflammation on magnetic resonance imaging (MRI) of the SI joints, disease activity, and treatment were investigated as potential predisposing factors. The main analysis consisted of the evaluation of the switch from nonradiographic to radiographic axial SpA, but other definitions of radiographic progression were also evaluated.Results. Of the 708 patients enrolled, 449 had baseline and 2-year pelvic radiographs. Of these patients, 47% were men. Their mean 6 SD age was 34 6 9 years, 61% were B27 positive, and 37% had inflammation of the SI joints on MRI. The percentages of patients who switched from nonradiographic to radiographic axial SpA (4.9% [16 of 326]) and from radiographic to nonradiographic axial SpA (5.7% [7 of 123]) were low. The mean 6 SD change in the total SI joint score (range 0-8) was small (0.1 6 0.8) but highly significant (P < 0.001). The potential baseline predisposing factors for meeting the modified New York criteria in the multivariate analysis were current smoking, HLA-B27 positivity, and inflammation of the SI joints on MRI, with odds ratios of 3.3 (95% confidence interval [95% CI] 1.0-11.5], 12.6 (95% CI 2.3-274), and 48.8 (95% CI 9.3-904), respectively.Conclusion. Our findings suggest that structural progression does exist in early SpA, but it is quite small and observed in a small number of patients, and that environmental (smoking status), genetic (HLA-B27 positivity), and inflammation (inflammation of the SI joints on MRI) markers might be independent predisposing factors for progression.
The recommended imaging investigations for patients with chronic inflammatory back pain (present for $3 months) occurring before the age of 45 years include both plain radiographs of the pelvis and (in the case of normal findings on radiography) magnetic resonance imaging (MRI) of the pelvis, since the suspected diagnosis in this case is axial spondyloarthritis (SpA) and the involvement of the sacroiliac (SI) joints seems to be a cornerstone of the disease. For epidemiologic studies and/or clinical trials, patients are enrolled according to existing classification criteria. The presence of structural damage of the SI joints observed on plain radiographs of the pelvis is mandatory in the first set of classification criteria (the New York criteria and the modified New York criteria) [bib_ref] Evaluation of diagnostic criteria for ankylosing spondylitis: a proposal for modification of..., Van Der Linden [/bib_ref]. These abnormalities on radiographs are wrongly called "sacroiliitis," since "-itis" means an inflammatory process in medicine and plain radiography cannot detect inflammation but only chronic change and structural damage, such as subchondral bone sclerosis and joint irregularities.
Axial SpA with radiographic damage is called either ankylosing spondylitis (AS) or radiographic axial SpA [bib_ref] Ankylosing spondylitis, spondyloarthropathy, spondyloarthritis, or spondylarthritis: what's in a name?, Claudepierre [/bib_ref]. Because the time lag between disease onset in terms of symptoms and the occurrence of structural damage may be quite long (estimated between 5 and 7 years) (4) and because some patients with SpA will never develop structural damage, other sets of criteria have been developed during the last 3 decades (e.g., the Amor criteria [bib_ref] Critères de classification des spondylarthropathies, Amor [/bib_ref] and the European Spondylarthropathy Study Group [ESSG] criteria [bib_ref] and the European Spondylarthropathy Study Group. The European Spondylarthropathy Study Group preliminary..., Dougados [/bib_ref]. The Assessment of Spondyloarthritis international Society (ASAS) developed classification criteria that include patients with nonradiographic axial SpA in addition to radiographic axial SpA and integrate MRI findings [bib_ref] The development of Assessment of Spondy-loArthritis international Society classification criteria for axial..., Rudwaleit [/bib_ref].
The distinction between radiographic and nonradiographic axial SpA based on the presence or absence of radiographic sacroiliitis is highlighted by the fact that pharmaceutical companies have developed their compounds, in particular tumor necrosis factor (TNF) inhibitors, with reference to the modified New York criteria [bib_ref] and the Ankylosing Spondylitis Study for the Evaluation of Recombinant Infliximab Therapy..., Van Der Heijde [/bib_ref] [bib_ref] for the Enbrel Ankylosing Spondylitis Study Group. Recombinant human tumor necrosis factor..., Davis [/bib_ref] [bib_ref] Efficacy and safety of golimumab in patients with ankylosing spondylitis: results of..., Inman [/bib_ref] and that these compounds have been approved only for patients with SI joint structural damage. Because of this restriction in the use of these compounds, pharmaceutical companies have more recently conducted specific studies in patients with nonradiographic axial SpA (i.e., excluding patients with structural damage) [bib_ref] Efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis: results..., Sieper [/bib_ref] [bib_ref] Symptomatic efficacy of etanercept and its effects on objective signs of inflammation..., Dougados [/bib_ref] [bib_ref] Efficacy of certolizumab pegol on signs and symptoms of axial spondyloarthritis including..., Landew E [/bib_ref]. The European Medicines Agency has approved TNF inhibitors for patients with nonradiographic axial SpA only if objective signs of inflammation, such as the presence of relevant SI subchondral bone marrow edema on MRI and/or an elevated Creactive protein (CRP) level, are present. Post hoc ana-lyses of the trials performed in nonradiographic axial SpA have detected these objective signs of inflammation as strong predictive factors for the effectiveness of anti-TNF treatment [bib_ref] Efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis: results..., Sieper [/bib_ref] [bib_ref] Effectiveness of tumor necrosis factor a blockers in early axial spondyloarthritis: data..., Molt O [/bib_ref].
However, the long-term natural history of patients with nonradiographic axial SpA is not well known. In particular, the rate of radiographic progression (i.e., the percentage of patients who will switch from having nonradiographic axial SpA to having radiographic axial SpA over time), the predisposing factors for such a switch, and whether the presence of objective signs of inflammation might predict this SI joint radiographic progression are not known. Moreover, categorization of the structural damage observed at the level of the SI joint (sacroiliitis according to the modified New York criteria [yes/no]) might not be the optimal way to evaluate the natural history of the disease, and other potential scoring systems of radiographic structural damage of the SI joint might be more appropriate.
The Devenir des Spondyloarthropathies Indiffer erenci ees R ecentes (DESIR) cohort, a French multicenter longitudinal observational study of patients with recent-onset inflammatory low back pain suggestive of axial SpA according to the treating rheumatologist [bib_ref] Clinical presentation of patients suffering from recent onset chronic inflammatory back pain..., Dougados [/bib_ref] , offered a unique opportunity to answer these questions. In the DESIR cohort, concomitantly with a clinical evaluation performed every 6 months, plain radiographs have been collected systematically at baseline and at the 2-year follow-up visit. Moreover, MRIs of the pelvis were obtained at baseline, so this could be evaluated as a possible predictive factor for progression.
# Patients and methods
Patients. For this analysis, the data collected during the first 2 years of follow-up in the DESIR cohort were used. The DESIR cohort has been described extensively [bib_ref] Clinical presentation of patients suffering from recent onset chronic inflammatory back pain..., Dougados [/bib_ref]. Briefly, consecutive patients ages 18-50 from 25 centers in France were included. Patients had inflammatory back pain in the thoracic spine, lumbar spine, and/or buttocks area based on either the Calin [bib_ref] Clinical history as a screening test for ankylosing spondylitis, Calin [/bib_ref] or Berlin (18) criteria with a duration of $3 months but ,3 years. They were included in the study if the treating rheumatologist considered the symptoms to be suggestive of axial SpA, with a score of $5 on a scale of 0-10, where 0 was not suggestive of axial SpA and 10 was very suggestive of axial SpA. Between December 2007 and April 2010, 708 patients were included.
The study was conducted according to good clinical practice guidelines and was approved by the appropriate medical ethics committee. A detailed description of the study protocol is available online at http://www.lacohortedesir.fr/desir-inenglish/. The research proposal for this particular analysis was approved by the scientific committee of the DESIR cohort.
Data collection. A database was built using a standardized case report form that included data from questionnaires, findings of physical examinations, ongoing treatments, comorbidities, and laboratory test results according to the DESIR protocol. The database used for this analysis was locked in October 2014.
Demographic and disease characteristics. At baseline, the following information was collected: age, sex, smoking status, HLA-B27 status, and duration of axial symptoms. At baseline and at the 6-month intermediate visits during the first 2 years of follow-up, the following parameters were also collected: Bath AS Disease Activity Index (BASDAI) [bib_ref] A new approach to defining disease status in ankylosing spondylitis: the Bath..., Garrett [/bib_ref] , Bath AS Functional Index (BASFI) [bib_ref] A new approach to defining functional ability in ankylosing spondylitis: the development..., Calin [/bib_ref] , CRP level, TNF therapy intake, and nonsteroidal antiinflammatory drug (NSAID) intake according to the ASAS NSAID score [bib_ref] ASAS recommendations for collecting, analysing and reporting NSAID intake in clinical trials/epidemiological..., Dougados [/bib_ref].
Radiographs of the pelvis. The radiographs of the pelvis obtained at baseline and after 2 years of follow-up were centrally stored in a specific software program after anonymization and blinding of the date of collection according to a prespecified list of randomization with 7 different letters (from A to G). For example, for a specific patient, the baseline radiograph could be coded as C and the 2-year radiograph as A.
The radiographs were evaluated by 2 central readers (RvdB and VVH; reader 1 and reader 2, respectively). A detailed description of the central reading has been published previously [bib_ref] Agreement between clinical practice and trained central reading in reading of sacroiliac..., Van Den Berg [/bib_ref]. Briefly, each reader evaluated each SI joint according to the modified New York method. In this method, the joint is graded on a scale of 0-4, where 0 5 a normal SI joint, 1 5 suspicious changes, 2 5 minimal abnormality, 3 5 unequivocal abnormality, and 4 5 severe abnormality (e.g., complete ankylosis of the SI joint). The 2 radiographs for each individual patient were evaluated at the same time. Readers were aware that the 2 radiographs were of the same patient but were unaware of the chronology of the radiographs. The readers were blinded with regard to all clinical and laboratory data and the other imaging modality (i.e., MRI). Agreement between the 2 readers with regard to fulfillment of the modified New York criteria for sacroiliitis at the patient level (i.e., at least unilateral grade 3 or bilateral grade 2 and/or a progression of at least 1 grade for each SI joint) was calculated. In cases in which the readers disagreed, a radiologist (MR) or a rheumatologist (PC) experienced in the field of SpA served as adjudicator. The adjudicator scored the pelvic radiograph as yes or no according to the modified New York criteria by assigning a grade for each SI joint based on the scale described above, and was blinded with regard to the assessment of the primary readers. An image was marked as positive if 2 of the 3 readers agreed.
MRI of the pelvis. The MRIs collected at baseline were stored centrally after anonymization. The images were evaluated by 2 central readers (RvdB and FT). A detailed description of the central reading has been published previously [bib_ref] Classification of axial SpA based on positive imaging (radiographs and/or MRI of..., Van Den Berg [/bib_ref]. Briefly, findings of MRI of the SI joint were considered positive according to the ASAS definition if bone marrow edema lesions highly suggestive of SpA were present (either $1 bone marrow edema lesion on $2 consecutive slices or severe bone marrow edema lesions on a single slice) [bib_ref] Defining active sacroiliitis on magnetic resonance imaging (MRI) for classification of axial..., Rudwaleit [/bib_ref]. All available baseline MRIs of the SI joint were read independently by the 2 readers, who were blinded with regard to all clinical and laboratory data and the other imaging modality (i.e., pelvic radiographs). Agreement on whether MRI findings were positive was calculated. In the case of disagreement, a senior radiologist (MR) served as adjudicator. The adjudicator scored the MRI of the SI joint according to the ASAS definition and was blinded with regard to the assessment of the primary readers. An image was marked as positive if 2 of the 3 readers agreed.
Statistical analysis. Since the primary objective of this study was to examine SI joint radiographic progression over the first 2 years, only patients with available radiographs at baseline and 2-year follow-up were analyzed. The first step of the analysis consisted of a descriptive comparison of the patients enrolled in the DESIR cohort and the patients who completed the first 2 years of the study.
With regard to the rate of radiographic progression, the main analysis was the evaluation of the percentage of patients who were classified at baseline as having nonradiographic axial SpA and subsequently developed radiographic axial SpA after 2 years (based on the modified New York criteria). Similarly, we evaluated the percentage of patients who were classified at baseline as having radiographic axial SpA and were subsequently classified as having nonradiographic SpA after 2 years. Radiographic progression was also evaluated in the entire population, considering the radiographic score as a continuous variable with a range of 0-8 (4 possible grades for each SI joint). This analysis was conducted separately for reader 1 and for reader 2 but was also done for the mean score of the 2 readers.
The final analysis evaluating radiographic progression consisted of the evaluation of the percentage of patients considered to be progressors based on additional definitions. The first proposed definition was a change of at least 1 grade in at least 1 SI joint after 2 years of follow-up. For this analysis, we were able to calculate the percentage of "progressors" (worsening) and similarly the percentage of "regressors" (improving). The second proposed definition was a change of at least 1 grade in at least 1 SI joint after 2 years of follow-up and an absolute score of the "worsened" joint at year 2 of at least 2 (i.e., at least minimal abnormality). The third definition was the percentage of patients with a change other than 0 in the total score of the 2 SI joints (mean of the 2 readers); for this analysis, we defined a "progressor" as a patient with a change in the total score of .0 and "regressor" as a patient with a change in the total score of ,0. These analyses were performed on the whole set of patients for whom pelvic radiographs were available but by presenting the data with regard to the presence or absence of baseline radiographic structural damage according to the modified New York criteria.
Possible predisposing factors for structural progression were evaluated according to different definitions of the dependent variable and to a similar list of potential independent variables. The potential independent variables included selected variables collected at baseline (i.e. age, sex, smoking status, HLA-B27 positivity, CRP level, presence of inflammation on MRI of the SI joints, BASDAI, and SI joint radiographic structural damage according to the modified New York criteria). The different definitions of the dependent variable were as follows: 1) switch from nonradiographic to radiographic axial SpA after 2 years of follow-up, 2) switch from radiographic to nonradiographic axial SpA after 2 years of follow-up, 3) changes (worsening) in at least 1 grade in at least 1 SI joint after 2 years of followup, 4) changes (improvement) in at least 1 grade in at least 1 SI joint after 2 years of follow-up, and 5) changes (worsening) in at least 1 grade in at least 1 SI joint after 2 years of follow-up and an absolute score for the "worsened" SI joint of at least 2 at year 2.
All of the analyses were performed using a multiple regression model that included all of the independent variables for which the univariate analysis revealed a statistical significance at a P level of at least ,0.20. Only the independent variables that had a P value of less than 0.05 in the multivariate analysis were considered to be statistically significant. No correction was performed because of the multiplicity of the tests, but the first analysis (i.e., the evaluation of the factors predicting the switch from nonradiographic to radiographic axial SpA) was considered the primary one.
# Results
Patients and study course. Of the 708 patients enrolled, 595 completed the first 2 years of follow-up. Radiographs of the pelvis at baseline and at 2 years were available for 449 patients. [fig_ref] Table 1: Baseline characteristics of the SpA patients* [/fig_ref] summarizes the baseline characteristics of the patients with regard to the completion of the study and the baseline SI joint radiographic status. There was no significant difference between the whole population of the cohort and the patients evaluated in this study.
Radiographic progression at year 2. New York criteria at baseline, 7 (5.7%) no longer fulfilled the modified New York criteria at year 2. A higher percentage of progressors, both in terms of absolute value and in terms of differences between the progressors and the regressors, was observed for the other definitions of progressors, in particular the definition of a progressor as a patient in whom a change in at least 1 grade in at least 1 SI joint was observed. According to this definition, 11.1% of the patients were defined as progressors and only 5.8% as regressors. If we define the "true" percentage of progression as the percentage of patients who experienced worsening minus the percentage of patients who experienced improvement, the progression can be estimated as 20.8% according to the modified New York criteria, 18.2% for changes in the total score of the 2 SI joints of .0, 15.3% for changes in at least 1 grade in at least 1 SI joint, and 17.1% for changes in at least 1 grade in at least 1 SI joint and an absolute score of $2 at year 2 in the worsened joint. The magnitude of true progression was always higher in the subgroup of patients with baseline radiographic structural damage.
Moreover, the evaluation of the changes in the mean total score (of the scores assigned by the 2 readers) of the 2 SI joints showed a minimal but significant worsening, with a mean 6 SD of 0.1 6 0.8 in the whole population (P , 0.001). Possible scores ranged from 28 (total disappearance at 2 years of a baseline bilateral grade 4) to 18 (appearance at 2 years of a bilateral grade 4 in a patient with baseline normal joints [bilateral grade 0]). This worsening was more pronounced in the patients with nonradiographic axial SpA at baseline (mean 6 SD 0.2 6 0.7; P , 0.001) than in the patients with radiographic axial SpA at baseline (mean 6 SD 0.0 6 0.8; P 5 0.10). Because of the previously demonstrated poor interreader reliability (22), we performed this analysis separately for each reader. The observed changes for reader 1 versus reader 2 were 10.2 6 0.9 versus 10.1 6 0.9, 10.2 6 0.9 versus 10.1 6 0.9, and 10.0 6 1.1 versus 20.1 6 0.9 in the entire population, the patients with nonradiographic axial SpA at baseline, and the patients with radiographic axial SpA at baseline, respectively.
Predisposing factors for structural progression. The main analysis was of the switch from nonradiographic to radiographic axial SpA based on the modified New York criteria. [fig_ref] Table 2: Radiographic progression during the 2-year follow-up period with regard to baseline and... [/fig_ref] summarizes the findings of the univariate analyses. Multiple logistic regression identified 3 variables: smoking status, HLA-B27 positivity, and inflammation observed on MRI of the SI joint [fig_ref] Figure 2: Risk of radiographic progression in the sacroiliac joint [/fig_ref] In order to check the validity of the observed results, we planned to perform a similar analysis defining the dependent variable as the switch from radiographic axial SpA to nonradiographic axial SpA. This analysis was not performed, however, because the number of patients was too small (only 7 of 123 patients experienced improvement).
The analysis defining radiographic progression as a worsening of at least 1 grade in at least 1 SI joint identified HLA-B27 positivity, positive findings on MRI, and the presence of baseline structural damage of the SI joint on radiographs of the pelvis as predisposing factors for radiographic progression (see [fig_ref] Table 1: Baseline characteristics of the SpA patients* [/fig_ref] , available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art. 39666/abstract). It should be noted that in the univariate analysis, there was a trend toward statistical significance for the variable "current smoker at baseline" (P 5 0.057). The percentage of progressors was 14.1% versus 6.9% for HLA-B27-positive patients versus HLA-B27-negative patients, 20.4% versus 6.4% for patients with positive findings on MRI versus those without positive findings on MRI, and 21.9% versus 7.4% for patients with radiographic structural damage versus those without radiographic structural damage according to the modified New York criteria. For this definition of change, we also performed an analysis defining radiographic regression as an improvement of at least 1 grade in at least 1 SI joint. According to this analysis, 26 of the 449 patients were considered regressors. This analysis identified only the variable "baseline structural damage of the SI joint on pelvic radiograph" as a predisposing factor, with an OR of 2.5 (95% CI 1.03-6.16) (P 5 0.043). The percentage of regressors was 11.4% versus 3.7% in the group with abnormal radiographic structural damage at baseline versus the group without abnormal radiographic structural damage at baseline.
The analysis defining radiographic progression as a worsening of at least 1 grade in at least 1 SI joint and a final absolute grade of at least 2 in the worsened joint identified the same predisposing factors (HLA-B27, inflammation of the SI joints on MRI, and baseline Finally, the multivariate analysis of the mean changes in the total score (of the scores assigned by the 2 readers) of the 2 SI joints identified only 2 variables as predisposing factors to radiographic progression: HLA-B27 positivity and baseline MRI positivity. HLA-B27 positivity was associated with an increase of 0.41 in the total score (P 5 0.014), and inflammation of the SI joints observed on MRI was associated with an increase of 1.03 in the total score (P , 0.001).
# Discussion
This study suggests that structural progression does occur during 2 years of follow-up in early axial SpA, but it is quite small and is only observed in a small number of patients. Our findings indicate that the total SI joint score and/or a change of at least 1 grade are the most sensitive definitions of progression. Moreover, this study also suggests that genetic factors (e.g., HLA-B27 positivity), environmental factors (e.g., smoking status), and inflammation (e.g., inflammation of the SI joints observed on MRI) are independent predictors of radiographic progression in the SI joint in early axial SpA.
The results regarding the percentage of patients switching from nonradiographic axial SpA to radiographic axial SpA (4.9%) and from radiographic axial SpA to nonradiographic axial SpA (5.7%) could suggest that the observed changes were related to a measurement error due to the poor intra-and interreliability of this outcome measure [bib_ref] Agreement between clinical practice and trained central reading in reading of sacroiliac..., Van Den Berg [/bib_ref] and not to true structural progression of the disease. However, our findings provide evidence of a true progression rate. The results found using the other definitions of progression support the notion of real progression (e.g., worsening in total SI joint score [in 20.9% of patients] versus improvement in total SI joint score [in 12.7% of patients] and changes of at least 1 grade in at least 1 SI joint [11.1% progressors versus 5.8% regressors]). In addition, the difference in mean change in total score was highly significant (mean 6 SD 0.1 6 0.8; P , 0.001). Moreover, similar findings have been reported in other cohorts [bib_ref] Defining active sacroiliitis on magnetic resonance imaging (MRI) for classification of axial..., Rudwaleit [/bib_ref]. In the German Spondyloarthropathy Inception Cohort (GESPIC), the percentage of patients with a change in at least 1 grade in at least 1 SI joint was 16.8% and 6.3% for the progressors and regressors, respectively [bib_ref] Rates and predictors of radiographic sacroiliitis progression over 2 years in patients..., Poddubnyy [/bib_ref].
It should be noted that the rate of progression observed in the DESIR cohort was very low. For example, 300 of the 449 patients evaluated had no change at all in the total SI joint score. Despite the fact that we have seen that there are several arguments in favor of the existence of true progression, it has to be recognized that the relatively high number of regressors makes the evaluation of the true rate of progression challenging. Our findings suggest that progression is a true phenomenon and that regression might reflect measurement error.
The results of the present study indicate that genetic factors (HLA-B27 positivity), environmental factors (smoking status), and inflammation (positive findings on MRI) could be considered independent factors for subsequent structural progression. Moreover, the analyses conducted in the entire baseline population (including patients with radiographic structural damage and those with nonradiographic structural damage) suggest that the presence of baseline structural damage is also a predisposing factor of structural progression.
The presence of objective signs of inflammation (either an abnormal CRP level or the presence of subchondral bone edema at the SI joint observed on MRI) have previously been reported as predisposing factors for subsequent radiographic progression in the SI joint [bib_ref] Severity of baseline magnetic resonance imaging-evident sacroiliitis and HLA-B27 status in early..., Bennett [/bib_ref]. Our study failed to show a clear relationship between an abnormal CRP level at baseline and radiographic progression. Such a relationship was previously reported in the German cohort, where MRIs of the SI joint were not available [bib_ref] Rates and predictors of radiographic sacroiliitis progression over 2 years in patients..., Poddubnyy [/bib_ref]. In our study, the relationship between the presence of inflammation on MRI and radiographic progression was very high, suggesting a very low risk of radiographic structural progression when no subchondral bone edema was observed on MRI of the SI joint.
A cross-sectional study of the DESIR cohort has previously emphasized the link between HLA-B27, radiographic structural damage, and the presence of subchondral bone edema observed on MRI of the SI joints [bib_ref] HLA-B27 positive patients differ from HLA-B27 negative patients in clinical presentation and..., Chung [/bib_ref]. Moreover, the risk of structural progression has been reported to be particularly high in the case of the coexistence of HLA-B27 positivity and inflammatory lesions of the SI joint in the Leeds cohort [bib_ref] Severity of baseline magnetic resonance imaging-evident sacroiliitis and HLA-B27 status in early..., Bennett [/bib_ref]. The findings of the present study support this link, since HLA-B27 positivity and inflammatory lesions of the SI joint on MRI were considered to be independent factors for structural progression. Objective signs of inflamma-tion in SpA have also been reported as predisposing factors for anti-TNF treatment response, especially in patients with nonradiographic axial SpA [bib_ref] Efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis: results..., Sieper [/bib_ref] [bib_ref] Efficacy of certolizumab pegol on signs and symptoms of axial spondyloarthritis including..., Landew E [/bib_ref] [bib_ref] Effectiveness of tumor necrosis factor a blockers in early axial spondyloarthritis: data..., Molt O [/bib_ref].
Our study also indicates that smoking might be an independent factor predisposing to subsequent structural progression. In the DESIR cohort, the cross-sectional analysis performed on the baseline data suggested a link between smoking status and both the activity and severity of the disease [bib_ref] Smokers in early axial spondyloarthritis have earlier disease onset, more disease activity,..., Chung [/bib_ref]. Smoking has also been reported to be related to the risk of structural progression at the level of the spine in the GESPIC cohort, with a potential doserelated effect (29) (i.e., the percentage of progressors defined by a change in the modified Stoke AS Spine Score of at least 2 points was 10.1%, 18.6%, and 28.6% in nonsmokers, those who smoked #10 cigarettes a day, and those who smoked .10 cigarettes a day, respectively). It should be noted that in the field of axial SpA, smoking has also been found to be related to a high incidence of the disease (30) and a poor response to biologic agents [bib_ref] on behalf of the Rheumatologists of Swiss Clinical Quality Management Program for..., Ciurea [/bib_ref].
Findings related to the baseline factors predisposing to subsequent structural progression should be interpreted with caution, since they were determined in a very small number of patients. For example, only 16 patients switched from a nonradiographic to a radiographic status according to the modified New York criteria.
Finally, the presence of baseline structural damage was identified as an important predisposing factor of subsequent structural progression when analyses were performed in the entire population (i.e., in patients with baseline radiographic or nonradiographic structural damage) (see [fig_ref] Table 1: Baseline characteristics of the SpA patients* [/fig_ref] , available on the Arthritis & Rheumatology web site at http:// onlinelibrary.wiley.com/doi/10.1002/art.39666/abstract). Such findings are consistent with previous data in rheumatology. For example, the presence of syndesmophytes is an important predisposing factor of subsequent radiographic structural progression at the level of the spine in axial SpA [bib_ref] Progression of radiographic damage in patients with ankylosing spondylitis: defining the central..., Baraliakos [/bib_ref] ; similarly, the presence of baseline erosion in rheumatoid arthritis is a strong predictor of subsequent radiographic progression [bib_ref] Matrix to predict rapid radiographic progression of early rheumatoid arthritis patients from..., Fautrel [/bib_ref]. Our study has some limitations. The relatively short duration of follow-up (2 years) could be considered a weakness of this study, since longer follow-up might be required to detect structural progression [bib_ref] Continuous long-term anti-TNF therapy does not lead to an increase in the..., Baraliakos [/bib_ref]. However, the short duration could be also seen as a strength, since despite this short time period, this study demonstrated a small but true structural progression.
The findings of the present study (a low rate of progression after a 2-year follow-up period) do not confirm the usual 10% rate (i.e., 10% of patients with non-radiographic structural damage will switch to radiographic structural damage after 2 years of follow-up) that is frequently mentioned in reviews [bib_ref] Radiographic progression in ankylosing spondylitis/axial spondyloarthritis: how fast and how clinically meaningful?, Poddubnyy [/bib_ref]. The low rate of progression observed in the present study has recently been confirmed in a population-based cohort study, with 6.4%, 17.3%, and 26.4% of patients switching from nonradiographic to radiographic disease after 5, 10, and 15 years of follow-up, respectively [bib_ref] Progression of nonradiographic axial spondyloarthritis to ankylosing spondylitis: a population-based cohort study, Wang [/bib_ref].
The relatively high number of missing values could also be considered a limitation. In our study, only 449 of the 708 patients enrolled were evaluated, mainly due to missing images. However, the similarity in the baseline clinical presentation between the 2 groups (the ones for whom we had a complete data image set and the ones for whom images were missing) is evidence that our results are valid and interpretable.
Our study also has many strengths, especially the high number of patients evaluated and the quality of the reading performed by 2 independent, trained readers. Additional studies with a longer follow-up period and studies in other cohorts of patients are needed to confirm these findings. Our findings also indicate the need for translational research studies to investigate the underlying mechanisms of radiographic progression in SpA.
[fig] Figure 1: Radiographic progression in the sacroiliac joint (SIJ) over a 2-year follow-up period in patients with recent-onset (,3 years) nonradiographic axial spondyloarthritis (nr-axial SpA) and patients with radiographic axial SpA (r-axial SpA). mNY 5 modified New York. [/fig]
[fig] Figure 2: Risk of radiographic progression in the sacroiliac joint (SIJ) after a 2-year follow-up period in patients with recent-onset axial spondyloarthritis (SpA) with regard to baseline parameters. Radiographic progression was defined as a switch from nonradiographic axial SpA to radiographic axial SpA according to the modified New York criteria. A, Smoking status at baseline. B, HLA-B27 positivity. C, Inflammation (subchondral bone edema according to the Assessment of SpondyloArthritis international Society/Outcome Measures in Rheumatology recommendations) of the SI joints on magnetic resonance imaging (MRI) at baseline, determined by a central reading procedure. Bars show the percent of patients. OR 5 odds ratio (with 95% confidence interval shown in brackets). [/fig]
[table] Table 1: Baseline characteristics of the SpA patients* [/table]
[table] Table 2: Radiographic progression during the 2-year follow-up period with regard to baseline and 2year characteristics in patients with recent-onset (,3 years) axial SpA* [/table]
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Depletion of Csk preferentially reduces the protein level of LynA in a Cbl-dependent manner in cancer cells
there are eight human Src-family tyrosine kinases (SfKs). SfK members c-Src, c-Yes, fyn, and Lyn are expressed in various cancer cells. SfK kinase activity is negatively regulated by csk tyrosine kinase.Reduced activity of csk causes aberrant activation of SfKs, which can be degraded by a compensatory mechanism depending on cbl-family ubiquitin ligases. We herein investigated whether all SfK members are similarly downregulated by cbl-family ubiquitin ligases in cancer cells lacking csk activity. We performed Western blotting of multiple cancer cells knocked down for csk and found that the protein levels of the 56 kDa isoform of Lyn (LynA), 53 kDa isoform of Lyn (LynB), c-Src, and Fyn, but not of c-Yes, were reduced by csk depletion. induction of c-cbl protein levels was also observed in csk-depleted cells.The reduction of LynA accompanying the depletion of Csk was significantly reversed by the knockdown for Cbls, whereas such significant recovery of LynB, c-Src, and Fyn was not observed. These results suggested that LynA is selectively downregulated by cbls in cancer cells lacking csk activity.
different members or splicing isoforms of SFKs in macrophages lacking the activity of Csk. In the present study, we examined whether particular members or splicing isoforms of SFKs were preferentially down-regulated by the protein degradation system when the activity of Csk was reduced in cancer cells where a different set of SFKs is expressed compared in macrophages.
# Results
LynA protein levels are preferentially reduced in csk-depleted cancer cells. In order to assess the effect of reduced activity of Csk on the protein levels of individual members of SFKs, we performed Western blotting of two types of cancer cells (HCT116 and HeLa S3 cells) transfected with small interfering RNA (siRNA) for Csk (siCsk_1, SASI_Hs02_00328637, and siCsk_2, SASI_Hs02_00328637) or control siRNA. HCT116 cells redundantly expressed c-Src, c-Yes, and Lyn []. In HCT116 cells, the depletion of Csk remarkably reduced LynA and mildly reduced c-Src. The protein level of LynB was slightly reduced upon the depletion of Csk in HCT116 cells, although this reduction was not statistically significant. The protein level of c-Yes was not altered by the depletion of Csk in HCT116 cells. HeLa S3 cells expressed c-Yes, Fyn, and Lyn []. HeLa S3 cells depleted of Csk exhibited reduced LynA, LynB, and Fyn levels. The reduction level of LynA was higher than that of LynB. Comparing the reduction level of Fyn with that of LynA or LynB was difficult because different reduction levels of Fyn were induced by two Csk siRNAs. Csk depletion did not affect the c-Yes protein levels in HeLa S3 and HCT116 cells. These results suggested that LynA protein levels are preferentially repressed via Csk depletion in cancer cells.
Kinase activity of SfKs is required for the depletion of csk to reduce the protein level of LynA. The depletion of Csk leads to the activation of SFKs by preventing the phosphorylation of the tyrosine residue at their C-terminal regulatory tail. To determine whether the activation of SFKs participated in the process by which the depletion of Csk reduced LynA, we examined whether an SFK inhibitor, PP2, was able to reverse the repressive effect of the depletion of Csk on the protein level of LynA. HCT116 cells were transfected with siRNA for Csk or control siRNA, and 24 h after transfection, these cells were treated with 10 μM PP2 or dimethyl sulfoxide (DMSO; control) for 24 h. These cells were analysed by Western blotting. We first tried to confirm whether treatment with PP2 repressed the kinase activity of SFKs. In cells treated with PP2, the tyrosine phosphorylation levels at the activation loop and the C-terminal negative regulatory tail were not correlated to the kinase activity of SFKs 25 ; thus, the effect of PP2 on the kinase activity of SFKs was not assessed by Western blotting with antibodies against phosphorylation at the conserved tyrosine in the activation loop [anti-p-SFKs (A-loop) antibody;] or in the C-terminal negative regulatory tail (anti-p-Src Y530 and anti-p-Lyn Y508 antibodies;. We therefore confirmed the effect of PP2 based on the reduction of the tyrosine phosphorylation of intracellular proteins detected by anti-phosphotyrosine (p-Tyr) antibody, p-Tyr panel). In cells transfected with control siRNA, no influence of PP2 on the protein levels of SFKs was observed. In cells depleted of Csk, treatment with PP2 reversed the reduction of LynA and also c-Src, whereas the protein level of c-Yes was comparable irrespective of whether cells treated with PP2. These results suggest that the activation of SFKs accompanying the depletion of Csk triggers the reduction of LynA.
constitutively active Src, v-Src, leads the reduction of LynA. To further verify whether the activation of SFKs leads the reduction of LynA, we examined whether constitutively active Src, v-Src 26 , leads the reduction of LynA. For this examination, we used HeLa S3/v-Src cells introduced with a system for the doxycycline (Dox)-inducible expression of v-Src. HeLa S3/v-Src cells were treated or not with 2 ng/mL Dox for 6 h and analysed by Western blotting. The Dox-induced expression of v-Src was confirmed using anti-Src and anti-p-SFKs (A-loop) antibodies. In cells expressing v-Src, the protein level of LynA was remarkably repressed. The protein levels of Fyn and LynB were mildly repressed. The protein level of c-Yes was not altered in cells expressing v-Src. Given that v-Src only slightly reduced the protein level of Csk, these results suggest that the aberrant activation of SFKs is sufficient to lead the reduction of LynA irrespective of whether Csk is depleted or not in cancer cells.
cbls are involved in csk-mediated reduction of LynA. A previous study showed that LynA was ubiquitinated by c-Cbl and subsequently degraded by the proteasome when mast cells were stimulated by FcεRI. We assessed whether Cbls are involved in the process through which the depletion of Csk reduces LynA in cancer cells. We first examined whether the depletion of Csk affected the protein levels of c-Cbl and Cbl-b. Western blot analysis of HCT116 or HeLa S3 cells transfected with Csk siRNA showed that the depletion of Csk increased the protein level of c-Cbl, whereas the protein level of Cbl-b was not affected(a-d),. We then examined whether the knockdown of Cbls was able to reverse the downregulation of LynA in Csk-depleted cells. In shows that the protein level of c-Cbl was comparably high irrespective of treatment with PP2 in HCT116 cells depleted of Csk. This result suggests that the activation of SFKs after the depletion of Csk is not required for the induction of c-Cbl. In HeLa S3 cells expressing v-Src, the protein levels of c-Cbl and Cbl-b were remarkably reduced ; thus, the activation of SFKs seems to exert negative, rather than positive, impact on the protein levels of Cbls.
LynA reduction accompanying Csk depletion does not affect epithelial or mesenchymal marker protein levels. LynA was reported to be a mediator of the epithelial-mesenchymal transition (EMT), decreasing and increasing epithelial and mesenchymal marker protein levels, respectively. This knowledge led us to hypothesize that LynA reduction accompanying Csk depletion serves as a mechanism to protect cancer cells from undergoing EMT. In order to test this hypothesis, we performed Western blotting using antibodies against an epithelial marker protein, E-cadherin, and a mesenchymal marker protein, vimentin.show the Western blot analysis of HCT116 and HeLa S3 cells transfected with control siRNA, siRNA for Csk, or a combination of siRNAs for Csk, c-Cbl, and Cbl-b. In HCT116 cells, the protein levels of E-cadherin were almost comparable among the three samples [], and vimentin was not detected in all three samples (data not shown). In HeLa S3 cells, the protein levels of vimentin were comparable among the three samples [], and E-cadherin was not detected in all three samples (data not shown). These results suggested that the reduction of LynA accompanying the depletion of Csk does not affect the protein levels of E-cadherin and vimentin in HCT116 and HeLa S3 cells. We further performed similar experiments using DLD1 and RKO colorectal cancer cell lines and MDA-MB231 and MCF-7 breast cancer cell lines []. In DLD1, RKO, and MDA-MB231 cell lines, we observed reduced LynA protein levels accompanying Csk depletion and recovery from LynA reduction upon the codepletion of c-Cbl and Cbl-b []. In DLD1 cells, E-cadherin was detected in all three samples [], whereas vimentin was not detected in all three samples (data not shown). In RKO cells, both E-cadherin and vimentin were not detected in all three samples (data not shown). In MDA-MB231 cells, the protein levels of vimentin were comparable among the three samples [], and E-cadherin was not detected in all three samples (data not shown). These results did not show a dynamic influence of LynA reduction and Csk depletion on EMT marker protein levels. In MCF-7 cells, we observed that Csk depletion enhanced c-Cbl protein levels but did not cause LynA reduction []. This result indicated that the upregulation of c-Cbl is not sufficient for Csk depletion to reduce LynA in MCF-7 cells. Taken together, our data suggested that LynA reduction accompanying Csk depletion does not serve as a mechanism to protect cancer cells from undergoing EMT. In future studies, we will try to elucidate the biological and physiological significance of LynA reduction in cancer cells lacking Csk activity.
# Discussion
A previous study showed that LynA was preferentially degraded, compared to LynB, Hck and Fgr, when Csk was inhibited in macrophages. In the present study, we examined the influence of the depletion of Csk on the protein levels of SFK members in cancer cells, which express a different set of SFK members compared to macrophages. We demonstrated that the aberrant activation of SFKs accompanying the depletion of Csk leads the preferential reduction of LynA, compared at least to c-Src, c-Yes and LynB, in a manner depending on Cbls in multiple . Activation of SFKs is not involved in the process by which the depletion of Csk increases the protein level of c-Cbl. Western blot analysis with antibodies against the indicated proteins. (a) Samples were prepared as described in. Samples were prepared as described in. Full-length blots are presented in. epithelial cancer cells. Given the knowledge that LynA drives migration and invasion in breast cancer cells, our results may suggest that the preferential reduction of LynA is a feedback mechanism for preventing cancer progression in cancer cells lacking the activity of Csk.
The requirement of Cbls in depleted Csk-mediated reduction of LynA in cancer cellsindicates the role of the ubiquitin-proteasome system in LynA reduction. In macrophages, the degradation of LynA accompanying the inhibition of Csk was demonstrated to be mediated by the ubiquitin-proteasome system, although the involvement of Cbls was not tested. Previous reports showed that Cbls can bind to and ubiquitinate Lyn. In RBL-2H3 mast cells, the stimulation of the high-affinity IgE receptor (FcεRI) resulted in the association of Lyn with c-Cbl and Cbl-b and subsequent ubiquitination of Lyn. The pull-down assay using GST-fused N-terminal regions of Lyn also supported the association of Lyn with c-Cbl 31,32 . Ubiquitination of Lyn was induced by the overexpression of c-Cbl in RBL-2H3 mast cells. These previous studies suggested that LynA is ubiquitinated by Cbls and subsequently degraded by the proteasome in immune cells lacking Csk activity. Our results suggested that this mechanism for reducing LynA in response to reduced Csk activity is commonly used not only in immune cells, but also in cancer cells, though not all cancer cells. At least in MCF-7 cells, the reduced protein level www.nature.com/scientificreports www.nature.com/scientificreports/ of LynA was not induced by the depletion of Csk []. We have not discerned the reason for the inability of MCF-7 cells to downregulate LynA after the depletion of Csk.
Freedman et al. posited the following reason for the observed preferential reduction of LynA compared to LynB: "LynA differs from LynB only in a 21 amino-acid insert in its unique region, and this unique insert contains a predicted ubiquitination site (K40)". LynA is speculated to be more susceptible to ubiquitination and subsequent proteolysis compared to LynB. We further searched predicted ubiquitination sites in the SFK members using the UbPred program. The predicted ubiquitination sites were present in all SFK members showing reduced protein levels after Csk depletion, that is, LynA (Lys20 and Lys40), LynB (Lys20), c-Src (Lys40), and Fyn (Lys13), whereas no predicted ubiquitination site was detected in c-Yes whose protein level is not reduced after the depletion of Csk. We speculated that the primary structure of c-Yes might not be targeted by ubiquitin ligases such as Cbls.
Unexpectedly, the reduction of c-Src and Fyn after the depletion of Csk was not reversed by the depletion of Cbls in cancer cells []. This suggested that c-Src and Fyn, in contrast to LynA, are not under the control of Cbls in cancer cells depleted of Csk. On the other hand, it has been reported in a number of studies that c-Src and Fyn, as well as Lyn, can be targeted by Cbls for ubiquitination and subsequent degradation; thus, selective targeting of Cbls to LynA may occur in limited situations, including under the condition in which Csk is depleted. Thus far, the mechanism of the reduction of c-Src and Fyn in cancer cells depleted of Csk is unclear. However, some questions still remain: (1) Is transcription or translation suppressed? (2) Is the degradation dependent on or independent of the ubiquitin-proteasome system? In order to assess the involvement of the ubiquitin-proteasome system in this mechanism, we attempted to examine whether the reduction of c-Src and Fyn after the depletion of Csk was reversed by a proteasome inhibitor, MG132, in HCT116 or HeLa S3 cells; however, we were not able to obtain convincing results because treatment with MG132 led to severe cell damage and death (data not shown).
Our results suggest that, to reduce LynA, the depletion of Csk accelerates the functions of Cbls by two mechanisms.reveals that one of the two mechanisms depends on the activation of SFKs accompanying the depletion of Csk. A number of reports have shown that the kinase activity of SFKs is required for their association with Cbls 23,34 and their ubiquitination by Cbls. Furthermore, SFKs have been suggested to phosphorylate Tyr371 of c-Cbl, which corresponds to Tyr363 of Cbl-b. Phosphorylation at this conserved tyrosine has been suggested to increase the E3 activity of Cbls. Crystal structural analyses of the N-terminal region of Cbls have revealed that phosphorylation at this conserved tyrosine changes the conformation of Cbls from a closed, inactive state into an open, active state. Although we have speculated that the depletion of Csk induces phosphorylation at this conserved tyrosine of Cbls, we have not yet tested this possibility because there is no commercially available antibody against Cbls that is phosphorylated at this conserved tyrosine.
Another mechanism by which the depletion of Csk accelerates the function of Cbls is the elevation of the protein level of c-Cbl. suggests that this mechanism is not mediated by the activation of SFKs. suggests that the activation of SFKs rather decreases the protein level of c-Cbl. It has been suggested in previous studies that the activation of SFKs leads to not only the ubiquitination of SFKs, but also the self-ubiquitination and subsequent degradation of c-Cbl. These results suggested that the depletion of Csk seems to promote intracellular activity to increase the protein level of c-Cbl that overcomes the activity of aberrantly activated SFKs to decrease c-Cbl. Thus far, we tested the involvement of protein kinase C (PKC) and transcription factor SP1 in this upregulation mechanism of c-Cbl, because previous studies indicated the involvement of these proteins in the regulation of the protein level of c-Cbl. Jeschke et al. showed that treatment of human preosteoclastic cells with a PKC activator, phorbol 12-myristate 13-acetate, enhanced the protein level of c-Cbl 41 . However, the involvement of PKC in the induction of c-Cbl accompanying the depletion of Csk may not be true, because the PKC inhibitor Gö6983 42 (1 μM) did not reverse the induction of c-Cbl. showed that the inhibition or knockdown of HDACs induced the expression of c-Cbl, and this induction was prevented by an SP1 inhibitor, mithramycin A 43 . We tested whether 1 μM mithramycin A prevented the depletion of Csk from increasing the protein level of c-Cbl in HCT116 cells. Mithramycin A decreased the protein level of c-Cbl irrespective of the depletion of Csk. Although this result may suggest the involvement of SP1 in the regulation of the protein level of c-Cbl, we cannot determine from this result whether the depletion of Csk promotes SP1 to increase the protein level of c-Cbl.
Although the expression of v-Src reduced LynA, this result does not mean that the activation of c-Src reduced LynA. Overexpression of HA-tagged c-Src in HeLa S3 cells did not reduce LynA . Knockdown of c-Src, c-Yes, or their combination did not prevent the reduction of LynA accompanying the depletion of Csk. These results indicated that the activation of c-Src and c-Yes do not mediate the reduction of LynA in cells depleted of Csk. Activation of LynA itself may be required to trigger its own reduction after Csk depletion.
Previous studies suggested that LynA seems to contribute to the aggressive behaviour of some types of cancer. Tornillo et al. reported that LynA promotes tumour cell invasion and that a high LynA/LynB isoform ratio is associated with poor prognosis of breast cancer patients. Two previous studies from different groups showed that Lyn mediates EMT. Although the authors of these two studies did not mention which contributes to the induction of EMT (whether LynA or LynB), at least one study showed results suggesting that the overexpression of LynA induced EMT. Given these results, the reduction of LynA may serve as a mechanism preventing aggressive behaviours such as invasion of cancer cells when the functions of Csk are compromised. However, this hypothesis was not supported by our results that the reduction of LynA was not associated with the reduction of E-cadherin or the induction of vimentin in cells depleted of Csk. We need further studies to demonstrate the physiological importance of the preferential reduction of LynA in cancer cells lacking the activity of Csk. |
Case Report: Simil-Appendicitis Presentation May Precede Cardiac Involvement in MIS-C Patient
[bib_ref] Multisystem inflammatory syndrome in U.S. Children and Adolescents, Feldstein [/bib_ref] [bib_ref] COVID-19 and multisystem inflammatory syndrome in children and adolescents, Jiang [/bib_ref] [bib_ref] Gastrointestinal symptoms as a major presentation component of a novel multisystem inflammatory..., Miller [/bib_ref] [bib_ref] SARS-CoV-2 and the gastrointestinal tract in children, Puoti [/bib_ref] [bib_ref] Features of intestinal disease associated with COVID-related multisystem inflammatory syndrome in children, Sahn [/bib_ref] [bib_ref] Acute abdomen in multisystem inflammatory syndrome in children: a systematic review, Rouva [/bib_ref] [bib_ref] Gastrointestinal symptoms as a major presentation component of a novel multisystem inflammatory..., Miller [/bib_ref] [bib_ref] Acute abdomen in multisystem inflammatory syndrome in children: a systematic review, Rouva [/bib_ref] [bib_ref] Abdominal imaging findings in critically ill children with multisystem inflammatory syndrome associated..., Morparia [/bib_ref] [bib_ref] Cardiac manifestations in SARS-CoV-2-associated multisystem inflammatory syndrome in children: a comprehensive review..., Sperotto [/bib_ref] [bib_ref] Acute cardiovascular manifestations in 286 children with multisystem inflammatory syndrome associated with..., Valverde [/bib_ref] [bib_ref] Cardiac manifestations in SARS-CoV-2-associated multisystem inflammatory syndrome in children: a comprehensive review..., Sperotto [/bib_ref] [bib_ref] Review of cardiac involvement in multisystem inflammatory syndrome in Children, Alsaied [/bib_ref] [bib_ref] Acute heart failure in multisystem inflammatory syndrome in children in the context..., Belhadjer [/bib_ref]
## Case description
We report the case of a 15-year-old adolescent who presented to the pediatric emergency department with a 2-day history of fever, vomiting and diarrhea and mild abdominal pain. His history was remarkable for a period of anosmia and ageusia experienced 1 month before presentation. In that occasion, two nasopharyngeal swabs for SARS-CoV-2 tested negative, while his mother's one tested positive.
At admission, he was febrile and reported a severe asthenia. Vital signs were normal, except for mild tachycardia (hearth rate 140 beats/min) and fever of 39 - C. Capillary refill time was lower Abbreviations: BNP, Brain natriuretic peptide; CDC, Center for disease control and prevention; CRP, C-reactive protein; ECG, Electrocardiogram; ESR, Erythrocyte sedimentation rate; IVIG, Intravenous immunoglobulin; LFEV, Left ventricular ejection fraction; MIS-C, Multisystem inflammatory syndrome in children; MRI, Magnetic resonance imaging; N.V., Normal value; US, ultrasound. than 2 s. The cardio-thoracic examination was unremarkable, while a mild diffuse tenderness on abdominal palpation was elicited. No rashes or cutaneous lesions were noted. Laboratory tests showed mild leukocytosis (white blood cells 10,480 mm 3 ), with lymphopenia (550 mm 3 ), elevation of C-reactive protein (CRP 137 mg/L, normal value < 5 mg/L), mild elevation of Ddimer (1,249 ng/ml; n.v. < 500 ng/ml) and fibrinogen within the normal values (430 mg/dl, n.v. 174-434 mg/dl). A nasopharyngeal swab to check the presence of SARS-CoV2 tested negative. Considering the history of recent ageusia and anosmia, the presence of fever, asthenia and gastrointestinal symptoms within elevation of inflammatory markers and lymphopenia, a diagnosis of MIS-C was suspected.
No signs of cardiac involvement were noted: myocardial markers were in normal range (cardiac troponin 2 ng/L [n.v. < 19 ng/L] and brain natriuretic peptide BNP 200 pg/ml [n.v. < 300 pg/ml]) with normal electrocardiogram and echocardiography. Nevertheless, the absence of cardiac involvement did not preclude a MIS-C diagnosis.
Within 24 h from admission the patient rapidly worsened, developing a progressive abdominal pain in the right lower quadrant, with local guarding and rebound tenderness. Abdominal ultrasound showed a thickening of the terminal ileus with ascites and mesenteric lymphadenopathy, while the appendix was not detected [fig_ref] FIGURE 1 |: Abdominal ultrasound showing transmural thickening of the terminal ileus [/fig_ref].
A laparoscopic exploration was performed to rule out acute appendicitis. The ileus and cecum appeared thickened and inflamed, while the appendix was normal. Broad spectrum antibiotic treatment was started.
Four days after admission and 2 days after surgery, despite antibiotic therapy the patient was still febrile and markedly asthenic. Thus, a second cardiological evaluation was performed, showing increased inflammatory and myocardial markers (CRP 250 mg/L, cardiac troponin 65 ng/L, BNP 9,195 pg/ml), negative T waves along with prolonged QT interval (490 ms) at ECG [fig_ref] FIGURE 2 |: Electrocardiogram showing sinus bradycardia [/fig_ref] and a reduced left ventricular ejection fraction (LVEF 55%) with a tricuspid regurgitation at echocardiography. According to the simultaneous presence of cardiac and abdominal involvement, a diagnosis of MIS-C was made and treatment with intravenous immunoglobulins (2 g/kg) and steroids (methylprednisolone 2 mg/kg) was started. Due to the concomitant myocarditis, he received a prophylactic anticoagulation (enoxaparin 4,000 IU/day) and antiplatelet therapy (acetylsalicylic acid 100 mg/day). After 24 h the patient had a prompt recovery with cessation of fever, abdominal pain and malaise. In few days inflammatory and cardiac markers progressively decreased to normal values, while ECGs and echocardiogram normalized in 3 weeks. The patient was kept in steroids for 1 month with a gradual reduction of the doses. Exercise restriction was recommended for 6 months, when the patient will undergo cardiac magnetic resonance imaging (MRI).
# Discussion and conclusion
Here, we reported the case of an adolescent with MIS-C in which gastrointestinal symptoms resembled an acute appendicitis. The rapid worsening of abdominal symptoms with simil-appendicitis presentation led to an unnecessary explorative laparoscopy and treatment delay. The subsequent cardiovascular involvement and the increasing inflammatory markers allowed the right diagnosis and treatment with prompt and complete recovery. Undoubtedly, MIS-C diagnosis was yet suspected at the onset of the disease due to the two organs involvement (abdominal and hematological systems), the fever and elevation of inflammatory markers. However, the progressive worsening of abdominal pain led to consider a surgical condition.
Despite gastrointestinal symptoms (abdominal pain, emesis and diarrhea) are common features in MIS-C patients, only in a few cases these manifestations resemble an acute appendicitis [bib_ref] Features of intestinal disease associated with COVID-related multisystem inflammatory syndrome in children, Sahn [/bib_ref] [bib_ref] Acute abdomen in multisystem inflammatory syndrome in children: a systematic review, Rouva [/bib_ref] [bib_ref] Gastrointestinal features in children with COVID-19: an observation of varied presentation in..., Tullie [/bib_ref] [bib_ref] A case of multisystem inflammatory syndrome in children mimicking acute appendicitis in..., Jackson [/bib_ref] [bib_ref] Severe SARS-CoV-2 infection in children with suspected acute abdomen: a case series..., Cabrero-Hernández [/bib_ref]. Moreover, when these manifestations precede other organ involvement a differential diagnosis between inflammatory bowel disease, abdominal surgical conditions or severe infections could be difficult [bib_ref] Gastrointestinal features in children with COVID-19: an observation of varied presentation in..., Tullie [/bib_ref] [bib_ref] A case of multisystem inflammatory syndrome in children mimicking acute appendicitis in..., Jackson [/bib_ref] [bib_ref] Severe SARS-CoV-2 infection in children with suspected acute abdomen: a case series..., Cabrero-Hernández [/bib_ref] [bib_ref] Case of multisystem inflammatory syndrome in children presenting as fever and abdominal..., Mahajan [/bib_ref]. Typical laboratory findings in MIS-C patients are lymphopenia with neutrophilia, increased PT and D-dimer, hypoalbuminemia, hypertransaminasemia, elevation of inflammatory (CRP, ESR, ferritin and fibrinogen) and cardiac (troponin and BNP) markers [bib_ref] Multisystem inflammatory syndrome in U.S. Children and Adolescents, Feldstein [/bib_ref]. As in our case, among all these values, increased inflammatory markers, lymphopenia and hypoalbuminemia are common findings in patients with gastrointestinal symptoms [bib_ref] Features of intestinal disease associated with COVID-related multisystem inflammatory syndrome in children, Sahn [/bib_ref]. Reviewing abdominal imaging in MIS-C patients, common US findings are ascites, acalculous cholecystitis, bowel wall thickening and mesenteric adenitis [bib_ref] Features of intestinal disease associated with COVID-related multisystem inflammatory syndrome in children, Sahn [/bib_ref] [bib_ref] Acute abdomen in multisystem inflammatory syndrome in children: a systematic review, Rouva [/bib_ref] [bib_ref] Abdominal imaging findings in critically ill children with multisystem inflammatory syndrome associated..., Morparia [/bib_ref]. Moreover, in a systematic review Rouva et al. reported an incidence of 30% of acute abdomen among MIS-C patient with gastrointestinal symptoms. The final diagnoses were mostly non-surgical (76.4%) and, as in our case, in around 25% of cases a terminal ileitis/ileocolitis was found [bib_ref] Abdominal imaging findings in critically ill children with multisystem inflammatory syndrome associated..., Morparia [/bib_ref]. Nevertheless, few authors even described MIS-C patients with acute abdomen presentation, in some cases simultaneous with cardiac involvement, for whom symptoms and ultrasound imaging were misleading. Lastly, a diagnosis of MIS-C should not preclude a possible diagnosis of appendicitis. Indeed, in some cases of acute abdomen in MIS-C, appendicitis was confirmed at macroscopic and histological evaluation [bib_ref] Acute appendicitis associated with multisystem inflammatory syndrome in children, Hofto [/bib_ref]. Moreover, few authors described a direct involvement of appendix in MIS-C, where the systemic inflammation led to vasculitis, thrombus formation and ischemic necrosis of the intestinal wall [bib_ref] Intestinal ischemia secondary to Covid-19, Khesrani [/bib_ref]. Therefore, in the first week of disease, gastrointestinal symptoms, laboratory exams and abdominal US could be misleading, especially if no other organ involvement is already present. Although cardiac involvement is not mandatory for MIS-C diagnosis, the absence of laboratory or functional markers of cardiac damage along with the quick development of acute abdomen in the first hours, led to an early misdiagnosis and delayed treatment. However, we cannot assume that an early treatment with steroid and IVIG would have avoided a cardiac involvement. Indeed, therapeutic strategy in MIS-C are driven to its similarity with Kawasaki disease.
In our case, retrospectively, the persistent asthenia could be seen as an early marker of cardiac involvement, a welldescribed symptom in MIS-C patient with cardiac manifestations [bib_ref] Acute heart failure in multisystem inflammatory syndrome in children in the context..., Belhadjer [/bib_ref]. Nevertheless, no elevation of cardiac enzymes or decreased ventricular function were noted in the patient until the second week of disease.
In regard to the risk of intensive care admission and inotropic support in MIS-C patient, a prompt and right diagnosis is mandatory. Up to 80% of patients develop cardiovascular involvement ranging from only mild elevation of cardiac markers (troponin and pro-BNP) to cardiogenic shock. If MIS-C shares some Kawasaki's disease features, the former usually results in more severe ventricular dysfunction and myocarditis. Cardiogenic shock has been proposed to be the result of either myocardial viral damage or "cytokine storm" vasodilatation. Other cardiovascular complications encompass coronary artery aneurism/dilatation and conduction abnormalities. According to a review of cardiac involvement in MIS-C, coronary artery dilatation is reported in up to 25% of patients, while heart conduction abnormalities showed a 7-60% prevalence. Typical arrhythmias are first-degree atrioventricular block, QTc prolongation and ST segment changes. The American Heart Association suggests repeated ECG during the acute phase, but telemetry is needed if any arrythmias occur. From several large studies, early immunomodulatory treatment seems to resolve cardiac damage in most of all cases, but little is known about cardiac sequelae in MIS-C patients. Once myocardial damage occurs, an expert consensus recommends exercise restriction for 6 months with a cardiac MRI at 3 or 6 months to evaluate heart function.
Up to now, no randomized trials have been developed for treatment and management of MIS-C, but due to the similarities between Kawasaki disease and MIS-C, an immunomodulatory approach with IVIG and steroids is recommended. Moreover, antiplatelet treatment and prophylactic anticoagulation are suggested once myocardial or coronary involvement are present. As in our case, several retrospective studies reported, in the majority of patients, normalization of myocardial markers, ECGs abnormalities and ventricular dysfunction after immunomodulatory treatment [bib_ref] Acute heart failure in multisystem inflammatory syndrome in children in the context..., Belhadjer [/bib_ref]. Nevertheless, longer studies and trials are needed to evaluate treatments and chronic sequalae.
In conclusion, our case highlights how gastrointestinal involvement in MIS-C could mimic acute appendicitis, and this presentation may precede cardiac involvement, leading to possible misdiagnosis and delayed treatment.
In patients with a recent exposure to SARS-CoV-2 a clinical presentation with fever, asthenia, and gastrointestinal symptoms should be seen as highly suggestive of MIS-C. This suspect may be supported by the presence of an increased inflammatory markers, lymphopenia and hypoalbuminemia and images suggestive of terminal ileitis on ultrasound. Even in the absence cardiac involvement at presentation the diagnosis of MIS-C should not be ruled-out and a strict cardiological follow-up should be performed.
# Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
# Ethics statement
Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.
[fig] FIGURE 1 |: Abdominal ultrasound showing transmural thickening of the terminal ileus. [/fig]
[fig] FIGURE 2 |: Electrocardiogram showing sinus bradycardia (HR 50 beats/min) with QT tract prolongation of 494 ms, diffuse T-waves alteration and minimal right branch block. [/fig]
|
Transcatheter Aortic Valve Replacement With Self‐Expandable Supra‐Annular Valves for Degenerated Surgical Bioprostheses: Insights From Transcatheter Valve Therapy Registry
If BMI < 30 kg/m 2 : No PPM ->0.85 cm 2 /m 2 ; Moderate PPM -0.85 -0.65 cm 2 /m 2 ; Severe PPM -<0.65 cm 2 /m 2 If BMI >= 30 kg/m 2 : No PPM ->0.70 cm 2 /m 2 ; Moderate PPM -0.60 -0.70 cm 2 /m 2 ; Severe PPM -<0.60 cm 2 /m 2
[table] Table S1: Clinical outcomes at 30 days and 1 year by post procedure patient prosthesis mismatch (PPM).Total number of patients from Evolut R and Evolut PRO Valves are represented. Data is represented as no. patients with event (Kaplan-Meier event rate as percentage). PPM is based of post-procedure and is calculated based on the following definition. [/table]
[table] Table S2: Patient prosthesis mismatch (PPM) by valve size for Evolut R and PRO valves. [/table]
[table] Table S3: Clinical outcomes overtime for Evolut R and Evolut PRO by valve size. [/table]
[table] Table S4: Echocardiographic overtime for Evolut R and Evolut PRO by valve size. [/table]
|
Dynamic External Fixation for Interphalangeal Comminuted Fractures With Mallet Injury
A 26 year-old man presented with gunshot wounds to the left ring and small fingers. The physical examination revealed open, comminuted fractures of the middle phalanges of the left ring and small fingers; an extensor lag at the distal interphalangeal joint (DIP) of the small finger suggested a terminal tendon injury. X-ray imaging demonstrated a pilon fracture of the ring finger middle phalanx base with mid-shaft comminution. The small finger middle phalanx head sustained a comminuted fracture with bicondylar involvement. After debridement in the operating room, we fabricated dynamic external fixation devices for stabilization of the fractures of the ring and small fingers; dermatotenodesis was performed to address the mallet injury. The devices were removed at 6 weeks, where minimal stiffness was noted at the ring DIP and proximal interphalangeal joint (PIP) of the small finger during active ranging. The small finger DIP demonstrated active flexion to 30 - at that time with no extensor lag. . Patient 6 weeks after dynamic distraction and external fixation device placement.
# Discussion
Dynamic distraction and external fixation (DDEF) is indicated for unstable PIP fracturedislocations and pilon fractures of the middle phalanx. This treatment is particularly useful when closed reduction yields a congruent joint that proves dynamically unstable (typically seen when >40% of the articular surface is involved). Open reduction internal fixation techniques may be employed adjunctively for large fracture fragments. 1,3,4 Volar plate or hemi-hamate arthroplasty may be performed concurrently to enhance dynamic stability of dorsal dislocations associated with volar lip fracture and comminution. Pilon-type fractures, defined by total articular surface involvement, and even severely comminuted fractures of the mid-shaft are well suited for DDEF, as it facilitates reduction of fracture fragments via ligamentotaxis from the surrounding soft tissues. Authors have also described the utility of DDEF for severely comminuted fractures of the DIP and metacarpophalangeal joints. DDEF is contraindicated for injuries associated with severe soft-tissue loss (compromising ligamentotaxis), chronic injury or severe arthritis, and segmental fractures involving the proximal or middle phalangeal head. DDEF maintains fracture alignment while allowing early active and passive ranging of the involved joints. This is paramount in severe phalangeal injuries prone to contracture with immobilization. Early motioning mitigates joint stiffness and promotes cartilage healing. The benefits of immediate motion for joint pliability compensate for intra-articular step-offs common in healed pilon injuries. Furthermore, the DDEF construct imposes minimal trauma on the soft tissues.Other benefits of DDEF include low cost, ease and quickness of device fabrication, and favorable functional outcomes. The authors prefer to use the push-traction DDEF design described by with tension provided by Kirschner wires only. After reduction has been achieved, one double-ended 0.045 in K-wire is placed at the center of rotation of the P1 head. It is critical to confirm accurate placement with lateral fluoroscopy before advancing the wire to prevent bone injury from multiple attempts. The wire should appear as a single dark dot in the center of the phalangeal head on lateral view. Advance the wire through the opposite cortex and soft tissue until the wire flanking the finger is equal lengths. Repeat this at the P2 head so that the wires are parallel. Use a heavy needle driver to bend the distal wire into a dorsal hook on each side; the first bend should be 0.5 to 1 cm distal to the DIP. The second bend of the hook should be 1 cm proximal to the first. Trim wires. Bend the proximal wire 90 - on each side and flip distally to engage the proximal wires into the dorsal hooks. The authors improvised a DDEF construct to simultaneously address the patient's small finger severely comminuted P2 head and shaft fracture (with total intra-articular surface involvement) and terminal tendon injury. Given the severity of comminution, the P2 fracture would benefit from longitudinal traction. The mallet finger required immobilization in extension.We first placed one 0.045 in K-wire transversely through the P2 head to stabilize the bicondylar fracture after open reduction. We then fashioned a DDEF construct as previously described with the proximal wire through the P1 head and the distal wire through the P3 base. The middle wire was affixed to the proximal wire dorsally. This technique promoted immediate motion of the PIP joint, while immobilizing the DIP after dermatotenodesis.
## Summary
The authors constructed a push-traction DDEF device to provide reduction stability while allowing early motion of the ring finger PIP for our patient's P2 pilon fracture. We created a novel DDEF device allowing PIP motion while immobilizing the DIP for the patient's concomitant P2 head/shaft comminuted fractures and mallet injury. |
What should we do to optimise outcome in twin pregnancy complicated with placenta percreta? A case report
Background: Patients with morbidly adherent placenta (MAP) are under risk of massive bleeding. It readily necessitates very complicated surgery and massive blood transfusion, and even leads to mortality. Cesarean hysterectomy (CH) is the procedure that is acknowledged worldwide, since it helps to minimize complications. Case presentation: A patient with dichorionic twin pregnancy underwent to cesarean section (CS) due to preliminary diagnosis of placenta percreta at her 35 th week of pregnancy. Both of the placentas were left in situ. The patient admitted with signs of infection. Emergency total abdominal hysterectomy was performed 7 weeks after CS. In the course of hysterectomy, 3 units of erythrocyte suspension and 2 units of fresh frozen plasma were transferred, whereas none was required during CS. Conclusion: Abandoning placenta in situ seems to be a logical alternative to the CH in patients with placenta percreta in order to minimize complications related to massive blood transfusion and surgical technique. However, it appears to increase maternal morbidity due to maternal infection in twin pregnancy.
# Background
Abnormal placental invasion, which is also called as morbidly adherent placenta (MAP), is considered as one of the most severe complications of pregnancy [bib_ref] Maternal outcome after conservative treatment of placenta accreta, Sentilhes [/bib_ref]. MAP is a potential life-threatening condition. Patients with MAP are under risk of massive bleeding due to spontaneous or forced separation of the placenta. Therefore, cesarean hysterectomy (CH) is the procedure that is acknowledged worldwide to prevent such complications in patients with diagnosis of MAP. However, Sentilhes et al. have tried an alternative approach and demonstrated that uterine conservation is possible in patients with MAP [bib_ref] Maternal outcome after conservative treatment of placenta accreta, Sentilhes [/bib_ref]. In this report, we present a case of MAP in a dichorionic (DC) twin pregnancy who is followed up with the retained placentas. This is the first reported case of a DC twin pregnancy in which both of the placentas were MAPs and were left in situ during cesarean section (CS). We also discuss the advantages and disadvantages of abandoning placenta in situ in such situations.
## Case presentation
Patient Thirty-three years old women with dichorionic diamniotic twin pregnancy admitted to our perinatology clinic at her 28 th gestational week with a preliminary diagnosis of complete placenta previa. She had two healthy-living children, one of which was delivered by CS, and one spontaneous abortion, which ended up with curettage.
Follow-up of the patient was done weekly until 35 th gestational age. Prior to the delivery, we were unable to determine the myometrial thickness at uterovesical contiguity by ultrasonography. Additionally, placental lacunes were prominent, and there were multiple tortuous vessels at uterovesical junction [fig_ref] Figure 1: Antepartum transvaginal ultrasonographic survey [/fig_ref]. Preliminary diagnosis was placenta percreta. Two-step surgery, first remaining the placenta in utero with intention of afterward-hysterotomy and -metroplasty, was planned to decrease complications due to surgery and massive blood transfusion.
## Procedures
CS was performed through a vertical midline abdominal incision. The incision was extended superiorly and inferiorly from the umbilicus in order to provide a major route to deliver fetuses without damaging the decidualplacental interface. Profuse, engorged, whorl-like patterned uteroplacental vessels were seen at the intraoperative evaluation at both parametra, particularly at the left side, and over the bladder [fig_ref] Figure 2: Uterus after the reparation [/fig_ref]. The lower uterine segment and corpus uteri were both invaded by the placentas. Therefore, it was a necessity to perform a fundal incision rather than a classical incision to the uterus. Each of the umbilical cords was tied for twice with no. 1 silk sutures after delivery of fetuses. Both of the placentas were abandoned in situ. Myometrium was sutured primarily with no. 1 vicryl sutures in two layers [fig_ref] Figure 2: Uterus after the reparation [/fig_ref]. Patient was not administered uterotonics during and after the procedure. Cefazolin was continued during post-operative period for 4 days. Intramuscular methotrexate was administered in 50 mg/m 2 dose to enhance placental involution at the postoperative day 1. Patient was discharged at the postoperative day 4. She was advised for regular visits for once in two weeks. Transabdominal and suprapubic ultrasonographic survey, serum quantitative measures for leukocytosis and C-reactive protein (CRP) were conducted at every visit. At postoperative first week, ultrasonography yielded a 70 × 101 mm residual placenta at the left lower segment of the uterine cavity, and the second placenta which is 57 × 99 mm in dimensions at the right lower segment of the uterine cavity. Patient did not encounter any sort of bleeding. Serum CRP was negative (<0.5 mg/dL), and serum leukocyte counts were normal. Serum beta-hCG value was 324.86 IU/ml. The subsequent calls during follow-up did not yield any symptoms and abnormal test results until post-operative 5 th week. Serum CRP was measured 2.8 mg/dL at that time. She was administered oral metronidazole twice a day and 3 rd generation cephalosporins once a day for the treatment of the infection. The ultrasonographic measurement yielded shrinkage in dimensions of both placentas. One week later, she admitted to our clinic with severe inguinal and lower abdominal pain, and leukorrhea. There was a discomfort on abdominal palpation. Additionally, tenderness was prominent at inguinal regions, particularly at the left side. Maximum body temperature was 38.7°C. Serum CRP was measured to increase 29.8 mg/dL. The second operation was planned. The operation was performed through the infraumbilical vertical midline incision. Uterus was pink to grayish in color. Distal part of the uterine corpus and bilateral adnexa were enlarged extremely due to uterine vasculature and mass of the placentas. Vesicouterine pouch was obliterated. Prophylactic ligation of bilateral hypogastric arteries was followed by routine technique for total hysterectomy . The division of uterine neovasculature at the boundary of lower uterine segment and bladder and dissection of vesicouterine space was accomplished by using electrosurgical vessel sealing equipment. Our measures yielded a 1100 ml total blood loss at intraoperative period. Three units of erythrocyte suspension and 2 units of fresh frozen plasma were delivered to the patient at the perioperative and postoperative period. She was administered 3 rd generation cephalosporin for 7 days. Postoperative follow-up was uneventful. The patient was discharged at the postoperative 7 th day.
## Histopathological findings
Placental sectioning showed extensive hemorrhage in the villous tissue with extensive inflammatory response and tissue necrosis at the microscopy.
# Conclusions
The incidence of morbidly adherent placenta (MAP) increased from one in 30.000 live births in 1930 to one in 533 live births recently [bib_ref] Clinical risk factors for placenta previaplacenta accreta, Miller [/bib_ref]. Causal factor is obvious endometrial defect related to previous surgeries, dilatation and curettages, previous placenta previa, advanced maternal age, multiparity, Asherman's syndrome, and submucous leiomyoma [bib_ref] The management and outcomes of placenta accreta, increta, and percreta in the..., Fitzpatrick [/bib_ref]. Primarily, the presence of placenta previa together with afore mentioned factors should raise physician's notice, since antenatal diagnosis of MAP and planning of the delivery could help to reduce morbidity and mortality [bib_ref] Maternal morbidity associated with multiple repeat cesarean deliveries, Silver [/bib_ref]. Obstetric magnetic resonance imaging (MRI) is a superior and a feasible diagnostic method in situations where the exact diagnosis could not be reached by sonography [bib_ref] Ultrasonographic evaluation of uteroplacental blood flow patterns of abnormally located and adherent..., Guy [/bib_ref]. In this case we did not perform MRI, because ultrasonographic features were vigorously suggestive of MAP. The timing for delivery could reasonably be postponed until 34 to 36 weeks, except for cases with massive vaginal bleeding and suspicion of extreme overgrowth to the adjacent organs.
Most of the cases of twin pregnancies with placenta percreta that were published so far reported uterine rupture at early gestational weeks of pregnancy [bib_ref] Spontaneous rupture of the uterus caused by placenta percreta at 28 weeks..., Nagy [/bib_ref] [bib_ref] Subsequent pregnancy outcome after conservative treatment of a previous cesarean scar pregnancy, Seow [/bib_ref] [bib_ref] Spontaneous uterine rupture at an unusual site due to placenta percreta in..., Topuz [/bib_ref]. Our case is interesting as it did reach to 35 th gestational week despite presence of increased placental burden of a twin pregnancy. Meanwhile, according to our best knowledge, there is no other case in the literature that both placentas were morbidly adherent and were remained in situ during the CS.
Detaching or making incision through adhesive placenta gives rise to massive blood loss, and complicates further steps of the surgery. Considering the prevention of hemorrhage, we preferentially performed a fundal rather than a classical incision to the uterus following a midline vertical incision to the skin. Therefore, as the first step of the treatment of MAP, we avoided even minor detachment of the placenta.
The following step in the optimal treatment usually addresses CH as the standard of care for MAP [bib_ref] Clinical risk factors for placenta previaplacenta accreta, Miller [/bib_ref]. After the fetus is delivered the uterus is just taken out while the placenta is still attached. This approach is widely resumed as having the best outcomes. As an alternative approach, here, we abandoned the placenta in situ instead of performing hysterectomy [bib_ref] The management and outcomes of placenta accreta, increta, and percreta in the..., Fitzpatrick [/bib_ref]. Patient's age, patient's desire to preserve her uterus, our belief to facilitate surgical outcomes and to decrease need for massive transfusion were the reasons to perform this type of surgery.
In patients with retained placenta, the concern of 'what to do with the placenta' arises. As it is theorized that placenta brakes down in time and would be pulled out partially, one can wait for the signs of expulsion of the placenta. Although the thought is reasonable, the journey to the summit is very long and troublesome. Regarding that theory, there are case series reporting favorable outcomes in patients with MAP in singleton pregnancies [bib_ref] Maternal outcome after conservative treatment of placenta accreta, Sentilhes [/bib_ref] [bib_ref] Planned caesarean hysterectomy versus "conserving" caesarean section in patients with placenta accreta, Amsalem [/bib_ref]. In this case, placenta did not brake down in a long period of time. Moreover, it caused metritis. Therefore, presence of uterine infection necessitated performing emergency surgery at a time before planned surgery for excision of the placental tissues together with metroplasty. Nevertheless, two of our main concerns were reached. Among them, first was to decrease the amount of transferred blood products, and decrease morbidity related to massive transfusion. The second was to decrease co-morbidities related to damaging the adjacent organs during emergency hysterectomy. The other and noble concern, which was to give a chance to preserve and recover the uterus, could not be reached. In such a circumstance, we advocate to ensure an exact control over demographics and medical Uterus, removed. Total size of the uterus is 20 cm in length. Both round ligaments are seen clamped. Scar of the previous uterine closure is seen between the clamps (white arrows). Border of the lower uterine segment and uterine corpus (black arrows). Grayish-yellow placental tissue is seen through the area of uterine perforation (white squares) condition of the patient, the extent of the invasion of placenta, the total volume or amount of the retained placenta, total uterine size comprising placental volume, and the hematoma in the uterine cavity together with the signs of cervical dilatation and expulsion of placenta. Ligation (LHA) or obliteration (OHA) of the hypogastric arteries were reported to be ineffective if performed without hysterectomy to control major pelvic hemorrhage in up to 60 % of cases of MAP [bib_ref] Maternal morbidity associated with multiple repeat cesarean deliveries, Silver [/bib_ref] [bib_ref] Failure of methotrexate and internal iliac balloon catheterization to manage placenta percreta, Butt [/bib_ref] [bib_ref] Hypogastric artery ligation for intractable pelvic hemorrhage, Papp [/bib_ref]. As it is known that bilateral LHA is a time consuming and ineffective procedure in patients with MAP, we did not intend to perform prophylactic LHA during CS in this case.
The data regarding the long-term reproductive outcomes after conservative treatment of patients with MAP are limited [bib_ref] Maternal outcome after conservative treatment of placenta accreta, Sentilhes [/bib_ref] [bib_ref] Spontaneous rupture of the uterus caused by placenta percreta at 28 weeks..., Nagy [/bib_ref] [bib_ref] Fertility and obstetric outcome after conservative management of placenta accreta, Provansal [/bib_ref] [bib_ref] Fertility and pregnancy outcomes following conservative treatment for placenta accreta, Sentilhes [/bib_ref]. One can assume that the physiology of the endometrium has not been corrected, and the theoretical risk of recurrent MAP rises in this population. However, we could improve implantation site within endometrial cavity by repairing the defective zone related to previous CSs owing to a popular theory of implantation of the embryo directly on the endomyometrial junction [bib_ref] The natural history of early first trimester pregnancies implanted in Caesarean scars:..., Zosmer [/bib_ref]. Despite this blurred picture and the increased risk of recurrent MAP, a chance to conceive should be considered in meticulously selected cases such as very young parturients.
In conclusion, leaving the placenta in situ seems to be a logical alternative to CH in patients with MAP. However, the surgeon should be aware of infectious, hemorrhagic, and psychological complications related to retained placenta. In presence of MAP with DC pregnancy, it appears to increase risk of maternal infection, and increase maternal morbidity. Therefore, we advocate the idea that uterine conservation approach should be personalized with meticulous patient selection.
## Consent
Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
[fig] Figure 1: Antepartum transvaginal ultrasonographic survey. Note the tortuous vessels anterior to the lower uterine segment just overneath the cervix [/fig]
[fig] Figure 2: Uterus after the reparation (Intraoperative view). Anterior part of the repaired uterine incision could be seen (white arrows). Incision continues superiorly and posteriorly (not shown). Upper borders of the DC placentas are prominent (transparent arrows). Black arrow indicates the left round ligament [/fig]
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Medicinal Plants from North and Central America and the Caribbean Considered Toxic for Humans: The Other Side of the Coin
The consumption of medicinal plants has notably increased over the past two decades. People consider herbal products as safe because of their natural origin, without taking into consideration whether these plants contain a toxic principle. This represents a serious health problem. A bibliographic search was carried out using published scientific material on native plants from Mexico, Central America, and the Caribbean, which describe the ethnobotanical and toxicological information of medicinal plants empirically considered to be toxic. A total of 216 medicinal plants belonging to 77 families have been reported as toxic. Of these plants, 76 had been studied, and 140 plants lacked studies regarding their toxicological effects. The toxicity of 16 plants species has been reported in clinical cases, particularly in children. From these plants, deaths have been reported with the consumption of Chenopodium ambrosioides, Argemone mexicana, and Thevetia peruviana. In most of the cases, the principle of the plant responsible for the toxicity is unknown. There is limited information about the toxicity of medicinal plants used in Mexico, Central America, and the Caribbean. More toxicological studies are necessary to contribute information about the safe use of the medicinal plants cited in this review.
# Introduction
The use of herbal medicine has increased around the world due to its presumptive efficiency, availability, and general acceptance. Approximately 80% of the general population, especially in developing countries, uses medicinal herbs for primary health care [bib_ref] Guidelines on the conservation of medicinal plants World Health Organization (OMS), Uicn [/bib_ref] [bib_ref] Acceptance and use of therapeutic medical plants in family medical care, Taddei-Bringas [/bib_ref]. Worldwide, the interest in medicinal plants by patients has increased over the past two decades. The global market for medicinal plants and plantderived drugs in 2015 was estimated at 25.6 billion dollars and is expected to rise to billion dollars in 2020. This clearly indicates that the consumption of medicinal plants is a current topic of interest. Despite the high consumption of medicinal plants and related products, their toxicity remains to be evaluated. In addition, many medicinal plants require scientific evidence for their medicinal use, particularly those that are sold as food supplements.
Some medicinal plants might trigger undesirable side effects for human health because of (a) pharmacodynamic interaction with prescribed drugs, (b) intrinsic effects, (c) pharmacokinetic interaction with prescribed drugs, and (d) the presence of contaminants and/or pathogenic microorganisms. Other factors that impact the toxicity of medicinal plants in humans include the age of the patient, nutritional status, and the presence of chronic diseases. The concentration of toxic metabolites in plants is determined by the season 2 Evidence-Based Complementary and Alternative Medicine of collection, nutrients in the soil, and growth stage, among others [bib_ref] Plantas tóxicas de México, Aguilar-Contreras [/bib_ref].
The main reasons for focusing this review on medicinal flora with supposed toxicological effects from Mexico, Central America, and the Caribbean are as follows: (a) the ancient importance of traditional medicine in this region, (b) their great biodiversity, and (c) the current use of herbal products. This review will be useful for physicians, toxicologists, pharmacologists, and general audiences. We have tried to describe in detail some toxic symptoms reported with the consumption of the medicinal plants covered in this review.
# Methodology
A bibliographic search was conducted from July 2016 to May 2017 of published scientific material on native plants from Mexico, Central America, and the Caribbean that describes the ethnomedical and toxicological information for medicinal plants reputed to be toxic for humans. The following keywords were searched in different scientific databases: plant extract, toxicity, Mexico, and Central America. Additional data were acquired from undergraduate and postgraduate theses, as well as published and electronic books. The admittance criteria for the selection of scientific information in this review were as follows: (i) plants native to North and Central America and the Caribbean, (ii) plants used for medicinal purposes with or without toxicological studies, (iii) plants with experimental reports on their in vitro and/or in vivo toxicity, (iv) plants thought to be toxic for humans, (v) studies where the concentrations were presented as weight/volume relationship in international units (mg/ml, g/ml), (vi) studies where the doses were presented as weight/weight relationship in international units (mg/kg, g/kg), and (vii) plants with information obtained from a clear source. Scientific studies reporting the combination of plant extracts were excluded. Medicinal plants considered toxic were classified into two categories: (1) plants with toxicological evidence reported in a scientific source and (2) plants without toxicological evidence. All plant names and their distributions were confirmed at the Missouri botanical garden. Many of the medicinal plants cited in this review have no common name in English. Therefore, the common names were given in Spanish [fig_ref] Table 1: Ethnobotanical information of medicinal plants from Mexico and Central America considered as... [/fig_ref].
## Medicinal plants considered to be toxic for humans
A total of 216 medicinal plants belonging to 77 families reported as toxic were recorded. Of these plants, [bib_ref] In vitro antibacterial, antifungal and cytotoxic activity of Scoparia dulcis L, Md [/bib_ref] had been studied, and 140 plants lacked studies regarding their toxicological effects [fig_ref] Table 1: Ethnobotanical information of medicinal plants from Mexico and Central America considered as... [/fig_ref]. Aristolochia (6 plant species), Euphorbia (6 plant species), Solanum (5 plant species), and Asclepias (5 plant species) are the plant genera most often reported to induce toxicity [fig_ref] Table 1: Ethnobotanical information of medicinal plants from Mexico and Central America considered as... [/fig_ref]. Chemotaxonomic studies should be performed to identify the toxic principle in these genera. The parts of the plants considered toxic are listed in the following order: aerial parts including branches, leaves and flowers (22%), whole plant (22%), leaves exclusively (15%), seeds (14%), roots (8%), fruits (8%), bark (4%), latex (3%), and other plant parts. The signs and symptoms of toxicity induced by medicinal plants are reported in [fig_ref] Table 1: Ethnobotanical information of medicinal plants from Mexico and Central America considered as... [/fig_ref]. The main toxic effects occur in the following order: nausea and vomiting (20%), dermatitis (14%), gastritis (9%), abdominal pain (9%), abortifacient (8%), skin burns (8%), hepatotoxicity (7%), severe diarrhea (6%), cardiotoxicity (5%), nephrotoxicity (2%), numbness (2%), dizziness (2%), and hallucinations (2%), among others.
## Dosages.
In most of the cases, the dose for the induction of toxic effects by medicinal plants is not indicated. Usually, consumers of medicinal plants believe that increasing the consumption of these products will increase the efficacy of the treatment. In these cases, the daily dosage is exceeded, which triggers toxicity. For instance, the roots of Ipomoea purga, a purgative agent, are used at a dose of 2 g/L/day. Administration of higher doses induces vomiting and abdominal pain. Fresh leaves of Prunus serotina, used for the treatment of cough, or Zanthoxylum fagara, an anxiolytic agent, each must be consumed in a maximum quantity of five leaves in 250 ml of water per day. Higher doses produce spasms and nausea. Approximately 5 mL of an infusion of Picrasma excelsa (10 g/L) should be administered three times per day. Higher doses induce hypotension. This infusion should not be prepared with ethanol and orally administered. If a person consumes the hydroalcoholic infusion, the consequences could be lethal. The maximum consumption of Manilkara zapota seeds should be 10 seeds per day. A higher consumption of these seeds might induce vomiting and gastroenteritis. On the other hand, Sosa-Gómezrecommends the preparation of an infusion using approximately 1-3 g Argemone mexicana leaves in 1 L of water. This infusion should be taken 3 times per day. A higher dose might induce immobilization.
Studies analyzing the range of doses considered safe for human consumption remain to be performed. The use of natural products needs scientific evidence to corroborate the medicinal uses attributed to different plant species. Many medicinal plants sold as "food supplements" lack warnings if the suggested dosage is exceeded.
## Toxic principles.
In some cases, the toxic principle is known. For instance, it is reported that cefalatin, the main toxic compound in Cephalanthus occidentalis bark, induces vomiting, anemia, and seizures, among other toxic effects. Similarly, hederagenin is the main toxic compound in Clematis dioica, which is a caustic substance [bib_ref] Plantas tóxicas de México, Aguilar-Contreras [/bib_ref]. Monocrotaline is the compound responsible for the toxic effects in Crotalaria sagittalis. Cianhidric acid, one of the most toxic compounds in plants, is found in Crescentia cujete fruit, Phaseolus lunatus whole plant, and Prunus serotina leaves and seeds [bib_ref] Plantas tóxicas de México, Aguilar-Contreras [/bib_ref]. In Phaseolus lunatus, the concentration of cianhidric acid ranges 6.8-533 mg/kg dw [bib_ref] The cyanogenic glycoside contents of raw and processed limabean varieties, Ologhobo [/bib_ref] [bib_ref] Food Additives and Contaminants -Part A Chemistry, Analysis, Control, Cressey [/bib_ref]. There is limited information on the major toxic compounds cited in this review. Therefore, the identification of toxic principles in medicinal plants is necessary.
Evidence-Based Complementary and Alternative Medicine 3 Evidence-Based Complementary and Alternative Medicine 5
## Toxicology
## In vitro studies.
The Artemia salina (brine shrimp) bioassay has been widely used for the analysis of acute toxicity in vitro. Although there are no range values to consider an extract or compound as toxic in the brine shrimp test, vincristine, the positive control for toxicity, has a lethal concentration 50 (LC 50 ) = 0.91 g/ml [bib_ref] In vitro antibacterial, antifungal and cytotoxic activity of Scoparia dulcis L, Md [/bib_ref]. Considering this value, plant extracts or compounds with LC 50 values 1000fold higher than vincristine could be considered nontoxic.
The following plant extracts have been tested for their in vitro toxicology using the brine shrimp test and had LC 50 values higher than 1000 g/ml. The ethyl acetate fraction of Solanum nigrescens aerial parts [bib_ref] Toxicity analysis, phytochemical and pharmacological study of the plant known as Mora..., Susana [/bib_ref] , the ethanol extract of Ambrosia peruviana whole plant [bib_ref] Toxicity of medicinal plants used in traditional medicine in Northern Peru, Bussmann [/bib_ref] , the aqueous extract of Jatropha gossypifolia aerial parts [bib_ref] Alkaloid presence and brine shrimp (Artemia salina) bioassay of medicinal species of..., Coe [/bib_ref] , the methanol extract of Jatropha dioica leaves [bib_ref] Antimicrobial activity and toxicity of plants from northern Mexico, Luis-Benjamín [/bib_ref] , the aqueous extract of Cnidoscolus urens whole plant [bib_ref] Alkaloid presence and brine shrimp (Artemia salina) bioassay of medicinal species of..., Coe [/bib_ref] , the ethanol extract of Crescentia cujete fruits [bib_ref] Chemistry and biology of ethanol extract from the epicarp of Crescentia cujete..., Espitia-Baena [/bib_ref] , the aqueous extract of Enterolobium cyclocarpum bark [bib_ref] Toxicological study of an aqueous extract from Enterolobium cyclocarpum (Jacq.) Griseb. duramen, Raya-González [/bib_ref] , and the ethanol extract of Cordia dentata leaves and their fractions [bib_ref] Perfil fitoquímico, actividad anti-Leishmania, hemolítica y toxicológica de Cordia dentata Poir. y..., Espitia-Baena [/bib_ref].
The plant extract and compounds that could be considered dangerous (LC 50 = 100-1000 g/ml) include the following: methanol and hexane proportions derived from a hexane extract of Gymnosperma glutinosum aerial parts [bib_ref] Antimicrobial and general toxicity activities of Gymnosperma glutinosum: A comparative study, Canales [/bib_ref] , the ethyl acetate extracts of Monstera deliciosa branches [bib_ref] Experiments on the toxicity of some ornamental plants in cattle, Tokarnia [/bib_ref] , and icosandrin, a cyclic homoflavonoid isolated from Phytolacca icosandra [bib_ref] Icosandrin, a novel peltogynoid from the fruits of Phytolacca icosandra (Phytolaccaceae), Montes [/bib_ref].
The plant extract and compounds that could be considered toxic (LC 50 = 10-100 g/ml) include the following: the ethyl acetate extracts of Monstera deliciosa leaves [bib_ref] Experiments on the toxicity of some ornamental plants in cattle, Tokarnia [/bib_ref] , the ethanol extract of Solanum americanum fruits [bib_ref] Toxicity from crude extracts and glycoalkaloid fractions of Solanum spp. Against Artemia..., Lopes [/bib_ref] , the ethanol extract of Scoparia dulcis aerial parts [bib_ref] In vitro antibacterial, antifungal and cytotoxic activity of Scoparia dulcis L, Md [/bib_ref] , the methanol extract of Enterolobium cyclocarpum leaves, the ethanol extract of Pimenta dioica leaves [bib_ref] Comparative study of the assay of Artemia salina L. and the estimate..., Parra [/bib_ref] , and the hydroalcoholic extract of Sanguinaria canadensis whole plant [bib_ref] Toxicity of Sanguinaria canadensis as compared to Aloe vera against brine shrimp..., Karim [/bib_ref]. None of the plant extracts or compounds included in this review were considered highly toxic (LC 50 < 10 g/ml).
## Cytotoxicity.
Other plant extracts and their compounds have been tested in other in vitro models, including cytotoxicity test in nontumorigenic cells, genotoxicity using the comet assay on lymphocytes, and the mutagenic test using lymphocytes or Salmonella spp. The positive controls for cytotoxicity in nontumorigenic cells include cisplatin and Taxol. These compounds have inhibitory concentration 50 (IC 50 ) values ranging from 0.1 to 4 g/ml [bib_ref] Antimicrobial and cytotoxic effects of Mexican medicinal plants, Jacobo-Salcedo [/bib_ref]. Some plants extracts have been reported to lack cytotoxic effects (IC 50 > 250 g/ml) in nontumorigenic cells. These include the ethanol extract of Phoradendron serotinum leaves tested on peripheral blood mononuclear cells [bib_ref] Antimicrobial and cytotoxic effects of Mexican medicinal plants, Jacobo-Salcedo [/bib_ref] , the aqueous extract of Cnidoscolus chayamansa leaves on baby hamster kidney (BHK) cells [bib_ref] Estudio toxicológico de Cnidoscolus chayamansa Mc Vaugh, Torrico [/bib_ref] , and the aqueous extract of Enterolobium cyclocarpum bark assayed on 3T3 murine preadipocytes [bib_ref] Toxicological study of an aqueous extract from Enterolobium cyclocarpum (Jacq.) Griseb. duramen, Raya-González [/bib_ref]. Additionally, the ethanol extract of Equisetum hyemale aerial parts evaluated on rabbit corneal fibroblasts (SIRC) [bib_ref] Antimicrobial activity and toxicity in vitro and in vivo of Equisetum hyemale..., De Queiroz [/bib_ref] , the methanol extract of Enterolobium cyclocarpum leaves evaluated on Vero cells (obtained from kidney epithelial cells extracted from the African green monkey (Cercopithecus aethiops), and the diterpene ent-kaur-16-en-19-oic acid, obtained from Annona cherimola, tested on rat embryo primary striatal cultures [bib_ref] Kaurenoic acid from pulp of Annona cherimolia in regard to annonaceae-induced parkinsonism, Guillopé [/bib_ref]. On the other hand, the hydroalcoholic extract of Hura crepitans leaves had an IC 50 = 107.7 g/ml in lung fibroblasts [bib_ref] Antimalarial activity and cytotoxicity of hydroalcoholic extracts from six plant species used..., Valdés [/bib_ref].
## Mutagenicity and genotoxicity.
Regarding mutagenicity, parthenin, isolated from Parthenium hysterophorus, lacked mutagenicity (0.19 to 19 M) but showed chromosomal aberrations at concentrations of 10-60 M in blood lymphocytes [bib_ref] Parthenin, a sesquiterpene lactone of Parthenium hysterophorus L. is a high toxicity..., Ramos [/bib_ref]. These results suggested genotoxic effects of parthenin. A methanol extract of Indigofera suffruticosa aerial parts (1.25-7.5 mg/plate) showed mutagenic activity in a Salmonella microsome assay [bib_ref] Mutagenic activity of Indigofera truxillensis and I. suffruticosa aerial parts, Varanda [/bib_ref]. The acetone extract of Heliopsis longipes roots (10-80 g/Petri dish) and its active compound affinin (6.25-50 g/Petri dish) were not mutagenic, as evaluated by the Ames test [bib_ref] Pharmacological and toxicological profile of extract from Heliopsis longipes and affinin, Déciga-Campos [/bib_ref]. Lobeline (5-10 mg/kg i.p.), an alkaloid isolated from Lobelia inflata, had no genotoxic or mutagenic effects in the comet assay, the micronucleus test in bone marrow, or the Salmonella/ microsome mutagenic assay [bib_ref] Evaluation of mutagenic and genotoxic activities of lobeline and its modulation on..., Costa [/bib_ref].
For genotoxicity, the ethyl acetate/n-hexane extract of Zinnia peruviana aerial parts tested using 5 and 20 mg/ml extracts showed genotoxic effects in PBMC compared to the positive control of copper sulfate (1%) [bib_ref] Potential genotoxicity of zinnia peruviana extract, Mattana [/bib_ref]. A butanol fraction of Urera baccifera roots at a 1.8 mg/g concentration of oxalic acid decreased leukocyte number significantly and increased cell death and DNA damage in primary cultures of leukocytes in comparison to the control treatment [bib_ref] Genotoxic evaluation, secondary metabolites and antioxidant capacity of leaves and roots of..., Gindri [/bib_ref]. The methanolic extract of Psittacanthus calyculatus aerial parts (200 and 400 mg/kg i.p.) did not induce chromosomal damage in peripheral blood erythrocytes obtained from mice after 72 h of exposure [bib_ref] Antihyperglycemic effect and genotoxicity of Psittacanthus calyculatus extract in streptozotocin-induced diabetic rats, Avila-Acevedo [/bib_ref]. An ethanol extract of Heliopsis longipes roots (3-100 mg/kg p.o.) did not produce genotoxic or cytotoxic effects on peripheral blood mononuclear cells obtained from mice 24-96 h after administration [bib_ref] Antinociceptive, genotoxic and histopathological study of Heliopsis longipes S.F. Blake in mice, Cariño-Cortés [/bib_ref]. Parthenin, isolated from Parthenium hysterophorus, showed genotoxic effects at 4-31 mg/kg i.p. in micronuclei in mouse peripheral blood after 48 and 72 h of treatment [bib_ref] Parthenin, a sesquiterpene lactone of Parthenium hysterophorus L. is a high toxicity..., Ramos [/bib_ref]. [bib_ref] Plantas medicinales de Ixhuatlán del Café, Veracruz, García-Fajardo [/bib_ref]. The guideline 423 of the Organization for Economic Cooperation and Development (OECD) establishes that substances with an LD 50 < 5 mg/kg are highly toxic, whereas LD 50 values from 5 to 50 mg/kg are very toxic, LD 50 values from 50 to 300 mg/kg are toxic, LD 50 values from 300 to 2000 mg/kg are dangerous, and LD 50 values higher than 2000 mg/kg are not dangerous.
## In vivo acute studies
## Lethal dose 50 (ld
Some plant extracts showed LD 50 > 2000 mg/kg p.o. in mice: ethanol extracts of leaves of Casimiroa edulis [bib_ref] New biological activities of Casimiroa edulis leaf extract and isolated compounds, Awaad [/bib_ref] and Cnidoscolus chayamansa [bib_ref] Cnidoscolus chayamansa Mc Vaugh, an important antioxidant, anti-inflammatory and cardioprotective plant used..., García-Rodríguez [/bib_ref] , ethanol extracts of aerial parts of Moussonia deppeana [bib_ref] Anti-inflammatory and toxicological evaluation of Moussonia deppeana (Schldl. & Cham) Hanst and..., Gutiérrez-Rebolledo [/bib_ref] , Equisetum hyemale [bib_ref] Antimicrobial activity and toxicity in vitro and in vivo of Equisetum hyemale..., De Queiroz [/bib_ref] , and Ruta chalepensis [bib_ref] Toxicity studies in mice of ethanol extracts of Foeniculum vulgare fruit and..., Shah [/bib_ref] , as well as methanol extracts of leaves of Chenopodium ambrosioides [bib_ref] Pharmacological screening of the methanolic extract of Chenopodium ambrosioides, Olajide [/bib_ref] and Rauvolfia tetraphylla [bib_ref] Acute toxicity evaluation of Rauvolfia tetraphylla leaf extract in rat by up..., Tamboli [/bib_ref]. The same pattern was also shown in aqueous extract of Cuphea aequipetala aerial parts [bib_ref] Evidence of the anti-Helicobacter pylori, gastroprotective and anti-inflammatory activities of Cuphea aequipetala..., Palacios-Espinosa [/bib_ref] , ethanol extract of Plumeria rubra flowers [bib_ref] Antioxidant and anti-inflammatory activities of flowers of plumeria rubra l. f. rubra..., Sirisha [/bib_ref] , aqueous extract of Larrea divaricata leaves [bib_ref] In vivo antitumoural activity and acute toxicity study of Larrea divaricata Cav...., Anesini [/bib_ref] , ethanol extract of Caesalpinia pulcherrima leaves and bark [bib_ref] Anticonvulsant effect of the ethanol extract of Caesalpinia pulcherrima (L.) Sw., Fabaceae,..., Kumar [/bib_ref] [bib_ref] Antifertility potential of the ethanolic extract of Caesalpinia pulcherrima Linn. leaves, Kumar [/bib_ref]. aqueous extract of Euphorbia prostrata whole plant [bib_ref] In vitro antimicrobial activity of Harungana madagascriensis and Euphorbia prostrata extracts against..., Kengni [/bib_ref] , aqueous-methanol extract of Ceiba pentandra leaves [bib_ref] Toxicological studies of Ceiba pentandra Linn, Sarkiyayi [/bib_ref] , petroleum ether, chloroform, and methanol extracts of Gelsemium sempervirens roots [bib_ref] Antianxiety activity of Gelsemium sempervirens, Dutt [/bib_ref] , acetone extract of Capsicum annum fruits [bib_ref] Antioxidant, antinociceptive, and anti-inflammatory effects of carotenoids extracted from dried pepper (Capsicum..., Hernández-Ortega [/bib_ref] , and aqueous and ethanol extract of Scoparia dulcis leaves and whole plant [bib_ref] Antisickling and toxicological evaluation of the leaves of Scoparia dulcis Linn (Scrophulariaceae), Abere [/bib_ref] [bib_ref] Evaluation of sedative and hypnotic activity of ethanolic extract of Scoparia dulcis..., Moniruzzaman [/bib_ref].
The following extracts have shown LD 50 > 2000 mg/kg p.o. in rats: aqueous extract of Pouteria sapota seeds [bib_ref] Acute toxicity and dermal and eye irritation of the aqueous and hydroalcoholic..., Dutok [/bib_ref] , methanol extract of Martynia annua leaves, ethanol extract of Flourensia cernua leaves [bib_ref] Evaluation of the acute toxicity of an alcoholic extract from tarbush leaves..., Zavala [/bib_ref] , aqueous extract of Enterolobium cyclocarpum bark [bib_ref] Toxicological study of an aqueous extract from Enterolobium cyclocarpum (Jacq.) Griseb. duramen, Raya-González [/bib_ref] , ethanol and aqueous extract C. pulcherrima aerial parts [bib_ref] Evaluation of Caesalpinia pulcherrima Linn. for anti-inflammatory and antiulcer activities, Sharma [/bib_ref] , aqueous extract of Passiflora edulis leaves [bib_ref] Effect of aqueous extract of Passiflora edulis on biochemical and hematological parameters..., Devaki [/bib_ref] , hydroalcoholic extract of Magnolia grandiflora seeds [bib_ref] Anticonvulsant effects of Magnolia grandiflora L. in the rat, Bastidas-Ramírez [/bib_ref] , and ethanol extract of Crescentia cujete fruits [bib_ref] Antivenom activity of ethanolic extract of Crescentia cujete Evidence-Based Complementary and Alternative..., Shastry [/bib_ref]. The same pattern was also shown in methanol extracts of leaves of Amaranthus spinosus [bib_ref] In vitro alpha-amylase inhibition and in vivo antioxidant potential of Amaranthus spinosus..., Kumar [/bib_ref] , and Rauvolfia tetraphylla [bib_ref] Bioactivity guided isolation of antipsychotic constituents from the leaves of Rauwolfia tetraphylla..., Gupta [/bib_ref] , chloroform-methanol extract of Cnidoscolus chayamansa leaves [bib_ref] Antiprotozoal, antimycobacterial, and anti-inflammatory evaluation of Cnidoscolus chayamansa (Mc Vaugh) extract and..., Pérez-González [/bib_ref] , aqueous extract of R. humilis fruits [bib_ref] Acute, subacute and subchronic safety assessment of betalains rich Rivina humilis L...., Khan [/bib_ref] , a chloroform fraction from an ethanol extract of Tagetes erecta flowers [bib_ref] Tagetes erecta Linn. and its mosquitocidal potency against Culex quinquefasciatus, Nikkon [/bib_ref] , aqueous extract of Karwinskia humboldtiana seeds [bib_ref] The ATP levels in kidneys and blood are mainly decreased by acute..., Jaramillo-Juárez [/bib_ref] , and hydroalcoholic extract of Senna occidentalis aerial parts [bib_ref] Acute and subacute toxicity of Cassia occidentalis L. stem and leaf in..., Silva [/bib_ref] , as well as lutein and lutein ester, obtained from Tagetes erecta [bib_ref] Toxicity profile of lutein and lutein ester isolated from marigold flowers (Tagetes..., Harikumar [/bib_ref] , and ethanol extract of Jatropha gossypiifolia aerial parts [bib_ref] Histopathological evaluation in rats after acute treatment with the ethanol extract from..., Mariz [/bib_ref] , and aqueous extract of Caladium bicolor [bib_ref] Antidiarrhoeal activity of aqueous leaf extract of Caladium bicolor (Araceae) and its..., Salako [/bib_ref].
Other plant extracts showed LD 50 > 2000 mg/kg i.p. in mice: hexane extract of Tilia mexicana inflorescences [bib_ref] Pharmacological evaluation of the anxiolytic and sedative effects of Tilia americana L...., Aguirre-Hernández [/bib_ref] , aqueous extract of Tagetes lucida aerial parts, ethanol extract of Mirabilis jalapa aerial parts [bib_ref] Antiinflammatory activity of aqueous extract of Mirabilis jalapa Linn. leaves, Singh [/bib_ref] , and aqueous extract of Urera baccifera leaves [bib_ref] Anti-inflammatory activity of aqueous extracts of five Costa Rican medicinal plants in..., Badilla [/bib_ref].
Some plant extracts and plant compounds had LD 50 values from 300 to 2000 mg/kg, which is considered dangerous. These plant extracts were intraperitoneally administered to mice: the ethanol extract of Tagetes lucida aerial parts (LD 50 = 970 mg/kg), the aqueous extract of Caladium bicolor leaves (LD 50 = 1778.28 mg/kg) [bib_ref] Antidiarrhoeal activity of aqueous leaf extract of Caladium bicolor (Araceae) and its..., Salako [/bib_ref] , and the ethanol extract of Tagetes lucida aerial parts (LD 50 = 970 mg/kg i.p.). On the other hand, the ethanol extract of Phoradendron serotinum leaves had an LD 50 = 375 mg/kg p.o. in mice [bib_ref] (Viscaceae) is associated with the release of immunity-related cytokines, Alonso-Castro [/bib_ref] , and sanguinarine, an alkaloid isolated from Sanguinaria canadensis, had an LD 50 = 1658 mg/kg p.o. in rats [bib_ref] Short-term toxicity studies of sangu1nar1ne and of two alkaloid extracts of Sanguinaria..., Becci [/bib_ref]. Methanol extracts of Tilia mexicana inflorescences had LD 50 values of 375 mg/kg i.p. in mice [bib_ref] Pharmacological evaluation of the anxiolytic and sedative effects of Tilia americana L...., Aguirre-Hernández [/bib_ref].
Other plant extracts and plant compounds had LD 50 values varying from 50 to 300 mg/kg, which is considered toxic. Ethanol extract of Phoradendron serotinum leaves had an LD 50 = 125 mg/kg i.p. in mice, [bib_ref] (Viscaceae) is associated with the release of immunity-related cytokines, Alonso-Castro [/bib_ref] , whereas capsaicin, the main active principle of Capsicum annum, had an LD 50 = 190 mg/kg p.o. [bib_ref] Non-mutagenicity of capsaicin in Albino mice, Muralidhara [/bib_ref]. The acetone extract of Heliopsis longipes roots had an LD 50 = 62.14 mg/kg p.o. in mice, whereas its active compound affinin had an LD 50 = 113.13 mg/kg p.o. in mice [bib_ref] Pharmacological and toxicological profile of extract from Heliopsis longipes and affinin, Déciga-Campos [/bib_ref]. The ethanol extract of Heliopsis longipes roots had an LD 50 = 288 mg/kg p.o. in mice [bib_ref] Antinociceptive, genotoxic and histopathological study of Heliopsis longipes S.F. Blake in mice, Cariño-Cortés [/bib_ref].
The following plant extracts and compounds can be considered very toxic (5-50 mg/kg): the free alkaloid fraction in hexane and methanol extracts from Erythrina americana seeds (LD 50 = 38.54 to 40.37 mg/kg i.p.) in mice [bib_ref] Effect of crude extracts of Erythrina americana Mill. on aggressive behavior in..., Garín-Aguilar [/bib_ref] , Jatropha curcas oil (LD 50 = 23.34 mg/kg p.o.) in mice [bib_ref] Toxicity of Jatropha curcas phorbol esters in mice, Li [/bib_ref] , and -solamargine, isolated from Solanum americanum, with an LD 50 = 42 ± 2 mg/kg i.p. in rats [bib_ref] Toxicological effects of -solamargine in experimental animals, Chami [/bib_ref]. The alkaloid Nmethylisocorydinium, obtained from Magnolia grandiflora trunk wood, had an LD 50 = 10 mg/kg i.p. in mice [bib_ref] Constituents of Magnolia grandiflora. III. Toxic principle of the wood, Rao [/bib_ref] andconiceine, isolated from Conium maculatum, had an LD 50 = 12 mg/kg p.o in mice [bib_ref] Pharmacological actions of hemlock (Conium maculatum) alkaloids, Bowman [/bib_ref]. Parthenin, the toxic compound of Parthenium hysterophorus, had an LD 50 = 42 mg/kg i.p. in rats [bib_ref] Guía de la flora medicinal: tóxica, aromática y condimenticia, Juscafresa [/bib_ref] Evidence-Based Complementary and Alternative Medicine [bib_ref] Characterization of a toxin from Parthenium hysterophorus and its mode of excretion..., Narasimhan [/bib_ref]. Capsaicin had an LD 50 = 8 mg/kg i.p. and 7.80 mg/kg i.m in mice [bib_ref] Acute toxicity of capsaicin in several animal species, Glinsukon [/bib_ref]. Sanguinarine, an alkaloid isolated from Sanguinaria canadensis, was toxic at 29 mg/kg i.v. in rats [bib_ref] Short-term toxicity studies of sangu1nar1ne and of two alkaloid extracts of Sanguinaria..., Becci [/bib_ref].
The following plant compounds had LD 50 values < 5 mg/kg, which is considered highly toxic. Capsaicin had an LD 50 = 0.56 mg/kg i.v. [bib_ref] Acute toxicity of capsaicin in several animal species, Glinsukon [/bib_ref].
## Biochemical and hematological parameters.
Treatment with plant extracts in short-term studies have effects on biochemical and hematological parameters, as well as the levels of the hepatic enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). An aqueous extract of Larrea divaricata aerial parts (0.5-200 mg/kg i.p.) did not affect the levels of ALT in mice after 2 days [bib_ref] In vivo immunomodulatory effects of aqueous extracts of Larrea divaricata Cav, Davicino [/bib_ref]. Aqueous extract of Karwinskia humboldtiana fruits (1250 mg/kg p.o.) administered to rats for 3 days increased the levels of hepatic enzymes compared to the untreated group [bib_ref] Hepatic and blood coagulation damage produced by administration of ripe fruit from..., Jaramillo [/bib_ref]. An aqueous extract of Passiflora edulis (30 mg/kg p.o.) did not affect motor coordination in mice or change the biochemical measurements in serum after 4 days [bib_ref] Assessment of the hypnotic/sedative effects and toxicity of Passiflora edulis aqueous extract..., Maluf [/bib_ref]. -Solamargine (15-35 mg/kg i.p.) did not affect hematological parameters or the levels of hepatic enzymes in rats after 5 days [bib_ref] Toxicological effects of -solamargine in experimental animals, Chami [/bib_ref] , whereas an aqueous extract of Karwinskia humboldtiana fruit (5000 mg/kg p.o.) in rats for 5 days induced weight loss (15%) in rats, as well as toxicity in the pancreas [bib_ref] Damage to pancreatic acinar cells and preservation of islets of langerhans in..., Carcano-Diaz [/bib_ref]. An aqueous extract of Passiflora edulis leaves (100-400 mg/kg p.o.) did not affect organ body weight or hematological parameters but decreased the levels of ALT in rats after 7 days of treatment at all doses [bib_ref] Effect of aqueous extract of Passiflora edulis on biochemical and hematological parameters..., Devaki [/bib_ref]. A methanol extract of R. tetraphylla leaves (1000 mg/kg p.o.) decreased body weight change and food consumption and increased total bilirubin in rats after 7 days [bib_ref] Subacute toxicity evaluation of Rauvolfia tetraphylla methanolic leaf extract in Sprague dawley..., Tamboli [/bib_ref]. An aqueous extract of K. humboldtiana (1000-2000 mg/kg p.o.) administered for 7 days in rats induced alterations in membrane fluidity and ATPase activity in liver submitochondrial particles [bib_ref] Mitochondrial atpase activity and membrane fluidity changes in rat liver in response..., Cid-Hernández [/bib_ref]. An ethanol extract of Euphorbia hirta leaves (60.4-483 mg/kg i.p.) [bib_ref] Antibacterial activities and toxicological potentials of crude ethanolic extracts of Euphorbia hirta, Ogbulie [/bib_ref] and a chloroform fraction from an ethanol extract of Tagetes erecta flowers (200-400 mg/kg p.o.) [bib_ref] Tagetes erecta Linn. and its mosquitocidal potency against Culex quinquefasciatus, Nikkon [/bib_ref] did not affect hematological or biochemical parameters in rats after 14 days. An aqueous extract of Euphorbia hirta whole plant administered at a single dose of 2000 mg/kg p.o. significantly decreased the levels of ALP and ALT after 14 days in broiler chickens [bib_ref] Growth performance, intestinal microflora, plasma fatty acid profile in broiler chickens fed..., Hashemi [/bib_ref]. Lobeline (5-10 mg/kg i.p.) did not affect the levels of AST, ALT, ALP, and LDH after 4 days of exposure [bib_ref] Evaluation of mutagenic and genotoxic activities of lobeline and its modulation on..., Costa [/bib_ref].
## Toxicity to reproduction and pregnancy.
-Solamargine (15-35 mg/kg i.p.) did not affect the number of spermatozoa or the weight of the testicles and epididymis of male rats after 24 h of treatment [bib_ref] Toxicological effects of -solamargine in experimental animals, Chami [/bib_ref]. Jervine (70-300 mg/kg p.o.), a steroidal alkaloid found in Veratrum californicum, administered on days 8-10 of gestation induced malformations in the offspring, including isolated cleft palate, mandibular micrognathia, and limb malformations in C57BL/6J and A/J mice [bib_ref] Expression of Veratrum alkaloid teratogenicity in the mouse, Omnell [/bib_ref]. The administration of an aqueous extract of Ruta chalepensis leaves (10 mg/kg p.o.) to pregnant rats from day 9 to day 17 of gestation decreased the uterine weight, the number of live fetuses, and the fetal weight.
The number of fetal resorptions was also increased, and the fetuses showed skeletal malformations [bib_ref] Efecto embriotóxico y teratogénico de Ruta chalepensis L. ruda, en ratón (Mus..., Gonzales [/bib_ref]. Additionally, an aqueous extract of R. chalepensis leaves (0.8 and 1.6 g/kg p.o.) administered to mice from day 1 to day 14 post coitum caused perinatal changes in mice such as righting reflex, cliff avoidance, and swimming ability, among others [bib_ref] Perinatal toxicology of Ruta chalepensis (Rutaceae) in mice, Zeichen De Sa [/bib_ref].
## Dermal
Tests. An aqueous extract of Pouteria sapota seeds lacked dermal irritation in rabbits and showed mild reversible eye irritation in rabbits [bib_ref] Acute toxicity and dermal and eye irritation of the aqueous and hydroalcoholic..., Dutok [/bib_ref]. Jatropha multifida sap did not induce skin lesions in rats after 14 days of treatment [bib_ref] Hemostatic activity screening and skin toxicity of sap of Jatropha multifida L...., Victorien [/bib_ref]. A diethyl ether extract of Jatropha multifida showed the presence of 16-hydroxyphorbol. This compound showed an irritant dose 50 (ID 50 ) of 0.05 g/ear [bib_ref] Irritant phorbol derivatives from four Jatropha species, Adolf [/bib_ref].
## In vivo subacute and chronic studies
## Biochemical and hematological parameters.
Some plant extracts and their active compounds have been tested for their effects on biochemical and hematological parameters in rodents for at least 18 days of exposure. A histological study has also been included in some reports. A hydroalcoholic extract of Senna occidentalis aerial parts (100-2500 mg/kg p.o.) [bib_ref] Acute and subacute toxicity of Cassia occidentalis L. stem and leaf in..., Silva [/bib_ref] and a hydroalcoholic extract of Sapindus saponaria leaves (44.76 mg/kg p.o.) and fruits (45.0 mg/kg p.o.) [bib_ref] Antiulcer activity of Sapindus saponaria L. in the rat, Meyer Albiero [/bib_ref] did not change the biochemical profile or hematological parameters or alter body weight and organ weight for 30 days in rats. An ethanol extract of the pod of Plumeria rubra (50-200 mg/kg p.o) [bib_ref] Effect of alcoholic pod extract of Plumeria rubra on biochemical and haematologicalparameters..., Dabhadkar [/bib_ref] and an aqueous-methanol extract of Ceiba pentandra leaves (250 and 500 mg/kg p.o.) [bib_ref] Toxicological studies of Ceiba pentandra Linn, Sarkiyayi [/bib_ref] did not alter hematological or biochemical parameters in rats and mice, respectively, after 21 days [bib_ref] Effect of alcoholic pod extract of Plumeria rubra on biochemical and haematologicalparameters..., Dabhadkar [/bib_ref]. A chloroform-methanol extract of Cnidoscolus chayamansa leaves (1000 mg/kg p.o.) [bib_ref] Antiprotozoal, antimycobacterial, and anti-inflammatory evaluation of Cnidoscolus chayamansa (Mc Vaugh) extract and..., Pérez-González [/bib_ref] and an ethanol extract of Moussonia deppeana aerial parts (1000 mg/kg p.o.) [bib_ref] Anti-inflammatory and toxicological evaluation of Moussonia deppeana (Schldl. & Cham) Hanst and..., Gutiérrez-Rebolledo [/bib_ref] did not affect biochemical or hematological parameters in mice after 28 days of daily administration. In addition, histological examinations of the spleen, kidney, and liver showed no abnormalities. Capsaicin (5-100 mg/kg p.o.) [bib_ref] A 4-week feeding study of ground red chilli (Capsicum annuum) in male..., Jang [/bib_ref] , obtained from Capsicum annum, and lutein and lutein ester (4-400 mg/kg p.o.) [bib_ref] Toxicity profile of lutein and lutein ester isolated from marigold flowers (Tagetes..., Harikumar [/bib_ref] , obtained from Tagetes erecta, did not affect hematological or biochemical parameters, growth, food consumption, or body weight in mice and rats, respectively, after 28 days. An aqueous extract of Rivina humilis fruits (2500 and 5000 mg/kg p.o.) administered daily for 35 days showed no changes in the hematological profile or in the relative organ weight, whereas the same extract administered daily for 90 days at 0.5-2 g/100 g in a powdered diet did not affect hematological parameters, biochemical determinations, or the levels of hepatic enzymes [bib_ref] Acute, subacute and subchronic safety assessment of betalains rich Rivina humilis L...., Khan [/bib_ref].
In contrast, some plant extracts have altered biochemical and/or hematological parameters. An aqueous extract of Abrus precatorius leaves (400-1600 mg/kg p.o.) was administered to rats for 18 days. Only the highest dose (1600 mg/kg p.o.) decreased levels of hematological parameters and increased the levels of hepatic enzymes [bib_ref] Studies on the toxicity of an aqueous extract of the leaves of..., Adedapo [/bib_ref]. An aqueous extract of Scoparia dulcis leaves (250-500 mg/kg p.o.) showed mild vascular and portal congestion in the heart and the liver, respectively, of rats treated daily with this extract for 30 days. Nevertheless, there were effects in the lungs and testis [bib_ref] Antisickling and toxicological evaluation of the leaves of Scoparia dulcis Linn (Scrophulariaceae), Abere [/bib_ref]. A methanol extract of Rauvolfia tetraphylla leaves did not affect hematological parameters. However, a significant decrease in the total bilirubin and glucose levels was observed in the mice treated at 100 and 300 mg/kg, with a significant increase in triglycerides at doses of 10-300 mg/kg after 28 days in mice [bib_ref] Bioactivity guided isolation of antipsychotic constituents from the leaves of Rauwolfia tetraphylla..., Gupta [/bib_ref]. An ethanol extract of the aerial parts from Jatropha gossypifolia (135 mg/kg p.o. or higher doses) reduced the activity in the central nervous system and showed hepatotoxicity, pulmonary damage, and digestive disturbances in rats over 13 weeks of treatment [bib_ref] Chronic toxicologic study of the ethanolic extract of the aerial parts of..., Mariz [/bib_ref]. The lethality was 46.6% and 13.3% among male and female rats under the highest tested dose (405 mg/kg), respectively [bib_ref] Chronic toxicologic study of the ethanolic extract of the aerial parts of..., Mariz [/bib_ref].
## Toxicity during reproduction and pregnancy.
Yao et al. [bib_ref] A reproductive screening test of goldenseal, Yao [/bib_ref] reported that an aqueous extract of Hydrastis canadensis (1.86 g/kg p.o.) containing 9.6 mg/ml of berberine and 8.4 mg/ml of hydrastine did not affect fetal development in pregnant rats over 20 days of treatment. A Prosopis juliflora seedcase added at 70% to the diet of pregnant rats resulted to be teratogenic (13-fold) compared to the untreated group. Aqueous and ethanol extracts of Plumeria rubra pods (200 mg/kg p.o.) had 51% and 100% abortifacient activity, respectively, in female albino rats from day 11 to day 15 of pregnancy [bib_ref] Abortifacient activity of Plumeria rubra (Linn) pod extract in female albino rats, Dabhadkar [/bib_ref]. The hydroalcoholic extract of Lantana camara leaves (1000-7000 mg/kg p.o.) administered during premating, mating, pregnancy, and lactation (56 days in total) in rats did not affect mating, pregnancy, delivery, and live birth. Nevertheless, the two highest doses tested (3000 and 7000 mg/kg p.o.) produced an increase in the resorption rate and parallel increases in the postimplantation loss index, as well as embryotoxicity characterized by skeletal abnormalities [bib_ref] Effects of Lantana camara (Verbenaceae) on general reproductive performance and teratology in..., Mello [/bib_ref].
## Carcinogenicity.
Only one plant extract has been tested for its carcinogenic effects. Rats (treated with doses ranging 136-1175 mg/kg p.o.) and mice (treated with doses ranging 375-3275 mg/kg) received an aqueous extract of Hydrastis canadensis root for 2 years (106 weeks). At the end of the treatment, the doses of 1175 mg/kg in rats and the doses varying from 1120 to 3275 mg/kg in mice showed hepatocarcinoma. [fig_ref] Table 2: Evaluation of causality and exclusion of alternate causes in clinical cases of... [/fig_ref]. The other two plants are described in Section 4.4.1. The Naranjo algorithm [bib_ref] A method for estimating the probability of adverse drug reactions, Naranjo [/bib_ref] , which consists of 10 questions that address the factors considered to determine the causal relationship in case reports, was used to assess causality. A score is obtained (maximum 13) and the results are classified as doubtful or unlikely (0), possible (from 1 to 4), probable (from 5 to 8), and clear or definite (socre > 9). The event must be definitive from a pharmacological or phenomenological point of view, using, if necessary, a conclusive procedure of reexposure.
Those cases that report hepatic damage were also evaluated using the method proposed by the Council for International Organizations of Medical Sciences/Roussel Uclaf Causality Assessment Method (CIOMS/RUCAM) [bib_ref] Causality assessment of adverse reactions to drugs-I: a novel method based on..., Danan [/bib_ref] , which is an organospecific instrument designed for the assessment of hepatotoxicity. This method evaluates the temporal relationship between the consumption of a substance (drug or natural remedy) and the appearance of hepatic damage, the absence or presence of risk factors, the exclusion of alternative causes of liver injury, among others. The sum of the scores leads to a final score between −8 and 14 points, which results in the following categories: highly probable or definite, probable, possible, or excluded. The amount of information of each clinical case considered for this review was classified as enough (number of criteria: 5-6), regular (number of criteria: 3-4), and poor (number of criteria: 1-2). The following criteria were used to evaluate the amount of information: (1) clear information regarding the intake and time elapsed for the onset of symptoms, (2) information of the dose ingested, (3) explanation and clinical management of the intoxication, (4) information for the exclusion of other causes that might induce the toxic effect, (5) information of the withdrawal of the plant substance, and (6) time of recovery from the toxicity or death of the patient.
The toxicity presented in clinical cases was mainly due to the accidental consumption of toxic medicinal plants, especially by children. In all the cases, the toxic effects occurred after the administration of the plant. The symptoms of toxicity were confirmed using objective evidence. None of the reports provided information about the presence of similar toxic effects compared to a previous experience. Improvement of symptoms occurred in some cases (i.e., . The information about the number of ingestion with the plant is only reported in some cases (i.e., [bib_ref] Rare Jatropha multifida intoxication in two children, Levin [/bib_ref] [bib_ref] Jatropha curcas -Poisoning, Kulkarni [/bib_ref] [bib_ref] Acute respiratory arrest following hemlock (Conium maculatum) intoxication [1], Biberci [/bib_ref] [bib_ref] Argemone mexicana poisoning: autopsy findings of two cases, Verma [/bib_ref] [bib_ref] Medical Memoranda. Toxic Effect of Podophyllum Application in Pregnancy, Chamberlain [/bib_ref] [bib_ref] Severe poisoning by plants used for traditional medicine in Mayotte, Durasnel [/bib_ref] [bib_ref] Poisoning due to Abrus precatorius, Fernando [/bib_ref].
## Case series.
Krenzelok et el. [bib_ref] Poinsettia exposures have good outcomes-Just as we thought, Krenzelok [/bib_ref] gathered information about Euphorbia pulcherrima exposure during an 8-year period in the United States of America. The results showed that children accounted for 93.3% of cases of exposure, which were accidental (98.9% of cases) and by ingestion (94.5% of cases). No deaths were reported. However, this study did not report the symptomatology. The toxicity of Cimicifuga racemosa has been extensively studied. The reviews of Borrelli and Ernst [bib_ref] Black cohosh (Cimicifuga racemosa): a systematic review of adverse events, Borrelli [/bib_ref] and Teschke et al. [bib_ref] Herb induced liver injury presumably caused by black cohosh: a survey of..., Teschke [/bib_ref] can be consulted for more information regarding the adverse effects of Cimicifuga racemosa in other clinical cases. The prevalence of allergy to Myroxylon pereirae resin has been reported in many countries, ranging from 5.4 to 11.8% (i.e., [bib_ref] Sensitivity to Myroxylon pereirae resin (balsam of Peru). A study of 50..., Avalos-Peralta [/bib_ref] [bib_ref] Contact allergy to fragrances: Frequencies of sensitization from 1996 to 2002. Results..., Schnuch [/bib_ref] [bib_ref] North American contact dermatitis group patch-test results, Marks [/bib_ref] [bib_ref] The significance of fragrance mix, balsam of Peru, colophony and propolis as..., Wöhrl [/bib_ref] [bib_ref] Patch testing with fine fragrances: Comparison with fragrance mix, balsam of Peru..., Trattner [/bib_ref]. From a total of 27815 patients recorded over 5 years in Croatia, 8.4% were positive to contact dermatitis upon exposure to Myroxylon pereirae bark [bib_ref] Contact allergy caused by fragrance mix and Myroxylon pereirae (balsam of Peru)..., Turić [/bib_ref]. In another case, the prevalence of toxicity by medicinal plants was also reported. Jatropha curcas, Andira inermis, and Canella winterana were the third, the fourth, and the fifth most cited plant species, respectively, associated with cases of toxicity in Cuba from 1998-2007 [bib_ref] Intoxicaciones por plantas tóxicas atendidas desde un servicio de información toxicológica, Macías-Peacok [/bib_ref]. Eddleston et al. [bib_ref] Acute yellow oleander (Thevetia peruviana) poisoning: Cardiac arrhythmias, electrolyte disturbances, and serum..., Eddleston [/bib_ref] reported 351 patients with a history of T. peruviana consumption for 2 years. No deaths were reported.
## General considerations
The identification of the compounds responsible for the toxicity has been reported only in some cases. Urushiol might be the compound responsible for the dermatitis reactions to Metopium brownei [bib_ref] Separation and characterization of Metopium brownei urushiol components, Rivero-Cruz [/bib_ref] , whereas sanguinarine is the compound associated with the toxicity of Argemone mexicana. Eddleston et al. [bib_ref] Epidemic of self-poisoning with seeds of the yellow oleander tree (Thevetia peruviana)..., Eddleston [/bib_ref] reported six fatalities in patients who ingested between 1 and 10 seeds of T. peruviana. These fatalities occurred due to high concentrations of cardiac glycosides (neriifolin, thevetin A, thevetin B, and oleandrin) in seeds. Three toxins (T-544, T-514, and T-496) have been reported in Karwinskia humboldtiana [bib_ref] Extraction and quantification of toxins from Karwinskia humboldtiana (Tullidora), Guerrero [/bib_ref]. Manihot esculenta, an important dietary staple, is toxic because of the presence of cyanogenic compounds. Linamarin, the predominant cyanogenic glycoside in Manihot esculenta, can be accumulated in a range of concentrations between 100 and 500 mg/kg in roots and leaves. The content of HCN in Manihot esculenta has been reported 0.1-1 mg/g fresh weight in the leaves [bib_ref] Cassava leaves as human food, Lancaster [/bib_ref]. Several intoxications have been described in humans. The clinical pattern consists of neuropathy and hyperthyroidism [bib_ref] Cyanogenesis in cassava (Manihot esculenta Crantz), Mcmahon [/bib_ref]. The mechanism of toxicity is also unknown in many cases. The mechanism of toxicity of Argemone mexicana oil might be explained by the inhibitory effects of sanguinarine on Na+/K+ATPase, induction of cell membrane damage by reactive oxygen species and lipid peroxidation, and inhibition of DNA polymerase activity, among other effects [bib_ref] Separation, identification and determination of sanguinarine in argemone and other adulterated edible..., Husain [/bib_ref]. Larrea tridentata and nordihydroguaiaretic acid, its active compound, generate acute hepatotoxicity by the inhibition of cyclooxygenase and cytochrome P-450 [bib_ref] Hepatotoxicity of herbal and dietary supplements: an update, Stickel [/bib_ref].
Special attention should be given in medicinal plants such as Argemone mexicana, Chenopodium ambrosioides, and Thevetia peruviana. Effects in humans have been reported due the consumption of these medicinal plants. Other plant species, including Abrus precatorius, Capsicum annum, Conium maculatum, Erythrina Americana, Heliopsis longipes, Hydrastis canadensis, Jatropha curcas, Jatropha gossypifolia, Karwinskia humboldtiana, Larrea tridentata, Magnolia grandiflora, Parthenium hysterophorus, Phoradendron serotinum, Plumeria rubra, Prosopis juliflora, Ruta chalepensis, Sanguinaria canadensis, Solanum americanum, and Veratrum californicum, have shown effects considered highly toxic, including hepatotoxicity, teratogenic, and cardiotoxicity, or with high toxicity in acute studies. Therefore, a total of 22 plants of 216 cited in this review should be extensively studied in terms of their toxicity. Regarding the hepatotoxicity induced by medicinal plants, Valdivia-Correa [bib_ref] Herbal medicine in Mexico: a cause of hepatotoxicity. A critical review, Valdivia-Correa [/bib_ref] reported 15 medicinal plants commonly used in Mexican traditional medicine that induce hepatotoxicity. Five (Equisetum hyemale, Tilia mexicana, Passiflora edulis, and Scoparia dulcis) of these fifteen medicinal are considered as toxic, according to our bibliographic research. The induction of hepatotoxicity induced by herbal products represents a serious problem in Mexico since the symptoms and signs might be confused with other diseases, and the diagnosis can be incorrect [bib_ref] Herbal medicine in Mexico: a cause of hepatotoxicity. A critical review, Valdivia-Correa [/bib_ref].
Some aspects that influence the toxicity of medicinal plants reported in this study are: (a) time of exposure, (b) misidentification of medicinal plants, and (c) adulteration of medicinal plants. Most of the acute symptoms reported in this review appear in the first 24 h after exposure. Nevertheless, more studies, including subacute and chronic assays, as well as the quantitation of hepatic enzymes, should be performed. In other cases, such as the intoxication of Crotalaria sagittalis, the toxic symptoms appear 2 to 6 months after the exposure [bib_ref] Plantas tóxicas de México, Aguilar-Contreras [/bib_ref]. However, chronic poisoning induced by medicinal plants is not easily detected since the symptoms are multiple and variable and a diagnosis cannot be made. Many poisonings caused by medicinal plants are classified as of unknown origin because most of the patients deny the consumption of herbal products. For instance, the clinical picture of intoxication with Karwinskia humboldtiana might be confused with poliomyelitis [bib_ref] Extraction and quantification of toxins from Karwinskia humboldtiana (Tullidora), Guerrero [/bib_ref]. In addition, in most of the cases, the plants are not taxonomically identified [bib_ref] Intoxicaciones por plantas tóxicas atendidas desde un servicio de información toxicológica, Macías-Peacok [/bib_ref]. The misidentification of medicinal plants represents a serious problem for human health. The adulteration of medicinal plants sold as food products should be considered as a risk of intoxication by medicinal plants.
Another aspect to consider for further studies is the evaluation of mixtures of medicinal plants and the combination of medicinal plants with allopathic medications. It is thought that the combination of medicinal plants might result in higher beneficial effects compared to those found with single preparations. Nevertheless, it might be the case that two toxic plants are combined and their toxic effects might result in a synergistic action. The self-medication of drugs along with the consumption of medicinal plants is a common practice among patients with chronic diseases [bib_ref] Effects of electroacupuncture of "Zusanli" (ST 36) on gastric mucosal blood flow,..., Lin [/bib_ref] , which can be considered as an alternative cause of intoxication. In the clinical record, it is not indicated whether the patient consumes medicinal plants. The interaction of herbal extracts and drugs remains to be studied. There are few documented cases that report the toxicity of the combination of plant extracts and drugs. For instance, the combination of Picrasma excelsa coumarins are reported to potentiate the activity of warfarin [bib_ref] Interactions between complementary medicines and warfarin, Myers [/bib_ref]. The toxicity of mixtures of medicinal plants and the combination of medicinal plants with allopathic medication requires further investigation.
# Conclusions
There is limited information about the toxicity of medicinal plants used in Mexico, Central America, and the Caribbean. The molecular mechanisms by which medicinal plants induce toxic effects should also be addressed. In many cases, intoxication by medicinal plants might be confused with other diseases. The detection of intoxication with medicinal plants could be difficult because the symptoms might be confused with other diseases.
The prevention of poisoning in humans can be avoided if the chemical composition of medicinal plants is known.
[fig] 4. 4: Clinical Cases. The toxicity of sixteen species plants has been reported in clinical cases. Fourteen of the sixteen plants are enlisted in [/fig]
[table] Table 1: Ethnobotanical information of medicinal plants from Mexico and Central America considered as toxic. [/table]
[table] Table 2: Evaluation of causality and exclusion of alternate causes in clinical cases of medicinal plants from Mexico and Central America considered as toxic. [/table]
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Association between Variant Y402H in Age-Related Macular Degeneration (AMD) Susceptibility Gene CFH and Treatment Response of AMD: A Meta-Analysis
Purpose: To investigate the association between polymorphism rs1061170 (T1277C, Y402H) in age-related macular degeneration (AMD) susceptibility gene Complement Factor H (CFH) and treatment response of neovascular AMD.Methods:We performed a literature-based meta-analysis including 10 published association studies involving 1,510 patients. Treatments included anti-VEGF (bevacizumab and ranibizumab) or photodynamic therapy. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using fixed-and random-effects models. Q-statistic test was used to assess heterogeneity.Results: Polymorphism rs1061170 showed a significant summary OR of 1.68 (95% CI, 1.09 to 2.60; P = 0.020; CC versus TT; random-effects) for treatment response of neovascular AMD with heterogeneity of 0.09. In subgroup analysis, rs1061170 was more likely to be a predictor of response to anti-VEGF therapy (P = 0.011). However, heterozygous TC genotype was not associated with altered treatment response (OR = 1.18, 95% CI, 0.95 to 1.47; P = 0.145; fixed-effects). Influence analysis indicated the robustness of our findings.Conclusions: rs1061170 might be associated with treatment response of neovascular AMD, especially for the anti-VEGF agents. It might be the first meta-analytically confirmed genetic marker predictive for AMD treatment response though a further validation in larger studies is needed.
# Introduction
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among elderly population. There are two types of AMD, one is non-exudative (dry or atropic) AMD, and the other is exudative (wet or neovascular). The later form, neovascular AMD, is characterized by the presence of choroidal neovascularization (CNV) beneath the fovea. [bib_ref] Patient predictors of histopathologic response after photodynamic therapy of Barrett's esophagus with..., Yachimski [/bib_ref] [bib_ref] Genetics of age-related macular degeneration, Ting [/bib_ref] CNV is a pathologic ocular occurrence in which aberrant blood vessels expand from the choroid to the retinal pigment epithelium, reaching the retina in a high proportion of patients. In addition, while traditionally neovascular AMD was characterized by CNV, advances in imaging technology have also identified retinal angiomatous proliferative (RAP) lesions, which in part originate in the retina in some patients. Neovascular AMD is caused by genetic and various environmental factors such as age, smoking, and serum level of cholesterol. [bib_ref] Patient predictors of histopathologic response after photodynamic therapy of Barrett's esophagus with..., Yachimski [/bib_ref] Over the past several years, some genes have been identified to be AMD susceptibility gene. [bib_ref] Patient predictors of histopathologic response after photodynamic therapy of Barrett's esophagus with..., Yachimski [/bib_ref] [bib_ref] Genetics of age-related macular degeneration, Ting [/bib_ref] Of these, the Complement Factor H (CFH) gene is one of the most important genes. [bib_ref] Complement factor H variant increases the risk of age-related macular degeneration, Haines [/bib_ref] [bib_ref] Complement factor H polymorphism in age-related macular degeneration, Klein [/bib_ref] [bib_ref] Complement factor H polymorphism and age-related macular degeneration, Edwards [/bib_ref] Variant rs1061170 (T1277C, Y402H) in the CFH gene located in the heparin and CRP-binding domain may cause complement dysregulation and lead to the pathogenesis of AMD. [bib_ref] Genetics of age-related macular degeneration, Ting [/bib_ref] [bib_ref] Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein, Laine [/bib_ref] Many independent studies as well as a meta-analysis have indicated that harboring the risk C-allele results in an approximately 2-fold increased risk of AMD. [bib_ref] Complement factor H variant increases the risk of age-related macular degeneration, Haines [/bib_ref] [bib_ref] Complement factor H polymorphism in age-related macular degeneration, Klein [/bib_ref] [bib_ref] Complement factor H polymorphism and age-related macular degeneration, Edwards [/bib_ref] [bib_ref] Prevalence of and Risk Factors for Age-Related Macular Degeneration in a Multiethnic..., Cheung [/bib_ref] The vision loss due to CNV development in neovascular AMD can be mainly managed by two different therapeutic strategies. One is photodynamic therapy (PDT) with verteporfin, and the other is intravitreal administration of drugs acting against vascular endothelial growth factor (VEGF), known as anti-VEGF treatment. Anti-VEGF agents (i.e., ranibizumab and bevacizumab) have shown efficacy in counteracting the damage owing to choroidal neovessels. Many efforts are underway to identify clinical, genetic, and pharmacologic biomarkers that could predict response to therapy, thereby providing important information to clinical decision making and treatment option. New pharmacogenetic knowledge provides data regarding the role of several single nucleotide polymorphisms (SNPs) as genetic predictors of treatment responsiveness to PDT as well as to anti-VEGF therapy. Since immune factors and inflammation are relatively important concepts linked to AMD and the complement system is a key component in the pathogenesis of AMD, [bib_ref] Molecular pathology of age-related macular degeneration, Ding [/bib_ref] they may play a major role in therapeutic interventions. Previous studies proposed that patients with the variant rs1061170 in the CFH gene have higher background levels of inflammation, may continue to affect the disease progression, and probably lead to more rapid recurrence of neovascularization. [bib_ref] Pharmacogenetics of complement factor H (Y402H) and treatment of exudative age-related macular..., Lee [/bib_ref] Patients with the different rs1061170 genotypes may also respond differently to treatment, and even require additional injections of agents.
Thus far, several association studies regarding the predictive role of rs1061170 in treatment response of neovascular AMD have been reported, [bib_ref] Pharmacogenetics of complement factor H (Y402H) and treatment of exudative age-related macular..., Lee [/bib_ref] [bib_ref] Association of complement factor H and LOC387715 genotypes with response of exudative..., Brantley [/bib_ref] [bib_ref] The effect of complement factor H Y402H polymorphism on the outcome of..., Seitsonen [/bib_ref] [bib_ref] CFH and LOC387715/ARMS2 genotypes and treatment with antioxidants and zinc for age-related..., Klein [/bib_ref] [bib_ref] Complement factor H Y402H and C-reactive protein polymorphism and photodynamic therapy response..., Feng [/bib_ref] [bib_ref] Genetic association with response to intravitreal ranibizumab in patients with neovascular AMD, Kloeckener-Gruissem [/bib_ref] [bib_ref] Complement factor H Y402H gene polymorphism and response to intravitreal bevacizumab in..., Nischler [/bib_ref] [bib_ref] Complement factor H and high-temperature requirement A-1 genotypes and treatment response of..., Tsuchihashi [/bib_ref] [bib_ref] CFH, VEGF and HTRA1 promoter genotype may influence the response to intravitreal..., Mckibbin [/bib_ref] [bib_ref] Association between high-risk disease loci and response to anti-vascular endothelial growth factor..., Orlin [/bib_ref] though the results were inconclusive yet. Therefore, we carried out a meta-analysis focusing on the relationship between polymorphism rs1061170 and treatment response of AMD in order to get a more convincing and precise conclusion.
# Methods
## Study identification and data extraction
Publication search of this meta-analysis was performed as described previously. [bib_ref] Age and gender variations in age-related macular degeneration prevalence in populations of..., Rudnicka [/bib_ref] [bib_ref] Systematic review and meta-analysis of the association between complement component 3 and..., Thakkinstian [/bib_ref] [bib_ref] Toll-like receptor 3 C1234T may protect against geographic atrophy through decreased dsRNA..., Zhou [/bib_ref] Briefly, relevant studies were searched in the PubMed, Medline, and Web of Science database (updated to February-10, 2012) using the following search terms: (''CFH'' or ''complement factor H'') and (age-related macular degeneration). There were 374 results, 155 of which regarding rs1061170 (T1277C, Y402H) of the CFH gene. Only those published studies in English language with full text articles were included in this meta-analysis; we did not define the minimum number of patients to be included for meta-analysis. The abstracts of those crudely identified 155 articles were reviewed. The inclusion criteria were: (i) evaluating the relationship between rs1061170 and treatment response of neovascular AMD, (ii) independent retrospective or prospective association study, and (iii) with sufficient available data to estimate an odds ratio (OR) with 95% confidence interval (CI). As a result, we identified 10 candidate studies for systematic review [fig_ref] Figure 1: The literature search process [/fig_ref]. The following variables were extracted from each study if available: first author's surname, publication year, ethnicity, numbers of cases, OR with 95% CI of response to treatment, treatment modality, and information of comparison. The information was collected independently by the two authors (C.H. and X.G.Z.), and any discrepancy were resolved by discussion.
# Statistical methods
For each study, OR with 95% CI were recorded or calculated to assess the relation strength between rs1061170 genotype and treatment response. The pooled OR was calculated by a fixedeffects model (using the Mantel-Haenszel method) or a randomeffects model (using the DerSimonian and Laird method) according to the heterogeneity. Heterogeneity assumption was checked by the Q test. If P-value was not more than 0.10, the inter-study heterogeneity was considered to be significant and we would choose the random-effects model to pool the ORs. Otherwise, the fixed-effects model was employed. Because of the limitation of original data, two types of OR were calculated in present meta-analysis: TC (YH) genotype versus TT (YY) genotype, and CC (HH) genotype versus TT (YY) genotype. The potential publication bias was examined visually in a funnel plot of ln[OR] against its standard error (SE), and the degree of asymmetry was tested using Egger's test. We also performed subpopulation analysis by treatment and ethnicity. Influence analysis (sensitivity analysis) was conducted by omitting each study to find potential outliers. All of the statistical analyses were performed using Stata/SE version 10.0 (Stata Corporation, College Station, TX).
# Results
## Systematic review of eligible studies
We identified 10 eligible studies for the present systematic review [fig_ref] Table 1: Characteristics of the studies included in the meta-analysis [/fig_ref] [bib_ref] Pharmacogenetics of complement factor H (Y402H) and treatment of exudative age-related macular..., Lee [/bib_ref] [bib_ref] Association of complement factor H and LOC387715 genotypes with response of exudative..., Brantley [/bib_ref] [bib_ref] The effect of complement factor H Y402H polymorphism on the outcome of..., Seitsonen [/bib_ref] [bib_ref] CFH and LOC387715/ARMS2 genotypes and treatment with antioxidants and zinc for age-related..., Klein [/bib_ref] [bib_ref] Complement factor H Y402H and C-reactive protein polymorphism and photodynamic therapy response..., Feng [/bib_ref] [bib_ref] Genetic association with response to intravitreal ranibizumab in patients with neovascular AMD, Kloeckener-Gruissem [/bib_ref] [bib_ref] Complement factor H Y402H gene polymorphism and response to intravitreal bevacizumab in..., Nischler [/bib_ref] [bib_ref] Complement factor H and high-temperature requirement A-1 genotypes and treatment response of..., Tsuchihashi [/bib_ref] [bib_ref] CFH, VEGF and HTRA1 promoter genotype may influence the response to intravitreal..., Mckibbin [/bib_ref] [bib_ref] Association between high-risk disease loci and response to anti-vascular endothelial growth factor..., Orlin [/bib_ref]. Other four candidate studies [bib_ref] An analysis of the CFH Y402H genotype in AMD patients and controls..., Goverdhan [/bib_ref] [bib_ref] Association of complement factor H and LOC387715 genotypes with response of exudative..., Brantley [/bib_ref] [bib_ref] CFH, VEGF, and PEDF genotypes and the response to intravitreous injection of..., Imai [/bib_ref] [bib_ref] Involvement of genetic factors in the response to a variable-dosing ranibizumab treatment..., Teper [/bib_ref] , although also investigating the relationship between neovascular AMD treatment response and rs1061170 genotype, failed to provide sufficient original data for OR estimation and were thus excluded from meta-analysis. Most reported studies were of a relative small sample size, with mean subject number of 151 (range, 86-265). We recorded the genotypic risks. If some data in the original papers were not presented in an appropriate form for meta-analysis, we estimated the ORs with 95% CIs from the raw data. Regarding ethnicity, seven studies were performed in Caucasians, one in Asians, and the remainders were unknown. The frequencies of variant C-allele of rs1061170 (T is the ancestral allele) were similar among Caucasian studies, from 50% to 58%. In contrast, the Asian study reported a much lower C-allele frequency of 7%, [bib_ref] Complement factor H and high-temperature requirement A-1 genotypes and treatment response of..., Tsuchihashi [/bib_ref] which however was consistent with that in Asian population according to the public dbSNP and HapMap databases. All the subjects included in the meta-analysis are patients, and we need to not check the departure from Hardy-Weinberg equilibrium (HWE). [bib_ref] Inspection of a deviation from Hardy-Weinberg equilibrium in familial breast cancer cases..., Yu [/bib_ref] [bib_ref] Investigating Hardy-Weinberg equilibrium in case-control or cohort studies or meta-analysis, Ziegler [/bib_ref] It is because that departure from HWE in patients might indicate a genetic association between studied locus and disease rather than a biased population selection. Testing for HWE in patients is not meaningful for quality control. Of note, due to heterogeneity between studies, some studies did not directly evaluate the treatment response by comparing responders with non-responders, but used the disease progression or recurrence as a surrogate of poor response. We clarified the OR of comparison and definition of good/poor response in [fig_ref] Table 1: Characteristics of the studies included in the meta-analysis [/fig_ref]. In terms of predictive role of rs1061170 in treatment response, when TC genotype versus TT genotype, six of the ten studies showed Callele tended to be a predictor of poor response; when CC including seven studies in Caucasians and one study reported by institution in Austria [bib_ref] Complement factor H Y402H gene polymorphism and response to intravitreal bevacizumab in..., Nischler [/bib_ref]. The study in Asians [bib_ref] Complement factor H and high-temperature requirement A-1 genotypes and treatment response of..., Tsuchihashi [/bib_ref] is not included; one study reported by institutions in South Korea and the USA [bib_ref] Association between high-risk disease loci and response to anti-vascular endothelial growth factor..., Orlin [/bib_ref] is also excluded because of a concern of the mixture of Korean patients in that study. doi:10.1371/journal.pone.0042464.t002 genotype versus TT genotype, seven of nine studies indicated a resistant role of C-allele in treatment.
# Meta-analysis
We subsequently meta-analyzed the ten studies for the pooled association between treatment response of neovascular AMD and rs1061170 genotype. The combined sample size was 1,510. [fig_ref] Table 2: Results of meta-analysis for the CFH rs1061170 [/fig_ref] presents the results of meta-analysis (ten studies for TC versus TT and nine studies for CC versus TT). The patients harboring homozygous for variant C-allele (CC genotype) seemed to be associated with a reduced response to treatment of neovascular AMD (pooled OR = 1.68, 95% CI, 1.09 to 2.60, random-effects,. However, heterozygous (TC genotype) was not associated with altered treatment response (pooled OR = 1.18, 95% CI, 0.95 to 1.47, fixed-effects). Due to limitation of original data, we could not do meta-analysis under the dominant or recessive genetic model. In subgroup analysis by treatment, we found that rs1061170 was more likely to be a . Forest plot of meta-analysis of association between CFH rs1061170 (T1277C, Y402H) and treatment response of neovascular AMD. A, Each study is shown by the point estimate of the odds ratio (OR) (the size of the square is proportional to the weight of each study) and 95% confidence interval (CI) for the OR (extending lines). Genotype comparison: CC versus TT. B, Forest plot of meta-analysis according to treatment modality. Anti-VEGF agents indicate bevacizumab and ranibizumab; PDT, photodynamic therapy. Genotype comparison: CC versus TT. doi:10.1371/journal.pone.0042464.g002 predictor of anti-VEGF therapy rather than a powerful predictor for PDT effectiveness (P = 0.011; OR = 1.42, 95% CI, 1.08 to 1.86). When we divided the patients according to ethnicity (Caucasians or not), the association of treatment response with rs1061170 was also observed. CC genotype was associated with a reduced response to treatment of neovascular AMD with an OR of 1.73 (95% CI, 1.05 to 2.86) in Caucasians. Since only one study performed in Asians was reported, we could not conduct a sub-meta-analysis in Asians.
In addition, we evaluated the influence of any individual study on the overall OR for rs1061170 [fig_ref] Figure 3: Influence analysis and publication bias plot for meta-analysis [/fig_ref]. The results showed that no study fundamentally changed the positive relationship between rs1061170 and treatment response. Moreover, Begg's funnel plot and Egger's test were performed to evaluate the publication bias of literature, and no significant publication bias was observed either in the CC versus TT comparison [fig_ref] Figure 3: Influence analysis and publication bias plot for meta-analysis [/fig_ref] , P = 0.27 for Egger's test) or the TC versus TT comparison (data not shown).
# Discussion
Because relatively small sample sizes in studies have hindered reliable assessment of the association between polymorphism rs1061170 (T1277C, Y402H) in the CFH gene and treatment response of neovascular AMD, we performed the current systematic meta-analysis of 10 relevant studies involving 1,510 patients. The results showed that polymorphism rs1061170 was a predictor of treatment response of neovascular AMD, especially for anti-VEGF agents. Individuals harboring homozygous for the variant risk C-allele corresponded to a decreased response to treatment by approximately 1.6-fold when compared with patients carrying homozygous for the ancestral T-allele. Sensitivity analysis indicated the robustness of our findings, and evidence of publication bias was not observed either. Our study for the first time provides evidence of an association between rs1061170 and treatment response of neovascular AMD. Complement dysregulation has emerged as an important pathogenetic factor in AMD. CFH is a critical negative regulator of complement activation. [bib_ref] Defective complement control of factor H (Y402H) and FHL-1 in age-related macular..., Skerka [/bib_ref] Previous biological studies have demonstrated that SNP rs1061170, which is located in the coding sequence, exerts allelic differences on the binding affinity to Creactive protein (CRP), with the risk allele showing reduced affinity. Previous observations support the functional relevance of rs1061170 to AMD pathogenesis as well as treatment response. A recent meta-analysis and many original papers have provided substantial evidence that rs1061170 is significantly associated with AMD risk. [bib_ref] Complement factor H variant increases the risk of age-related macular degeneration, Haines [/bib_ref] [bib_ref] Complement factor H polymorphism in age-related macular degeneration, Klein [/bib_ref] [bib_ref] Complement factor H polymorphism and age-related macular degeneration, Edwards [/bib_ref] [bib_ref] Prevalence of and Risk Factors for Age-Related Macular Degeneration in a Multiethnic..., Cheung [/bib_ref] In the present study, our results solidly expand the predictive role of rs1061170 in neovascular AMD treatment response, especially for anti-VEGF therapy.
However, considering the relative low OR of 1.7 with 95% CI of 1.1 to 2.6, using rs1061170 only to predict AMD treatment response is not accurate enough, probably coupling with high false-positivity and high false-negativity. Therefore, combining a series of predictive loci as well as clinical and pathological markers would be a better way. Thus far, some polymorphisms in the CRP (rs2808636, rs876538), VEGF (rs699947, rs2146323), pigment epithelium derived factor (PEDF, rs12603825), HTRA1 (rs11200638), and Factor XIII-A (rs5985) genes were also involved in prediction of neovascular AMD treatment response. Additionally, other polymorphisms (such as rs1410996, rs2274700) and haplotypes in the CFH gene may be also helpful to determine the treatment outcome. [bib_ref] Complement factor H and high-temperature requirement A-1 genotypes and treatment response of..., Tsuchihashi [/bib_ref] [bib_ref] Phenotype and genotype characteristics of age-related macular degeneration in a Japanese population, Mori [/bib_ref] However, because of limited study number, those SNPs are not available for meta-analysis yet and further molecular epidemiological studies are needed. In addition, methodologic issues such as publication bias could have particular impact when combining results of observational studies. [bib_ref] Metaanalysis of observational studies in epidemiology: a proposal for reporting. Metaanalysis Of..., Stroup [/bib_ref] Our results are purely based on literature that has been published and are not validated by our own data. Although we did not observe apparent publication bias by Eggers test or funnel plots, these analyses however cannot rule out this problem. Further validation in larger studies is needed.
In conclusion, our analysis might provide a piece of evidence that polymorphism rs1061170 is associated with treatment response of neovascular AMD, especially for the anti-VEGF agents (i.e., ranibizumab and bevacizumab). Our results for the first time meta-analytically confirmed a genetic marker predictive for AMD treatment response, and these results, if validated, might promote the potential for clinical treatment response prediction by combining it with other potential clinicopathological and genetic markers in the future.
[fig] Figure 1: The literature search process. doi:10.1371/journal.pone.0042464.g001 Y402H and AMD Treatment Response PLOS ONE | www.plosone.org [/fig]
[fig] Figure 3: Influence analysis and publication bias plot for meta-analysis. A shows the influence of individual studies on the summary OR (CC genotype versus TT genotype). The horizontal axis represents the odds ratio. Every circle indicates the pooled OR when the left study is omitted in this meta-analysis. The two ends of every broken line represent the respective 95% CI. B shows the Begg's funnel plot of studies included in the metaanalysis (genotype comparison: CC versus TT). The vertical axis represents ln[OR] and the horizontal axis means the standard error of ln[OR]. Horizontal line and sloping lines in funnel plot represent summary OR and expected 95% CI for a given standard error, respectively. Area of each circle represents the contribution of each study to the pooled OR. doi:10.1371/journal.pone.0042464.g003 [/fig]
[fig] Figure 2: Forest plot of meta-analysis of association between CFH rs1061170 (T1277C, Y402H) and treatment response of neovascular AMD. A, Each study is shown by the point estimate of the odds ratio (OR) (the size of the square is proportional to the weight of each study) and 95% confidence interval (CI) for the OR (extending lines). Genotype comparison: CC versus TT. B, Forest plot of meta-analysis according to treatment modality. Anti-VEGF agents indicate bevacizumab and ranibizumab; PDT, photodynamic therapy. Genotype comparison: CC versus TT. doi:10.1371/journal.pone.0042464.g002 [/fig]
[table] Table 1: Characteristics of the studies included in the meta-analysis.Abbreviations: AA, Asian; AMD, age-related macular degeneration; AO&Zn, antioxidants and Zinc; Bev., bevacizumab; CA, Caucasian; CI, confidence interval; ETDRS, Early Treatment Diabetic Retinopathy Study; Frq, frequency; MAF, minor allele frequency; MTA., meta-analysis; NA, not available; OR, odds ratio; PDT, photodynamic therapy; Progr., progression; Ran., ranibizumab; Recur., recurrence; Rspr, responders; UD, Undeclared; VA, visual acuity.*Frequency of the variant C-allele of rs1061170 (T1277C, Y402H). The ancestral allele of rs1061170 is T according to dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs = 1061170). doi:10.1371/journal.pone.0042464.t001 [/table]
[table] Table 2: Results of meta-analysis for the CFH rs1061170 (T1277C, Y402H) and treatment response of neovascular AMD. [/table]
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Trend and distribution of mesothelioma in Denmark.
[bib_ref] Cancer risk of asbestos exposure, Selikoff [/bib_ref] [bib_ref] Asbestos and mesothelioma: a review, Kannerstein [/bib_ref] [bib_ref] Epidemiology of asbestos related tumours, Newhouse [/bib_ref] [bib_ref] Fiber carcinogenesis: epidemiologic observations and the Stanton hypothesis, Havington [/bib_ref]
# Material and methods
Since 1942 new cases of cancer in the population of Denmark have been notified to the National Cancer Registry. This registration is based on reports from hospital departments, pathology institutes and notifications from practising physicians supplemented with information from death certificates. The tumour classification used since 1942 is in accordance with the 7th Revision of the International Classification of Diseases, though with an addition of a suffix-number in case of mesothelioma, rendering this subgroup of malignancies easily identifiable.
All cases of mesothelioma were extracted from the files of the Cancer Registry and separated according to site of tumour, but leaving out cases notified during the first year of registration, i.e. 1942. Age and sex-specific incidence rates were calculated and adjusted to the European standard population in order to eliminate the effects of changes over time in the age structure of the Danish population (direct standardisation) . The time trend of the adjusted incidence rates has been assessed by calculating the slope of the regression line and subsequently tested for significant deviation from zero using the programmes developed by Within selected geographical areas characterized by the existence or absence of known asbestos consuming industries, cases of mesothelioma were identified and the expected number of cases calculated by using the age and sex-specific rates for the total population of Denmark for the year 1972 (indirect standardization). The relative risks (RR) were constructed and 95% confidence limits were derived from the exact Poisson distribution.
# Results
From 1943-1980 a total of 685 male and 338 female cases of malignant mesothelioma were notified to the Cancer Registry. The peritoneal and pleural localisations are by far the most common and mesothelioma of the pericardium account for only 1% of the tumours. They are excluded in the following description. shows highly significant increases in the age-adjusted incidence rates of malignant mesothelioma both of the pleura and of the peritoneum; even the rate of peritoneal mesothelioma becomes steady for both sexes from the mid-1960s onward and the steepness of the rate increase of pleural mesothelioma declines throughout the 1970s. The magnitude of peritoneal mesothelioma incidence is quite similar for men and women while the pleural mesothelioma rates are 3 times higher among males than among females.
The calculated relative risks for males compared to females for mesothelioma of the pleura and peritoneum shown in [fig_ref] Table I: Male-female and pleural-peritoneal incidence ratio for mesothelioma inDenmark 1943Denmark -1980 [/fig_ref] and the pattern of the graphs shown in [fig_ref] Figure 1: Age-adjusted incidence rates of malignant mesothelioma of the pleura and the peritoneum... [/fig_ref] demonstrate that the increase in incidence of pleural mesothelioma occurs 10-15 years earlier among males than females, while simultaneous increases are observed for peritoneal mesothelioma of the two sexes. shows the incidence of malignant mesothelioma in five separate age-groups during the period 1943-1980. It will be seen that this type of cancer is extremely rare in the under 30 age-group, increasingly common in age-groups 30-44, 45-59, 60-74, with the highest incidence in the 75+ agegroup. The stagnation in the incidence increase though the 1970s noted in [fig_ref] Figure 1: Age-adjusted incidence rates of malignant mesothelioma of the pleura and the peritoneum... [/fig_ref] is hardly recognized among the oldest age-group in olds. Among the 45-59 years old an actual fall in the incidence of mesothelioma is observed. shows the incidence of the malignant mesothelioma in relation to age at diagnosis classified into four separate birth cohorts according to the year of birth: 1860-1879, 1880-1899, 1900- . It is noticed that the graphs are shifting to the left with increasing year of birth, indicating the overall increase in incidence by calendar time.
For a restricted time period 1968-1977, [fig_ref] Table I: Male-female and pleural-peritoneal incidence ratio for mesothelioma inDenmark 1943Denmark -1980 [/fig_ref] shows the age-adjusted risk of mesothelioma relative to that of the whole population of Denmark for three selected geographical areas, i.e. capital including capital suburbs, provincial towns and rural areas. A statistically significant deviation in the relative risk is seen among male inhabitants of the capital (Copenhagen) and suburbs, RR= 1,76 and among men living in rural areas, RR = 0,44 indicating a risk association to degree of urbanization. The same pattern is seen among females but the deviations from unity are not statistically significant. When dividing the group of provincial towns (capital excluded) into towns with major ship-building industries (Helsing0r, Nakskov, Odense, Svendborg, Aarhus, Aalborg, Frederikshavn) -and towns without major shipyards a significant deviation from unity again appears. This deviation is not seen when towns and rural districts are divided into coastal and inland areas. [bib_ref] Validity of Danish death certificates for cancer patients in Denmark, Storm [/bib_ref] and that the reporting procedure to the Cancer Registry has remained unchanged during the period. Throughout the registration period, 1943-1980, .90% of the cases have been histologically verified quite uniformly but major problems are notorious in making the correct diagnosis [bib_ref] Pathology of human malignant mesothelioma, Suzuki [/bib_ref] often necessitating histochemical analysis and electronmicroscopy. Large inter-observer differences also exist [bib_ref] Diffuse mesotheliomas: observer variation in histological diagnosis, Mccaughey [/bib_ref] and experience from Canada indicate that overdiagnosing may occur [bib_ref] Mesothelioma registries in identifying asbestos hazards, Mcdonald [/bib_ref] partly because of rising interest and awareness among pathologists and clinicians. The diagnosis of malignant mesothelioma, on the other hand, has been known and accepted by Danish pathologists at least since 1931 so it would seem unlikely that the observed large increase in the incidence is merely spurious since the rise is seen before 1960 when the first indications of the asbestos relationship were published. That the incidence of all other primary malignancies of pleura among both sexes is virtually constant and low (-.2-3 per million inhabitants) during the whole period (not shown in the Figures) is incompatible with major changes in the diagnostic practice. Studies from other European countries and North American have demonstrated a rise in incidence, too, [bib_ref] Mesothelioma registries in identifying asbestos hazards, Mcdonald [/bib_ref] [bib_ref] Trends in mortality of diffuse malignant mesothelioma of pleura, Ahlmark [/bib_ref]. However, from Table III it appears that the incidence of mesothelioma in Denmark today (age-adjusted incidence rates among males of 13-15 and among females of 5-7 per million) is extremely high even when comparable countries are considered (e.g. . This may in part be caused by a high autopsy frequency and by a high proportion of the diagnosed cases being reported to the Cancer Registry, but the importance of Denmark's ship-building industries and other major asbestos consuming plants may also be a significant factor. As can be seen from [fig_ref] Figure 4: Annual average import of raw asbestos to Denmark in theyears 1913-1980 [/fig_ref] the import of raw asbestos to Denmark was of considerable dimension already in the 1930s and after the Second World War there was a very large increase until 1980 when the import and the use of croccidolite was abandoned and strong restrictions were put on the use of other types of asbestos. The increase in the total incidence rates is stagnating and a fall of incidence rate is noted for the younger age-groups while that for the oldest is still rising and that for the middle-aged are in between. There exist no measurements of dust levels at asbestos consuming plants from the 1930s and 1940s but it seems reasonable to presume that the working conditions have improved as a consequence of precautions in the use of asbestos already introduced in the 1930s and further developed in the early 1950s until the advent of prohibitive restrictions. This presumed decrease in dust concentration (despite a vast increase in the import of asbestos) could offer partial explanation at least of the stagnation in the increase of incidence observed in the early 1970s.
The crowding of cases in towns with major shipbuilding industries which is seen from [fig_ref] Table I: Male-female and pleural-peritoneal incidence ratio for mesothelioma inDenmark 1943Denmark -1980 [/fig_ref] , could partly be explained by a higher degree of urbanization in these areas. Similar observations, however, have been made in e.g. USA, Canada and the UK [bib_ref] Epidemiology of mesothelioma from estimated incidence, Mcdonald [/bib_ref] and the high risks are more likely to be a reflection of occupational asbestos exposure. [bib_ref] Malignant mesothelioma in North America, Mcdonald [/bib_ref] has calculated relative risks for persons employed in shipbuilding (but not with insulation) to be 3, and for insulators, 46. The unequal geographical distribution among men, in fact, seems surprising considering an annual intermunicipal migration rate of 5% and the very long latency period between exposure and diagnosis. The much more equal distribution of the female cases may indicate a lesser professional exposure to asbestos, also supported by the smaller incidence rates and the slower increase of incidence.
In conclusion, the pattern of distribution and the time-trend of malignant mesothelioma in Denmark is similar to that of other western countries with the important exception that the incidence rates in the 1970s are among the highest ever recorded on a national basis together with an indication of a stagnating tendency of the increase of incidence.
[fig] Figure 1: Age-adjusted incidence rates of malignant mesothelioma of the pleura and the peritoneum among males (a) and females (b) in Denmark in the years 1943 [/fig]
[fig] 0, Figure 2, Figure 3: ~, os "*-> 's "->> "Age-adjusted incidence rates of malignant mesothelioma in relation to age at diagnosis among males (a) and (b) in Denmark in the years 1943-80. Age-adjusted incidence rates of malignant mesothelioma male and female birth cohorts in the period 1860-1939. [/fig]
[fig] Figure 4: Annual average import of raw asbestos to Denmark in theyears 1913-1980. [/fig]
[table] Table I: Male-female and pleural-peritoneal incidence ratio for mesothelioma inDenmark 1943Denmark -1980 [/table]
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Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart
SynopsisA successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50 % of its activity, promising thermal stability up to 90 - C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness.
# Introduction
Electrochemical etching of single crystalline silicon in hydrofluoric acid (HF)-based electrolytic solutions leads to the formation of various pore arrays, known as porous silicon [bib_ref] Electrochemical formation of a novel porous silicon/polypyrrole hybrid structure with enhanced electrical..., Harraz [/bib_ref]. The pores are generated by means of anodic electrochemical or photoelectrochemical etching of a silicon wafer under galvanostatic conditions. Porous silicon material with different optical features can be produced by varying the etching parameters, including Abbreviations: CL, cathode luminescence; EDS, energy-dispersive X-ray spectroscopy; FE-SEM, field emission SEM; FT-IR, Fourier transform IR spectroscopy; XPS, X-ray photoelectron spectroscopy. [bib_ref] Electrochemical formation of a novel porous silicon/polypyrrole hybrid structure with enhanced electrical..., Harraz [/bib_ref] To whom correspondence should be addressed (email: [email protected]).
etching time and anodization current density [bib_ref] Lanthanide luminescence enhancements in porous silicon resonant microcavities, Jenie [/bib_ref]. The unique features of porous silicon make this material a frontline candidate for enzyme immobilization [bib_ref] Enzyme immobilization on porous silicon surfaces, Letant [/bib_ref] [bib_ref] Porous silicon as an entrapping matrix for the immobilization of urease, Chaudhari [/bib_ref] [bib_ref] Conductivity-based catechol sensor using tyrosinase immobilized in porous silicon, Tembe [/bib_ref] [bib_ref] Applications of microstructured porous silicon as a biocatalytic surface, Bengtsson [/bib_ref] [bib_ref] Quantatitive assessment of enzyme immobilization capacity in porous silicon, Delouise [/bib_ref] [bib_ref] Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on..., Ressine [/bib_ref] , drug delivery [bib_ref] Microfluidic assisted one-step fabrication of porous silicon@acetalated dextran nanocomposites for precisely controlled..., Liu [/bib_ref] [bib_ref] Facile synthesis of biocompatible superparamagnetic mesoporous nanoparticles for imageable drug delivery, Nissinen [/bib_ref] , energyharvesting devices [bib_ref] Swift heavy ion irradiation reduces porous silicon thermal conductivity, Massoud [/bib_ref] [bib_ref] Hydrothermal growth of flower-like ZnO nanostructures on porous silicon substrate, Eswar [/bib_ref] [bib_ref] Copper deposition in microporous silicon using supercritical fluid, Jin [/bib_ref] [bib_ref] Novel mesoporous silicon nanorod as an anode material for lithium ion batteries, Zhou [/bib_ref] [bib_ref] Si/C composite lithium-ion battery anodes synthesized using silicon nanoparticles from porous silicon, Park [/bib_ref] [bib_ref] CO 2 detection with CN x thin films deposited on porous silicon, Zouadi [/bib_ref] and biosensing [bib_ref] Electrochemical impedance spectroscopy of oxidized porous silicon, Mula [/bib_ref] [bib_ref] Conductivity and relaxation time of porous silicon using the Kramers-Kronig relation, Darian [/bib_ref] , due to its large internal surface area, prodigious pore volume, biodegradability, and tunable pore geometry via variation of both porosity and crystallite size [bib_ref] Thermal conductivity of partially amorphous porous silicon by photoacoustic technique, Isaiev [/bib_ref] [bib_ref] Antibody modified porous silicon microparticles for the selective capture of cells, Guan [/bib_ref] [bib_ref] In situ synthesis of peptide nucleic acids in porous silicon for drug..., Beavers [/bib_ref] [bib_ref] Enantioselective extraction mediated by a chiral cavitand-salen covalently assembled on a porous..., D'urso [/bib_ref] [bib_ref] A nanostopper approach to selectively engineer the surfaces of mesoporous silicon, Xu [/bib_ref]. The biodegradation of porous silicon results in the formation of orthosilicic acid, which can easily be absorbed from the gastrointestinal tract and excreted from the body [bib_ref] Facile synthesis of macroporous silicon photocathodes with enhanced photoelectrochemical performance, Li [/bib_ref]. In addition, porous silicon can easily be made with the biomolecules and used in imaging and tumour targeting, although the detection mechanism is based on variation in either the photoluminescence spectra or the diffraction patterns [bib_ref] Porous silicon formation during Au-catalyzed etching, Vega [/bib_ref] [bib_ref] Intravitreal controlled release of dexamethasone from engineered microparticles of porous silicon dioxide, Wang [/bib_ref] [bib_ref] In vivo evaluation of porous silicon and porous silicon solid lipid nanocomposites..., Kallinen [/bib_ref]. As a result of the outstanding biocompatibility of porous silicon, it is popular in the biotechnology field for catalytic functions and enzyme immobilization [bib_ref] A new electrochemical sensor based on porous silicon supported Pt-Pd nanoalloy for..., Ensafi [/bib_ref]. Enzyme immobilization on a solid host might be favoured over its free counterpart [bib_ref] Glucose oxidase enzyme immobilized porous silica for improved performance of a glucose..., Khan [/bib_ref] [bib_ref] Porous silicon-cyclodextrin based polymer composites for drug delivery applications, Montelongo [/bib_ref] because immobilized enzyme has several advantageous features as a result of its reusability, prolonged shelf-life, better thermal and storage stability, and ease of separation of the enzyme from the reaction mixture with no enzyme contamination of the product .
In the present study, physical adsorption methodology was used to immobilize acetylcholinesterase on porous silicon architecture and the hydrolytic response towards acetylthiocholine iodide was assessed using a spectrophotometric bioassay. The porous silicon-immobilized acetylcholinesterase generates synergistic effects, which provide convenient enzyme handling, dexterous segregation from the reaction mixture, easy recycling protocols and reusability of the biocatalyst. The photoluminescence properties of a mesoporous silicon surface before and after enzyme immobilization suggest that enzyme localized on the porous silicon surface shows a slight enhancement in the photoluminescence emission intensity of the bare porous silicon rather than destruction of the visible photoluminescence of porous silicon. The cathode luminescence (CL), alongside fluorescence excitation and emission spectra before and after enzyme immobilization, can be used as an efficient reporting platform for successful enzyme adsorption on to the porous silicon surfaces with retention of immobilized enzyme activity being easily assessed by spectrophotometric bioassay.
## Experimental
# Materials and instrumentation
During the experiment the following were used: acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7, acetylcholinesterase from human erythrocytes), acetylthiocholine iodide, 5,5 -dithio-bis(2-nitrobenzoic acid), neostigmine methyl sulfate and MgCl 2 , purchased from Sigma-Aldrich, NaCl (Daejung Chemical and Metals Co., Ltd), ethanol, water (Samchun Chemicals), HF (48 %, w/w; Merck) and boron-doped p-type silicon wafers with a resistivity of 1-20 cm and thickness 500-550 μm (obtained from Cree Co.). The photoluminescence spectra and relative photoluminescence intensities were measured on an FS-2 fluorescence spectrometer (Scinco) and Lab-Ram HR-800 spectrometer (Horiba Jobin Yvon) with a He/Cd laser source (325 nm). Enzyme immobilization on the surface of porous silicon was confirmed by photoluminescence measurement through study of the changes in the photoluminescence intensity of porous silicon before and after enzyme adsorption. Pore size and porous film thickness were determined by field emission (FE)-SEM images (MIRA3 LMH, TES-CAN). Cross-sectional views were obtained by etching on the reverse of the slide and breaking the sample. The Fourier transform IR (FT-IR) spectra were recorded using a PerkinElmer FT-IR spectrometer connected to an Auto IMAGE FT-IR microscope. The elemental analysis was performed with energydispersive X-ray spectroscopy (EDS) analysis, using FE-SEM, and X-ray photoelectron spectroscopy (XPS) analysis, using a VG surface analysis system (Thermo VG Scientific, ESCA Lab. 2000).
## Enzyme activity measurements
The inhibitory activity of acetylcholinesterase (acetylcholinesterase from human erythrocytes, 0.03 units/ml) was determined spectrophotometrically using acetylthiocholine iodide as a substrate and the reported method of Ellman et al. [bib_ref] A new and rapid colorimetric determination of acetylcholinesterase activity, Ellman [/bib_ref]. Briefly, the assay solution consisted of 180 μl of 50 mM Tris/HCl buffer, pH 8.0, containing 0.1 M sodium chloride, 0.02 M magnesium chloride, 20 μl of enzyme and 15 μl of 14.9 μM neostigmine methyl sulfate, and pre-incubated for 30 min at 4 - C. To this reaction mixture 5,5 -dithio-bis(2-nitrobenzoic acid) (0.3 mM, 20 μl) and acetylthiocholine iodide (1.8 mM, 20 μl) were added and incubated for 10 min at 37 - C, followed by measurement of the absorbance at 412 nm. The same assay procedure was followed for immobilized enzyme. The assay measurements were carried out using a micro-plate reader (OptiMax, Tunable Micro plate Reader; wavelength range 340-850 nm for 96-well plates). The reaction rates of immobilized enzyme were compared with its free counterpart, and the percentage inhibition was calculated using the formula 100 − (Abs testwell /Abs control )×100 [bib_ref] Identification of novel urease inhibitors by high-throughput virtual and in vitro screening, Abid [/bib_ref] , where Abs testwell is the absorbance of the well being tested and Abs control the absorbance of the control sample.
## Preparation of porous silicon surface
The silicon wafer (boron-doped p-type silicon wafers with resistivity 1-20 cm and thickness 500-550 μm) was cut into chips sized 1×1 cm 2 and degreased with an ultrasonic bath of acetone for 5 min, and then rinsed in deionized water. After drying with nitrogen gas, silver paste was simply sputtered on to the back of the wafer to provide a back ohmic contact for anodization. Adhesive tape (ASF-110) was used to bind wire and a 0.25-cm 2 paper section was used on the front of the silicon wafer. The paper section was removed to expose 0.25 cm 2 of the silicon wafer surface, which was then rinsed with trichloroethylene, acetone and deionized water. The porous layer was prepared on a p-type silicon wafer by using electrochemical anodization. The silicon wafer was anodized at a current density of 20 mA/cm 2 using a Keithley 2400 source meter in HF (48 %, w/w; Merck) electrolyte solution (HF:H 2 O:C 2 H 2 OH = 1:1:2, by vol.) for 5, 10, 15, 20, 25, 30 and 35 min, respectively, in dark conditions to obtain an appropriately structured sample. The counter electrode used during the etching process was platinum. Immediately after anodization, the sample was taken out from the cell, rinsed in deionized water and left to dry in nitrogen gas.
## Immobilization of the enzyme on the porous silicon surface
A total of 20 μl (0.03 units/ml) of acetylcholinesterase was dropped on to the surface of the porous silicon and allowed to dry. The enzyme loading was optimized by using different volumes of enzyme in the range 10-30 μl on the porous silicon sample. Porous silicon samples obtained by varying the etching time from 5 min to 35 min, with constant current density, were tested for immobilization of acetylcholinesterase, and the efficiency of the immobilization was confirmed by studying the retention of activity on the surface of the porous silicon spectrophotometrically. However, the samples prepared with a current density of 20 mA/cm 2 for 30 min showed optimum results and were therefore used for further measurements.
# Results and discussion
To investigate the development of a nano-porous silicon layer during the etching process, the surface morphology was examined using FE-SEM analysis before and after enzyme immobilization. [fig_ref] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of [/fig_ref] shows the nano-porous silicon surface before enzyme immobilization whereas the micrograph in [fig_ref] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of [/fig_ref] spectrophotometric bioassay utilizing neostigmine methyl sulfate as a standard acetylcholinesterase inhibitor [bib_ref] Structural insights into substrate traffic and inhibition in acetylcholinesterase, Colletier [/bib_ref] [bib_ref] Effects of TAK-802, a novel acetylcholinesterase inhibitor, on distension-induced rhythmic bladder contractions..., Nagabukuro [/bib_ref] [bib_ref] Image-contrast technology based on the electrochemiluminescence of porous silicon and its application..., Emmerling [/bib_ref].
The cross-sectional FE-SEM images were obtained to discriminate the porous surface from the enzyme-immobilized porous surface. In the cross-sectional FE-SEM micrograph, the porous silicon layer appears to be similar to a sponge-like thin layer, and there is adsorption of enzyme on to that sponge-like porous surface through the creation of a second microlayer of enzyme [fig_ref] Figure 2: Cross-sectional FE-SEM viewCross-sectional FE-SEM of [/fig_ref]. The enzyme was observed to be retained on the porous layer with no penetration into the bulk of the porous surface. The major trapping forces for a thin enzyme layer on the porous surface were considered to be van der Waals' forces and hydrogen bonding between the enzyme's hydroxyl group and the porous layer's hydrogen atoms.
# Ft-ir analysis
The FT-IR analysis was done to determine the chemical composition of the samples. The presence of enzyme in the porous silicon material was indicated in the FT-IR spectra by the appearance of a new broad signal at 3364 cm − 1 , assigned to acetylcholinesterase's free hydroxyl stretching mode, whereas no such signal was detected in the highlighted area on the bare porous silicon spectrum. The sharp signal at 2962 cm − 1 for both bare and enzyme-immobilized porous silicon surfaces was attributed to C-H stretching vibrations due to adsorbed hydrocarbons on the surface. The appearance of a sharp as well as a broad-band signal, with less growth in the intensity at the spectral span from 2335 cm − 1 to 2119 cm − 1 , is characteristic of the Si-H bond, which partially vanished after acetylcholinesterase immobilization. The broad-band signal at 1641 cm − 1 was also observed on the enzyme-immobilized surface associated with the NH bending and scissoring mode, although no such signal was harvested for bare porous silicon. Meanwhile, a sharp signal in the range 1451-1410 cm − 1 was assigned to the C= =C stretching vibration arising from the immobilized enzyme skeleton present on the porous surface [fig_ref] Figure 3 FT: -IR spectra FT-IR spectra of bare porous silicon surface and enzyme-immobilized porous... [/fig_ref].
# Eds analysis
The elemental analyses of the bare porous silicon surface, as well as the acetylcholinesterase-immobilized porous silicon surface, were done to gain insight into the presence of enzymes on the porous silicon surface using EDS analysis. There was an appreciable appearance of new elements, including chlorine, magnesium and sodium, which are attributed to the enzyme skeleton adsorbed on to the porous silicon surface. Moreover, the weight percentage of silicon, fluorine and carbon considerably varied in the EDS spectrum after enzyme immobilization, giving further evidence of the presence of enzyme on the porous silicon surface, as shown in .
# Xps analysis
To diagnose the particular chemical species and the electronic environments at the surface, the XPS analysis was performed us- ing VG surface analysis system (Thermo VG Scientific, ESCA Lab. 2000) before and after the enzyme was immobilized on the porous silicon surface. During XPS analysis, the sample was irradiated with monoenergetic X-rays, causing photoelectrons to be emitted from the sample surface. An electron energy analyser determines the binding energy of the photoelectrons and, from this and the intensities of the photoelectron peak, the element's identity, chemical state and quantity were determined. The information from XPS analysis was used for analysis of bare and acetylcholinesterase-immobilized surface layers. The original XPS spectrum obtained by analysing bare porous silicon and enzyme-immobilized porous silicon for several elements present on the surface is shown in [fig_ref] Figure 5: XPS analysis data XPS analysis for the bare porous silicon [/fig_ref].
The results of XPS analysis are consistent with EDS analysis. The detected elements on the surface and their weight percentage for bare and immobilized porous silicon samples are shown in . There was a considerable increment in the weight percentage of carbon from 6.21 % to 24.62 % after enzyme immobilization, indicating the hydrocarbon structure of the enzyme trapped on the porous silicon, whereas there was a remarkable decline in the silicon weight percentage and complete elimination of fluorine on the enzyme-immobilized porous silicon, indicating the surface compensation by enzyme. Meanwhile, the expected high weight percentage of oxygen due to acetylcholinesterase's free hydroxyl group and manifestation of extra elements, including magnesium, chlorine, sodium and nitrogen, was expected from the adsorbed acetylcholinesterase skeleton.
## Photoluminescence and cl measurements
The porous silicon surface displayed variable dynamic features in the tuning surface chemistry, especially, in the case of . . photoluminescence on porous silicon, where the surface adsorbent may just block the photoluminescence process or cause a retardation of electronic excitation; however, some chemical species may act as electron donors or acceptors, thus leading to quenching or enhancement of surface luminescence [62]. In the present study, the photoinduced visible light emission from lightly oxidized porous silicon showed photoluminescence emission maxima at 643 nm and fluorescence excitation maxima at 579 nm. There was a sudden increment in the intensity of porous silicon excitation and emission spectra after enzyme immobilization. The enzyme was trapped on the porous silicon, with no destruction of its photoluminescence and slight enhancement in the photoluminescence intensity, whereas free enzyme in solution did not exhibit any photoluminescence. However, following enzyme immobilization on porous silicon architecture, the enzyme aggregates on porous silicon were found via luminescence, assessed by photoluminescence as well as CL using a fluorescence spectrometer and FE-SEM, respectively. In the CL spectrum, the brightness from the area of enzyme aggregation (inset in is due to luminescence from the immobilized enzyme on the porous silicon surface. These CL spectra, alongside photoluminescence excitation and emission spectra before and after enzyme immobilization, can be used as an efficient reporting platform for successful enzyme adsorption on to the porous silicon surface. Moreover, the immobilized enzyme is retained on the porous silicon surface without obliteration of its luminescence behaviour at room temperature, as shown in .
## Acetylcholinesterase inhibition assay for immobilized and free enzyme
To assess the hydrolysis response of acetylcholinesterase towards acetylthiocholine iodide, the spectrophotometric bioassay was performed using the standard acetylcholinesterase inhibitor (neostigmine methyl sulfate, 14.9 μM). For a comparative study, both free and porous silicon-immobilized enzymes were exploited in the spectrophotometric bioassay. The 50 % enzyme inhibition was observed at 0.03 μg/ml of neostigmine methyl sulfate for the free enzyme, whereas porous silicon-immobilized acetylcholinesterase exhibited 50 % activity at a drug concentration of 0.033 μg/ml, which was almost the same as that of the soluble enzyme. An activity close to that of the porous silicon-immobilized enzyme, with its free counterpart, indicates successful enzyme adsorption on to the porous silicon surface, alongside retention of its hydrolysis response towards acetylthiocholine iodide, as shown in .
## Figure 6 photoluminescence excitation spectra
Photoluminescence excitation spectra (left side spectra) of bare porous silicon, as well as enzyme-immobilized porous silicon surface, and photoluminescence emission spectra (right side spectra) of bare and acetylcholinesterase-immobilized porous silicon surface, whereas a CL spectrum (inset) was obtained to get further assistance in understanding the photoluminescence enhancement from a porous silicon surface after enzyme immobilization (the scale bar for the inset is 200 μm); a.u., absorbance units.
## Figure 7 comparison of ic 50 values
Comparison of IC 50 (μg/ml) values for free and porous silicon-immobilized acetylcholinesterase.
## Reusability
One of the most important properties of the immobilized enzyme, primarily due to its reusability, is the prerequisite that commercialization of biocatalysts demands that the enzymatic process be economically cost saving; this makes it superior to the free counterpart by assembling an enzymatic process that is economically favourable and deeply rooted in the field of biotechnology [bib_ref] Immobilization of endo-inulinase on non-porous amino functionalized silica nanoparticles, Karimi [/bib_ref] [bib_ref] Comparison of the performance of commercial immobilized lipases in the synthesis of..., Martins [/bib_ref]. To find out how reusable the enzyme is, the spectrophotometric assay was conducted using varying concentrations of neostigmine methyl sulfate and half-maximal inhibitory con- centration (IC 50 ) values were calculated to achieve 50 % enzyme inhibition with the drugs used. From [fig_ref] Figure 8: The IC50 values The IC 50 [/fig_ref] , it is clear that, for the immobilized enzyme, the enzyme inhibition activity was quite similar, compared with the free enzyme, for up to three batches. However, during the third cycle, a slight reduction in immobilized enzyme activity was observed, because 50 % enzyme activity was obtained at a slightly higher drug consumption (0.04 μg/ml of neostigmine methyl sulfate) compared with the first and second cycles. However, at the fourth cycle of reusability, the enzyme activity was reduced by up to 36 % and the IC 50 values could not be calculated. From these results, it was concluded that porous silicon-immobilized enzyme can be used for up to three cycles, with a promising hydrolytic response towards acetylthiocholine iodide, although afterwards a gradual decrease in enzyme activity would make it unfeasible to obtain 50 % enzyme inhibition with the standard treatment drug. However, the immobilized enzyme is completely inactivated after five batches.
## Storage stability
To determine the storage stability of porous silicon-immobilized acetylcholinesterase, the immobilized enzyme, as well as its free counterpart, were stored at 4 - C and their hydrolytic response towards acetylthiocholine iodide was evaluated after regular 4day intervals by spectrophotometric bioassay. The free enzyme showed very similar activity, as well as the porous siliconimmobilized acetylcholinesterase, for up to 8 days; there was then a continuous decrease in the activity of the free enzyme and it was completely inactivated after 20 days. However, the immobilized enzyme retained 50 % activity, comparable to that of free enzyme over a longer period of time, and preserved its 50 % activity at slightly higher drug concentrations for up to 44 days. Thereafter, the activity of the immobilized enzyme was reduced below 50 %, as shown in [fig_ref] Figure 9: Comparison of IC 50 valuesComparison of IC 50 [/fig_ref]. These results lead us to conclude that the immobilized enzyme possesses significant advantages over the free enzyme because of its longer shelf-life, improved stability, convenient handling, inactivity when separated from the reaction mixture and prevention of contamination by the enzyme's products.
## Thermal stability
To investigate the effect of temperature on the inhibition potential of immobilized enzyme towards neostigmine methyl sulfate, the free and porous silicon-immobilized enzyme were incubated at different temperatures ranging from 20 - C to 120 - C for 1 h, followed by measurement of the enzyme activity spectrophotometrically. The IC 50 values for free and immobilized enzyme were calculated using different concentrations of neostigmine methyl sulfate, ranging from 0.0091 μg/ml to 0.294 μg/ml. The free acetylcholinesterase exhibited 50 % activity with varying concentrations of neostigmine methyl sulfate up to 50 - C; latterly there was complete inactivation of the free enzyme. Meanwhile, the porous silicon-immobilized enzyme displayed 50 % inhibitory activity over a bigger temperature span, up to 90 - C, as shown in [fig_ref] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of [/fig_ref]. The immobilized enzyme resists thermal denaturation over much higher temperatures compared with the free enzyme, and this interesting operational feature of immobilized enzyme under harsh reaction conditions could contribute to the enzyme's longer shelf-life.
## The ph effect
The effect of pH was evaluated in the pH range 4-9 to figure out the optimal pH span for the immobilized enzyme. The results [fig_ref] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of [/fig_ref] Thermal stabilities Thermal stability of free enzyme and porous silicon-immobilized enzyme by incubating both at a variable temperature for 1 h. [fig_ref] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of [/fig_ref] The pH stability The pH stability of free and immobilized enzyme for assessment of the optimal pH for enzymatic activity.
showed that the enzyme's activity strongly depended on the buffer's pH. The free and immobilized enzymes work well at pH 8, but there was less than 50 % enzyme activity left at pH <6, as well as pH >9 for the free enzyme, whereas the immobilized enzyme retained 50 % activity over a broad pH span -4-9. In the meantime, 50 % enzyme activity was obtained with slightly larger amounts of drug for the free enzyme at a pH below and above 8, whereas the immobilized enzyme remained stable, with a better activity in the pH range shown in [fig_ref] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of [/fig_ref]. This property of the immobilized enzyme makes it a reliable source for enzyme catalysis reactions over a broad pH spectrum.
# Conclusion
Acetylcholinesterase has been successfully immobilized on the nano-porous silicon surface; the porous surface suitable for enzyme immobilization was found by changing the etching parameters. The hydrolytic response of the immobilized enzyme towards acetylthiocholine iodide was assessed using a spectrophotometric bioassay. The percentage inhibitions of the immobilized as well as the free enzyme-catalysed reactions were monitored using the standard acetylcholinesterase inhibitor neostigmine methyl sulfate. The immobilization processes considerably enhance storage stability for up to 44 days, with retention of 50 % enzymatic activity and application of the enzyme in slightly harsh conditions (thermal stability up to 90 - C), easy recovery from the media with no contamination of the reaction mixture, reusability for up to three cycles and pH stability over the broad span 4-9. This finding suggests that the immobilized enzyme can be capitalized on as a most promising technique in organic and biomolecule synthesis, with satisfactory technological and economical advantages over the free enzyme due to its recycling, ease of separation from the reaction mixture and superficial handling strategy. The simple adopted immobilization procedure provides very good enzyme adsorption on to the nano-channel of a mesoporous silicon surface, with minimal leaching and an abundant hydrolytic response towards acetylthiocholine iodide, comparable with soluble enzyme, while the enzyme adsorption on to the porous silicon architecture can easily be inspected using photoluminescence measurements, spectrophotometric assay, FE-SEM, FT-IR, EDS analysis, CL measurement and XPS analysis.
# Author contribution
Muhammad Saleem and Muhammad Rafiq carried out the experiments. Sung-Yum Seo gave advice on the experiments. Ki Hwan Lee conceived and supervised the study.
# Funding
This work was supported by the research grant of the Kongju National University in 2015.
[fig] Figure 1: Plan-view FE-SEMPlan-view FE-SEM of (a) bare porous silicon surface and (b) acetylcholinesterase-immobilized porous silicon surface. [/fig]
[fig] Figure 2: Cross-sectional FE-SEM viewCross-sectional FE-SEM of (a) bare porous silicon surface and (b) acetylcholinesterase-immobilized porous silicon surface. [/fig]
[fig] Figure 3 FT: -IR spectra FT-IR spectra of bare porous silicon surface and enzyme-immobilized porous silicon surface. [/fig]
[fig] Figure 5: XPS analysis data XPS analysis for the bare porous silicon (first-row spectra) and acetylcholinesterase-immobilized porous silicon (secondand third-row spectra), along with their binding energy values -obtained for carbon, fluorine, oxygen and sodium at 1s core level, and silicon, magnesium and chlorine at 2p core level. [/fig]
[fig] Figure 8: The IC50 values The IC 50 (μg/ml) values of free enzyme and porous silicon-immobilized acetylcholinesterase after repeated use for up to three cycles. [/fig]
[fig] Figure 9: Comparison of IC 50 valuesComparison of IC 50 (μg/ml) values of free enzyme with those of porous silicon-immobilized acetylcholinesterase, with the passage of time, to assess storage stability by keeping both of them at 4 • C, followed by continuous measurement of their activity at regular 4-day intervals. [/fig]
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Neural Resources Supporting Language Production vs. Comprehension in Chronic Post-stroke Aphasia: A Meta-Analysis Using Activation Likelihood Estimates
In post-stroke aphasia, language tasks recruit a combination of residual regions within the canonical language network, as well as regions outside of it in the left and right hemispheres. However, there is a lack of consensus as to how the neural resources engaged by language production and comprehension following a left hemisphere stroke differ from one another and from controls. The present meta-analysis used activation likelihood estimates to aggregate across 44 published fMRI and PET studies to characterize the functional reorganization patterns for expressive and receptive language processes in persons with chronic post-stroke aphasia (PWA). Our results in part replicate previous meta-analyses: we find that PWA activate residual regions within the left lateralized language network, regardless of task. Our results extend this work to show differential recruitment of the left and right hemispheres during language production and comprehension in PWA. First, we find that PWA engage left perilesional regions during language comprehension, and that the extent of this activation is likely driven by stimulus type and domain-general cognitive resources needed for task completion. In contrast to comprehension, language production was associated with activation of the right frontal and temporal cortices. Further analyses linked right hemisphere regions involved in motor speech planning for language production with successful naming in PWA, while unsuccessful naming was associated with the engagement of the right inferior frontal gyrus, a region often implicated in domain-general cognitive processes. While the within-group findings indicate that the engagement of the right hemisphere during language tasks in post-stroke aphasia differs for expressive vs. receptive tasks, the overall lack of major between-group differences between PWA and controls implies that PWA rely on similar cognitive-linguistic resources for language as controls. However, more studies are needed that report coordinates for PWA and controls completing the same LaCroix et al.Language Production vs. Comprehension Meta-Analysis tasks in order for future meta-analyses to characterize how aphasia affects the neural resources engaged during language, particularly for specific tasks and as a function of behavioral performance.
In post-stroke aphasia, language tasks recruit a combination of residual regions within the canonical language network, as well as regions outside of it in the left and right hemispheres. However, there is a lack of consensus as to how the neural resources engaged by language production and comprehension following a left hemisphere stroke differ from one another and from controls. The present meta-analysis used activation likelihood estimates to aggregate across 44 published fMRI and PET studies to characterize the functional reorganization patterns for expressive and receptive language processes in persons with chronic post-stroke aphasia (PWA). Our results in part replicate previous meta-analyses: we find that PWA activate residual regions within the left lateralized language network, regardless of task. Our results extend this work to show differential recruitment of the left and right hemispheres during language production and comprehension in PWA. First, we find that PWA engage left perilesional regions during language comprehension, and that the extent of this activation is likely driven by stimulus type and domain-general cognitive resources needed for task completion. In contrast to comprehension, language production was associated with activation of the right frontal and temporal cortices. Further analyses linked right hemisphere regions involved in motor speech planning for language production with successful naming in PWA, while unsuccessful naming was associated with the engagement of the right inferior frontal gyrus, a region often implicated in domain-general cognitive processes. While the within-group findings indicate that the engagement of the right hemisphere during language tasks in post-stroke aphasia differs for expressive vs. receptive tasks, the overall lack of major between-group differences between PWA and controls implies that PWA rely on similar cognitive-linguistic resources for language as controls. However, more studies are needed that report coordinates for PWA and controls completing the same tasks in order for future meta-analyses to characterize how aphasia affects the neural resources engaged during language, particularly for specific tasks and as a function of behavioral performance.
# Introduction
Aphasia is an acquired communication disorder in which individuals have difficulty with the production and/or comprehension of language, typically following a left hemisphere stroke. Recovery from aphasia is highly variable but largely dependent on stroke specific characteristics such as lesion size and location (particularly the extent of white matter involvement;. Each of these characteristics also impacts the neural resources which support language functions poststroke. For example, large left hemisphere lesions are generally associated with increased right hemisphere activation compared to smaller, more focal left hemisphere lesions. However, focal damage can also lead to widespread disruptions of functionally and/or anatomically connected brain regions that support language; for example by disrupting critical white matter tracts. Both lesion size and location have been associated with behavioral outcomes including overall aphasia severity and specific language abilities (e.g., auditory comprehension, verbal expression;. Additionally, the exact neural resources engaged during language tasks in persons with aphasia (PWA) is known to be influenced by task demands, and dependent upon the location of the lesion that resulted in language impairments.
Language recovery in post-stroke aphasia is likely driven by a combination of functional reorganization and activation of residual regions within the canonical language network 1 . Functional reorganization involves recruitment of brain regions outside of the canonical language network to varying degrees; these can include left perilesional regions 2 , bilateral cognitive 1 For the purposes of this paper, we define the canonical language network as the left inferior frontal and precentral gyri, left insula, left temporal-parietal junction, left anterior temporal lobe (middle and inferior temporal gyri), bilateral mid superior temporal gyri, and bilateral mid-posterior middle temporal gyri. 2 "Perilesional tissue" and "perilesional activations" are routinely used in the extant literature to describe fMRI/PET activations which are adjacent to the area defined as lesioned in studies with access to a PWA's exact lesion location. However, the term perilesional is also used in meta-analyses similar to the present study (e.g.,. In these instances, perilesional refers to the residual tissue surrounding the lesion which is activated, and thus suggests that the tissue is not lesioned in the majority of participants included in the analysis. Therefore, we will networks, and/or right hemisphere homologs of the left lateralized portions of the canonical language network. While left perilesional regions have consistently been shown to support residual language functions (e.g.,, the role of the right hemisphere in language recovery is less clear, particularly since right hemisphere activation has been associated with both betterand poorer language outcomes . Small sample sizes and other inconsistencies across studies (e.g., variable tasks and number of trials, differences in thresholding and analysis approaches) likely have contributed to these seemingly mixed results, leaving many unanswered questions regarding neural reorganization and language recovery in aphasia.
Activation likelihood estimation (ALE) is a meta-analysis technique that can be used to overcome the limitations of individual experiments. ALE can identify brain regions which are consistently activated across all imaging studies of interest. The algorithm then determines whether this convergence is higher than what would be expected from a spatially random distribution. In their 2011 ALE metaanalysis of activation in PWA during any type of language task, Turkeltaub and colleagues found PWA activate a combination of spared regions within the canonical language network, left perilesional regions, and right hemisphere homologs during language. A secondary analysis comparing PWA with and without lesions to the left inferior frontal gyrus (IFG) revealed those with lesions to the left IFG activate the right IFG more during language tasks than those with a lesion sparing the left IFG. These results provide some evidence that anatomically and/or functionally homologous regions may be engaged to support language through compensatory processes in PWA. For example, the right inferior frontal gyrus may be more engaged during language tasks when the left inferior frontal gyrus is lesioned, due to shared domain-general cognitive functions.
However,meta-analysis almost exclusively included production tasks (75%, 9/12 studies; e.g., picture naming, verb generation) and did not consider possible differences in functional reorganization for production vs. comprehension. Thus, this approach likely missed important use the term perilesional to discuss the results of this paper in accordance with this definition. insights into the relative contributions of perilesional vs. right hemisphere engagement for language processing post-stroke; particularly since expressive and receptive language processes are typically supported by distinct (but interacting) neural resources that have different lateralization patterns. For instance, language production is associated with a left lateralized dorsal stream in frontal and parietal cortices, while language comprehension is generally supported by bilateral ventral streams in temporal and parietal regions. It is therefore likely that the left and right hemispheres are recruited differently for expressive and receptive language following a left hemisphere stroke. Thus, the seemingly mixed results across individual studies may actually represent separate functional reorganization patterns for productive vs. receptive language following a left hemisphere stroke. For example, damage to the left dorsal stream may result in activation of residual left hemisphere tissue during language production tasks, while other studies show recruitment of their right hemisphere homologs. For the more bilateral ventral stream, some previous work in PWA suggests increased right ventral stream activation during language comprehension tasks, whereas other studies have found activation predominately within the perilesional tissue of the left ventral stream, or even activation of domain-general regions in right frontal cortex. Thus, there is a need to conduct meta-analyses investigating language production and comprehension studies separately in order to better understand how functional reorganization may differ for expressive vs. receptive language abilities, particularly regarding perilesional vs. right hemisphere involvement.
Several more recent reviews have also taken critical steps toward summarizing the literature related to the neural resources supporting language functions following a left hemisphere stroke. Two of these papers primarily focus on providing comprehensive reviews of theories related to language recovery from post-stroke aphasia, and also how different person-and stroke-specific characteristics impact language recovery.review primarily focuses on longitudinal studies of language recovery and how neurostimulation may impact language recovery in the acute and sub-acute recovery stages. In a partial extension of this work,conducted a systematic review in which they critically appraised the strength of the literature regarding the neural resources supporting language in the chronic stage of aphasia recovery.also sought to identify brain regions that were associated with increased or decreased activation for PWA during language tasks. While their review and analysis is incredibly informative, it included multiple methods for reporting findings (i.e., coordinates, figures, text descriptions were all included), thus, the spatial resolution of their findings of activation (or deactivation) is somewhat limited. Furthermore, activation and deactivations were not discussed in terms of differences between expressive and receptive language processes. Therefore, questions remain regarding the neural resources supporting language production vs. comprehension in poststroke aphasia.
Comparing the neural resources which are engaged by language production and comprehension may be helpful in characterizing the mechanisms which drive right hemisphere engagement during language in post-stroke aphasia. A direct comparison of language production and comprehension tasks in post-stroke aphasia is warranted since several individual studies indicate that production and comprehension recruit partially distinct neural resources in PWA. This direct comparison can also help elucidate whether the right hemisphere is engaged in language in PWA due to speech and language-specific processes or more domain-general cognitive processes, which are known to recruit similar, yet distinct neural resources as language (e.g.,. For example, voxels in the right hemisphere which are activated by production and also more significantly activated for production than comprehension, may be performing similar computations as regions within the left dorsal stream, including those involved in motor speech planning for language. Alternatively, finding right hemisphere regions that are equally activated by both production and comprehension, may instead suggest that the right hemisphere is engaged by language through domain-general processes related to attention, executive control, or alertness, or perhaps through language resources shared by production and comprehension tasks (e.g., phonological processes, decision-making).
The present meta-analysis expands upon previous reviews by using activation likelihood estimation (ALE;, which provides greater spatial resolution than region of interest approaches, to separately investigate the neural resources engaged by language production vs. comprehension in persons with chronic aphasia. This direct comparison between production and comprehension is now sufficiently powereddue to the substantial increase in published fMRI/PET studies over the last 10 years. We focus on chronic aphasia here as longitudinal studies of language recovery in aphasia indicate that in the acute stage (<6 months post-stroke), there is a gradual transition from the initial recruitment of right hemisphere resources to left perilesional regions as language recovers. The present meta-analysis sought to determine what neural resources are consistently engaged across studies by language production and/or comprehension in PWA, and how this compares to controls. We additionally aimed to explore whether the neural resources engaged by language differ for specific tasks (e.g., picture naming, word generation) in PWA and controls.
# Methods
## Literature search
This meta-analysis was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) framework. A PRISMA flow diagram for the literature search is documented in. The search criteria for each database are reported in. PubMed and Google Scholar were periodically searched between August 2015 and December 2020 to locate peer-reviewed articles published prior to December 2020 that used fMRI or PET to measure brain activations to speech and language stimuli in PWA. The PubMed search yielded a total of 759 articles. For Google Scholar, we extracted the first 150 citations for each search criteria, which resulted in 1,500 citations (150 citations x 10 search criteria). The combined lists resulted in 1,944 articles after duplicates were removed. The titles and abstracts of these 1,944 articles were reviewed to determine if they met the following criteria: (1) publication was written in English, (2) adult participants with a history of stroke, and (3) use of fMRI or PET methodologies. We identified 173 citations meeting these three criteria and extracted the full-text articles for further review. Of the 173 citations meeting our first three criteria, 137 were excluded for the reasons reported inand. This left 36 articles which met our additional inclusion criteria: (4) studies reported peak
## Database
Search terms
PubMed Google Scholar speech production aphasia fMRI; speech comprehension aphasia fMRI; speech processing aphasia fMRI; expressive language aphasia fMRI; receptive language aphasia fMRI; speech production aphasia PET; speech comprehension aphasia PET; speech processing aphasia PET; expressive language aphasia PET; receptive language aphasia PET coordinates from a whole-brain analysis, (5) compared language production or comprehension tasks to a non-speech baseline (e.g., rest, noise, tones, visual stimuli), (6) in persons with chronic aphasia (>6 months post-stroke). In addition, we manually searched Wilson and Schneck's (2021) recent systematic review and meta-analysis of functional neuroimaging studies in poststroke aphasia and identified an additional eight articles meeting our inclusion criteria 3 . Our combined searches resulted in a total of 44 articles being included in the meta-analysis (Supplementary . From these 44 articles, we extracted 50 tasks, 29 production (207 PWA and 194 control subjects) and 21 comprehension tasks (228 PWA and 207 control subjects) for analysis; five articles included both production and comprehension tasks. Of these 50 tasks, 33 tasks, 16 production (164 PWA and 194 control subjects) and 17 comprehension (205 PWA and 207 control subjects), included PWA and control participants completing the same task in the same study; these tasks were analyzed separately to compare activations between the two groups. Since not all studies included control data, each section within the results is structured to first present ALEs that included all PWA data, followed by ALEs for the control participants, and lastly group comparisons using the subset of data that included both PWA and controls in the same study completing the same task. Production tasks were overt or covert and included picture naming, word generation (noun, verb), repetition (syllable, word), and spontaneous language production. Comprehension tasks could be auditory or visual and included listening to sentences, semantic decision, reading (single words, pseudowords), and rhyming tasks.
## Activation likelihood estimate meta-analysis
Activation likelihood estimates (ALEs), a coordinate-based metaanalysis method, were calculated for each single condition and contrast of interest using GingerALE Version 3.0.2. All MNI coordinates were transformed into Talairach space using GingerALE's stereotaxic coordinate converter. Talairach coordinates were then combined to create 3D maps depicting the likelihood of activation within each voxel in a MRI template. Significant areas were identified depending on whether the identified area was more likely to occur in comparison to spatially random distributions. Number of subjects in each study was input into the analysis and used to calculate the amount of blurring and uncertainty around the coordinates, which is accounted for in the FWHM of the Gaussian curve. It is also recommended that ALEs include at least 8-17 studies to avoid clusters that are primarily driven by one study. Single condition analyses were thresholded using a cluster-level correction for multiple comparisons at p = 0.05 (10,000 permutations) with a cluster forming threshold of p < 0.001. The following single condition analyses were computed separately for PWA and controls: a combined analysis for language production and comprehension, language production only, and language comprehension only. We additionally computed separate sub-analyses for PWA and controls using coordinates associated with tasks frequently used to measure language abilities in PWA: picture naming, auditory sentence listening, semantic decisions, and word generation.
Contrast analyses were computed to identify common and distinct neural resources involved in language production and comprehension within PWA and controls (within-subject analyses). Contrast analyses were also used to conduct betweengroup comparisons for the combined language production and comprehension, language production only, language comprehension only, and four task-specific analyses (picture naming, auditory sentence listening, semantic decisions, and word generation). Contrast analyses use ALE maps thresholded for multiple comparisons, therefore the contrast threshold was set to uncorrected p = 0.05 (10,000 permutations) with a 200 mm 3 minimum volume. ALE statistical maps were rendered onto the ch2.nii template brain using MRIcron.
# Results
## Neural resources engaged by language
We first computed an ALE which combined language production and comprehension in a single analysis to identify neural resources which are generally engaged by language in PWA and controls, regardless of task.
## All pwa
The combined ALE included all 50 tasks (29 production, 21 comprehension) and identified five significant clusters for PWA in bilateral fronto-temporal regions. Peak activations within the temporal lobe included the right posterior superior temporal gyrus and left posterior middle temporal gyrus. In the frontal lobe, peak activations were in the left superior and middle frontal gyri, as well as the right anterior insula (largest cluster). These peak activations additionally extended into the bilateral inferior frontal gyri, left medial frontal gyrus, left cingulate gyrus, left posterior insula, and right middle and precentral gyri (p < 0.001 corrected; Table 2;. We also conducted this analysis using the 33 tasks that included PWA and control data; the coordinates associated with this analysis for PWA are reported inand depicted in.
## Controls
The combined language production and comprehension ALE for controls included 33 tasks (16 production, 17 comprehension). This ALE identified seven significant clusters in controls. The largest cluster's peak was in the left inferior frontal gyrus (pars opercularis). Subsequent peaks were identified in the left superior frontal gyrus, left precentral gyrus, left middle temporal gyrus, left superior temporal gyrus, right superior temporal gyrus (two peaks), and left superior parietal lobule (p < 0.001 corrected; Supplementary Table 3;.
## Pwa vs. controls
The ALE conjunction analysis using the 33 tasks which included PWA and control data revealed nine significant clusters activated in PWA and controls. Six clusters were in the left hemisphere with the largest peak being in the left inferior frontal gyrus (pars triangularis). Smaller activations were found in the left superior frontal gyrus, left medial frontal gyrus, and left posterior superior temporal gyrus (three peaks). The remaining three peaks were in the right hemisphere and included the right medial frontal gyrus, right anterior insula, and right posterior middle temporal gyrus. As expected, the contrast analysis revealed.
## Neural resources engaged by language production
All PWA
The language production ALE included all 29 production tasks with aphasia data. We also conducted this analysis using the 16 production tasks that included PWA and control data; the coordinates associated with this analysis for PWA are reported inand depicted in. The ALE with all 29 production tasks identified five significant clusters in PWA. The largest peak was in the right inferior frontal gyrus (pars triangularis). Smaller peaks were identified in the right posterior superior temporal gyrus, left cingulate gyrus, left The x, y, z coordinates are in Talairach space and refer to the peak voxel activated in each cluster. All contrast ALEs are thresholded at p = 0.05 uncorrected. Asterisks indicate anatomical location of peak voxel.
middle frontal gyrus, and left posterior middle temporal gyrus. These peak activations extended into the right precentral gyrus, right superior frontal gyrus, right medial frontal gyrus, and right anterior insula (p < 0.001 corrected;;. We additionally decided to compute post-hoc ALE analyses for each aphasia subtype. However, due to a paucity of aphasia type-specific activations reported in literature, posthoc ALEs were only computed for the two most frequent aphasia types for which data was reported: Broca's aphasia (7 studies with 15 PWA) and Wernicke's aphasia (3 studies with 8 PWA). However, these ALEs should be interpreted with caution due to having fewer than eight studies included. The findings from both the Broca's and Wernicke's aphasia ALEs align with the production results from the larger PWA sample. The Broca's aphasia ALE identified four significant clusters with peaks in the right precentral gyrus (largest peak), right middle frontal gyrus, right medial frontal gyrus, and left postcentral gyrus. The Wernicke's aphasia ALE identified three significant clusters with peaks in the right inferior frontal gyrus (pars opercularis; largest), right middle temporal gyrus, and left caudate (Supplementary
## Controls
Sixteen production tasks included control data. Six clusters were significantly activated by controls during language production.
The control group's largest peak was in the left inferior frontal gyrus (pars opercularis). Additional peaks were identified in the left superior frontal gyrus, left middle temporal gyrus, left superior temporal gyrus, right insula, and right superior temporal gyrus (p < 0.001 corrected; Supplementary Table 3;.
## Pwa vs. controls
Sixteen production studies included PWA and controls completing the same task. Three significant clusters were activated by both PWA and controls during language production: peak activations were identified in the left middle frontal gyrus (largest cluster), right posterior superior temporal gyrus, and right anterior insula. PWA did not significantly activate any regions more than controls during language production. However, the analysis identified seven clusters significantly more active in controls than PWA: the largest cluster was in the left inferior frontal gyrus (pars opercularis), and smaller clusters were identified in the left superior frontal gyrus, bilateral posterior middle temporal gyri, right anterior superior temporal gyrus, and right inferior frontal gyrus (pars triangularis; p = 0.05 uncorrected;;. It was not possible to compare persons with Broca's aphasia or persons with Wernicke's aphasia to controls completing the same task as there were only two studies for each aphasia diagnosis that also included control data for the same task.
## Neural resources engaged by language comprehension
All PWA
The language comprehension ALE included all 21 comprehension tasks with aphasia data. We also conducted this analysis using the 17 comprehension tasks that included PWA and control data; the coordinates associated with this analysis for PWA are reported inand depicted in. For all 21 comprehension tasks with aphasia data, the ALE identified nine significant clusters activated by PWA. Seven clusters were located in the left hemisphere with the largest peak being in the left middle frontal gyrus. Smaller peaks were found in the left inferior frontal gyrus (pars orbitalis), left medial frontal gyrus, left precentral gyrus, left posterior superior temporal gyrus, and left middle temporal gyrus (one anterior and one posterior peak). Additional peak activations in the right hemisphere were found in the right middle frontal gyrus and right claustrum (p < 0.001 corrected; Table 2;. We were not able to conduct post-hoc analyses for the aphasia subtypes as only two comprehension studies provided aphasia type-specific activation data (one for Broca's aphasia and one for Wernicke's aphasia). However, post-hoc analyses dividing the comprehension tasks by sensory modality (visual reading vs. auditory; Supplementary Table 5) indicate that these findings are likely driven by the auditory comprehension tasks because the peak activations for the auditory comprehension tasks are quite similar to the PWA comprehension ALE, and the visual comprehension tasks did not elicit any significant activations at the threshold of p < 0.001 corrected.
## Controls
Seventeen comprehension tasks included control data. The control comprehension ALE identified three clusters to be significantly activated during comprehension tasks. The largest cluster's peak was in the left middle frontal gyrus. Two additional peaks were identified in the left inferior frontal gyrus (pars triangularis) and left middle temporal gyrus (p < 0.001 corrected; Supplementary Table 3;.
## Pwa vs. controls
Seventeen comprehension tasks included PWA and control data. Four significant clusters were identified in both PWA and controls during language comprehension: three peaks were in the left inferior frontal gyrus (pars opercularis, pars triangularis, pars orbitalis) with the largest cluster being in the left pars orbitalis. One additional peak, in the left posterior middle temporal gyrus, was also observed to be activated by both PWA and controls during language comprehension. PWA significantly activated the left precentral gyrus more so than controls, while controls significantly activated the left middle frontal gyrus more than PWA (p = 0.05 uncorrected;;.
## Neural resources engaged by language production vs. comprehension
To further explore the contributions of the right hemisphere to language in post-stroke aphasia, we contrasted activation for language production and comprehension in PWA and controls. Here we report the ALE findings for production greater than comprehension, comprehension greater than production, and their conjunction.
## All pwa
The ALE included data from all 29 production and 21 comprehension tasks (50 total) that reported aphasia data. The conjunction analysis identified seven significant clusters activated for language production and comprehension in PWA. These regions included the left middle, superior, and medial frontal gyri, left middle temporal gyrus (three clusters), and right anterior insula (largest cluster). The production greater than comprehension ALE in PWA identified the right precentral gyrus and right posterior superior temporal gyrus (largest cluster) to be more activated during language production than comprehension. For comprehension greater than production, PWA activated the left inferior frontal gyrus (pars triangularis) the most, but also the left superior frontal gyrus, left precentral gyrus, left posterior middle temporal gyrus (two peaks), left anterior superior temporal gyrus, right middle frontal gyrus, and right putamen (lentiform nucleus; p = 0.05 uncorrected; Table 2;. This same analysis was conducted using the 33 tasks (16 production, 17 comprehension) which included PWA and control data; the coordinates associated with this analysis for PWA are reported in Supplementary Table 2 and depicted in.
## Controls
The ALE included data from the 16 production and 17 comprehension tasks (33 total) that included control data. The conjunction analysis identified eight clusters activated by production and comprehension in controls. These clusters had peaks in the left inferior frontal gyrus (pars orbitalis; largest cluster), left inferior frontal gyrus (pars triangularis; three peaks), left middle frontal gyrus (two peaks), left insula, and left middle temporal gyrus. The production greater than comprehension analysis identified five significant clusters. The largest cluster's peak was in the left medial frontal gyrus. Subsequent peaks were identified in the left middle frontal gyrus, right inferior frontal gyrus (pars triangularis), and right superior temporal gyrus (two peaks). Controls significantly activated two clusters for comprehension greater than production, the largest cluster was in the left middle temporal gyrus, and the other in the left inferior frontal gyrus (pars orbitalis) (p = 0.05 uncorrected; Supplementary Table 3;.
## The effects of task type on the neural resources engaged by language 4
Picture Naming Tasks All PWA Fifteen articles included a total of 106 PWA completing a picture naming task during scanning, for which an ALE was computed. We also conducted this analysis using the seven picture naming tasks that included both PWA and control data; the coordinates associated with this analysis for PWA are reported inand depicted in. The ALE using all 15 picture naming tasks identified two significant clusters in PWA. The largest cluster's peak was in the right posterior superior temporal gyrus and extended into the right 4 ALEs that include fewer than eight tasks should be interpreted with caution due to the small sample size. However, we report the corresponding ALEs as these tasks are most commonly reported in the aphasia literature (e.g.,posterior middle temporal gyrus. The smaller cluster's peak was in the right precentral gyrus and extended into the right inferior frontal gyrus (pars triangularis; p < 0.001 corrected;;.
To explore activations related to naming task performance, we additionally conducted post-hoc ALEs to explore whether picture naming activation patterns differed for correctly and incorrectly named items. The ALE for correct responses included nine studies with 78 PWA. This ALE identified two significant clusters with peaks in the right posterior superior temporal gyrus and right precentral gyrus; this finding aligns with the results of the main picture naming ALE; p < 0.001 corrected). The ALE for incorrectly named items should be interpreted with caution as it only included four studies with 12 PWA. Nonetheless, this ALE did identify activation of voxels in the right inferior frontal gyrus (pars opercularis) and right posterior middle temporal gyrus when items were incorrectly named; p < 0.001 corrected). The x, y, z coordinates are in Talairach space and refer to the peak voxel activated in each cluster. All single condition ALEs are thresholded at p < 0.001 corrected and contrast ALEs at p = 0.05 uncorrected. Asterisks indicate anatomical location of peak voxel.
No brain regions were significantly more active during correct naming than incorrect naming, but voxels in the right inferior frontal gyrus (pars triangularis) were found to be significantly more activated during incorrect naming than correct naming; p = 0.05 uncorrected).
## Controls
Seven articles included 105 control participants completing a picture naming task during scanning. The control ALE identified one cluster in the left inferior frontal gyrus (pars triangularis; p < 0.001 corrected;;.
## Pwa vs. controls
Seven articles included 89 PWA and 105 control participants completing the same picture naming task during scanning. The conjunction ALE identified no significant clusters to be activated by both PWA and controls. PWA did not activate any brain region more than controls. However, the control greater than PWA ALE identified significant peaks in the left inferior frontal gyrus (pars orbitalis; largest cluster), left middle frontal gyrus, left medial frontal gyrus, and right superior temporal gyrus (p = 0.05 uncorrected;.
## Word generation tasks all pwa
Eight articles included word generation tasks while scanning 62 PWA. The ALE for word generation resulted in five significant clusters with the largest peak being in the right posterior superior temporal gyrus, and smaller peaks in the right anterior insula, left posterior middle temporal gyrus, left cingulate gyrus, and left middle frontal gyrus (p < 0.001 corrected;;. We also conducted this analysis using just the six word generation tasks that included PWA and control data; the coordinates associated with this analysis for PWA are reported inand depicted in Supplementary(p < 0.001 corrected). A post-hoc ALE of performance-related activations for word generation tasks could not be computed because no word generation study reported whether the coordinates were associated with correct or incorrect responses.
## Controls
Six articles included 63 control participants completing a word generation task during scanning. The control ALE identified four significant clusters with peaks in the left inferior frontal gyrus (pars triangularis; largest cluster), left superior frontal gyrus, left precentral gyrus, and right inferior frontal gyrus (pars triangularis, pars orbitalis; p < 0.001 corrected;;.
## Pwa vs. controls
Six articles included 43 PWA and 63 control participants completing the same word generation task. PWA and controls both activated the right inferior frontal gyrus (pars orbitalis) during word generation. PWA activated no brain regions more than controls. The control greater than PWA ALE identified four significant clusters including the left inferior frontal gyrus (pars triangularis; largest cluster), left middle frontal gyrus, left superior frontal gyrus, and right inferior frontal gyrus (pars triangularis; p = 0.05 uncorrected;.
## Semantic decision tasks all pwa
Eight articles included 91 PWA completing a semantic decision task during scanning. The ALE for semantic decisions identified two significant clusters; the largest cluster peaked in the left posterior middle temporal gyrus and the smaller peak was in the left inferior frontal gyrus (pars triangularis; p < 0.001 corrected;;. We also conducted this analysis using just the six semantic decision tasks that included PWA and control data; the coordinates associated with this analysis are reported inand depicted in Supplementary(p < 0.001 corrected). A post-hoc ALE of performance-related activations for semantic decision tasks could not be computed because only two studies reported whether the coordinates were associated with correct or incorrect responses.
## Controls
Six articles included 70 control participants completing a semantic decision task during scanning. The control ALE identified no significant clusters (p < 0.001 corrected; Table 5;; because of this the contrast comparing PWA and controls could not be computed.
## Auditory sentence listening tasks all pwa
In seven studies, 109 PWA listened to auditory sentences during scanning. The ALE for auditory sentence listening identified six clusters. Five clusters were located in the left hemisphere: the largest peak was in the left inferior frontal gyrus (pars orbitalis), and smaller peaks were identified in the left middle frontal, superior frontal, posterior middle temporal, and anterior superior temporal gyri. The sixth peak was in the right claustrum (p < 0.001 corrected;;. We also conducted this analysis using just the six sentence listening studies that included PWA and control data; the coordinates associated with this analysis for PWA are reported inand depicted in Supplementary(p < 0.001 corrected). A post-hoc ALE of performance-related activations for auditory sentence listening tasks could not be computed because no study FIGURE 7 | Representative sagittal slices for the ALEs associating picture naming activation with correct responses (red) and incorrect responses (blue; p < 0.001 corrected). Only the right hemisphere is depicted. Sample size denotes the number of tasks included in the ALE.
reported whether the coordinates were associated with correct or incorrect responses.
## Controls
Six articles included 91 control participants completing an auditory listening task during scanning. The control ALE identified one significant cluster in the left middle temporal gyrus (p < 0.001 corrected;;.
## Pwa vs. controls
Six articles included 108 PWA and 91 control participants completing the same auditory listening task during scanning. The ALE conjunction analysis identified PWA and controls to both activate the left middle temporal gyrus, however, neither PWA nor controls activated any brain region more than the other group (p = 0.05 uncorrected;.
# Discussion
The present meta-analysis investigated how the left and right hemispheres are engaged during language production and comprehension in persons with chronic aphasia. We further sought to characterize how the neural resources engaged by language production and comprehension in PWA compares to what is observed in control subjects, as well as how the neural resources may differ based on task type. As expected, we found that PWA activated bilateral frontal and temporal cortices during language production and comprehension, similar to controls.
However, unlike what has been previously reported in controls (e.g.,, we find PWA to demonstrate greater overall activation of the right hemisphere for language production compared to comprehension, while the left hemisphere exhibited greater activation for comprehension than production. The implications of our findings regarding the functional reorganization of language in post-stroke aphasia, and how they may be related to the recruitment of domain-general cognitive resources are discussed below.
## Neural resources engaged by language in pwa vs. controls
As expected, our combined analysis investigating the neural resources engaged by language in PWA, across all production and comprehension tasks, identified a bilateral fronto-temporal network, coinciding with dual-stream models of speech processing (e.g.,. Specifically, PWA activated the canonical language network including the left middle frontal and temporal gyri, as well as left perilesional (e.g., tissue adjacent to the canonical language network) and right hemisphere regions (e.g., right insula). These findings align with a previous meta-analysis, in which PWA activated spared regions within the left lateralized language network, plus left perilesional and right hemisphere homologs including the right inferior frontal gyrus (pars orbitalis), right middle frontal gyrus, right insula, and right middle temporal gyrus. This same analysis in our control group revealed a similar bilateral fronto-temporal network, with additional activations being observed in the left superior and inferior parietal lobes. We further found that both PWA and controls activate left frontal regions (e.g., left inferior frontal gyrus) and bilateral superior and middle temporal gyri in response to all language tasks; this activation pattern was expected based on the dual stream models. In addition, PWA and controls both engaged bilateral regions implicated in multiple cognitive-linguistic functions, including the bilateral superior and medial frontal gyri and insula, across all language tasks. While the current and previous meta-analyses cannot fully account for behavioral performance, previous work associates left hemisphere activation (see Wilson and Schneck, 2021 for a review), and to a lesser extent, activation of the right middle temporal gyrus with better language outcomes in PWA. Resting-state fMRI studies have also found increased functional connectivity within the left hemisphere to be associated with greater language abilities. These previous results, in addition to our ALE contrast indicating that only a small cluster in the right inferior frontal gyrus (pars triangularis) was more activated in PWA than controls across all language tasks, suggest that spared regions within and adjacent to the canonical language network, as well as more domain-general regions typically activated in controls are engaged during language tasks in PWA. Although lesion volume could not be accounted for in our study, previous work indicates that increased activation of the right hemisphere results in better language outcomes, particularly when large portions of the left hemisphere are lesioned. Thus, right hemisphere activations in PWA, such as in the right inferior Only studies which included both PWA and control data are included in these analyses. The x, y, z coordinates are in Talairach space and refer to the peak voxel activated in each cluster. All contrast ALEs are thresholded at p = 0.05 uncorrected. Asterisks indicate anatomical location of peak voxel.
frontal gyrus, may reflect compensation for widespread damage to portions of the left lateralized canonical language network.
## Language production engages the right hemisphere in pwa
Bilateral fronto-temporal cortices were activated by PWA during language production tasks. The ALE in control subjects indicated a similar network, but more left lateralized. Although the direct contrast of PWA vs. controls indicates that PWA did not activate any regions more than controls, the within-group ALEs depict large swaths of activation in right motor and pre-motor cortices in PWA, but much smaller right hemisphere frontal activations in controls. This discrepancy in findings could be because only half of the production studies included control data, so the between-group contrasts had reduced power compared to the within-group comparisons. The bilateral organization of the neural resources engaged by production in PWA was somewhat unexpected as it is well-established that language production is highly left lateralized (e.g.,. To gain more insight into the mechanisms that may be driving right hemisphere engagement during language production in poststroke aphasia, we examined not only the brain regions activated during language production, but also those activated more for production than comprehension (section Neural Resources Engaged by Language Production vs. Comprehension). We posit that regions significantly activated in both analyses may reflect involvement in computations specific to speech production, as opposed to domain-general functions (e.g., general alertness or effort) or language production or comprehension processes. To this end, we identified the right precentral gyrus and the right superior temporal gyrus to be significantly activated in both analyses in PWA; no left hemisphere regions were identified. It is possible that these regions are compensating for damage to regions which support motor speech planning for language, but certainly further work is needed to determine the specificity of these activations. This same analysis in controls identified a similar, but bilateral activation pattern: controls activated the right superior temporal gyrus and the right inferior frontal gyrus, but also, as expected based on previous work, left inferior, middle, and superior frontal gyri. The shared right superior temporal gyrus activation for PWA and controls is likely associated with processing one's own speechas the majority of language production tasks (22/29) required an overt response. The right frontal activations are likely tied to articulatory processes. The overlap in findings in the right hemisphere for PWA and controls indicates that PWA likely engage right hemisphere resources already involved in language production to compensate for damage to the canonical language network, and that these right hemisphere resources appear to be involved in functions specific to speech production, rather than more domain-general functions. Our ALE exploring the effect of task on language production activations suggests that not all right hemisphere activation during language production in PWA is tied to language-specific processes. Instead, the production task sub-analysis suggests that some right hemisphere activation, particularly in the frontal lobes, may depend on cognitive demands inherent to the task. For example, the lexical retrieval aspects of picture naming have been associated with the left anterior and posterior temporal cortex, and the articulatory planning and programming components with frontal regions, including the left inferior frontal gyrus; though others implicate this region with semantic processing; e.g.,.
Though underpowered with seven articles, the controls' picture naming ALE associated the left inferior frontal gyrus (pars triangularis) with picture naming. The PWA's picture naming ALE was adequately powered with 15 studies and identified a similar pattern of activation to controls, but in the right hemisphere: PWA activated the right precentral gyrus and right superior temporal gyrus. Notably, this right hemisphere activation pattern in PWA during naming tasks appears to be driven by correct responses, coinciding with previous studies indicating that increased activation of the right precentral gyrus is associated with improved naming abilities in post-stroke aphasia (e.g., .
In contrast to picture naming tasks, word generation tasks additionally require executive functions to select words that meet task constraints (e.g., naming words that begin with the letter "M" without including proper nouns or repeating the same word with a different ending;. During word generation tasks, we find PWA to activate the left middle frontal gyrus, right inferior frontal gyrus (pars triangularis), and bilateral cingulate gyri, all of which have been associated with executive functions in controls in the present study, but also in past work (e.g.,. The inherent differences in the use of executive functions for word generation vs. picture naming likely explains the more bilateral frontal activation PWA and controls demonstrate during word generation tasks compared to picture naming tasks. Thus, these task-specific ALEs during language production suggest that motor speech planning for language production is supported by right hemisphere homologs in post-stroke aphasia, while activations beyond these regions, in either hemisphere, are likely driven to some extent by more domain-general cognitive functions.
The relationship between behavioral performance and neural activation was sparsely reported in the studies that were possible to include in the present meta-analysis-see the limitations section below for more discussion. However, we did compute ALEs for task-related activations when possible, i.e., for correct and incorrect picture naming responses (although these findings should be interpreted with caution given the small sample size, i.e., nine and four studies, respectively). Correct and incorrect responses both activated right fronto-temporal regions: correct responses activated the right precentral gyrus and right superior temporal gyrus, and incorrect responses activated the right inferior frontal gyrus (pars opercularis) and right middle temporal gyrus. Contrasting correct and incorrect responses further linked the right inferior frontal gyrus (pars triangularis) with unsuccessful naming. Although activation of the right inferior frontal gyrus has generally been associated with better language abilities, particularly when the left inferior frontal gyrus is lesioned, it seems that the right pars triangularis and pars opercularis should be examined separately as previous work indicates that the right pars opercularis functions similarly to the left pars opercularis, but that the right pars triangularis functions differently than the left pars triangularis show that the right hemisphere has a multifaceted contribution to spoken language production, but that activation of the right pars triangularis appears to be particularly detrimental to language recovery in post-stroke aphasia. Future work using correlation or regression analyses are needed to better identify activations which significantly predict successful language production abilities in PWA.
## Language comprehension engages the left hemisphere in pwa
During language comprehension tasks, PWA and controls activated several left hemisphere regions including large clusters in the posterior and anterior middle and superior temporal gyri and left inferior frontal gyrus-coinciding with dual-stream models. However, findings in the right hemisphere deviated from our expectations based on previous findings of bilateral temporal activations during language comprehension: in the right hemisphere, PWA activated the right middle frontal gyrus and right claustrum but no right temporal regions, and controls did not significantly activate any right hemisphere regions. The left ventral stream activation in PWA and controls coincides with previous lesion-symptom mapping studies indicating that left temporal cortices are critical to single wordand sentence comprehension in PWA. However, dual stream models (e.g.,and fMRI/PET studies in controls (e.g.,also reliably implicate the right hemisphere in receptive language tasks, so the lack of right temporal activations identified by the comprehension ALEs was surprising. The lack of right temporal lobe findings for comprehension may be an artifact of the types of tasks used in the studies meeting our inclusion criteria, i.e., approximately one-third were sentencelevel tasks and one-third were semantic decision tasks, and both sentence-level comprehension and lexical-semantic processes are left-dominant. Thus, it is possible that the more consistent involvement of the left hemisphere for language comprehension in our study (in PWA and controls) may be driven to some extent by stimulus type. The ALEs exploring the effect of task type on the neural resources engaged by language comprehension lend some additional support to this possibility as the auditory sentence listening ALE results highly overlap with the overall language comprehension ALE, particularly in PWA.
Cognitive processes associated with task completion may explain recruitment of the left frontal cortex during language comprehension in PWA and controls. PWA and controls both activated the left inferior and middle frontal gyri during the comprehension tasks, as well as the left precentral gyrus, left middle temporal gyrus, and right claustrum. While it is unclear from the present study whether engagement of domain-general resources improves behavioral performance or not, the relationship between frontal regions and domaingeneral computations is well-established (e.g.,, as is the relationship between the claustrum and attention (e.g.,. There is also a small body of work that links the left middle temporal gyrus with working memory (e.g.,, however, it is more commonly linked to passive listening (e.g.,. Notably, out of all these shared activations, only the left precentral gyrus was more activated in PWA compared to controls. This increased activation of the left precentral gyrus, in a region of motor cortex closer to the hand than mouth area, is likely due to increased effort of PWA in their motor responses, not domain-general cognitive processes, as 18 of the 21 studies included in the comprehension ALE required participants to make an overt judgment about the stimulus, typically through a button press. The only region more activated by controls than PWA during comprehension tasks was a small cluster in the left inferior frontal gyrus (pars opercularis), an area that PWA also reliably activated. This finding suggests that non-lesioned frontal resources may play some role in language comprehension in post-stroke aphasia, but that their recruitment is not unique to PWA, and therefore may not be compensatory. However, previous work does propose that activation of domain-general resources may upregulate the remaining intact portions of the canonical language network. Thus, future studies are needed to better characterize the contributions of domain-general cognitive resources to the functional reorganization of language functions in post-stroke aphasia.
## Limitations and future directions
While the ALE methodology has several strengths, which allow it to overcome some of the limitations of individual studies (e.g., small sample size, inadequate power, differences in task), there are nonetheless limitations. First, our inclusion criteria were limited to PWA in the chronic recovery stage. Thus, our findings cannot be extended to the acute and sub-acute phases. In the acute and sub-acute phases, the current evidence indicates that language is initially supported by right hemisphere resources before gradually transitioning to left perilesional regions as language recovers. For example,found auditory comprehension performance to be positively correlated with acute right inferior frontal gyrus activation, but in the chronic stage, left hemisphere activation was associated with better comprehension. While a meta-analysis of acute and sub-acute language recovery in post-stroke aphasia is beyond the scope of this paper, it would certainly further our understanding of the trajectory of functional activation differences in post-stroke language recovery.
This meta-analysis is also limited in that it only partially accounts for the relationship between neural activation and behavioral performance. This is not due to a methodological limitation of the current study, but rather a function of what studies are available in the published literature for us to input into our meta-analysis. While there was (marginally) sufficient data to conduct post-hoc ALEs that examine activations related to correctly and incorrectly named items during picture naming, we were not able to do the same for the other production or comprehension tasks due to the multitude of ways in which performance (if reported) was described in each study (e.g., coordinates, correlations, text descriptions, figures). In total, 11/50 tasks reported coordinates associated with behavioral performance of some kind. Of these 11 studies, nine were the picture naming tasks we computed ALEs for, and two were comprehension tasks (both semantic decision tasks). To overcome this limitation, future fMRI and PET studies of language recovery in aphasia should ideally include correlations between behavioral performance and brain activations, and separately report coordinates for correct and incorrect responses. This type of consistency in the literature will strengthen our understanding of the effectiveness of compensatory strategies to post-stroke language recovery.
Our inclusion criteria were further limited to whole brain analyses and tasks that utilized a non-speech baseline task (e.g., listening to tones, rest). While each of these criteria was necessary to balance sufficient power and homogeneity, it did result in the exclusion of several articles. However, with these criteria, we still identified a sufficient number of studies to achieve appropriate power for our main analysis of interest: how the neural resources engaged by language production vs. comprehension differ in PWA. Nonetheless, we excluded 12 studies which did not report a whole-brain analysis (e.g., ROI, VOI) and 16 studies that contrasted a language task with a language baseline (e.g., spontaneous speech vs. repeated speech). We also excluded 51 studies that did not report any, or only partial, functional activation coordinates. This heterogeneity within functional neuroimaging studies of aphasia recovery suggests a need for general fMRI and PET reporting guidelines. We suggest, that at a minimum, functional imaging studies of language recovery in post-stroke aphasia should report coordinates from whole-brain analyses where the primary task of interest is compared to rest, in addition to their primary analyses of interest, with the caveats noted that rest does not adequately control for non-language related activations and may actually subtract activation related to semantic processing from semantic decision tasks. We also second recommendations made byregarding mechanisms to reduce task performance confounds and improve contrast validity. The adoption of general result reporting guidelines will reduce bias within the field as there will be greater homogeneity across studies for future meta-and megaanalyses of functional imaging studies, which will be instrumental in furthering our understanding of language recovery in poststroke aphasia.
A final limitation of the present work is our focus on studies reporting coordinates not related to treatment. Thus, from the present meta-analysis, it is difficult to infer how treatment affects the neural resources supporting language. The heterogeneity of treatment interventions and methodological approaches makes it difficult to include these studies in an ALE meta-analysis, yet there is certainly evidence that behavioral treatments can influence the neural resources that are recruited during language tasks. For example,report on a case involving a PWA with a left frontal and anterior temporal lesion. Prior to treatment, the PWA had no activation in either hemisphere during an oral reading task, however, following treatment, activation of right hemisphere homologs was observed. These results further indicate that the right hemisphere appears better able to support language production when the left language network is damaged, and that this recruitment may be driven by treatment. Other studies show similar changes in the neural resources supporting language following treatment. Since it is well-established that language can continue to improve following therapy administered in the chronic stage (e.g.,, there is a continued need to investigate how therapy impacts the neural resources supporting language recovery. This is particularly important since therapy-induced changes in the brain likely impact all neuroimaging studies of language recovery since most PWA receive some treatment in the acute and/or chronic stages that is not accounted for in many fMRI studies. Thus, continued work is needed to investigate likely differences in the brain regions supporting spontaneous language recovery and those that may be recruited secondary to treatment in order to better understand each process separately, as well as their interaction.
# Conclusion
Our exhaustive meta-analysis of language activations in PWA identified different intra-and inter-hemispheric functional organization patterns for production and comprehension. As expected, PWA activated the left middle and superior temporal gyri during language comprehension. We found additional intra-hemispheric activations in the left frontal gyri, but notably none in the right temporal lobes, which contrasted with what was expected based on predictions from the dual stream models. Further analyses suggest that the left lateralized perilesional engagement during comprehension may be driven by a combination of stimulus complexity and recruitment of domain-general cognitive resources. In contrast to the comprehension results, production was associated with activation of the right frontal and temporal cortices in PWA. We also found that activation of regions known to support domain-general resources, such as the right inferior frontal gyrus, particularly the pars triangularis, were associated with unsuccessful naming, while activation of regions involved in motor speech planning for language production, such as the right precentral gyrus, were linked to successful naming. Overall, the within-group findings indicate that the neural resources engaged by language in post-stroke aphasia, and the engagement of the right hemisphere, differ for expressive and receptive language processes, with more right hemisphere involvement seen for production than comprehension. However, the overall similarities in areas activated by PWA and controls indicates that PWA likely engage similar neural resources during language tasks as controls, rather than recruiting unique resources.
# Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. |
Multimodal Investigations of Reward Circuitry and Anhedonia in Adolescent Depression
Depression is a highly prevalent condition with devastating personal and public health consequences that often first manifests during adolescence. Though extensively studied, the pathogenesis of depression remains poorly understood, and efforts to stratify risks and identify optimal interventions have proceeded slowly. A major impediment has been the reliance on an all-or-nothing categorical diagnostic scheme based solely on whether a patient endorses an arbitrary number of common symptoms for a sufficiently long period. This approach masks the well-documented heterogeneity of depression, a disorder that is highly variable in presentation, severity, and course between individuals and is frequently comorbid with other psychiatric conditions. In this targeted review, we outline the limitations of traditional diagnosis-based research and instead advocate an alternative approach centered around symptoms as unique dimensions of clinical dysfunction that span across disorders and more closely reflect underlying neurobiological abnormalities. In particular, we highlight anhedonia-the reduced ability to anticipate and experience pleasure-as a specific, quantifiable index of reward dysfunction and an ideal candidate for dimensional investigation. Anhedonia is a core symptom of depression but also a salient feature of numerous other conditions, and its severity varies widely within clinical and even healthy populations. Similarly, reward dysfunction is a hallmark of depression but is evident across many psychiatric conditions. Reward function is especially relevant in adolescence, a period characterized by exaggerated reward-seeking behaviors and rapid maturation of neural reward circuitry. We detail extensive work by our research group and others to investigate the neural and systemic factors contributing to reward dysfunction in youth, including our cumulative findings using multiple neuroimaging and immunological measures to study depressed adolescents but also trans-diagnostic cohorts with diverse psychiatric symptoms. We describe convergent evidence that reward dysfunction: (a) predicts worse clinical outcomes, (b) is associated with functional and chemical abnormalities within and beyond the neural reward circuitry, (c) is linked to elevated peripheral levels of inflammatory biomarkers, and (d) manifests early in the course of illness. Emphasis is placed on high-resolution neuroimaging techniques, comprehensive immunological assays, and data-driven analyses to fully capture and characterize the complex, interconnected nature of these systems and their contributions to adolescent reward dysfunction.
# Introduction
Depression in children and adolescents is associated with significant distress, family burden, and functional impairment including academic failure, social dysfunction, and substance use. Critically, pediatric depression also significantly increases the risk of suicide, which ranks as the second leading cause of death between ages 10-34 years. Recent epidemiological surveys indicate that up to 20% of adolescents experience at least one depressive episode before entering adulthood. The prevalence of major depressive disorder also rises sharply during adolescence, with 12-months positivity rates increasing from 2.7% at ages 8-15 years (4) to 8.2% by ages 12-17 years. Yet, only one in three depressed adolescents currently receives disorder-specific treatment. Adolescent depression is also a strong predictor of depression in adulthood, which is ranked by the World Health Organization as the leading cause of years lived with disability.
Efforts to address this major public health challenge are complicated by the considerable variation in adolescent depression presentation, severity, and especially course. While approximately 40% of depressed youth will fully recover by adulthood (7), others will experience persistent and progressive illness, often despite apparently successful treatment. For example, Birmaher et al. followed 107 adolescents treated with psychotherapy and demonstrated that, despite high short-term remittance rates of around 80%, 38% of these patients later experienced symptom recurrence. Worse still, showed that 62% of depressed adolescents treated with psychotropic medications experienced a relapse within 78 weeks, despite ongoing treatment. Similarly, the Treatment for Adolescents with Depression Study found that 47% of remitted patients and 67% of non-responders reported depression recurrence. Yet, despite concerted and increasingly sophisticated research in recent decades, early biomarkers to reliably predict adolescent depression trajectory have remained elusive.
## Shortcomings of the categorical diagnostic system
Although progress has been slow in identifying definite biobehavioral predictors of depression outcomes in youth, mounting evidence has highlighted the prevailing categorical diagnostic framework itself as a major impediment to understanding the neurobiology of depression and other psychiatric conditions. At issue are the heterogeneous and largely arbitrary criteria used to define clusters of symptoms as particular psychiatric diagnoses in the Diagnostic and Statistical Manual of Mental Disorders (DSM)and similar diagnostic schema such as the International Statistical Classification of Diseases and Related Health Problems (ICD). As the presence and severity of specific symptoms varies widely among psychiatric patients as well as the general population (see section Shortcomings of the Categorical Diagnostic System below), the DSM relies on severity "cut-offs" and endorsement of minimum numbers of super-threshold symptoms to achieve definitive diagnoses. Though convenient for clinicians, the resulting labels obscure crucial inter-individual differences by collapsing numerous disparate clinical presentations into a single putative disorder.
In the case of major depressive disorder, current DSM-5 guidelines identify nine core symptoms, many of which encompass multiple further variations. Adults who experience at least five of these nine symptoms for a sufficient period (most of the day, nearly every day for a minimum of 2 weeks) meet criteria for a major depressive episode. Under this paradigm, a total of 227 possible symptom combinations fulfill the clinical definition of "major depressive disorder, " and it is possible for people with no overlapping symptoms to nevertheless share the same depression diagnosis. Nor is this merely a hypothetical concern; a recent survey of psychiatric symptomatology among 1,500 depressed adults identified 170 distinct symptom combinations fulfilling diagnostic criteria for major depression, with variable rates of occurrence.
The DSM-5 criteria for diagnosing major depressive disorder in children and adolescents include all the symptoms listed for adults inas well as two possible alternatives: irritable mood may be substituted for depressed mood (item 1), and failure to achieve expected weight gains may be substituted for significant weight changes or appetite disturbances (item 3). In other words, diagnostic criteria for depression are even less consistent in youth than they are in adults. Additional factors that may drive variability in pediatric depression diagnoses include: (a) potential difficulty identifying or reporting the type, intensity, or chronology of symptoms for young patients; (b) the internalized nature of several symptoms limiting insights from informants such as parents or teachers, often resulting in contradictory information; (c) symptoms of major depressive episodes potentially varying over time, settings, or developmental stages; and (d) the confounding effects of common comorbidities such as anxiety and substance use. Accordingly, reliable diagnosis of adolescent depression requires experienced clinicians to accurately discern the presence and severity of numerous symptoms, synthesize potentially conflicting reports from patients and informants, and verify or revise their conclusions through sustained longitudinal observation. These involved procedures represent a serious burden on limited community mental health resources, can delay access to potentially time-sensitive interventions, and perpetuate reductive illness classifications with limited neurobiological validity.
## Better understanding of adolescent depression via dimensional approaches
In response to these challenges, our group and a growing number of others have shifted toward a dimensional investigative approach that focuses on continuous measures of specific psychiatric symptoms rather than binary classification into broad diagnoses. The dimensional approach views psychiatric symptoms as narrowly defined clinical manifestations of underlying neurobiological abnormalities that may be shared across multiple categorically defined disorders (e.g., anhedonia is a hallmark of both depression and schizophrenia). Whereas, studies centered around categorical diagnoses treat symptom heterogeneity and comorbidity as limitations to be overcome, the dimensional framework treats inter-individual variability as a strength to be leveraged. Symptom severity can be readily quantified through standard clinician-rated and selfreported assessments, including many specifically designed for use with adolescents. Additionally, the straightforward conceptual mapping of many symptoms onto core functional domains (see section Anhedonia as a Clinical Manifestation of Reward Dysfunction) facilitates translation of insights from animal models and vice versa. Another key facet of dimensional research is recruitment of clinically diverse cohorts in order to capture the full range of symptom severity. In marked contrast to the restrictive inclusion criteria used in categorical research to limit variability, dimensional investigations aim to enroll individuals with a variety of psychiatric conditions, including comorbid and sub-threshold symptomatology, resulting in more representative samples and more generalizable findings. In recognition of these advantages, the National Institute of Mental Health (NIMH) has promulgated the Research Domain Criteria (RDoC), a set of guidelines designed to promote the adoption of dimensional investigative methods and provide a unified framework for interpreting results. The RDoC calls for researchers to focus on identifying specific, biologically meaningful indices of psychiatric phenomena in order to identify shared and distinct etiological mechanisms and inform the next generation of targeted interventions.
Here, we provide a targeted review of these developing trends in research methodology and their application to the study of reward dysfunction in adolescent depression and beyond. We discuss the emergence of the dimensional investigative approach over the past decade, as exemplified by our group's evolving corpus of work as well as a selection of landmark studies by other researchers. Particularly, we aim to demonstrate how a focus on anhedonia has enabled us to leverage multimodal neuroimaging and biological measures and identify shared mechanisms of reward dysfunction across youth with diverse clinical profiles, including depression but also anxiety and behavioral issues. Section Reward Dysfunction as a Promising Predictor of Adolescent Depression Trajectory highlights anhedonia as a direct reflection of deficits in reward processing and an ideal candidate for the dimensional approach advocated in this review. Section Neuroimaging Reward Dysfunction in Adolescent Depression discusses results from our comprehensive adolescent neuroimaging program, including task-based functional magnetic resonance imaging (fMRI) to probe reward processes directly, resting-state fMRI to study key subcortical reward circuitry as well as wholebrain networks, proton magnetic resonance spectroscopy ( 1 H MRS) to investigate potential neurochemical alterations within the reward system, and diffusion-weighted imaging (DWI) to examine white matter integrity in reward-related tracts. Section Immune Function and Inflammation in Depression and Reward Dysfunction presents findings from our studies of immunological and inflammatory biomarkers using detailed assays of peripheral cytokines and kynurenine pathway metabolites in youth. Throughout, we emphasize how integration of multiple research modalities through data-driven techniques can further enhance understanding of adolescent depression and advance the goals of identifying objective outcome predictors and targets for personalized early interventions.
## Reward dysfunction as a promising predictor of adolescent depression trajectory
Across species, adolescence is often defined as a period of development when reward-seeking behaviors are dominant, attributed to the rapid maturational changes within the corticolimbic reward circuitry . At the same time, adolescence is a critical period when symptoms of many prodromal psychiatric conditions, including depression, anxiety, substance abuse, and psychosis, first emerge . This increased incidence has been attributed to rapid changes in the brain during adolescence, involving synaptic pruning, myelination, neurotransmission, and the formation of intrinsic functional circuits found in the adult brain. As such, deviations from the typical maturation of reward circuitry may underlie the emergence of depressive symptomatology in youth regardless of categorical diagnosis. At the same time, the increased neuroplasticity of reward circuits during adolescence provides a unique developmental window for preventive therapies and early interventions targeting reward dysfunction. It is therefore crucial to identify such pathophysiological processes early, ideally before they progress to a full-blown depression episode, in order to limit the effects of chronicity and development of maladaptive behaviors.
## Anhedonia as a clinical manifestation of reward dysfunction
Anhedonia, defined as the reduced capacity to experience pleasure, is a salient clinical feature of several psychiatric disorders but plays an especially prominent role in depression, where it is considered a core diagnostic symptom (see. Several features make anhedonia an appealing target for a dimensional investigative approach. Clinically, anhedonic patients may report reduced motivation to pursue potentially pleasurable activities (anticipatory anhedonia), reduced experience of pleasure during such activities (consummatory anhedonia), or both. As such, anhedonia appears to directly reflect deficits in reward function, particularly the key processes of reward anticipation and reward attainment. In adolescents, anhedonia often presents as a prodromal symptom prior to the onset of well-defined psychopathologyand is associated with less favorable treatment responses, including persistence of depression symptoms into adulthood. Importantly, anhedonia is also linked to increased suicide risk in both pediatric and adult populations.
Anhedonia can be readily quantified using a variety of questionnaires, including the 14-item Snaith-Hamilton Pleasure Scale (SHAPS)and the more detailed Temporal Experience of Pleasure Scale (TEPS), which includes 10 items related to anticipatory anhedonia and 8 items related to consummatory anhedonia. Though originally developed with adult populations in mind, both scales have subsequently been evaluated for use in adolescent samples. A 2006 validation study of the SHAPS in a cohort of 585 high-school freshmen (age ∼14.5) reported high construct validity and supported use of the SHAPS in adolescent populations. While the TEPS has not been explicitly validated in adolescents, both anticipatory and consummatory TEPS subscales were found to have high internal consistency (ordinal α > 0.8) in a sample of 449 adolescents (agein a recent evaluation of an unrelated social anhedonia questionnaire that included the TEPS for external validation. Additionally, a series of studies in university undergraduates reported high measurement and structural invariance for the TEPS across time, gender, and culture, particularly when anticipatory and consummatory anhedonia were further subdivided into abstract and contextual components [but see].
## Anhedonia predicts worse adolescent depression outcomes
Several studies have now concluded that anhedonia accounts for a significant proportion of the variance in adolescent depression outcomes. A large cross-sectional analysis by our group examined clinical correlates of two core symptoms, anhedonia and irritability, in a cohort of 90 adolescents (ages 12-20, M ± SD 16.4 ± 2.2 years, 51 female). All participants presented with a current depressive episode of ≥6 weeks duration and moderate or greater severity, defined as a score of ≥35 on the Children's Depression Rating Scale-Revised (CDRS-R) (55), a standard clinician-rated scale to assess depression severity in youth. As illustrated in, both anhedonia and irritability exhibited broad dimensional variability with approximately normal distributions across subjects, underscoring the high degree of variation in clinical presentation even among youth diagnosed with a significant current depressive episode.
However, we found that only anhedonia, not irritability, was associated with poorer clinical outcomes, including greater overall illness severity, longer episode duration, and increased suicidality, as well as a larger number of depressive episodes.
Moreover, we recently reported initial results from a followup study examining longitudinal outcomes after ∼2 years in a mixed group of 29 adolescents with mood and anxiety symptoms as well as 14 healthy controls. In line with our earlier results, we found that only anhedonia, but not irritability, at baseline was associated with more severe clinical prognosis, including future depression severity, anhedonia severity, and suicidality. This study also illustrates a key principle of our dimensional analysis approach, namely that symptom severity lies along a continuum from typical (low severity) to potentially pathological (high severity) even among nominally healthy individuals, denoted by circles in. Additional findings from this study relating fMRI measures of reward function to future clinical outcomes are detailed in section Reward Function Predicts Adolescent Depression Outcomes below.
Several independent lines of evidence further support our conclusion that anhedonia, as an index of reward dysfunction, plays a uniquely predictive role in the trajectory of depression. A multivariate analysis of 334 depressed youth in the Treatment of Resistant Depression in Adolescents (TORDIA) study found that, out of five symptom dimensions, only anhedonia predicted longer remission time and fewer depression-free days over 24 weeks; findings remained significant even when accounting for overall depression severity. A study of 75 depressed adult psychiatric inpatients similarly found that anhedonia Frontiers in Psychiatry | www.frontiersin.org predicted treatment non-response among depressed inpatients, while our study of 135 adults hospitalized for acute suicide attempt or high suicide risk documented that anhedonia as well as entrapment were independently associated with suicidality scores.
Taken together, these findings support the notion that anhedonia reflects key aspects of reward dysfunction across ages and may map particularly well onto underlying neural processes that contribute to the development and maintenance of depression in youth.
## Linking reward function to reward circuitry
Reward function is inherently complex, involving multiple discrete processes and phases, including reward anticipation, attainment, and valuation (60, 61). Neuroanatomically, however, many reward processes engage a common circuit linking regions of the dopaminergic midbrain, basal ganglia, and frontal cortex. In animals ranging from rodents to non-human primates, the processes of reward anticipation and, particularly for novel stimuli, reward attainment rapidly engage midbrain populations of dopaminergic neurons in the ventral tegmental area and substantia nigra (62-65). These reward-encoding signals propagate via ascending projections to the ventral striatum (mesolimbic pathway) and dorsal striatum (nigrostriatal pathway) en route to cortical targets, including the ventromedial prefrontal cortex (PFC), subgenual and pregenual anterior cingulate cortex (ACC), and supplementary motor area. As will be discussed further in section Neuroimaging Reward Dysfunction in Adolescent Depression, a substantial body of literature now ties the clinical phenomenology of anhedonia to disturbances in this core reward circuit. At the same time, neuroimaging studies of anhedonia and related constructs frequently report abnormalities beyond the canonical reward circuitry, highlighting the need for an integrative, empirically driven approach to fully characterize the interdependent neural processes supporting reward function.
## Neuroimaging reward dysfunction in adolescent depression
Non-invasively studying the human brain in vivo is a major technical challenge. Nevertheless, a robust research community has emerged around the study of adolescent depression using a variety of increasingly sophisticated neuroimaging techniques. Since no single technique can adequately capture all aspects of neural anatomy and function, combining evidence from complementary modalities is crucial for verifying and clarifying results. For example, several longitudinal studies using functional magnetic resonance imaging (fMRI) have found that altered neural activation profiles during reward anticipation and attainment in non-depressed adolescents predicted the onset of future depression. While these fMRI studies suggest that detectable changes in reward-related neural activity precede the onset of adolescent depression symptoms, this conclusion is strengthened considerably by corroborating evidence from electroencephalography (EEG), a completely different neuroimaging technique. In an EEG study of 444 healthy female adolescents (age 13-15), blunted reward positivity and reduced delta band amplitude during reward attainment predicted the onset of depressive disorders and greater depression severity at 18-months follow-up, even when controlling for baseline depressive symptoms, lifetime psychiatric history, and parental psychiatric history. By bringing together multimodal results from both fMRI and EEG studies, a recent meta-analysis was able to identify a set of reward system alterations that consistently preceded the onset of adolescent depression symptomatology. However, studies employing multiple neuroimaging techniques within the same cohort are rare.
In this section, we review findings in adolescent depression based on a variety of imaging methods, focusing particularly on advanced MRI techniques employed by our group to examine the neural underpinnings of reward dysfunction. These include task-based and resting-state fMRI to assay neural activity across the brain, proton magnetic resonance spectroscopy ( 1 H MRS) to detect neurochemical concentrations in targeted regions, and diffusion-weighted imaging (DWI) to model structural connectivity in white matter. As we seek to demonstrate, combining data from multiple neuroimaging modalities, as well as other biological measures (see section Immune Function and Inflammation in Depression and Reward Dysfunction), within a single cohort substantially enhances our ability to make inferences about brain function and its role in the emergence of psychiatric illness.
## High-resolution neuroimaging and high-fidelity analysis approach
Functional MRI is a powerful, though indirect, method to measure neural activity. Through a phenomenon known as neurovascular coupling, localized increases in neural activity trigger increased perfusion with oxygen-rich blood to meet metabolic demands. Thanks to the magnetic properties of hemoglobin, these shifts in blood flow give rise to an endogenous contrast mechanism known as the blood-oxygenation-leveldependent (BOLD) signal, which can be detected and tracked over time using rapid, inhomogeneity-sensitive fMRI sequences. The combination of relatively high spatial fidelity, whole-brain coverage, and increasingly rapid sampling rates through the use of accelerated imaging techniques make fMRI an attractive method for studying human cognition in healthy and diseased states. Over the past two decades, the body of basic and clinical neuroscience literature utilizing fMRI has grown exponentially.
Despite these efforts, considerable uncertainty remains regarding in vivo reward function, particularly in youth. Much of the difficulty in mapping reward circuitry can be attributed to limitations in MRI methodology. This is particularly true for subcortical structures such as the ventral tegmental area (source of dopaminergic reward signals) and the habenula (inhibits dopamine signals in response to negative outcomes). These and other key reward-related structures have limited anatomical tissue contrast and have proven difficult to reliably segment, with the most accepted methods rely on heuristic anatomical landmarks. These issues are compounded in fMRI studies, where the combination of low spatial resolution, signal contamination from poorly delineated tissue boundaries, and high physiological noise make subcortical reward structures especially challenging to study.
Our laboratory has therefore pursued a high-resolution imaging acquisition and processing approach modeled after the Human Connectome Project (HCP)and designed to systematically address these limitations. As early adopters of multiband acceleration, we have now collected hundreds of whole-brain fMRI datasets at 3T with high spatial resolution (2.3 mm isotropic vs. ≥3 mm isotropic for traditional sequences) and sampling rates (repetition time = 1 s vs. ≥ 2 s for traditional sequences). These high-resolution fMRI sequences allow for substantially improved signal localization and enable us to fully capitalize on advanced tools such as the HCP Pipelines to perform sophisticated preprocessing procedures, ICA-FIX to automatically classify and remove structured noise (100-102), and multimodal surface matching (MSMAll) to robustly align corresponding cortical areas and maximize spatial fidelity (103-106). Wherever possible, our analyses employ thresholdfree cluster enhancement (TFCE) for adaptive, data-driven identification of meaningful fMRI effects (107) in conjunction with non-parametric permutation-based statistics for precise control of family-wise error (FWE) rateswith minimal assumptions about data distribution, in line with current best practices. Finally, we ascribe to a transparent "open science" philosophy and have endeavored to make study preprints, final results datasets, and analysis code freely and readily available to the community. While our neuroimaging procedures have evolved over the past years and will continue to do so as improved techniques become available, we emphasize the importance of a holistic approach to high-fidelity neuroimaging at each stage of acquisition and processing to study neural function as accurately and reproducibly as possible.
## Task-based functional magnetic resonance imaging
A major use-case for fMRI is to examine neural activation during specific activities or mental processes. In task-based fMRI, participants can complete a wide range of behavioral task paradigms while being scanned to capture corresponding neural activity, including reward processing. One of the earliest and most prominent researchers to use fMRI tasks to specifically examine adolescent reward processing and associated deficits was Erika Forbes. Using a probabilistic reward task, Forbes and colleagues documented reduced activation in the bilateral striatum and portions of the orbitofrontal cortex (OFC), key reward processing regions, during reward anticipation and reward attainment in 14 adolescents/pre-adolescents (ages 8-17) with major depressive disorder vs. 17 age-matched healthy controls. The finding of reduced striatal reward response was corroborated in a follow-up study with an expanded sample of 15 depressed adolescents/pre-adolescents and 28 healthy controls, which also reported that individuals with less caudate activation had lower overall positive affect, assessed by telephone outside the laboratory setting. In subsequent research, Forbes et al. found that the degree of striatal activation to rewards positively correlated with positive affect while medial PFC activation positively correlated with depression symptomatology in a large sample of young people with no history of psychiatric illness (N = 77, ages 11-13). Crucially, they also found evidence that striatal reward reactivity predicted subsequent improvement in mood and anxiety symptoms following 8 weeks of open-label treatment (behavioral or behavioral + antidepressant therapy) in a sample of adolescents (ages 10-16) with major depression. As detailed below, our group has likewise made extensive use of task-based fMRI to probe key aspects of reward function and their relevance to adolescent mood and anxiety disorders.
## The reward flanker task
As discussed in section Reward Dysfunction as a Promising Predictor of Adolescent Depression Trajectory, reward dysfunction and anhedonia in adolescent depression entail deficits in multiple reward processes. For example, some patients may lack the motivation to pursue potentially pleasurable activities (i.e., deficits in reward anticipation), while others may seek out such activities but find that they experience little or no pleasure as a result (i.e., deficits in reward attainment). To better delineate neural activity during these distinct phases of reward processing, our group has developed the Reward Flanker Task (RFT). Using the RFT, we are able to directly examine neural responses to reward anticipation (monetary cues) and reward attainment (monetary outcomes), but also a number of important additional constructs related to outcome uncertainty and reward prediction errors.
Implementation of the RFT is described extensively in our previous publication. Briefly, the RFT combines elements of the earlier Incentive Flankerand Monetary Incentive Delay (117) tasks, both of which are widely used in psychological as well as neuroimaging research to examine reward responses. Trials in the RFT consist of: 1) Cue indicating high (50%), low (10%), no (0%), or unknown (?) trial type for 4-6 s 2) Flanker stimulus (short string with a unique letter, e.g., CCHCC) for 300 ms 3) Calibrated response window with a blank display for ≤1,700 ms 4) Feedback indicating high (50%), low (10%), or no (0%) reward earned for 2 s 5) Inter-trial-interval with a blank display for 4-6 s. Unknown cues yield high, low, and no reward outcomes equally. During the response window, participants attempt to identify the unique target letter from the flanker stimulus using button presses. To keep the task challenging and ensure that some errors are made, the amount of time a participant is allotted to make a response is set at 0.8-1.5 times their mean reaction time during a pre-scan practice session. Correct responses to flanker stimuli result in winning a high, low, or no reward outcome based on the trial type. A total of 120 trials (30 of each type) are presented in pseudo-random order across four 4-min fMRI runs.
Compared to other reward paradigms such as the Incentive Flanker and Monetary Incentive Delay, the RFT has several novel features. First, cues used to signal the reward value of each trial are presented with varying durations and for sufficient lengths to allow for the separation of brain activity during reward expectancy from attainment despite the slow hemodynamic response. Second, the task uses three levels of reward value (high, low, and no reward) to examine brain activity in response to increasing reward value for both expectancy and attainment. Third, the task includes unknown reward cues, allow us to dissociate responses to certain vs. uncertain reward outcomes. In our studies, the contrast of reward attainment following uncertain vs. certain cues is referred to as positive prediction error. Finally, as cues elicit reward anticipation such that a specific, effortful response is required to obtain the reward, rather than passive anticipation of a definite reward, the RFT is particularly well-suited to probe motivational deficits in anhedonia.
Following the development of the RFT, we conducted a pilot study to examine brain activation during distinct reward processes in a sample of 22 psychotropic medication-free adolescents, including 16 with primarily mood and anxiety symptoms and 6 healthy controls. Results from our wholebrain analyses, controlled for false discovery rate (FDR), are displayed in(all p TFCE−FDR < 0.05). We found that reward anticipation activated a large bilateral network including default-mode, salience, and limbic regions important for reward appraisal and conflict resolution. Reward attainment engaged a more restricted network including the hippocampus, medial temporal lobe, dorsal caudate, and occipital areas related to memory and emotion processing but notably not the ventral striatum. Additionally, we found that positive prediction errors yielded widespread cortical activation in many of the same regions identified in during reward anticipation/attainment, including the ventral striatum. Exploratory analyses at a relaxed threshold (uncorrected p < 0.001) further identified several positive correlations between anhedonia severity and RFT activation, specifically in the right angular gyrus during reward anticipation and in the left precuneus during positive prediction errors. These results support the ability to differentiate reward processes and identify links to clinical reward dysfunction in adolescents using the RFT. A follow-up RFT study in an expanded adolescent cohort is currently in preparation, with findings largely corroborating our initial pilot results; additionally, three reward networks derived from this followup study served as the basis for our recent graph theory analysis using resting-state fMRI, described in section Using Graph Theory to Bridge Resting-State and Task-Based fMRI Studies below.
## Reward function predicts adolescent depression outcomes
As described previously in section Task-Based Functional Magnetic Resonance Imaging, we recently published findings from a longitudinal study of 43 adolescents indicating that anhedonia, but not irritability, predicted depression severity and suicidal ideation at ∼24 months (56), corroborating our earlier cross-sectional results. In additional to clinical associations, we examined whether measures of neural reward function at baseline would predict future clinical outcomes in a subset of 22 depressed and 10 healthy adolescents with RFT fMRI data. In the full sample, we found that activation during positive prediction error in the dorsal ACC, mid-cingulate, anterior insula, operculum, and motor cortex significantly (all p TFCE−FWE < 0.05) predicted future anhedonia severity. Activation during positive prediction error in the left angular gyrus further predicted future depression severity in the full sample. In the depressed-only cohort, analyses indicated that positive predication error activation in the dorsal ACC/mid-cingulate again predicted future anhedonia, while activation in the medial precuneus/posterior cingulate during reward attainment predicted future suicidality (not assessed in controls due to minimal range). Thus, altered activation during reward processing, specifically in cingulate areas related to evaluating reward outcomes as well as regions important to pain and salience processing, was linked to future reward deficits, indexed by anhedonia.
## Altered emotion processing and self perception in adolescent depression
While the majority of our work has used monetary rewards to probe reward circuitry, as in the RFT, happy facial expressions are also commonly used as rewarding social stimulus in studies of adolescent reward processing. Studies have also consistently reported emotion processing deficits in people with depression, who are less accurate and more negative in their interpretation of facial affect than healthy individuals. This pattern is reflected in neuroimaging data, with depressed adults showing stronger responses to sad/angry faces and weaker activation to happy faces in the striatum and amygdala relative to healthy controls. Notably, a study of children at high risk for developing depression (N = 38) yielded largely consistent findings of amygdala hyperactivation to fearful faces and ACC hypoactivation to happy faces relative to low-risk controls (N = 23) (120). Using a similar approach, we sought to examine neural activation during emotional valence judgements in depressed adolescents. A total of 19 psychotropic-medication-free depressed and 18 healthy control adolescents completed a 3T fMRI task where they were shown faces with neutral, happy, sad, or fearful expressions and were asked to rate the sadness of the expressions. Analyses indicated that depressed adolescents relative to controls had reduced activation in the left superior temporal gyrus while judging sad and neutral faces. Within the depressed group, greater illness severity was associated with stronger neural activation in major reward and aversion processing regions while judging sad faces (e.g., putamen, ventromedial PFC, amygdala, anterior insula) and fearful faces (e.g., dorsal ACC, anterior insula). Anhedonia scores in the depressed group showed similar positive correlations with activation while judging sad faces (e.g., putamen, caudate, and amygdala) but were uniquely anticorrelated with activation while judging happy faces (e.g., dorsal ACC, anterior insula). While evidence supports a negative bias when evaluating the emotional valence of others, excessively negative selfperception is even more characteristic of depression. We therefore conducted a separate 3T fMRI study of selfreferential word processing in a group of 23 depressed and 18 healthy control adolescents. Subjects were presented with words describing positive and negative personal traits and were asked to either judge whether the trait: (1) applied to themselves, or (2) was a good trait to have in general. Adolescents with depression endorsed more negative traits but exhibited stronger activation in the posterior cingulate while making positive self-judgements than their healthy counterparts. Within the depressed group, positive self-perception was negatively correlated with multiple dimensional measures of symptom severity, including self-and clinician-rated depression severity, social and general anxiety, irritability, and anhedonia. Additionally, a psychophysiological interaction analysis of task-evoked functional connectivity among four cortical midline regions of interest revealed diminished connectivity of the dorsomedial PFC-the same region identified in our earlier study of striatal resting-state connectivity in adolescent depression (section Resting-State Functional Connectivity of the Striatum in Adolescent Depression)-during self-judgement.
Naturally, these studies should be interpreted with caution given their small sample sizes. Nevertheless, they offer insight into the perception of self and others in adolescent depression, suggesting that neural reward circuitry and clinical reward deficits can influence seemingly distinct cognitive processes related to internally and externally directed judgements. Interestingly, a recent study in a larger sample of depressed and healthy adolescents (N = 120) combined these approached by having subjects identify their own emotional expressions from photos taken 1-2 weeks prior to the task. Results indicated that, unlike when processing the emotional expressions of others, recognition accuracy among depressed adolescents was highest for happy faces. Analysis of fMRI data further revealed a that elevated amygdala connectivity with regions of the ACC and PFC during the task was associated with increased suicidality and was highest in youth with a history of suicide attempts.
## Resting-state functional magnetic resonance imaging
While task-based fMRI seeks to identify brain activity in response to external stimuli, resting-state fMRI is a complementary approach that provides unique information on brain function, particularly its organization into large-scale networks. In restingstate fMRI, BOLD signals are measured in the absence of external task stimuli, with subjects free to let their minds wander. The most common class of resting-state fMRI analyses focus on resting-state functional connectivity (RSFC), which measures correlations in low-frequency BOLD fluctuations (typically ∼0.1-0.01 Hz) between difference regions. Networks identified using RSFC can reflect not only known anatomical connectivity between regions but also areas with no direct anatomical links, implying that correlated activity is instead driven by shared function. Several features make resting-state fMRI well-suited for research: findings can be compared readily across studies and populations, the low cognitive demand allows for participation of individuals who may not be able to complete complex tasks, and many wellcharacterized networks can be readily identified even at the single-subject level using hypothesis-free techniques such as independent components analysis.
Numerous studies have now documented resting-state network alterations in adolescent depression and related conditions. Brain structures most frequently implicated in these studies include the anterior cingulate cortex, insula, and especially the default-mode network, which encompasses regions of the medial PFC, posterior cingulate, lateral temporal lobes, and hippocampus that are suppressed during fMRI tasks. Dimensional studies further indicate that changes in resting-state network properties are correlated with adolescent depression severity. For example, Roselinde Kaiser, Diego Pizzagalli, and colleagues recently found that co-activation of the default-mode network and the (typically distinct) fronto-insular network during rest was associated with more severe mood symptoms in a sample of 45 depressed adolescents (ages 13-19) (132); a similar pattern of insula/default-mode hyperconnectivity during an emotional memory task was related mood status in over the subsequent 2 weeks. Furthermore, evidence from children/early adolescents indicates that RSFC abnormalities arise very early in the course of illness and may precede the emergence of overt clinical symptoms. Susan Whitfield-Gabrieli's group, for instance, reported default-mode network hyperconnectivity with the subgenual ACC and OFC in 27 children (ages 8-14) with a parental history of major depression vs. 16 age-matched controls with no parental depression history. A subsequent study based on a community sample of 54 children assessed longitudinally at ages seven and 11 found that reduced RSFC between the subgenual ACC and dorsolateral PFC at baseline was associated with the development of mood and anxiety symptoms at 4-years follow-up. Moreover, this connectivity pattern predicted future depression symptomatology better than a standard parent-rated clinical scale. Increased RSFC between the medial and dorsolateral PFC was separately associated with future attentional symptoms.
As detailed below, our group has utilized resting-state fMRI extensively to examine reward circuitry in adolescent depression, both through seed-based RSFC analyses of a priori reward areas (sections Resting-State Functional Connectivity of the Striatum in Adolescent Depression and Habenula Functional Connectivity and Subclinical Depression in Young Adults) and data-driven analyses using graph theory (section Using Graph Theory to Bridge Resting-State and Task-Based fMRI Studies).
## Resting-state functional connectivity of the striatum in adolescent depression
We conducted an early study of striatum-based RSFC in a cohort of 42 psychotropic-medication-free adolescents, 21 with a current major depressive episode and 21 healthy controls with no history of psychiatric illness. Mean timeseries were extracted using spherical seeds (∼4 mm radius) from six striatal regions per hemisphere (dorsal caudate, ventral caudate, dorsal caudal putamen, dorsal rostral putamen, ventral rostral putamen, nucleus accumbens) created as part of an earlier study. Secondary analyses examined RSFC from three template seeds per hemisphere corresponding to the major divisions of the striatum (caudate, putamen, nucleus accumbens), as defined by the Harvard-Oxford Structural Atlas. At the group level, depressed adolescents manifested increased RSFC between all striatal seeds bilaterally and the dorsomedial PFC as well as between the right ventral caudate and the subgenual ACC compared to controls. Controls exhibited stronger RSFC between striatal seeds and extrastriate visual areas in the occipital lobe, between the left dorsal caudate and the superior temporal lobe, and between the ventral caudate and postcentral gyrus. Within the depressed group, overall depression severity was associated with RSFC between the striatum and midline structures including the precuneus, posterior cingulate cortex, and dorsomedial PFC. However, anhedonia severity was associated with distinct striatal RSFC patterns involving the pregenual ACC, subgenual ACC, supplementary motor area, and supramarginal gyrus, even after controlling for depression severity. Symptom correlation results are presented in. Although the sample size was modest, these findings provided early evidence for anhedonia as a unique clinical variable capturing facets of reward dysfunction beyond depression severity alone.
## Habenula functional connectivity and subclinical depression in young adults
While the dopaminergic reward circuit described in section Linking Reward Function to Reward Circuitry is strongly engaged by positive prediction errors (i.e., receipt of unexpected rewards), reward-related dopamine signaling is inhibited by negative prediction errors (i.e., non-receipt of expected rewards). Preclinical work over the past decade indicates that this pattern is driven by a highly conserved circuit centering on the lateral habenula, a small epithalamic nucleus that sends extensive regulatory projections to the dopaminergic midbrain and other monoamine systems . The habenula responds vigorously to aversive stimulibut is inhibited by rewards. Further, excitatory habenula stimulation induces depression-and anhedonia-like behaviors in animal models similar to those produced by lesions of the ventral tegmental area and nucleus accumbens. As such, the habenula is considered a highly promising candidate structure in the study of clinical reward dysfunction. However, the small size (∼30 mm 3 per hemisphere) and limited anatomical contrast of the habenula with nearby thalamic tissue have severely limited in vivo research.
In response to these challenges, our team has worked extensively to refine habenula imaging methodology, codeveloping an automated segmentation scheme based on 3T anatomical MRI. The technique entails taking ratios of T1-weighted (T1w) to T2-weighted (T2w) images to selectively enhance in vivo myelin contrast. As the habenula contains more myelin than the surrounding thalamus, this method significantly improves tissue contrast relative to T1w or T2w MRI alone. A region-growing algorithm then automatically identifies and assigns probabilistic weights to voxels containing habenula tissue.
Using this technique, we performed the first whole-brain study of human habenula RSFC using high-resolution fMRI data from 50 young adults (ages in the HCP, 25 with low and 25 with high subclinical depression scores. In line with animal literature, we found extensive RSFC with reward-and aversionrelated areas in the full sample, including the ventral tegmental area, nucleus accumbens, dorsal ACC, and periaqueductal gray (p TFCE−FWE < 0.05, controlled for subclinical depression group). At a relaxed exploratory threshold (uncorrected p < 0.001, k ≥ 10), group contrasts further revealed reduced habenula RSFC with the mid-cingulate, right amygdala, and right anterior insula, regions implicated in mood disorders and aversion processing, in subjects with high subclinical depression scores.
Building on this work, we developed an optimized method for downsampling anatomical-resolution (∼0.8 mm isotropic) habenula segmentations to fMRI resolution (∼2 mm isotropic) for use as RSFC seeds. These individual-specific seeds account for tissue probability weights, partial volume effects, and template normalization, significantly improving BOLD sensitivity compared to other seeding methods. In a follow-up study also incorporating neuroanatomically accurate surface analysis, we created the most detailed maps to date of habenula RSFC in a representative sample of 68 healthy young adults from the HCP. Findings corroborated our initial results and further revealed positive habenula RSFC with the insula, pregenual ACC, and striatum as well as a pattern of weakly negative RSFC throughout task-negative regions of the default-mode network. A forthcoming manuscript will detail findings using the same . Plots show seed-to-cluster RSFC values (mean ± 95% CI) for three clusters (mid-cingulate, amygdala, anterior insula) where habenula RSFC differed between groups (exploratory p uncorrected < 0.001, k ≥ 10). No group differences in RSFC were detected for adjacent seeds in the dorsomedial or centromedian thalamus with the same clusters. Excerpted from. sophisticated mapping approach in a large, clinically diverse adolescent sample.
## Using graph theory to bridge resting-state and task-based fmri studies
As described in section The Reward Flanker Task, our task-based fMRI findings using the RFT have identified distinct activation networks engaged by the processes of reward anticipation, reward attainment, and positive prediction error. Building on this work, we conducted a data-driven graph theory analysis to identify resting-state network features associated with psychiatric symptomatology in a sample of 68 clinical adolescents with predominantly mood and anxiety symptoms as well as 19 healthy adolescent controls. This analysis capitalized on the Cole-Anticevic Brain-wide Network Partition (157), a recent whole-brain extension of the landmark HCP cortical parcellation (103) that identified 358 additional subcortical parcels on the basis of RSFC patterns. By applying the Cole-Anticevic atlas to task activation maps derived from our updated RFT analysis, we generated network templates corresponding to Reward Anticipation (114 nodes), Reward Attainment (103 nodes), and Reward Prediction Error (117 nodes), reprinted as .
Graph theoretical analyses were performed using the Brain Connectivity toolboxto derive three descriptive metrics within the three RFT-derived networks as well as a Whole Brain network (750 nodes) derived from the Cole-Anticevic atlas. Strength Centrality represented the sum of edge weights per node and captured the overall influence of each node within the network. Eigenvector Centrality represented the largest magnitude eigenvector per node and captured the influence of each node over highly influential nodes within the network. Local Efficiency represented the inverse shortest path length between each node and its neighborhood and captured how readily information could propagate to other nodes in the network.
As detailed in our manuscript now in press (156), dimensional symptom analyses revealed that depression severity significantly correlated with Strength Centrality in two ventral striatum nodes as well as with Strength Centrality and Local Efficiency measures in the right inferior pallidum within the Reward Attainment network. Anticipatory anhedonia, meanwhile, correlated with Local Efficiency in numerous reward-related nodes across networks, including the subgenual ACC within Whole Brain and Reward Attainment networks, dorsal ACC within Reward Attainment and Reward Prediction Error networks, OFC within the Reward Attainment network, ventral striatum within the Reward Prediction Error network, and the dorsal caudate within Reward Anticipation and Reward Prediction Error networks. Notably, significant correlations were not identified with consummatory anhedonia and were much more limited for total anhedonia (i.e., anticipatory + consummatory scores), highlighting the need for detailed measures of reward dysfunction in dimensional research. Similarly, no significant correlations were identified with anxiety severity, and no significant group differences were detected between clinical and healthy control groups, again underscoring the central role of reward dysfunction in adolescent depression symptomatology.
## Neurochemical abnormalities revealed by magnetic resonance spectroscopy
Proton magnetic resonance spectroscopy ( 1 H MRS) is a nuclear magnetic resonance technique closely related to MRI that can distinguish protons from various tissue chemicals, allowing for in vivo measurement of human biochemistry. Localized 1 H MRS has gained popularity in psychiatric research due to its ability to directly assess the concentrations of many important neurochemical metabolites, including glutamate, glutamine, γ-aminobutyric acid (GABA), N-acetylaspartate, and myoinositol. Of particular interest are GABA and glutamate, respectively, the most common inhibitory and excitatory neurotransmitters in the brain. Interest in the role of excitatory and inhibitory neurotransmission in depression has been stimulated by the discovery that ketamine, a glutamatergic NMDA receptor antagonist, exhibits rapid antidepressant activity through an apparently distinct mechanism from classic serotonergic antidepressant drugs. A recent metaanalysis of 49 1 H MRS glutamate studies found the strongest support for decreased glutamate levels in the medial PFC across 1,180 depressed vs. 1,066 healthy adults, with limited evidence supporting a similar pattern of glutamate dysregulation in depressed youth. Studies in animal models, meanwhile, suggest that GABA is involved in regulation of striatal dopamine release, while both 1 H MRS and lumbar puncture studies in adults have specifically reported GABA reductions in adults with melancholic depression, a severe subtype of the disorder characterized by high anhedonia levels. Using the dimensional approach advocated throughout this review, our laboratory sought to understand the possible link between GABA and reward dysfunction in youth. As spatial coverage using 1 H MRS is limited, our investigations have focused on the ACC and the striatum, key reward-related regions frequently implicated in our studies of adolescent depression using other modalities.
Our first 1 H MRS study utilized a case-control, cross-sectional design to examine GABA levels in a sample of 20 depressed and 21 healthy control adolescents. Analyses revealed that adolescents with depression had significantly lower ACC GABA than age-matched controls (p < 0.003). When we split depressed adolescents into subgroups based on the presence of clinically significant anhedonia, only anhedonic subjects were found to have significantly lower GABA levels than controls. ACC GABA was also found to be negatively correlated with anhedonia severity in the depressed cohort (r = −0.50, p < 0.03) and the whole sample (r = −0.54, p < 0.001). These findings were the first evidence for an important association between ACC GABA and anhedonia severity in adolescent depression.
Our initial ACC GABA results were subsequently corroborated by a follow-up 1 H MRS study in an expanded cohort, now including 44 depressed and 36 healthy control adolescents. Analyses were performed using analysis of covariance (ANCOVA) to control for age, sex, ethnicity, and cerebrospinal fluid content in the ACC voxel. Again, we found that ACC GABA levels were significantly lower in the depressed group (p = 0.003) relative to controls. When depressed FIGURE 8 | Parcels derived from the Cole-Anticevic Brain-wide Network Partition (black lines) most strongly activated during the RFT by reward anticipation (green), reward attainment (blue), reward prediction errors (red), or some combination thereof (mixed colors) and used as reward networks in our graph theory study. Reprinted from. subjects were classified based on anhedonia, analyses revealed a significant difference in ACC GABA levels across anhedonic depressed, non-anhedonic depressed, and healthy control adolescents (p = 0.003); as shown in, pairwise follow-up tests indicating that GABA levels were decreased only in the anhedonic depressed subgroup relative to controls (p = 0.002). Similar to our earlier results, anhedonia severity was inversely correlated with ACC GABA levels in the depressed group, even when controlling for overall depression severity (r = −0.33, p = 0.03). These confirmatory findings support our conclusion that deficits in ACC GABA may serve as a biomarker for reward dysfunction in depressed adolescents.
Interestingly, we found the opposite relationship with striatal GABA levels in a related 1 H MRS study of 20 depressed and 16 healthy control adolescents. In this case, our analyses revealed depressed youth to have higher GABA levels in the striatum compared to controls. Moreover, combining 1 H MRS measures from both regions indicated that higher striatal GABA was associated with lower ACC GABA in adolescents with depression. While striatal GABA was not found to be associated with symptom severity, including anhedonia, the inverse relationship between ACC and striatal GABA levels hints at a possible protective role against the development of reward dysfunction. More than anything, this study highlights the need for further integrative 1 H MRS research to fully map regional differences in GABA levels and their role in the etiology of adolescent depression.
In addition to studying GABA, our team has conducted preliminary 1 H MRS investigations of glutathione, the primary antioxidant found in brain tissue. Glutathione plays a central role in cellular oxidative balance through non-enzymatic scavenging of free radicals and enzyme-catalyzed detoxification of hydrogen peroxide via glutathione peroxidase. Studies have documented decreased glutathione and glutathione peroxidase in both depressed humans and animal models of depression. In an exploratory study of 11 adults with major depressive disorder and 10 healthy controls, we found that anhedonia, but not fatigue, was negatively correlated with occipital glutathione levels indexed by 1 H MRS. More recently, we published initial results from a study of occipital glutathione in 19 unmedicated adolescents (ages 12-21) with major depressive disorder and 8 age-matched healthy controls. Results indicated that cortical glutathione levels were significantly reduced in the depressed vs. control group, although correlations with anhedonia and overall depression severity did not meet statistical significance. Forthcoming, work from our group will evaluate glutathione and GABA levels in a larger cohort of adolescents to enable us to more definitively interpret the role of these important metabolites in adolescent depression and reward dysfunction. (B) Within the combined sample of depressed adolescents (N = 44), anhedonia was negatively associated with ACC GABA after controlling for depression severity (partial r = −0.33, p = 0.03). Excerpted from. * Significant at the Šidák-corrected p < 0.05 level.
## White matter integrity and adolescent depression symptomatology
In 2013, our group published a pilot investigation into the role of white matter in adolescent depression using DWI. Specifically, diffusion-weighted data were collected at 3T using 1,000 s/mm 2 gradients along 12 directions, with each direction sampled four times and averaged. Subjects comprised 17 depressed adolescents with a current major depressive episode of at least 8 weeks duration as well as 16 agematched healthy controls; all participants were psychotropicmedication-naïve. Analyses were performed using FSL's Tract-Based Spatial Statistics pipelineto model three principle diffusion directions per voxel and project the inferred white matter fibers onto a template "skeleton" representing major white matter tracts. Four standard measures derived from diffusion tensor modeling were examined: fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD). Results were reported at an exploratory threshold (uncorrected p < 0.001, k ≥ 10) using permutationbased non-parametric statistics.
Relative to healthy controls, we found that depressed adolescents had altered diffusivity metrics in four clusters: reduced anterior cingulum FA, anterior corona radiata AD, and posterior cingulum RD as well as increased posterior cingulum FA. In line with our other neuroimaging work and supporting our emphasis on dimensional symptom severity analyses, though, we found much more extensive associations with clinical symptomatology.
Depression severity correlated with a total of 16 clusters across diffusivity metrics, including negative correlations with FA in the anterior thalamic radiation near the left pallidum, genu of the corpus callosum near the ACC, and anterior cingulum bundle within the left precuneus; positive correlations with RD were observed in several overlapping clusters, including the anterior thalamic radiation bilaterally and the left genu of the corpus callosum.
Anhedonia severity was linked to altered diffusivity metric in 14 clusters, including reduced FA in the anterior limb of the internal capsule adjacent to the right basal ganglia; increased MD in the external capsule adjacent to the left putamen and in fibers projecting to the right OFC; and increased RD in the left anterior thalamic radiation, right anterior limb of the internal capsule, and projection fibers to the right OFC.
Unlike in our studies of clinical outcomes (section Anhedonia Predicts Worse Adolescent Depression Outcomes) and reward function using the RFT (section Task-Based Functional Magnetic Resonance Imaging), we also found associations between irritability scores and diffusivity metrics in a total of 14 clusters. These included positive correlations with MD in a cluster spanning the anterior corona radiata and anterior thalamic radiation near the left putamen, with RD in a cluster spanning the anterior limb of the internal capsule and anterior thalamic radiation near the right putamen, and with AD in the anterior corona radiata near the left putamen.
While preliminary, these results indicate that reduced white matter integrity in tracts projecting to or through major reward processing regions is associated with adolescent depression symptomatology. Unlike our resting-state and task-based fMRI results, we found that tracts with altered diffusivity metrics partially overlapped between different depression symptoms. This observation suggests that different disease mechanisms may be at work in gray vs. white matter, with the former exhibiting more specific abnormalities linked to reward dysfunction and the latter exhibiting more generalized loss of integrity in adolescent depression.
## Immune function and inflammation in depression and reward dysfunction
Though the research discussed thus far emphasizes the neural underpinnings of depression and reward dysfunction, substantial evidence also implicates the immune system in these disease processes. Converging data from preclinical and clinical studies document that disturbances in reward circuitry are induced by inflammation, possibly reflecting an evolutionary adaptation to conserve energy and facilitate the healing process by limiting reward-seeking behavior. In animal models, immunological challenges such as exposure to lipopolysaccharide, a bacterial endotoxin and potent inflammatory agent, result in "sickness behavior" characterized by decreased social exploration, sleep disorders, and, in female rats, inhibition of sexual behavior . In other words, the sickness behavior phenotype reflects broad-based deficits in reward processingand mirrors many aspects of depression phenomenology in humans (see section Shortcomings of the Categorical Diagnostic System).
At the same time, mounting evidence has documented activation of oxidative and nitrosative pathways, which are known to be induced by inflammatory responses, in depression. Reported abnormalities include decreased antioxidant levels, lowered antioxidant enzyme activity, increased circulatory oxygen radicals, and oxidative damage to lipids in depressed adults and in animal models of depression. As noted in section Neurochemical Abnormalities Revealed by Magnetic Resonance Spectroscopy, our group found a negative association between cortical glutathione and anhedonia in a pilot 1 H MRS study in adults. Simultaneously, expression of multiple neurotrophic factors (e.g., brain derived neurotrophic factor, BCL-2 proteins) is suppressed in depression.
Based on these observations, a leading theory posits that the pathophysiology of depression and reward dysfunction can be traced an imbalance between neurotrophic and neurotoxic factors, resulting in a cumulative loss or atrophy of neurons and glia. Supporting evidence includes significant volume reductions in brain volume, altered metabolic rates, and neurochemical abnormalities in specific brain regions including the PFC, amygdala, hippocampus, and basal ganglia, along with disturbances in limbic-cortical-striatal-pallidal-thalamic circuits. Postmortem studies provide additional evidence of neurodegenerative processes in depression, with decreased cortical thickness, neuronal size, and neuronal/glial density reported in the left rostral OFC, ACC, dorsolateral PFC, and hippocampus. However, the direct mechanisms giving rise to the proposed neurotrophic/neurotoxic imbalance in depression remain largely unknown, especially during the critical neurodevelopmental period of adolescence (section Reward Dysfunction as a Promising Predictor of Adolescent Depression Trajectory). Here, we describe our work to elucidate key metabolic and immunological pathways and their role in reward dysfunction early in the course of illness.
## The kynurenine pathway links immune and reward function
Our laboratory has conducted extensive research into the kynurenine pathway, a series of catabolic reactions that accounts for the breakdown of approximately 99% of free tryptophan (TRP), the rate-limiting amino acid precursor in serotonin (5-HT) synthesis. Due to the excitotoxic properties of several biomolecules synthesized via the kynurenine pathway, together with the reduction in TRP availability for 5-HT synthesis, the kynurenine pathway has been hypothesized to play an important role in linking peripheral inflammatory processes to central nervous system oxidative stress, atrophy, and cell death in depression and other disorders. The major enzymes, metabolites, and branches of the kynurenine pathway, as well FIGURE 12 | Overview of the kynurenine pathway and its position as a link between peripheral inflammation and central reward circuitry disruption. See list of acronyms at the start of main text. Reprinted from. * Metabolites capable of crossing the blood-brain barrier.
as proposed links to neuroinflammatory disease processes, are illustrated in.
The rate-limiting enzyme in the pathway is indoleamine 2,3-dioxygenase (IDO), which is induced by pro-inflammatory cytokinesand metabolizes TRP into kynurenine (KYN). KYN is then compartmentally metabolized in the brain via two major branches. In the neurotoxic branch, KYN is sequentially converted into 3-hydroxykynurenine (3-HK) and 3hydroxyanthranilic acid (3-HAA), neurotoxins that contribute to free radical generation, en route to forming quinolinic acid (QUIN), an excitotoxic agonist of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Alternatively, in the neurotrophic branch, KYN is metabolized into kynurenic acid (KA), an NMDA receptor antagonist with neuroprotective properties. Thus, induction of kynurenine pathway metabolism, especially the neurotoxic branch, may contribute to multiple neurodegenerative mechanisms in depression.
Converging lines of research have indeed linked increased IDO activity to anhedonia and reward dysfunction. In mice, peripheral inhibition of IDO has been shown to block transcription of IDO in the brain and prevent the development of depression-and anhedonia-like behaviors following immunological stimulation. In humans, clinical studies have reported increased urinary excretion of KYN and 3-HK in anhedonic depressed women (220) as well as relationships between kynurenine pathway activity and anhedonia in adults with depression. Moreover, we recently reported increased kynurenine pathway activity in suicidal depressed youth, further emphasizing the clinical significance of TRP metabolism.
## The kynurenine pathway in adolescent depression and anhedonia
An earlier series of studies by our group provided the first evidence of kynurenine pathway activation in adolescent depression and specifically in anhedonia. Our first study examined a cohort of 50 adolescents with major depressive disorder and 22 healthy controls, with the depressed cohort divided into 20 melancholic subjects, characterized by more severe illness and prominent anhedonia, and 30 non-melancholic subjects. We found that adolescents with depression had significantly lower plasma TRP concentrations and higher KYN/TRP ratios, a proxy for IDO activity, than either nonmelancholic depressed or healthy control subjects. Moreover, within the melancholic cohort, KYN levels were negatively correlated with depression severity, while 3-HAA/KYN ratios were positively associated with depression scores, indicating preferential engagement of the neurotoxic KYN → 3-HK → 3-HAA branch of the kynurenine pathway.
Building on these results, we subsequently examined the possible role of the kynurenine pathway in anhedonia, the hallmark symptom of melancholia. In a sample of 36 adolescents with major depressive disorder, including 22 medication-free participants and 20 healthy controls, we found that plasma concentrations of kynurenine pathway metabolites correlated with anhedonia severity while controlling for overall depression severity. Interestingly, IDO activity (i.e., KYN/TRP ratio) positively correlated with anhedonia severity in medication-free adolescents with depression alone (r = 0.42, p = 0.05) and in combination with controls (r = 0.44, p = 0.004), but this correlation was reduced when the combined sample included medicated depressed subjects (r = 0.30, p = 0.02) and did not reach significance in the depressed cohort alone when medicated subjects were included (r = 0.31, p = 0.06). This study was the first to link reward dysfunction to kynurenine pathway induction in youth. Additionally, our findings highlight the important, but frequently underappreciated, impact that treatment can have on biological measures in psychiatric research, suggesting that psychotropic medication may have a normalizing effect on the neurometabolic abnormalities observed in unmedicated participants.
## Preliminary findings from kynurenine pathway neuroimaging
Several of our neuroimaging studies further corroborate the link between immunological disruptions and reward deficits in adolescents. In a pilot study of seven melancholic depressed, seven non-melancholic depressed, and six healthy control adolescents, we found that peripheral KYN levels correlated with total choline, a spectroscopic biomarker for cell membrane turnover, in the right caudate (rho = 0.93, p Bonferroni = 0.03), while peripheral 3-HAA correlated with total choline in the left putamen (rho = 0.96, p Bonferroni = 0.006) only within the melancholic (i.e., highly anhedonic) depressed subgroup. While the small sample of this preliminary investigation is obviously a limitation, the magnitude of the observed correlations was remarkably high, suggesting a possible link between the global TRP metabolism and regional cell membrane metabolism within the striatum. A more recent pilot analysis of resting-state fMRI and kynurenine pathway metabolites in 14 unmedicated adolescents with depression and seven healthy controls similarly found preliminary evidence of associations between several kynurenine pathway metabolites and RSFC within reward-and salience-related neurocircuitry.
## Comprehensive cytokine profiling reveals extensive associations with anhedonia
As the primary signaling molecules mediating human immune response, cytokines have been frequent target of investigation across disease states, and the link between elevated levels of pro-inflammatory cytokines and depression is robustly documented. This includes a substantial body of evidence supporting a direct, causal link between exposure to pro-inflammatory cytokines or inflammatory triggers and reward dysfunction. In rhesus monkeys, chronic administration of interferon-alpha (IFN-α), a major proinflammatory cytokine, results in decreased dopaminergic neurotransmission along with anhedonia-like behavior. Extremely similar results are described in humans treated with IFN-α as part of immunotherapy regimes (228), who report a "flu-like"' syndrome characterized by anhedonia, fatigue, anorexia, and hypersomnia-again, all hallmarks of depression. Another study of healthy individuals who received lipopolysaccharide vs. saline placebo injections likewise reported that exposure to the endotoxin induced motivational changes in a behavioral task. However, the vast majority of clinical evidence supporting a link between inflammation and depression has come from studies in adult cohorts, and the relationship between these phenomena in pediatric populations remains less clear.
To address this gap, initial studies by our group examined plasma levels of major pro-inflammatory cytokines in the context of adolescent depression. We documented significantly higher levels of interferon-gamma (IFN-γ) in medically healthy adolescents with major depressive disorder compared to healthy controls; unexpectedly, however, we found that suicidal depressed adolescents had decreased levels of tumor necrosis factor-alpha (TNF-α) compared to non-suicidal adolescents with depression. Building on this initial work, we performed a novel in vitro experiment using peripheral blood mononuclear cells to study the role of cytokine induction in the emergence of adolescent depression symptomatology without exposing this vulnerable population to a potentially aggravating immunological challenge. These cells share a common mesodermal lineage with microglia and have an overlapping gene expression profile, thus expressing many of the same surface receptor and signaling proteins as their inaccessible counterparts in the brain. Blood samples were obtained from a clinical sample of 54 adolescents with predominantly mood and anxiety symptoms as well as 22 healthy control adolescents. Peripheral blood mononuclear cells were cultured for 6 h in the presence of lipopolysaccharide, after which the supernatant fluid was collected and assayed. To better capture the complex, multivariate nature of the immune system, comprehensive panels were performed using multiplex fluorescence assays to quantify 41 distinct pro-and anti-inflammatory cytokines. Analyses were controlled for multiple comparisons across all 41 measures (p FDR < 0.05) as well as overall depression severity, body mass index, age, and sex. Even at this stringent threshold, we found that anhedonia was positively correlated (rho = 0.33-0.57) with 19 of the cytokines included in our panel.
Cytokines linked to anhedonia were primarily pro-inflammatory and/or hematopoietic growth factors (N = 15), with a small number of chemokines (N = 2) and anti-inflammatory cytokines (N = 2) also identified. In line with our other dimensional work, no significant group differences were observed between depressed adolescents and healthy controls, and no significant association was detected with any other clinical measure (i.e., depression severity, anxiety, suicidality, fatigue). These findings provide strong evidence that inflammatory responses are tied to reward deficits in adolescents regardless of diagnostic status and again showcase the central role of reward dysfunction in early depression symptomatology.
## Data-driven analysis of inflammatory factors and reward function
Neuroimaging work further documents the specific effects of inflammatory processes within the dopaminergic reward circuitry. Several positron emission tomography (PET) studies in adults have demonstrated changes in striatal glucose metabolism subsequent to immunotherapy. Using fMRI, decreased striatal activation in response to rewarding stimuli was reported in patients undergoing treatment with IFNα (237). Similarly, exposure to bacterial endotoxins in healthy volunteers resulted in decreased ventral striatum activation during a reward task (238) but increased pain sensitivity, elevated activation in the anterior insula (a region involved in pain perception), and reduced activation in the rostral ACC (a region involved in pain regulation) during a noxious pressure paradigm. Typhoid vaccination in healthy volunteers has also been shown to alter activity in the substantia nigra, part of the dopaminergic midbrain, in relation to psychomotor slowing. Additionally, multiple neuroanatomical studies in depressed adults have documented relationships between peripheral kynurenine pathway metabolites and volume changes in reward-related brain regions.
One of our latest projects combined elements of our detailed cytokine panel analysis, described in section Comprehensive Cytokine Profiling Reveals Extensive Associations With Anhedonia, with our task-based fMRI studies using the RFT, presented in section Task-Based Functional Magnetic Resonance Imaging, to map between inflammation and neural reward processes in adolescents. Subjects consisted of 34 unmedicated adolescents with significant clinical symptoms predominantly related to mood and anxiety conditions and 12 age-matched healthy controls who completed both a blood draw and the RFT fMRI task. Multiplex fluorescent panels to measure 41 cytokines and fMRI analyses to model activation during reward anticipation and reward attainment were performed as described above. However, to address the high degree of correlation between many cytokines and limit the issue of multiple comparisons, we performed an additional dimensionality reduction step using factor analysis, a data-driven technique to identify shared sources of variance in complex, multivariate datasets. This approach yielded four inflammatory factors that together explained 76.4% of variance across all cytokines. As shown in, one of these factors was found to negatively correlate with activation in three bilateral posterior cingulate/inferior precuneus clusters during reward anticipation, while another was negatively correlated with activation in a single right angular gyrus cluster during reward attainment (all whole-brain p FWE < 0.05). Remarkably, 16 of the 19 cytokines found to be associated with anhedonia severity in our earlier dimensional studywere also identified in this fMRI analysis, implying that a common set of inflammatory modulators influence both clinical and neural reward function.
## Conclusions and future directions
The ongoing transition from traditional, diagnosis-based research to modern, dimensional paradigms marks a sea change in psychiatry and holds great promise to overcome longstanding barriers to understanding depression. Our group has pursued this dimensional approach to examine anhedonia as a complex behavioral construct that reflects alterations in developing reward circuitry. Early on, we showed that anhedonia severity varied widely and was associated with worse outcomes in adolescents with moderate-to-severe depression. Our subsequent clinical investigations have confirmed and extended this conclusion, indicating that higher anhedonia specifically predicted future depression severity and suicidality. At the same time, we documented substantial individual variation in anhedonia severity that cut across disorders and was present even in healthy adolescents. Our research program has therefore targeted anhedonia as a window to studying the neurobiological underpinnings of reward dysfunction in youth.
To this end, we employ an array of in vivo neuroimaging modalities to examine the mechanisms driving reward dysfunction in adolescent depression and across psychiatric conditions. With novel fMRI tasks, we were able to parse distinct phases of reward processing and related cognitive functions in both clinical and healthy adolescent cohorts. We found that reward anticipation, reward attainment, and reward prediction errors differentially elicited brain activity within and beyond the dopaminergic reward system. Subsequent research revealed that altered activation during these reward processes was linked to future reward deficits in the form of anhedonia. Using complementary resting-state fMRI techniques to map subcortical RSFC networks, we identified circuit-level alterations that were associated with anhedonia and depression severity as well as with subclinical depression. Bridging task-based and resting-state fMRI with data-driven graph theoretical analyses, we detailed differential relationships between anticipatory vs. consummatory anhedonia with RSFC features of whole-brain and reward-taskderived networks, highlighting the need for detailed dimensional measures to fully elucidate reward-related constructs. In conjunction with fMRI, we employed 1 H MRS to measure regional GABA levels within key reward regions, providing biochemical evidence linking cortical GABA deficits to clinical reward deficits and more severe depression in youth. Most importantly, findings across different neuroimaging modalities converged on a relatively small number of structures, namely the FIGURE 13 | Two factors identified by data-driven principal component analysis (PCA) of cytokine panels were found to significantly correlate with neural reward function in adolescents. Both factors were dominated by markers of inflammation and hematopoietic growth. Reprinted from. Scale bar indicates z-score.
ACC and striatum, that appear to play a disproportionate role in the emergence of depression symptomatology in youth.
Our group has also systematically examined inflammation and its contributions to pediatric reward dysfunction. We found evidence connecting activation of the kynurenine pathway, which degrades tryptophan into a series of neurometabolically active toxic species, to reward deficits (anhedonia), restingstate network anomalies, and altered neurochemical profiles. Employing detailed cytokine panels to capture a wide spectrum of immune biomarkers as well as an in vitro immunological challenge to induce inflammatory responses, we documented a unique relationship between anhedonia and immune activity in adolescents. Capitalizing on our concurrent collection of neuroimaging data in the same cohort, we employed a datadriven dimensionality reduction and identified a pair of immune factors that correlated with neural measures of reward function. In summary, the use of dimensional investigative techniques together with rigorous methodology and an integrative data collection strategy has enabled our group to characterize key aspects of reward dysfunction in youth that cut across disorders.
It is important to note that, while the dimensional approach offers many advantages over categorical diagnostics for understanding disease mechanisms, it also presents its own challenges and limitations. Of particular concern given our focus on reward dysfunction are the nuances of "anhedonia." While far more specific than "depression, " insofar as anhedonia represents a well-defined clinical phenomenon (i.e., reduced capacity to experience pleasure), efforts to develop reliable anhedonia scales indicate that it is not a monolithic construct. Indeed, the TEPS questionnaire employed in many of our studies is subdivided into items related to reward anticipation (i.e., pleasure at the thought of future rewards) vs. reward consumption (i.e., pleasure experienced when a reward is obtained). Subsequent work has suggested that two subscales may be inadequate to fully capture anhedonia phenomenology, particularly in cross-cultural settings. The TEPS was originally developed by researchers in the University of California system and validated based on responses of undergraduate volunteers from the Berkeley campus. A team of researchers from the University of the Chinese Academy of Sciences, however, found that a four-factor model that further subdivided pleasure experience into abstract components (i.e., items regarding beliefs about pleasure in general) and contextual components (i.e., items regarding specific pleasurable events) was a better fit for Chinese undergraduate students as well as ethnically Asian undergraduate students from an American sample, whereas the both models fit the overall American model equally well. Conversely, another group from the University of Melbourne examined TEPS responses from university students in the United Kingdom and Australia and reported that, although a two-factor model did not adequately model the response data, this was due to the high correlation between items across subscales, and a two-factor solution still outperformed the four-factor version. At present, the question of how to best discriminate between aspects of reward dysfunction remains unresolved; it is likely that additional, objective metrics such as the Probabilistic Reward Task developed by Diego Pizzagalli and colleaguesare needed to supplement subjective questionnaires and reliably quantify hedonic capacity. A related concern is that reward dysfunction may have distinct etiological mechanisms in different psychiatric contexts. It is possible, e.g., that anhedonia associated with chronic substance abuse may result from unique circuit-level changes compared to anhedonia associated with depression, which presumably arises from issues with endogenous reward signaling. Finally, while this review has attempted to illustrate the emerging discipline of dimensional psychiatric research by highlighting key findings from across the field in conjunction with our own work, it should not be taken as a comprehensive overview of all the important and exciting work being done to map the origins of reward dysfunction in adolescents.
Moving forward, we aim to build on the foundation of knowledge detailed above and extend our work further by incorporating powerful new resources that are just becoming available to the community through large, multisite neuroimaging initiatives. Prominent examples include the Nathan Kline Institute-Rockland Sample (NKI-RS) and HCP Lifespan Initiative, which aims to create large (N > 1,000) community profiles across age groups, as well as the Adolescent Brain Cognitive Development (ABCD) study, which aims to track >10,000 participants from pre-adolescence (age ∼10) to early adulthood (age ∼18) in the largest multi-site study currently operating in the United States. These projects are designed to overcome the inherent limitations of smaller-scale cross-sectional and longitudinal studies in childhood or adolescence, which frequently suffer from insufficient power, high attrition, and inadequate follow-up periods, as well as study designs being restricted to a specific neural system. The dynamic and sometimes ephemeral nature of depression symptomatology in adolescents makes it especially critical to compile multiple observations within a large and diverse cohort over time. Incorporating high-quality data from these public sources in conjunction with our ongoing, targeted investigations provides an exciting new avenue to pursue the neurobiological origins of reward dysfunction in youth and ultimately identify reliable predictors of illness trajectory into adulthood. |
Puberty timing and markers of cardiovascular structure and function at 25 years: a prospective cohort study
Background: Whether earlier onset of puberty is associated with higher cardiovascular risk in early adulthood is not well understood. Our objective was to examine the association between puberty timing and markers of cardiovascular structure and function at age 25 years. Methods: We conducted a prospective birth cohort study using data from the Avon Longitudinal Study of Parents and Children (ALSPAC). Participants were born between April 1, 1991, and December 31, 1992. Exposure of interest was age at peak height velocity (aPHV), an objective and validated growth-based measure of puberty onset. Outcome measures included cardiovascular structure and function at age 25 years: carotid intima-media thickness (CIMT), left ventricular mass index (LVMI) and relative wall thickness (RWT), pulse wave velocity (PWV) and systolic blood pressure (SBP). Multiple imputation was used to impute missing data on covariates and outcomes. Linear regression was used to examine the association between aPHV and each measure of cardiac structure and function, adjusting for maternal age, gestational age, household social class, maternal education, mother's partner's education, breastfeeding, parity, birthweight, maternal body mass index, maternal marital status, maternal prenatal smoking status and height and fat mass at age 9. All analyses were stratified by sex.Results: A total of 2752-4571 participants were included in the imputed analyses. A 1-year older aPHV was not strongly associated with markers of cardiac structure and function in males and females at 25 years and most results spanned the null value. In adjusted analyses, a 1-year older aPHV was associated with 0.003 mm (95% confidence interval (CI) 0.00001, 0.006) and 0.0008 mm (95% CI − 0.002, 0.003) higher CIMT; 0.02 m/s (95% CI − 0.05, 0.09) and 0.02 m/s (95% CI − 0.04, 0.09) higher PWV; and 0.003 mmHg (95% CI − 0.60, 0.60) and 0.13 mmHg (95% CI − 0.44, 0.70) higher SBP, among males and females, respectively. A 1-year older aPHV was associated with − 0.55 g/m 2.7 (95% CI − 0.03, − 1.08) and − 0.89 g/m 2.7 (95% CI − 0.45, − 1.34) lower LVMI and − 0.001 (95% CI − 0.006, 0.002) and − 0.002 (95% CI − 0.006, 0.002) lower RWT among males and females.Conclusions: Earlier puberty is unlikely to have a major impact on pre-clinical cardiovascular risk in early adulthood.
# Background
Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide, with 7.8 million premature CVD deaths estimated in 2025 if current trajectories are not altered [bib_ref] Demographic and epidemiologic drivers of global cardiovascular mortality, Roth [/bib_ref] [bib_ref] Estimates of global and regional premature cardiovascular mortality in 2025, Roth [/bib_ref]. CVD risk originates in early life and tracks through the life course [bib_ref] Early childhood hospitalisation with infection and subclinical atherosclerosis in adulthood: the Cardiovascular..., Burgner [/bib_ref] [bib_ref] A review on the genetic, environmental, and lifestyle aspects of the early-life..., Kelishadi [/bib_ref]. Onset of puberty is a transitional period between childhood and adulthood with intense hormonal activity, including the release of gonadotropins, leptin, sex-steroids and growth hormone, leading to physical bodily changes and the appearance of secondary sexual characteristics. The most striking feature of puberty is a spurt in height which occurs in males in late puberty and is highly correlated with secondary sexual characteristics such as enlargement of larynx, deepening of voice and genitalia development [bib_ref] Timing of voice breaking in males associated with growth and weight gain..., Ong [/bib_ref]. Conversely, growth spurts tend to start earlier in girls, often coinciding with breast development and menarche, but with shorter duration in comparison to boys [bib_ref] A mixed effects model to estimate timing and intensity of pubertal growth..., Cole [/bib_ref] [bib_ref] Pubertal development and regulation, Abreu [/bib_ref] [bib_ref] The effect of gonadotropin-releasing hormone analogue on final adult height in children..., Khawaja [/bib_ref]. Age at puberty onset has been decreasing for several decades, with increasing childhood adiposity (a condition of being severely overweight, or obese) thought to play a substantial role [bib_ref] Link between body fat and the timing of puberty, Kaplowitz [/bib_ref].
Several studies to date have examined the association between earlier puberty timing and CVD risk, with conflicting findings [bib_ref] Age at menarche, total mortality and mortality from ischaemic heart disease and..., Jacobsen [/bib_ref] [bib_ref] Early age at menarche associated with cardiovascular disease and mortality, Lakshman [/bib_ref] [bib_ref] Age at menarche and risks of coronary heart and other vascular diseases..., Canoy [/bib_ref] [bib_ref] A prospective study of age at menarche, parity, age at first birth,..., Colditz [/bib_ref] [bib_ref] Association between age at menarche and cardiovascular disease: a systematic review on..., Luijken [/bib_ref] [bib_ref] Age at menarche and risk of major cardiovascular diseases: evidence of birth..., Yang [/bib_ref] [bib_ref] Association of age at menarche with cardiovascular risk factors, vascular structure, and..., Kivimäki [/bib_ref]. A key limitation of many studies including observational cohort studies and Mendelian randomisation (MR) designs has included lack of adjustment for early childhood adiposity or use of indirect measures of adiposity such as BMI for adjustment, resulting in residual confounding of the puberty timing-CVD risk associations by early life adiposity [bib_ref] Age at menarche, total mortality and mortality from ischaemic heart disease and..., Jacobsen [/bib_ref] [bib_ref] Early age at menarche associated with cardiovascular disease and mortality, Lakshman [/bib_ref] [bib_ref] A prospective study of age at menarche, parity, age at first birth,..., Colditz [/bib_ref] [bib_ref] Association between age at menarche and cardiovascular disease: a systematic review on..., Luijken [/bib_ref] [bib_ref] Age at menarche and adult body mass index: a Mendelian randomization study, Gill [/bib_ref] [bib_ref] Identifying potential causal effects of age at menarche: a Mendelian randomization phenome-wide..., Magnus [/bib_ref] ]. In addition, though age at menarche offers a reliable marker of puberty timing in females, most previous studies have assessed puberty timing using self-reported measures among males such as voice change, facial hair and pubic hair [bib_ref] Puberty timing associated with diabetes, cardiovascular disease and also diverse health outcomes..., Day [/bib_ref] [bib_ref] Pubertal timing and Cardiometabolic markers at age 16 years, Berentzen [/bib_ref] , leading to measurement error [bib_ref] validity of self-assessment of pubertal maturation, Rasmussen [/bib_ref]. Thus, studies examining the association between objectively measured puberty timing and CVD risk in males and females, while adjusting for direct measures of childhood adiposity are required to better understand the aetiology of puberty timing and CVD risk in early life.
Using the objective growth-based measure of puberty onset (age at peak height velocity [aPHV]), we aimed to better understand the association between puberty timing and pre-clinical cardiovascular risk in early adulthood using markers of cardiovascular structure and function at age 25 years (carotid intima-media thickness, left ventricular mass index and relative wall thickness, pulse wave velocity and systolic blood pressure).
# Methods
## Study participants
We used data from the Avon Longitudinal Study of Parents and Children (ALSPAC), a prospective birth cohort study based in Southwest England [bib_ref] Cohort profile: the 'children of the 90s'--the index offspring of the Avon..., Boyd [/bib_ref] [bib_ref] Cohort profile: the Avon Longitudinal Study of Parents and Children: ALSPAC mothers..., Fraser [/bib_ref] [bib_ref] The Avon Longitudinal Study of Parents and Children (ALSPAC): an update on..., Northstone [/bib_ref]. The women invited to participate in this study were pregnant with an expected delivery date between , and living in one of the three Bristol-based health districts. A detailed description of this study is available elsewhere [bib_ref] Cohort profile: the 'children of the 90s'--the index offspring of the Avon..., Boyd [/bib_ref] [bib_ref] Cohort profile: the Avon Longitudinal Study of Parents and Children: ALSPAC mothers..., Fraser [/bib_ref] [bib_ref] The Avon Longitudinal Study of Parents and Children (ALSPAC): an update on..., Northstone [/bib_ref].
The initial number of pregnancies enrolled was 14,541. When the oldest children were approximately 7 years of age, an attempt was made to increase the initial sample with eligible children who did not join the study originally. This resulted in an additional 913 children being enrolled. Therefore, the total sample size was 15,454 pregnancies. Of these 14,901 were alive at 1 year of age. In the three decades since enrolment, ALSPAC has used questionnaires completed by both parents and children, routine medical data and research clinics as methods of follow-up. The clinics took place when the participants were 7, 9, 10, 11, 13, 15, 17 and 25 years old [bib_ref] Cohort profile: the 'children of the 90s'--the index offspring of the Avon..., Boyd [/bib_ref] [bib_ref] Cohort profile: the Avon Longitudinal Study of Parents and Children: ALSPAC mothers..., Fraser [/bib_ref] [bib_ref] The Avon Longitudinal Study of Parents and Children (ALSPAC): an update on..., Northstone [/bib_ref] [bib_ref] Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing..., Harris [/bib_ref] [bib_ref] The REDCap consortium: Building an international community of software platform partners, Harris [/bib_ref].
Ethical approval for the ALSPAC study was obtained from the ALSPAC Law and Ethics Committee and Local Research Ethics Committees. Informed consent for the use of data collected via questionnaires and clinics was obtained from participants following the recommendations of the ALSPAC Law and Ethics Committee at the time. The study website contains details of all the data that is available through a fully searchable data dictionary and variable search tool: http://www.bristol.ac.uk/ alspac/researchers/our-data/.
## Exposure
## Assessment of timing of puberty
Puberty is a period of intense hormonal activity and rapid growth, of which the most striking feature is the spurt in height [bib_ref] A mixed effects model to estimate timing and intensity of pubertal growth..., Cole [/bib_ref]. aPHV is a validated measure of puberty timing [bib_ref] A mixed effects model to estimate timing and intensity of pubertal growth..., Cole [/bib_ref] captured using Superimposition by Translation and Rotation (SITAR), a non-linear multilevel model with natural cubic splines which estimates the population average growth curve and departures from it as random effects [bib_ref] SITAR--a useful instrument for growth curve analysis, Cole [/bib_ref] [bib_ref] Modelling height in adolescence: a comparison of methods for estimating the age..., Simpkin [/bib_ref]. Using SITAR, PHV was identified in ALSPAC participants using numerical differentiation of the individually predicted growth curves, with aPHV being the age at which the maximum velocity is observed [bib_ref] SITAR--a useful instrument for growth curve analysis, Cole [/bib_ref] [bib_ref] Modelling height in adolescence: a comparison of methods for estimating the age..., Simpkin [/bib_ref] [bib_ref] Using SITAR (SuperImposition by Translation and Rotation) to estimate age at peak..., Frysz [/bib_ref]. Repeated height data included measurements from research clinics and were used here to derive aPHV using SITAR. Individuals with at least one measurement of height from 5 years to < 10 years, 10 years to < 15 years and 15 years to 20 years were included. Data were analysed for males and females separately. The model was fitted using the SITAR package in R version 3.4.1, as described elsewhere [bib_ref] Using SITAR (SuperImposition by Translation and Rotation) to estimate age at peak..., Frysz [/bib_ref]. Further details on how aPHV was derived are described elsewhere [bib_ref] Using SITAR (SuperImposition by Translation and Rotation) to estimate age at peak..., Frysz [/bib_ref] [bib_ref] Puberty timing and adiposity change across childhood and adolescence: disentangling cause and..., O'keeffe [/bib_ref].
## Outcomes
## Assessment of cardiovascular risk
Carotid intima-media thickness, left ventricular mass index and relative wall thickness, pulse wave velocity and systolic blood pressure were measured at research clinics at age 25 years. Participants fasted for 6 h before the clinic, with the exception of those participants with a diagnosis of diabetes or a condition that would not allow fasting. Carotid intima-media thickness scans of the left and right common carotid arteries were performed using a CardioHealth Panasonic system with a 13-5 MHz linear array broadband transducer according to a standardised protocol. Carotid intima-media thickness has been validated in several studies as a strong predictor of CVD-risk [bib_ref] Cardiovascular risk factors in childhood and carotid artery intimamedia thickness in adulthood:..., Raitakari [/bib_ref] [bib_ref] Common carotid intima-media thickness and risk of stroke and myocardial infarction: the..., Bots [/bib_ref] [bib_ref] Association of coronary heart disease incidence with carotid arterial wall thickness and..., Chambless [/bib_ref]. Participants lay on a couch with their arms by their side, while a trained researcher performed the ultrasound test on both sides of their neck. Right and left carotid intima-media thickness measurements were taken to be the average of 3 end-diastolic measurements of the far-wall of the common carotid artery over a length of 5-10 mm, and 10 mm adjoining the bifurcation. The mean of both right and left carotid intimamedia thickness measures was calculated and used here.
Echocardiography was performed by two experienced echo-cardiographers using a Philips EPIQ 7G Ultrasound System equipped with a X5-1 transducer in accordance with American Society of Echocardiography guidelines; these techniques are described elsewhere [bib_ref] Hypertensive disorders of pregnancy and offspring cardiac structure and function in adolescence, Timpka [/bib_ref]. Left ventricular mass, assessed by ultrasound, was indexed to height 2.7 to adjust for body surface area. Relative wall thickness was calculated using left ventricular internal diameter in diastole and thickness of the left ventricular posterior wall and septal wall. Pulse wave velocity was measured using a Vicorder device (Skidmore Medical, Bristol, UK) at femoral and carotid artery level which has been validated in previous studies in adolescents [bib_ref] Validating a new oscillometric device for aortic pulse wave velocity measurements in..., Kracht [/bib_ref]. Ten pulse wave velocity measurements were taken within ≤ 0.5 m/s of each other. These were averaged to give a measurement of arterial stiffness. Systolic blood pressure was measured (typically from the right arm) with the subject in a sitting position using an Omron 705-IT machine [bib_ref] Hypertensive disorders of pregnancy and offspring cardiac structure and function in adolescence, Timpka [/bib_ref].
## Covariates
We selected potential confounders a priori and used a directed acyclic graph (DAG) to encode this causal knowledge of this research question. In summary, we have included only covariates in our model, which we believe to be common causes of the exposure and outcome and have excluded any variables that might be potential mediators of the association [bib_ref] Principles of confounder selection, Vanderweele [/bib_ref]. Therefore, we considered the following as possible confounders of the association between aPHV and cardiovascular structure and function at age 25 years; maternal age, gestational age at birth, household social class, maternal education, mother's partner's education, breastfeeding of baby until 3 months, parity, birthweight, maternal body mass index (BMI), maternal marital status, maternal smoking status during first 3 months of pregnancy, and height and DXA-determined fat mass at age 9.
Maternal age was reported in the mother's antenatal questionnaires. Gestational age at birth was estimated from clinical records. Household social class was measured as the highest of the mother's or her partner's occupational social class using data on job title and details of occupation collected about the mother and her partner from the mother's questionnaire at 32 weeks gestation. Social class was derived using the standard occupational classification (SOC) codes developed by the UK Office of Population Census and Surveys and classified as I professional, II managerial and technical, IIINM non-manual, IIIM manual and IV&V part skilled occupations and unskilled occupations.
A questionnaire at 32 weeks gestation asked mothers to report on educational attainment, which was categorised as below O-Level (Ordinary Level; exams taken in different subjects usually at age 15-16 at the completion of legally required school attendance, equivalent to today's UK General Certificate of Secondary Education), O-Level only, A-Level (Advanced-Level; exams taken in different subjects usually at age 18) or university degree or above.
Breastfeeding information was collected via questionnaires administered at 4 weeks, 6 months and 15 months. Parity was defined as the number of previous pregnancies that had resulted in a live or stillborn infant collected at 18 weeks gestation. Birthweight was extracted from medical records. Maternal height and weight data were self-reported from a questionnaire administered at 12 weeks gestation; these were used to calculate maternal BMI. Marital status was obtained from antenatal questionnaires and classified as never married, married and widowed, divorced, or separated. Smoking in the first trimester of pregnancy was self-reported by mothers at 18 weeks gestation; responses to smoking any tobacco (cigarettes, cigars, pipes, or other) were grouped as follows: no smoking, < 10 per day, 10-19 per day or greater than 19 per day, and were re-categorised as a dichotomous variable (smoking: yes/no).
Height and fat mass of offspring was measured at clinics at age 9 years. Standing height was measured to the last complete mm using the Harpenden Stadiometer. Fat mass (in kg, less head) was derived from whole body DXA scans performed using a GE Lunar Prodigy (Madison, WI, USA) narrow fan beam densitometer.
# Statistical analysis
Statistical analysis was performed using Stata MP 14.2. Linearity of association between aPHV and cardiovascular structure and function in males and females was assessed by comparing fit of models regressing carotid intima-media thickness, left ventricular mass index and relative wall thickness, pulse wave velocity, and systolic blood pressure on fourths of aPHV (treated as a continuous exposure) to models regressing carotid intimamedia thickness, left ventricular mass index and relative wall thickness, pulse wave velocity and systolic blood pressure on fourths of aPHV (treated as a categorical exposure); models were then formally compared using a likelihood ratio test. Data on aPHV and all cardiovascular outcomes were normally distributed. We used linear regression to examine the association between aPHV and each measure of cardiac structure and function. All analyses were stratified by sex to examine whether associations of aPHV and each outcome differed for males and females.
## Dealing with missing data
There were 1197-2193 participants with complete data for aPHV, each outcome and all covariates. Cardiac structure and function were also measured at the 18 year ALSPAC clinic. To increase efficiency and minimise selection bias, we used multivariate multiple imputation to impute missing data on covariates and outcomes in all participants that had a measure of aPHV and had a measure of the outcome at the age 18 year or age 25 year research clinic (N = 4339 for carotid intima-media thickness, N = 2752 for left ventricular mass index, N = 2776 for relative wall thickness, N = 3964 for pulse wave velocity and N = 4571 for systolic blood pressure). We carried out 20 cycles of regression switching and generated 20 imputation datasets [bib_ref] Multiple imputation of missing values, Royston [/bib_ref]. We then examined associations of aPHV and outcomes in these multiple imputed datasets; results are averaged across the results from each of these 20 datasets using Rubin's rules, taking account of uncertainty in the imputation so that the standard errors for any regression coefficients (used to calculate 95% confidence intervals) take account of uncertainty in the imputations and uncertainty in the estimate [bib_ref] Multiple imputation of missing values, Royston [/bib_ref]. See Additional file 1: [fig_ref] Table 1: Distribution/proportions of baseline characteristics by fourths of age at peak height velocity... [/fig_ref] for a list of the variables included in the multiple imputation models and how they were entered into the models.
We repeated the main adjusted analysis in the observed dataset, among participants with complete-case data on exposure, outcome and covariates (N = 1199 for carotid intima-media thickness, N = 1197 for left ventricular mass index, N = 1203 for relative wall thickness, N = 1394 for pulse wave velocity and N = 2193 for systolic blood pressure).
# Results
The distribution/proportions of baseline characteristics by fourths of age at peak height velocity are shown in [fig_ref] Table 1: Distribution/proportions of baseline characteristics by fourths of age at peak height velocity... [/fig_ref] , and the characteristics of the cohort included in the analysis (by sex) are shown in Additional file 1: [fig_ref] Table 2: Associations between age at peak height velocity and cardiac structure and function... [/fig_ref]. A total of 2752-4571 participants were included in the imputed analyses. The mean aPHV was 13.54 years in males and 11.73 years in females. Findings from linearity tests of the association between aPHV and cardiac structure and function demonstrated little evidence of departures from linearity, allowing aPHV to be examined as a continuous exposure (Additional file 1: . The distribution of variables in observed and imputed data is shown in Additional file 1: Tables S4-S8; distributions of most variables were broadly similar between observed and imputed datasets with some minor differences only for socio-economic position indicators which were slightly higher in the observed datasets.
## Age at peak height velocity and cardiovascular structure and function males
There was little evidence of association between aPHV and cardiac structure and function in males, with results mostly spanning the null value. For example, in confounder-adjusted analyses, a one-year older aPHV was associated with 0.003 mm (95% confidence interval (CI) 0.00001, 0.006) higher carotid intima-media thickness, 0.02 m/s (95% CI − 0.05, 0.09) higher pulse wave velocity and 0.003 mmHg (95% CI − 0.60, 0.60) higher systolic blood pressure at 25 years. A 1-year older aPHV was associated with a − 0.55 g/m 2.7 (95% CI − 0.03, − 1.08) lower left ventricular mass index and − 0.001 (95% CI − 0.006, 0.002) lower relative wall thickness at 25 years [fig_ref] Table 2: Associations between age at peak height velocity and cardiac structure and function... [/fig_ref].
## Females
Similar to males, there was little evidence of association observed between aPHV and cardiac structure and function in females, with results mostly spanning the null value. In confounder-adjusted analyses, a one-year older aPHV was associated with 0.0008 mm (95% CI − 0.002, 0.003) higher carotid intima-media thickness, 0.02 m/s (95% CI − 0.04, 0.09) higher pulse wave velocity and 0.13 mmHg (95% CI − 0.44, 0.70) higher systolic blood pressure at 25 years. A one-year older aPHV was associated with a − 0.89 g/m 2.7 (95% CI − 0.45, − 1.34) lower left ventricular mass index and − 0.002 (95% CI − 0.006, 0.002) lower relative wall thickness at 25 years [fig_ref] Table 2: Associations between age at peak height velocity and cardiac structure and function... [/fig_ref].
Unadjusted and confounder adjusted results were not materially different for each outcome in males and females [fig_ref] Table 2: Associations between age at peak height velocity and cardiac structure and function... [/fig_ref].
Adjusted associations of aPHV with measures of cardiac structure and function among participants with complete-case data on exposure, outcome and covariates were comparable to the main results obtained using imputed datasets (Additional file 1: .
# Discussion
This study aimed to better understand the association between puberty timing and cardiovascular structure and function at age 25 years using aPHV as an objective growth-based measure of puberty onset and carotid intima-media thickness, left ventricular mass index and relative wall thickness, pulse wave velocity and systolic blood pressure as pre-clinical measures of cardiovascular risk. There was little evidence of association between aPHV and cardiovascular structure and function at 25 years, with results spanning the null in all but left ventricular mass index for which the association was inverse.
Previous studies have shown that a ∼ 5 g/m 2 higher left ventricular mass index can be predicted to correspond to a 7-20% increase in CVD morbidity and mortality [bib_ref] Midlife blood pressure change and left ventricular mass and remodelling in older..., Ghosh [/bib_ref] [bib_ref] LV mass assessed by echocardiography and CMR, cardiovascular outcomes, and medical practice, Armstrong [/bib_ref] [bib_ref] Framingham score and LV mass predict events in young adults: CARDIA study, Armstrong [/bib_ref]. Given the small differences in left ventricular mass index per year older aPHV found here, our findings suggest that earlier puberty is unlikely to have a major impact on pre-clinical cardiovascular risk in early adulthood.
## Comparison with other studies
Several studies to date have examined the association between puberty timing and clinical endpoints such as CVD and traditional cardiovascular risk factors such as blood pressure and adiposity, with conflicting findings . However, these studies used different measures of puberty timing (objective vs. selfreport) [bib_ref] Influence of puberty timing on adiposity and cardiometabolic traits: a Mendelian randomisation..., Bell [/bib_ref] [bib_ref] The relationship between pubertal timing and markers of vascular and cardiac structure..., Hardy [/bib_ref] and had varying degrees of adjustment for confounding (particularly early childhood adiposity) [bib_ref] Age at menarche, total mortality and mortality from ischaemic heart disease and..., Jacobsen [/bib_ref] [bib_ref] Early age at menarche associated with cardiovascular disease and mortality, Lakshman [/bib_ref] [bib_ref] Age at menarche and risks of coronary heart and other vascular diseases..., Canoy [/bib_ref] [bib_ref] Age at menarche and adult body mass index: a Mendelian randomization study, Gill [/bib_ref] and different ranges in follow-up and age of measurement of outcomes [bib_ref] Association of age at menarche with cardiovascular risk factors, vascular structure, and..., Kivimäki [/bib_ref] [bib_ref] Pubertal timing and Cardiometabolic markers at age 16 years, Berentzen [/bib_ref] [bib_ref] Reproductive factors, intima media thickness and carotid plaques in a crosssectional study..., Stöckl [/bib_ref].
In contrast, fewer studies have examined associations of puberty timing with measures of cardiac structure and function, which are used clinically to assess arterial stiffness, left ventricular hypertrophy and cardiac remodelling [bib_ref] The relationship between pubertal timing and markers of vascular and cardiac structure..., Hardy [/bib_ref]. Hardy and colleagues found evidence of an association between later puberty timing (measured using age at menarche) and lower left ventricular mass and relative wall thickness in females in the National Study of Health and Development (NSHD) (N = 1385), though results attenuated after adjustment for childhood or adult adiposity [bib_ref] The relationship between pubertal timing and markers of vascular and cardiac structure..., Hardy [/bib_ref]. In contrast, there was no strong evidence of an association between puberty timing (measured via physical examination) and any measure of cardiac structure or function in males in the NSHD, a finding comparable to ours [bib_ref] The relationship between pubertal timing and markers of vascular and cardiac structure..., Hardy [/bib_ref]. Our findings are also comparable to results from the Young Finns Cohort (N = 794) which demonstrated no strong evidence of associations between puberty timing (measured via age at menarche) and carotid intima-media thickness in females at age 30-39 years [bib_ref] Association of age at menarche with cardiovascular risk factors, vascular structure, and..., Kivimäki [/bib_ref]. Similarly, in a study of 800 women aged 50 to 81 years in Southern Germany, age at menarche was not associated with carotid intima-media thickness in both unadjusted and confounderadjusted analyses [bib_ref] Reproductive factors, intima media thickness and carotid plaques in a crosssectional study..., Stöckl [/bib_ref].
# Strengths and limitations
Strengths of this study include use of an objective measure of puberty timing (aPHV) based on prospective, repeated measures of height from age 5 years to 20 years which is a more accurate marker of puberty timing than self-reported measures. We also used measures of preclinical CVD, which strongly predict later cardiovascular risk. We adjusted for childhood adiposity using DXA fat mass at age 9, allowing us to adjust for directly measured pre-pubertal adiposity, a key limitation in several previous studies [bib_ref] Age at menarche, total mortality and mortality from ischaemic heart disease and..., Jacobsen [/bib_ref] [bib_ref] Early age at menarche associated with cardiovascular disease and mortality, Lakshman [/bib_ref] [bib_ref] Age at menarche and risks of coronary heart and other vascular diseases..., Canoy [/bib_ref] [bib_ref] Age at menarche and adult body mass index: a Mendelian randomization study, Gill [/bib_ref]. However, there are also several limitations; a limitation of our adiposity adjustment may include the possibility that DXA fat mass is measured after pubertal onset for a small proportion of participants (N = 1 for males, N = 41 for females) giving rise to the possibility of collider bias if DXA fat mass at age 9 is a mediator of the puberty timing-cardiac risk association. However, we believe that any bias introduced due to this is minimal, given that prior work in this cohort has shown that puberty has little impact on postpubertal fat mass gain [bib_ref] Puberty timing and adiposity change across childhood and adolescence: disentangling cause and..., O'keeffe [/bib_ref] (suggesting fat mass is an unlikely mediator of any associations of puberty timing with health outcomes) and that analyses without adjustment for fat mass were similar to confounder-adjusted results. Nevertheless, though we have shown that our approach to adjustment for adiposity here is not likely to have introduced bias to our results, the direction of causality of the relationship between adiposity and puberty timing is complex given the potential shared genetic architecture between the traits. In addition, the outcome measures used may be subject to measurement error which may have led to biased estimates. However, any measurement error is likely to be non-differential and would therefore bias our results towards to the null [bib_ref] Misclassification in 2 X 2 tables, Bross [/bib_ref] [bib_ref] Proper interpretation of non-differential misclassification effects: expectations vs observations, Jurek [/bib_ref]. Further limitations include potential for selection bias due to missing data and attrition from the Adjusted for maternal age, gestational age, household social class, maternal education, mother's partner's education, breastfeeding of baby until 3 months, parity, birthweight, maternal body mass index, maternal marital status, maternal smoking status during first 3 months of pregnancy, and height and fat mass of offspring at age 9 cohort. However, the use of multivariate multiple imputation aimed to minimise bias and a loss of statistical power by imputing missing covariate and outcome data, and results in multiply imputed and complete-case data were similar. However, replication of findings in studies with larger sample sizes may be needed in order to provide more precise estimates of associations. Moreover, our outcomes were measured at age 25 years, and while the pre-clinical measures of CVD used here are strong predictors of later cardiovascular risk [bib_ref] Cardiovascular risk factors in childhood and carotid artery intimamedia thickness in adulthood:..., Raitakari [/bib_ref] [bib_ref] The relationship between systolic blood pressure and cardiovascular risk--results of the Brisighella..., Borghi [/bib_ref] , young adulthood might be too early to detect changes in cardiac structure and function that progress with age. Therefore, future studies examining a puberty timing and CVD risk relationship in older populations may be worthwhile. Finally, the vast majority of the ALSPAC cohort are of White ethnicity [bib_ref] Cohort profile: the 'children of the 90s'--the index offspring of the Avon..., Boyd [/bib_ref] , and a key limitation of our study is the generalisability of the findings to non-White ethnicities.
# Conclusion
Age at peak height velocity was not strongly associated with measures of cardiac structure and function among males and females at age 25 years. Earlier puberty is unlikely to have a major impact on pre-clinical cardiovascular risk in early adulthood.
## Supplementary information
The online version contains supplementary material available at https://doi. org/10.1186/s12916-021-01949-y.
Additional file 1: [fig_ref] Table 1: Distribution/proportions of baseline characteristics by fourths of age at peak height velocity... [/fig_ref]. [Variables used in multivariable multiple imputation models]. [fig_ref] Table 2: Associations between age at peak height velocity and cardiac structure and function... [/fig_ref]. [Characteristics of ALSPAC participants included in the analysis by sex and based on imputed data]. .
[Likelihood ratio test examining linearity of association between age at peak height velocity and cardiac structure and function outcomes by sex]. . [Distributions of imputed characteristics in the imputation datasets and in observed data (i.e. without imputation) for carotid intimamedia thickness in males and females]. . [Distributions of imputed characteristics in the imputation datasets and in observed data (i.e. without imputation) for left ventricular mass index in males and females]. . [Distributions of imputed characteristics in the imputation datasets and in observed data (i.e. without imputation) for relative wall thickness in males and females]. . [Distributions of imputed characteristics in the imputation datasets and in observed data (i.e. without imputation) for pulse wave velocity in males and females]. .
[Distributions of imputed characteristics in the imputation datasets and in observed data (i.e. without imputation) for systolic blood pressure in males and females]. . [Adjusted associations of age at peak height velocity with measures of cardiac structure and function among participants with complete-case data on exposure, outcome and covariates]. [fig_ref] Table 1: Distribution/proportions of baseline characteristics by fourths of age at peak height velocity... [/fig_ref]. [Pearson's correlation coefficient examining the association between age at voice breaking and age at peak height velocity in males, and age at menarche and age at peak height velocity in females].
[table] Table 1: Distribution/proportions of baseline characteristics by fourths of age at peak height velocity based on imputed data [/table]
[table] Table 2: Associations between age at peak height velocity and cardiac structure and function in males and females at age 25 [/table]
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Impact of Cadmium on Intracellular Zinc Levels in HepG2 Cells: Quantitative Evaluations and Molecular Effects
Cadmium is classified as a human carcinogen, and its disturbance in zinc homeostasis has been well established. However, its extent as well as molecular mechanisms involved in cadmium carcinogenesis has yet to be fully clarified. To this end, we used the zinc specific probe Zinquin to visualize and to quantitatively evaluate changes in the concentration of labile zinc, in an in vitro model of human hepatic cells (HepG2) exposed to cadmium. A very large increase (+93%) of intracellular labile zinc, displaced by cadmium from the zinc proteome, was measured when HepG2 were exposed to 10 M cadmium for 24 hrs. Microarray expression profiling showed that in cells, featuring an increase of labile zinc after cadmium exposure, one of the top regulated genes is Snail1 (+3.6), which is included in the adherens junction pathway and linked to cancer. In the same pathway MET, TGF-R, and two members of the Rho-family GTPase, Rac, and cdc42 all implicated in the loss of adherence features and acquisition of migratory and cancer properties were regulated, as well. The microRNAs analysis showed a downregulation of miR-34a and miR-200a, both implicated in the epithelial-mesenchymal transition. These microRNAs results support the role played by zinc in affecting gene expression at the posttranscriptional level.
# Introduction
Cadmium is a highly persistent pollutant harmful to humans and animals, listed as one of the top ten hazardous substances by the Agency for Toxic Substances and Disease Registry, and classified as a human carcinogen, based on epidemiological studies and animal experiments, by the International Agency for Research on Cancer. The widespread presence of Cd 2+ makes it a severe environmental health problem that needs to be considered thoroughly. Apart from natural sources, major fonts of exposure are cadmium-contaminated food and water, providing around 30 g per day for adults, cigarette smoke [bib_ref] Cadmium toxicity in animal cells by interference with essential metals, Martelli [/bib_ref] , and cosmetic products [bib_ref] Toxic metals contained in cosmetics: a status report, Bocca [/bib_ref].
Cadmium enters the cells by utilizing transport pathways typically evolved for essential metals. Varieties of pathways have been suggested and recently reviewed [bib_ref] Catch me if you can! Novel aspects of cadmium transport in mammalian..., Thévenod [/bib_ref]. In mammalian cells well established pathways involve receptor-mediated endocytosis (megalin/cubilin) of Cdmetallothionein complex and cotransport mechanisms by divalent metal ion transporter 1 (DMT1) and ZIP proteins (ZIP8 and ZIP14A/B). A recently discovered candidate pathway for Cd 2+ entry into cells is through the T-Type calcium channels Ca v 3.1 [bib_ref] Cd 2+ block and permeation of CaV3.1 ( 1G) T-type calcium channels:..., Lopin [/bib_ref]. Due to its molecular mimicry, cadmium follows a Trojan horse strategy leading to interference with essential metals homeostasis. In mammalian cells cadmium toxicity has been closely related to zinc homeostasis. Once inside the cells, cadmium is expected to displace zinc in proteins and enzymes in which zinc has a sulphur-dominated coordination sphere, such as the metallothioneins, the zinc sensor MTF-1, and multiple zinc-finger proteins [bib_ref] Cadmium toxicity in animal cells by interference with essential metals, Martelli [/bib_ref] [bib_ref] Cadmium induces conformational modifications of wild-type p53 and suppresses p53 response to..., Méplan [/bib_ref] [bib_ref] Toxic metal proteomics: reaction of the mammalian zinc proteome with Cd 2+, Namdarghanbari [/bib_ref].
However, even though the disturbance of cadmium in zinc homeostasis has already been recognized, its extent has not been a matter of study yet.
In this context, the aim of our work was (1) to demonstrate that the exposure of a human hepatic model system (HepG2 cells) to cadmium leads to an increase of the intracellular pool of labile Zn(II) andto quantitatively measure this increase. To this end, we have used a fluorescent probe (Zinquin) specifically developed for the visualization and measurement of intracellular labile zinc ions [bib_ref] Correlation of apoptosis with change in intracellular labile Zn(II) using Zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic..., Zalewski [/bib_ref]. A further aim was to analyze the consequences at molecular level of this increase, as zinc is a recognized second messenger and transcriptional regulator [bib_ref] Functions of zinc in signaling, proliferation and differentiation of mammalian cells, Beyersmann [/bib_ref] [bib_ref] Zinc is a novel intracellular second messenger, Yamasaki [/bib_ref].
# Materials and methods
## Cell cultures and treatments.
Human hepatoblastoma cells (HepG2) represent a useful in vitro model for cadmium accumulation and toxicity studies, as previously reported [bib_ref] Characterization of the human hepatocellular carcinoma (HEPG2) cell line as an in..., Dehn [/bib_ref] [bib_ref] Different induction of metallothioneins and Hsp70 and presence of the membrane transporter..., Urani [/bib_ref] [bib_ref] Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure, Urani [/bib_ref]. Furthermore HepG2 are, among others, a widely used alternative cell system to primary hepatocytes as they retain, under specific cultivation conditions, many metabolic functions of normal liver cellsThe HepG2cells were routinely cultured in Opti-MEM medium (Life Technologies, Monza, Italy) supplemented with 10% inactivated fetal bovine serum and 1% antibiotics (streptomycin/penicillin). Cells were kept in incubator at constant 37 ∘ C under a humidified 5% CO 2 atmosphere.
For fluorescence microscopy visualization and spectrofluorimetric measurements the cells were cultured either in complete medium (control) or in complete medium containing CdCl 2 (Cd, 0.1 and 10 M) or ZnSO 4 (Zn, 10, 50, and 170 M) for 24 hrs.
The viability of HepG2 cells exposed to the highest CdCl 2 (Sigma-Aldrich, Milan, Italy) concentration (10 M) for 24 hrs was between 80 and 90% and above 80% in ZnSO 4 (Sigma-Aldrich, Milan, Italy) treated cells, as previously published [bib_ref] Copper and zinc uptake and hsp70 expression in HepG2 cells, Urani [/bib_ref] [bib_ref] Cytotoxicity and induction of protective mechanisms in HepG2 cells exposed to cadmium, Urani [/bib_ref] , and confirmed herewith (data not shown).
## Fluorescent visualization of free
Zinc. Cells were seeded on sterile glass coverslips in 35 mm culture plates (80.000 cells/plate) in complete culture medium. The medium was removed 24 hrs after seeding and substituted with Zn-or Cdcontaining medium at concentrations indicated in par. 2.1. The cells were left with the control or treatment media in incubator for further 24 hrs.
At the end of treatment time, the cells were processed for fluorescent experiments with Zinquin according to Sarwar Nasir et al. [bib_ref] The chemical cell biology of zinc: structure and intracellular fluorescence of a..., Nasir [/bib_ref] with some modifications.
Zinquin [ethyl (2-methyl-8-p-toluenesulphonamido-6quinolyloxy)acetate] is a fluorescent probe used to detect labile intracellular Zn and to reveal Zn in cultured cells. Zinquin is permeable and is readily taken up by the cells and is essentially nonfluorescent until it complexes Zn with a high selectivity [bib_ref] Correlation of apoptosis with change in intracellular labile Zn(II) using Zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic..., Zalewski [/bib_ref] [bib_ref] Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship..., Coyle [/bib_ref] , thus providing a means for labile zinc detection and visualization. At the end of all treatments, the medium was removed and the cells were rinsed twice with warm PBS and fixed in formaldehyde (3.7% in PBS) for 30 min at 37 ∘ C. After rinsing with PBS, the cells were incubated with Zinquin to a final concentration of 25 M in PBS and left in the dark for 30 min at 37 ∘ C for binding with zinc. Samples incubated with PBS alone were used for autofluorescence evaluations of HepG2 cells. The solution was removed and the coverslips were washed twice with PBS and once with distilled water. The dried coverslips were subsequently mounted on glass slides using glycerol and PBS (9 : 1), containing 2.5% (v/v) 1,4-diazabicyclo[2.2.2]octane solution (DABCO) to reduce fluorescence quenching.
Slides were examined with a fluorescent microscope (Zeiss Axioplan) utilizing a standard UV filter set. All images were acquired by a digital camera (CoolSnap-ProColors Media Cybernetics, Bethesda, MA, USA) and stored by Image Proplus software (Media Cybernetics).
## Spectrofluorimetry.
Spectrofluorimetry experiments were performed essentially according to Coyle and coworkers [bib_ref] Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship..., Coyle [/bib_ref] with minor modifications. HepG2 cells were seeded in 165 cm 2 flasks (3 × 10 6 cells/flasks) and left to recover for 24 hrs before treatments with zinc or cadmium, as described in par.
## Microarray expression profiling.
In this study we reanalyze the gene expression data that we recently published [bib_ref] Whole genome analysis and microRNAs regulation in HepG2 cells exposed to cadmium, Fabbri [/bib_ref] and submitted to NCBI repository (accession number GSE3128).
The whole gene expression of HepG2 cells treated with 10 M Cd was analyzed by Agilent microarray. In order to detect expression changes between treated and control cells the moderated test was applied. Moderated statistics were generated by Limma Bioconductor package. Modulated genes were chosen as those with log 2 fold change greater than 2 and a false discovery rate (Benjamini and Hochberg's method) corrected value smaller than 0.05 [bib_ref] Linear models and empirical Bayes methods for assessing differential expression in microarray..., Smyth [/bib_ref]. All of the above computations were conducted using the statistics programming environment.
A validation of ten genes regulated by 10 M Cd was done by a real-time quantitative PCR (qPCR) analysis on the same RNA samples that were used for the microarray. The trends of all validated genes were confirmed as presented in our recent paper [bib_ref] Whole genome analysis and microRNAs regulation in HepG2 cells exposed to cadmium, Fabbri [/bib_ref].
The regulated genes have been mapped on the KEGG (Kyoto Encyclopedia of Genes and Genomes) PATHWAY, a collection of manually drawn pathway maps representing molecular interactions and reaction networks [bib_ref] KEGG for representation and analysis of molecular networks involving diseases and drugs, Kanehisa [/bib_ref].
The heat map for visualization of expression profiles was performed with TM4 system for microarray data analysis. In the map, red and green colours means higher and lower expression levels [bib_ref] TM4: a free, opensource system for microarray data management and analysis, Saeed [/bib_ref].
## Micrornas expression
Profiling. RNA was extracted using the MIRVANA kit (AMBION) and was reverse transcribed with Taqman MicroRNA Reverse Transcription Kit using Megaplex RT Primers (Applied Biosystems).
MicroRNA expression was measured and quantified using microfluidic cards (Taqman ArrayMicroRNA Cards, set A, V2.2 and set B, V3, Applied Biosystems) allowing the detection of about 754 unique assays specific and four candidate endogenous control assays (Applied Biosystems, Foster City, CA). Samples of control and 10 M Cd were tested. Analyses were carried out on the ABI Prism 7900HT Sequence Detection System (SDS) (Applied Biosystems). MicroRNA levels were normalized to endogenous control U6 (Applied Biosystems). MiRNAs with a threshold cycle <33 that showed a log 2 fold change greater than two in samples treated with cadmium as compared to control samples were considered as induced.
# Statistical analysis.
All experiments were performed in triplicate, and two or three different technical replicates were analyzed in each experiment. Data are expressed as mean ± SD. Student's t-test was used for sample comparison, and the software package Statgraphics Plus version 5.0 was used (Statistical Graphics Corp., Manugistic Inc., Rockville, MD, USA).
# Results and discussion
## Cd exposure in hepg2 cells increases intracellular labile zn: qualitative evaluations and intracellular distribution.
To provide evidence that exposure of HepG2 cells to Cd induces an increase of intracellular zinc, we have used the zincspecific fluorescent probe Zinquin to visualize by microscopy and to measure by spectrofluorimetry the fluorescence intensities. "Free" zinc concentration (where the term free is referred to as freely available, zinc readily bound to chelating agents) is maintained by a complex buffering system in the picomolar to nanomolar range in cells grown under normal conditions, while the total zinc in these cells is evaluated in the high M [bib_ref] Zinc-buffering capacity of a eukaryotic cell at physiological pZn, Krężel [/bib_ref] [bib_ref] Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis, Outten [/bib_ref] [bib_ref] Genetically encoded FRET sensors to monitor intracellular Zn 2+ homeostasis, Vinkenborg [/bib_ref]. In fact, the main pool of intracellular zinc is bound to a vast population of metalloproteins, referred to as the zinc proteome of the cells, or its excess is stored in zinc-containing vesicles or organelles [bib_ref] Zinc transporters and the cellular trafficking of zinc, Eide [/bib_ref]. In this respect, it should be noted that Zinquin not only binds free zinc (reaction (1)), but can also access Zn-protein with open coordination sites to form fluorescent ternary adducts (reaction (2)), or chelating Zn 2+ from the proteome (reaction (3)) [bib_ref] Reactions of the fluorescent sensor, zinquin, with the zinc-proteome: adduct formation and..., Nowakowski [/bib_ref] [bib_ref] Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection..., Figueroa [/bib_ref] :
[formula] Zn 2+ + 2ZQ → Zn (ZQ) 2(1) [/formula]
Zn-protein + ZQ → ZQ-Zn-protein
Zn-protein + 2ZQ → Zn (ZQ) 2 + apo-protein (3) However, most of the zinc bound to proteins is unreactive toward Zinquin. The term "labile" will be used in the following to indicate all of the Zn(II) sensed by Zinquin. The displacement of Zn(II) from the Zn-proteome by Cd(II) and its labilization has been recently demonstrated on the isolated proteome extracted by pig kidney LLC-PK1 cells by using Zinquin as a fluorescent probe [bib_ref] Toxic metal proteomics: reaction of the mammalian zinc proteome with Cd 2+, Namdarghanbari [/bib_ref]. Here, we are investigating the effect of Cd exposure on the Zn-homeostasis in intact human cells. HepG2 cells were exposed to noncytotoxic Cd concentrations (0.1 and 10 M) and to Zn concentrations representing an overload of the metal (50 and 170 M). Food and water are major sources of Cd intake for nonoccupationally exposed population. This metal cannot undergo metabolic degradation, is poorly excreted, and is retained in the liver with a very long biological half life (20-30 years), thus providing us with the fact that the maximum concentration used (10 M) may be compatible with bioaccumulation in the liver. On the other hand, since zinc is an essential element, the cells can buffer high concentrations of this metal. Thus, with the goal of inducing in our cells an observable effect, we exposed HepG2 to high nonphysiological zinc concentrations, up to 170 M, that was shown not to be cytotoxic [bib_ref] Copper and zinc uptake and hsp70 expression in HepG2 cells, Urani [/bib_ref] [bib_ref] Cytotoxicity and induction of protective mechanisms in HepG2 cells exposed to cadmium, Urani [/bib_ref]. Moreover, the exposure to 10 M Zn provided the results for a direct comparison to the equimolar concentration of Cd (10 M). Low physiological labile zinc in the control HepG2 (grown in complete medium) incubated with Zinquin was confirmed as shown by fluorescence microscopy undetectable zinc levels [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref]. Cells without Zinquin probing were also analyzed for the evaluation of the background autofluorescence [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref].
The growth of HepG2 cells for 24 hrs in medium containing 10 or 50 M Zn [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref] and 1(e), resp.) or containing 0.1 M Cd [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref] led to a very low and almost comparable to that of controls or slightly increased fluorescence related to intracellular labile Zn. On the contrary, a dramatic increase in fluorescence intensity was observed when the cells were exposed for 24 hrs to the highest Zn concentration [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref] , 170 M Zn), as expected. In these samples, the fluorescence was predominantly distributed in the cytoplasm and in the perinuclear region and had a punctate appearance, as previously reported in other cell types [bib_ref] Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship..., Coyle [/bib_ref] [bib_ref] Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection..., Figueroa [/bib_ref]. Notably, an intense and punctuate fluorescence was observed in HepG2 cells grown in culture medium containing 10 M Cd [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref] , showing the high increase of intracellular labile zinc when HepG2 cells are exposed to Cd. These punctuate, vescicular-like structures of high fluorescence intensity were previously designated "zincosomes, " possibly representing late endosomes, at least in certain cells type. These structures may have a role in detoxifying excess zinc increased by toxic agents [bib_ref] Functions of zinc in signaling, proliferation and differentiation of mammalian cells, Beyersmann [/bib_ref] [bib_ref] Zinc transporters and the cellular trafficking of zinc, Eide [/bib_ref] , as in our cells by Cd.
## Quantification of intracellular labile zinc in cd-and zn-treated cells.
The assessment of the amount of intracellular labile Zn in HepG2 cells exposed to excess of Zn or to Cd was carried out by spectrofluorimetric measurements. The fluorescence enhancement after Zinquin binding to intracellular labile Zn was measured as arbitrary units and expressed as a relative increase of the signal with respect to basal control values (cells grown in complete medium). Indeed, it was necessary to express the fluorescence intensities as a Δ% versus controls due to fluctuations of basal conditions possibly due to variations in zinc content amongst different experiments. However, despite this basal difference, the increase was fairly constant within a specific zinc or cadmium treatment, as demonstrated by the relatively small standard deviations obtained in the different biological replicates. [fig_ref] Figure 2: a summary of all fluorescence intensities expressed as mean ± SD of... [/fig_ref] gives an overview of the increment of intracellular labile Zn ions in all treated samples, expressed as fluorescence intensities, while, in intracellular labile Zn in cells grown in the presence of 10 M Cd: a mean value of +93% versus basal fluorescence intensities was obtained. To the best of our knowledge, this is the first time that the amount of labile zinc is visualized and quantitatively evaluated in Cd-exposed mammalian cells. In our previous works [bib_ref] Different induction of metallothioneins and Hsp70 and presence of the membrane transporter..., Urani [/bib_ref] [bib_ref] Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure, Urani [/bib_ref] the total intracellular amount of zinc and cadmium in HepG2 cells exposed to 170 M Zn and 10 M Cd for 24 hrs was measured by an analytical technique (ICP-AES). The increment in the total concentrations with respect to the basal values was about 0.075 ppm/10 6 cells for zinc in cells exposed for 24 hrs to 170 M Zn and 0.050 ppm/10 6 cells for cadmium in samples exposed to 10 M Cd. Thus, it can be noted that, at the same exposure time, the uptake of cadmium is only slightly smaller than that of zinc, although cells have grown in the presence of a concentration of cadmium which is about 20 times smaller than that of zinc. This result clearly indicates that the uptake of cadmium, that uses aspecific transport pathways [bib_ref] Catch me if you can! Novel aspects of cadmium transport in mammalian..., Thévenod [/bib_ref] , occurs to a significantly larger extent to that of zinc, whose concentration is, on the contrary, strictly regulated. This result is consistent with the large increment of the fluorescence intensity of HepG2 cells exposed to 10 M Cd as due to cadmium to zinc replacement in proteins and mobilization of the latter metal ion, a mechanism previously proposed [bib_ref] Cadmium induces conformational modifications of wild-type p53 and suppresses p53 response to..., Méplan [/bib_ref] and recently demonstrated in a prokaryotic zinc-finger domain [bib_ref] Zinc to cadmium replacement in the prokaryotic zinc-finger domain, Malgieri [/bib_ref] , and proteome extracted from pig kidney LLC-PK1 cells [bib_ref] Toxic metal proteomics: reaction of the mammalian zinc proteome with Cd 2+, Namdarghanbari [/bib_ref].
## Biological effects of labile zinc increase in hepg2 cells:
Genes and MicroRNAs Regulation. Prompted by the results discussed above, we have reanalyzed the molecular data and pathways to better understand the mechanisms of cadmium biological effects and to correlate the role of zinc in gene and molecular regulation. The elevated concentrations of Zn(II) visualized and measured in HepG2 cells after 24 hrs of Cd(II) exposure activated a number of zinc-related processes. One of the top regulated genes in our samples, as identified by the Microarray expression profiling [fig_ref] Figure 3: The genes regulated by 10 M Cd treatment and belonging to adherens... [/fig_ref] , is Snail1, with an upregulation of +3.6 fold change with respect to controls. Snail1 is included in many biological pathways, one of which is the adherens junctions, represented in [fig_ref] Figure 4: Representation of the adherens junction pathway map from KEGG [/fig_ref]. Snail1 belongs to a superfamily of zinc-finger transcription factors involved, among others, in the acquisition of invasive and migratory properties during tumor progression [bib_ref] The Snail superfamily of zinc-finger transcription factors, Nieto [/bib_ref]. The mechanism of Snail1 activation has been recently related in breast cancer cells to zinc influx and intracellular zinc increase, either directly or indirectly [bib_ref] A mechanism for epithelial-mesenchymal transition and anoikis resistance in breast cancer triggered..., Hogstrand [/bib_ref] , thus strongly supporting our data.
Other upregulated genes in the adherens junction pathway [fig_ref] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot... [/fig_ref] , TGF-R (1 fold change), and the two members of the Rho-family GTPase, Rac (1.5 fold change), and cdc42 (1.5 fold change). The MET tyrosine kinase receptor (known also as the HGF receptor) promotes, among others, cancer growth and metastasis by conveying proliferative, antiapoptotic, and promigratory signals (see for a review [bib_ref] MET signalling: principles and functions in development, organ regeneration and cancer, Trusolino [/bib_ref]. The transforming growth factor receptor (TGF-R) is a member of the TGF-signalling pathway, involved in cell proliferation and cell migration functions. In addition, TGF-is a signalling molecule implicated in Snail1 activation and in our samples exposed to Cd is upregulated with a 1.4 fold change [bib_ref] TGF in cancer, Massagué [/bib_ref]. The Rac and the cdc42 genes promote lamellipodia and filopodia formation, thus regulating cell migration through cytoskeleton remodelling (see for a review [bib_ref] Rho GTPases-masters of cell migration, Sadok [/bib_ref].
The role of elevated zinc concentrations was demonstrated to affect gene expression in cancer cells at posttranscriptional level [bib_ref] Zinc at cytotoxic concentrations affects posttranscriptional events of gene expression in cancer..., Zheng [/bib_ref]. Thus, intriguing results linking zinc levels and gene expression can emerge from microRNAs (miRNA) analysis in HepG2 cells exposed to cadmium. The miRNA are small noncoding RNAs initially transcribed and processed in the nucleus as precursors. These molecules are then exported to the cytoplasm where they become mature miRNA of about 23 nucleotides whose main function is to negatively regulate gene expression at the posttranscriptional level by repression of protein translation or promotion of mRNA degradation [bib_ref] MicroRNAs: target recognition and regulatory functions, Bartel [/bib_ref]. In addition, miRNAs by targeting multiple transcripts play a crucial role in tumorigenesis and cancer progression [bib_ref] Aberrant regulation and function of microRNAs in cancer, Adams [/bib_ref]. Even if the role played by zinc in cell growth and proliferation is well known [bib_ref] Functions of zinc in signaling, proliferation and differentiation of mammalian cells, Beyersmann [/bib_ref] , the involvement of this metal in the regulation of gene expression at posttranscriptional level is still to be explored. Recently it was demonstrated that elevated intracellular zinc concentrations modulate the miRNA expression in mammalian cells, with miR-223 among the top downregulated miRNA [bib_ref] Zinc at cytotoxic concentrations affects posttranscriptional events of gene expression in cancer..., Zheng [/bib_ref]. It is important to highlight that miR-223 is involved in cell cycle regulation, proliferation, and survival [bib_ref] MiR-223: infection, inflammation and cancer, Haneklaus [/bib_ref] , thus suggesting the relevance of zinc concentrations in the epigenetic mechanisms of cancer, at least those known up to now.
Two major miRNAs were downregulated in our samples: a miR-34 family member (−1.1 fold change) and a miR-200 family member (−1.2 fold change).
Very interestingly, a decrease in miR-34, which normally antagonizes Snail1, was recently described as part of the p53/miRNA-34 axis. Namely, in the absence of a functional p53 and of a decrease of miRNA-34, Snail1 is upregulated, as we found in our samples. This axis promotes the epithelialmesenchymal transition (EMT) and the invasion program in neoplastic cells [bib_ref] A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial-mesenchymal transition, Kim [/bib_ref]. Further supporting this mechanism are our results in HepG2 cells on miR-200 family. The miR-200 family has been related to the suppression of the epithelial-mesenchymal transition as well. A correlation between downregulation of the miR-200 family member and induction of TGF-1 in renal proximal tubule epithelial cells (NRK-52E) was observed and are responsible for the epithelial-mesenchymal transition [bib_ref] The miR-200 family regulates TGF-1-induced renal tubular epithelial to mesenchymal transition through..., Xiong [/bib_ref]. On the other hand, an overexpression of the family member miR-200b was demonstrated to significantly reduce cellular proliferation and inhibition of cell migration and metastasis in gastric carcinoma cells [bib_ref] MicroRNA-200b regulates cell proliferation, invasion, and migration by directly targeting ZEB2 in..., Kurashige [/bib_ref]. All these data strengthen the correlation between high zinc levels, Sanil1 upregulation, miR-34, and miR-200 family members downregulation.
# Conclusions
We have previously demonstrated [bib_ref] Whole genome analysis and microRNAs regulation in HepG2 cells exposed to cadmium, Fabbri [/bib_ref] [bib_ref] Regulation of metallothioneins and ZnT-1 transporter expression in human hepatoma cells HepG2..., Urani [/bib_ref] that Cd exposure in HepG2 cells modulates genes and proteins responsible for metal homeostasis, such as the metallothioneins (MT) and membrane transporters responsible for the elimination of zinc excess (i.e., ZnT-1). However, we posed [bib_ref] Regulation of metallothioneins and ZnT-1 transporter expression in human hepatoma cells HepG2..., Urani [/bib_ref] a question on ZnT-1 regulation by Cd exposure: how does Cd regulate ZnT-1 levels? We proposed a mechanism of activation by elevated labile zinc concentrations possibly due to the replacement of Zn(II) from the Zn-proteome by Cd(II) and its corresponding labilization. As known, both MT and ZnT-1 are activated by the metal transcription factor-1 (MTF-1) that functions as a metal sensor. MTF-1 in vitro is robustly regulated by zinc but not by cadmium ions, as just published [bib_ref] Zinc'ing sensibly: controlling zinc homeostasis at the transcriptional level, Choi [/bib_ref] ,and further confirming that labile zinc displaced by cadmium is the primary mediator in MTF-1 activation. The regulation of MTF-1 by metals, such as Cd, could be a direct consequence of zinc to cadmium displacement from zinc-containing proteins. The Cd-Zn exchange and the possible substitution of Cd in Znbinding sites were previously described [bib_ref] Cadmium induces conformational modifications of wild-type p53 and suppresses p53 response to..., Méplan [/bib_ref] [bib_ref] Cadmium-zinc exchange and their binary relationship in the structure of Znrelated proteins:..., Tang [/bib_ref]. The results presented here demonstrate the increment of labile zinc levels in the presence of cadmium, supporting the validity of the mechanism proposed above. Further work is underway by our research group to investigate the gene expression and the biological implications of the modulation of these genes, due to the displacement of zinc with cadmium as a function of their concentrations.
Taking into account our present and previously published data, the following mechanisms emerge intracellular labile zinc level, which is a known proliferation signal [bib_ref] Functions of zinc in signaling, proliferation and differentiation of mammalian cells, Beyersmann [/bib_ref]. This response leads to the activation of Snail1, that along with the upregulation of other genes responsible for loss of adherence, represents a signal promoting cell migration and metastasis (present work, [fig_ref] Figure 4: Representation of the adherens junction pathway map from KEGG [/fig_ref] adherens junction); (iii) miR-34 downregulation contributes to the abnormal expression of Snail1, which is normally antagonized by miR-34 and whose pathological expression has been linked to cancer cell epithelial-mesenchymal transition; (iv) miR-200 downregulation contributes to the epithelial-mesenchymal transition as well.
Therefore, our results provide new information on the existing proposed mechanisms for Cd toxicity and biological effects and along with those already reported in the literature open new intriguing interpretations on Cd-induced carcinogenicity.
## Highlights
(i) HepG2 cells exposed to Cd show an increase of intracellular labile zinc. (ii) Labile zinc is visualized and measured by the zinc specific probe Zinquin. (iii) Genes belonging to loss of adherence and cell migration pathways are activated. (iv) Cd exposure affects the expression of miR-34a and miR-200a. - miR-200a
Rac/cdc42 +93% : Overview of cellular and molecular effects of 10 M Cd in HepG2 cells. Cd enters the cells through aspecific sites. Intracellular Cd accumulation determines an increase of labile zinc, a known proliferation signal, and second messenger. The replacement of Zn with Cd in the zinc proteome [bib_ref] Cadmium-zinc exchange and their binary relationship in the structure of Znrelated proteins:..., Tang [/bib_ref] is the hypothetical process underlying the above described mechanism. Genes (Snail, MET, TGF-R, and Rac/cdc42) involved in the loss of cell adherence and also comprised in the mechanism of epithelial-mesenchymal transition are disregulated. MicroRNAs (miR-34a, miR-200a) with tumor-suppressor functions are downregulated. Cd-exposed HepG2 cells have a nonfunctional p53 [bib_ref] Cadmium Impairs p53 Activity in HepG2 Cells, Urani [/bib_ref] that along with miR-34 downregulation represents the disregulated axis in Snail1-dependent epithelial-mesenchymal transition [bib_ref] A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial-mesenchymal transition, Kim [/bib_ref].
[fig] 2. 1: Treated and control cells were harvested by tripsinization, and a small aliquot from the cell suspension was counted by a Coulter counter to have an estimate of cells number in each sample. All cell suspensions were centrifuged (200 ×g, 10 min) and the pellets resuspended in 2 mL of Hanks Balanced Salt Solution (HBSS: 137 mM NaCl, 5.4 mM KCl, 0.3 mM KH 2 PO 4 , 0.33 mM Na 2 HPO 4 , 1.23 mM CaCl 2 × 2H 2 O, 0.81 mM MgSO 4 × 7H 2 O, 4.2 mM NaHCO 3 , and 5.6 mM D-Glucose). The cells in suspension were incubated with Zinquin (final concentration 25 M) in HBSS for 40 min at 37 ∘ C. Then the cells were washed three times with HBSS, and fluorescence was measured at an excitation wavelength of 370 nm and emission wavelength of 490 nm (slit width 5-10 nm) in a Jasco FP-777 spectrofluorimeter. Fluorescence intensities are expressed as arbitrary units and are normalized on a fixed number of cells (100.000 cells). [/fig]
[fig] Figure 1: Microscopy images of HepG2 cells loaded with 25 M of the zinc specific fluorescent probe Zinquin for intracellular visualization and distribution of labile zinc. HepG2 cells grown in control medium without Zinquin were used for autofluorescence visualization (a). Cells grown in control medium show undetectable basal levels of loosely bound zinc (b). HepG2 cells grown in medium containing, respectively, 0.1 (c) and 10 M (f) Cd for 24 hrs; (d, e, and g): cells exposed to increasing zinc concentrations (10, 50, and 170 M, resp.). Microscope magnification 400x. [/fig]
[fig] Figure 2: a summary of all fluorescence intensities expressed as mean ± SD of Δ% of basal fluorescence is presented.At low zinc concentrations (10 and 50 M), the intensity of Zinquin fluorescence is relatively low and features mean values of +21% versus basal fluorescence intensities, in agreement with a strict regulation of the cellular zinc content and distribution[10]. Zinquin fluorescence was significantly raised (+225% versus controls, < 0.05) in HepG2 grown in the presence of the highest zinc concentration (170 M), used as a positive control for zinc excess. The most interesting result is the fluorescence intensity of Zinquin bound to * Spectrofluorimetric quantification of intracellular labile zinc. Samples are expressed as Δ% ± SD versus controls (cells grown in control medium in the presence of 25 M Zinquin). * < 0.05 significantly different (Student's -test) from the corresponding Cd or Zn treatment at lower concentration. [/fig]
[fig] Figure 3: The genes regulated by 10 M Cd treatment and belonging to adherens junction pathway represented in Figure 4 are displayed. (a) Heat map graphically representing the gene regulation. Each row represents a mRNA (green = low expression, red= high expression). (b) In the table the data are expressed as log 2 FC (fold change), comparing HepG2 treated with 10 M Cd to control cells. [/fig]
[fig] Figure 4: Representation of the adherens junction pathway map from KEGG. Upregulated genes in HepG2 cells by 10 M Cd treatment for 24 hrs are colored in the signaling pathway map. [/fig]
[table] Table 1 Cd 0: Fluorescence intensities (arbitrary units) of labile zinc in cadmium-or zinc-treated HepG2 cells. The results are expressed as increment of basal (control) values ± SD of at least three independent biological replicates. * P < 0.05 significantly different (Student's -test) from the corresponding Cd or Zn treatment at lower concentration. [/table]
|
Interaction of drought‐ and pathogen‐induced mortality in Norway spruce and Scots pine
Pathogenic diseases frequently occur in drought-stressed trees. However, their contribution to the process of drought-induced mortality is poorly understood. We combined drought and stem inoculation treatments to study the physiological processes leading to drought-induced mortality in Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) saplings infected with Heterobasidion annosum s.s. We analysed the saplings' water status, gas exchange, nonstructural carbohydrates (NSCs) and defence responses, and how they related to mortality. Saplings were followed for two growing seasons, including an artificially induced 3-month dormancy period. The combined drought and pathogen treatment significantly increased spruce mortality; however, no interaction between these stressors was observed in pine, although individually each stressor caused mortality. Our results suggest that pathogen infection decreased carbon reserves in spruce, reducing the capacity of saplings to cope with drought, resulting in increased mortality rates. Defoliation, relative water content and the starch concentration of needles were predictors of mortality in both species under drought and pathogen infection. Infection and drought stress create conflicting needs for carbon to compartmentalize the pathogen and to avoid turgor loss, respectively. Heterobasidion annosum reduces the functional sapwood area and shifts NSC allocation patterns, reducing the capacity of trees to cope with drought.
# | introduction
Global change is posing increasing challenges for forest ecosystems.
Emergent and/or invasive pests and diseases can be the primary cause of extensive tree death (e.g., [bib_ref] Chestnut blight: the classical problem of an introduced pathogen, Anagnostakis [/bib_ref] [bib_ref] The invasive forest pathogen Hymenoscyphus fraxineus boosts mortality and triggers niche replacement..., Díaz-Yáñez [/bib_ref] [bib_ref] Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and..., Rizzo [/bib_ref]. Diseases not only emerge following the introduction of pathogens to new environments but also by modifying their virulence due to climate-driven processes [bib_ref] Drivers of emerging fungal diseases of forest trees, Ghelardini [/bib_ref]. Therefore, it is increasingly important both to predict the risk of an acceleration in the development of existing diseases linked to climate change and to understand pathogen-induced tree death. In turn, drought is a well-recognized driver of large-scale tree mortality and long-term ecosystem dynamics [bib_ref] A global overview of drought and heatinduced tree mortality reveals emerging climate..., Allen [/bib_ref] [bib_ref] On underestimation of global vulnerability to tree mortality and forest die-off from..., Allen [/bib_ref] [bib_ref] Hanging by a thread? Forests and drought, Brodribb [/bib_ref] [bib_ref] Research frontiers for improving our understanding of drought-induced tree and forest mortality, Hartmann [/bib_ref]. Although droughtinduced tree mortality has been intensively studied over the past two decades (for a review see [bib_ref] A multi-species synthesis of physiological mechanisms in drought-induced tree mortality, Adams [/bib_ref] [bib_ref] Triggers of tree mortality under drought, Choat [/bib_ref] , the pathways leading to tree mortality are not completely understood yet. Often tree mortality arises from an interaction between biotic (pests and pathogens) and abiotic stress agents, rather than being the result of a single disturbance factor [bib_ref] Temperate forest health in an era of emerging megadisturbance, Millar [/bib_ref] [bib_ref] Water stress predisposition to disease, and overview, Schoeneweiss [/bib_ref] [bib_ref] Interacting effects of global change on forest pest and pathogen dynamics, Simler-Williamson [/bib_ref] [bib_ref] Which trees die during drought? The key role of insect hosttree selection, Stephenson [/bib_ref]. This adds complexity to our understanding of mortality pathways, and hence, modelling mortality and future forest dynamics remains challenging [bib_ref] Tree mortality submodels drive simulated long-term forest dynamics: assessing 15 models from..., Bugmann [/bib_ref] [bib_ref] Why is tree drought mortality so hard to predict?, Trugman [/bib_ref].
Tree mortality under drought conditions may be a consequence of hydraulic failure, the exhaustion of carbohydrate reserves, impaired phloem transport or a combination of these factors [bib_ref] A multi-species synthesis of physiological mechanisms in drought-induced tree mortality, Adams [/bib_ref] [bib_ref] Triggers of tree mortality under drought, Choat [/bib_ref] [bib_ref] Greater focus on water pools may improve our ability to understand and..., Martinez-Vilalta [/bib_ref] [bib_ref] Drought impacts on tree phloem: from cell-level responses to ecological significance, Salmon [/bib_ref]. In the presence of severe drought, dramatic hydraulic failure occurs, with tree mortality typically associated with a loss of hydraulic conductivity of at least 60% [bib_ref] A multi-species synthesis of physiological mechanisms in drought-induced tree mortality, Adams [/bib_ref] ; however, the mechanistic link remains to be established [bib_ref] Hydraulic failure and tree mortality: from correlation to causation, Mantova [/bib_ref]. An impaired conductive system interrupts the soil-root hydraulic continuum, which jeopardizes the delivery of sufficient water to the canopy, leading to critical cell dehydration [bib_ref] No need for pipes when the well is dry-a comment on hydraulic..., Körner [/bib_ref] [bib_ref] Greater focus on water pools may improve our ability to understand and..., Martinez-Vilalta [/bib_ref]. Carbohydrates are required for osmoregulation to avoid turgor loss and may have a relevant role in maintaining the tree's water status under declining water availability [bib_ref] How do trees die? A test of the hydraulic failure and carbon..., Sevanto [/bib_ref]. Even within a species, the role of NSCs in the pathway to mortality may vary depending on the tree's strategy for mitigating drought stress, with some trees suffering fast, catastrophic hydraulic failure but unaltered carbohydrate reserves, while others show a clear contribution of hydraulic constraints and carbohydrate use in the mortality process .
When necrotrophic fungi infect the stem or roots, they can destroy the cambium and vascular tissue of the infected host, potentially inducing hydraulic impairment and affecting carbon and water transport in the tree [bib_ref] The effect of fungal pathogens on the water and carbon economy of..., Oliva [/bib_ref]. Trees defend their sapwood from necrotrophic fungal pathogens by forming Cexpensive barriers [bib_ref] Do trees use reserve or newly assimilated carbon for their defense reactions?..., Guérard [/bib_ref] , resulting in areas of nonconducting sapwood. As stated by Alex L. [bib_ref] Compartmentalization: a conceptual framework for understanding how trees grow and defend themselves, Shigo [/bib_ref] : 'Trees do not cast off dead and dying parts. Trees form boundaries between dying parts and the healthy frame of the wood and the inner bark'. In the sapwood, an injury or an infection may be compartmentalized by the formation of a reaction zone [bib_ref] Tansley Review No. 87: antimicrobial defences in the wood of living trees, Pearce [/bib_ref]. In the reaction zone, the sapwood is both physically and chemically modified, involving de novo synthesis of secondary metabolites from NSCs, which relies on the availability of C at the site of infection [bib_ref] Do trees use reserve or newly assimilated carbon for their defense reactions?..., Guérard [/bib_ref]. When drought coincides with pathogens or pest attacks, the role of C depletion in the pathway to mortality is thought to be amplified [bib_ref] Drought predisposes piñon-juniper woodlands to insect attacks and mortality, Gaylord [/bib_ref] [bib_ref] Mechanisms of piñon pine mortality after severe drought: a retrospective study of..., Gaylord [/bib_ref] [bib_ref] Mechanisms of plant survival and mortality during drought: why do some plants..., Mcdowell [/bib_ref] [bib_ref] The effect of fungal pathogens on the water and carbon economy of..., Oliva [/bib_ref]. On the one hand, droughtinduced stomatal closure decreases C uptake. On the other hand, C may be allocated to distal tissues suffering pathogen infection.
Thus, in addition to the pathogen-induced loss of sapwood, the increased C demand for defence reactions can potentially accelerate drought-induced tree death in the case of necrotrophic pathogens infecting the stem or roots. However, there is little empirical evidence to support these hypotheses.
The necrotrophic pathogenic fungi of the Heterobasidion annosum sensu lato species-complex cause root and butt rot in most species of conifers trees in the northern hemisphere . Both Heterobasidion parviporum and Heterobasidion annosum sensu stricto, two of the European species that belong to this complex, are strong pathogens on Norway spruce (Picea abies (L.)
Karst.). Heterobasidion parviporum is specialized in Norway spruce, whereas H. annosum s.s. is usually associated with Scots pine (Pinus sylvestris L.) but can also infect and damage Norway spruce [bib_ref] Biology, epidemiology, and control of heterobasidion species worldwide, Garbelotto [/bib_ref]. The species in this complex primarily enter the tree via wounds and colonize the sapwood of living trees.
Conifers differ in the way that they respond to these pathogens. In Norway spruce, H. annosum s.s. attacks the sapwood and colonizes the heartwood of the root system and the lower part of the stem.
From the inner sapwood, the pathogen spreads outwards and can reach and damage the phloem. Norway spruce compartmentalizes the pathogen by creating a reaction zone impregnated with secondary metabolites. By contrast, in Scots pine, the pathogen cannot colonize the heartwood and occurs only in the sapwood and the phloem [bib_ref] Biology of Heterobasidion annosum, Korhonen [/bib_ref]. Scots pine typically shows a weaker defence response to infection by H. annosum s.s. than spruce [bib_ref] Anatomical-based defense responses of Scots pine (Pinus sylvestris) stems to two fungal..., Nagy [/bib_ref] , and the reaction zone may include resin inclusions and discolouration but is not always visible [bib_ref] Incidence and impact of root infection by Heterobasidion spp., and the justification..., Wang [/bib_ref]. Analyses of the interaction between H. annosum s.l. and drought in conifers are scarce. Heterobasidion parviporum has been suggested to increase drought stress in Norway spruce [bib_ref] Fungal root pathogen (Heterobasidion parviporum) increases drought stress in Norway spruce stand..., Gori [/bib_ref]. In addition, drought has been shown to lead to larger necrotic lesions in trees inoculated with either H. parviporum or H. annosum s.s. compared with nondrought conditions [bib_ref] Low water availability increases necrosis in Picea abies after artificial inoculation with..., Terhonen [/bib_ref]. However, the effect of drought on the development of H. annosum s.s. decay in the sapwood and the effects of these combined stresses on the mortality process in Norway spruce and
Scots pine remain to be tested. The drought responses of both tree species have been described. They both present strict stomatal control and progressive leaf loss under limiting water availability [bib_ref] The influence of soil moisture on water potential, transpiration and photosynthesis of..., Havranek [/bib_ref]. Therefore, the two tree species, together with their shared pathogen H. annosum s.s., provide a suitable system for studying the interaction between drought and necrotrophic pathogens.
Here, we used the host species Norway spruce and Scots pine and the pathogen H. annosum s.s. as models to study the interaction between pathogenic infection and drought as drivers of tree mortality in conifers, focusing on the effects of these stressors on the C and water balance of saplings. We tested the following Hypotheses: (i) that the interaction between drought and infection by a necrotrophic pathogen has an additive effect and increases the risk of mortality compared with either stressor alone; (ii) this increased mortality risk is due to the three following nonexclusive, interactive processes: (1) drought reduces C reserves and impairs the capacity of trees to build up defence barriers to compartmentalize the pathogen;
(2) defence needs associated with pathogenic infection reduce the amount of C available to cope with drought, which eventually impairs water status; and (3) pathogen infection affects hydraulic performance due to a reduction in the sapwood cross-sectional area.
## | materials and methods
## | plant material and experimental design
A total of 46 P. abies (L.) Karst. and 47 P. sylvestris L. open-pollinated saplings that had been grown in a field at a commercial nursery and produced following standard procedures (Härnevi Nursery) were transferred to 40-L pots and grown in a thermostatically controlled greenhouse (SLU) from May to August 2017. The air temperature fluctuated between 16°C and 30°C during the day and between 8°C and 18°C during the night, depending on the weather conditions. Saplings were 6 years old and 1.5-2.0 m tall with a mean diameter of ca. 5 cm (at 10 cm from the collar). On 22 August 2017, half of the saplings were inoculated by inserting infected wood plugs into holes drilled in the trunk, which were then sealed with silicon. Six 5-mm holes 2-cm apart were drilled in each sapling. Inoculum was produced by placing autoclaved wood plugs in sealed Petri dishes colonized with the H. annosum s.s. isolate Mj 87, which has been shown to be highly pathogenic on both Norway spruce and Scots pine in previous trials [bib_ref] Intraspecific variation in Heterobasidion annosum for growth in sapwood of Picea abies..., Swedjemark [/bib_ref] ; H. annosum s.s. is referred to as the Pintersterility group of H. annosum s.l. in this publication). The Petri dishes were incubated at 20°C in the dark. The other half of the saplings served as a control and were treated by inserting sterile plugs into the holes. The saplings were watered once a week both before (May-August 2017) and during the experiment (August 2017-August 2018). Two different watering regimes were used corresponding to a gravimetric soil water content of ca. 20% (drought) or 40% (well-watered). The resulting treatment combinations were: well-watered/noninoculated (W/NI; N = 10 for spruce and 7 for pine); well-watered/inoculated (W/I; N = 9 for each species); drought/noninoculated (D/NI; N = 10 for spruce and 8 for pine); and drought/inoculated (D/I; N = 10 for spruce and 9 for pine).
Soil moisture was assessed the day before watering with a Decagon GS1 sensor coupled to a ProCheck Handheld Reader. Soil moisture was measured in mV and was converted to volumetric soil water content (VWC) by a calibrated curve obtained from soil samples with known VWC. Transformation from VWC to pF (i.e., the logarithm of the absolute value of the soil matric potential) and then to water potential was obtained from a characteristic soil curve conducted by the METER Group AG.
The growing conditions during the experiment (from 22
August 2017 until 2 August 2018) were 16 h of light at 22°C and 8 h of dark at 18°C. We also simulated a 10-week dormancy period with the following conditions: 3 weeks at temperatures between 10°C and 15°C and no light from 11 December 2017 to 2 January 2018; 6 weeks at 5°C and no light from 3 January to 18 February 2018; and 1 week with light and temperatures between 10°C and 15°C from 19 to 26 February 2018 (Supporting Information: .
## | physiological measurements
The saplings' physiological status and defoliation were monitored once before performing the treatments and then from 17 October 2017, onwards, that is, 2 months after the treatments were performed, and then every 2 weeks for a year. During the 10-week dormancy period, only two measurements were made (see Supporting Information: for an overview of measurements). We measured the net assimilation rate (A), stomatal conductance (g s ) and
transpiration rate (E) on fully expanded current-year needles using an LI-COR LI-6400XT gas-exchange analyser system (LI-COR). Chamber conditions were kept at 400 μmol CO 2 m −2 s −1 , 1700 μmol photons m −2 s −1 , 40% relative humidity and 28°C block temperature.
Measurements were taken once steady-state gas exchange was achieved. Instantaneous photosynthetic WUE was calculated as the ratio A/E. In addition, we assessed defoliation by assigning a grade from 0 to 4 (0, no defoliation; 1, 25% defoliated; 2, 50% defoliated; 3, 75% defoliated; 4, completely defoliated). We considered defoliation caused by the treatments to be any excess defoliation relative to the control treatment. Hence, we interpreted that a treatment caused defoliation when this was significantly higher than that of the control treatment. To analyse the risk of mortality associated with each treatment and species (Hypothesis i), we also recorded mortality every 2 weeks based on complete defoliation, cambial death (presence of necrotic cambium after removing the bark) and extremely low (undetectable) gas exchange.
On each sampling date, a branch at mid-height on each sapling was chosen. One twig on this branch was used to measure midday water potential (Ψ md ) and a fascicle was sampled to measure the needle relative water content (RWC) (see Supporting Information: for sampling dates). In addition, one spruce twig was sampled on four sampling dates and one pine twig was sampled on two sampling dates to determine the NSC level of a fascicle. One spruce and one pine twig were also sampled on two dates to obtain measurements of xylem hydraulic conductivity (K h ) and native embolism. For the RWC measurements, needles were first placed in vials on ice and weighed in the laboratory to obtain the fresh weight and then rehydrated overnight at 4°C in darkness, blotted dry on a paper towel and weighed again to obtain the turgid weight. Finally, needles were dried in the oven at 70°C for 24 h and reweighed to obtain the dry weight. The RWC was calculated as: (fresh weight − dry weight)/(turgid weight − dry weight) × 100. For Ψ md measurements, sampled twigs were placed immediately in plastic bags and stored in a cold container until they were measured (within 1-2 h) using a Scholander-type pressure chamber (Digital 80 Bar Portable Plant Moisture System; Skye Instruments Ltd.).
Xylem conductivity and native embolism were measured twice: before treatments were applied and 3.5 months after treatment. For K h measurements, twigs were placed inside plastic bags with a damp cloth, which were then placed in a cooler. In the laboratory, twig segments were recut underwater to obtain a segment of ca. 10 cm with no lateral branches. We debarked the proximal end and put it in a degassed perfusion solution (10 mM KCl and 1 mM CaCl 2 in ultrapure filtered water), which was placed ca. 50 cm above the segment to generate a pressure gradient of ca. 5 kPa, and let water flow for ca. 10 min. We collected and weighed the solution at the distal end for the next 10 min and then repeated this procedure until consecutive measurements changed by less than 20%. We then calculated K h (m 4 MPa -1 s -1 ) as the water flow multiplied by the segment length divided by the pressure gradient. We rehydrated the samples for 48 h and then repeated the procedure to determine the maximum hydraulic conductivity (K h, max ), and sapwood-specific maximum conductivity (K s, max ) by dividing K h, max by the crosssection. We calculated the percentage loss of conductivity (PLC) as 100 × (1 − K h /K h, max ).
## | nsc measurements
NSC measurements were only performed on living saplings. Each sampled twig and its current-year needles (see Section 2.2 for details of twigs used for NSC sampling) were microwaved immediately after sampling for 90 s at 600 W to stop the enzymatic activity and then subsequently oven-dried for 72 h at 70°C. Spruce stem and root samples were taken from surviving trees at the end of the experiment. Stem samples were obtained by cutting out a ca. 1 cm 3 piece of sapwood ca. 8 cm above the uppermost hole of the inoculation treatments. Root samples were obtained by cutting off pieces of roots that were less than 0.5 cm in diameter. Stem and root samples were immediately frozen until processed. All samples were milled in a ball mill (Retsch M400) to obtain a fine and homogeneous powder. Soluble sugars (SS) were extracted with 80% (v/v) ethanol in a water bath at 60°C and their concentration was determined colorimetrically as described by [bib_ref] An improved colorimetric method to quantify sugar content of plant tissue, Buysse [/bib_ref]. Starch and complex sugars remaining in the undissolved pellet after ethanol extractions were enzymatically reduced to glucose using 0.5% amyloglucosidase (Fluka 10115) and analysed following [bib_ref] Seasonal dynamics of non-structural carbohydrates in two species of Mediterranean sub-shrubs with..., Palacio [/bib_ref]. NSCs measured after ethanol extraction were considered to be SS and carbohydrates measured after enzymatic digestion were considered to be starch.
Both components were expressed in glucose equivalents. The total NSC concentration of a sample was considered to be the sum of SS and starch.
## | infection assessments
We monitored lesion length 2 and 3 months after treatments were performed (see Supporting Information: . The outer bark was slightly scratched to reveal the colonized phloem tissue. The radius of the ellipsoid-shaped necrosis was measured horizontally in all spruce saplings. Most of the inoculated pine saplings were early girdled, and hence, we did not monitor lesion length in pine saplings.
To test the relationship between C stores and the defence reaction in spruce sapwood, which involves the formation of a reaction zone to try to compartmentalize the infection, we analysed three cross-sections of the treated part of the stem (i.e., the top, middle and bottom of the inoculated section) per sapling (Supporting Information: . We sprayed these sections with the pH indicator 2,6 dichlorophenolindophenol to discriminate the reaction zone (tissues with a pH above 6.0 were dyed blue) from decayed zones (tissues with a pH of less than 5.5 were dyed red) [bib_ref] Changes in sapwood of roots of Norway spruce, attacked by Fomes annosus...., Johansson [/bib_ref] [bib_ref] Physiochemical and molecular features of the necrotic lesion in Heterobasidion-Norway spruce pathosystem, Liu [/bib_ref] [bib_ref] Transcriptional responses of Norway spruce (Picea abies) inner sapwood against Heterobasidion parviporum, Oliva [/bib_ref]. Dyed and undyed images were used to calculate the proportion of each section corresponding to functional sapwood, decayed sapwood, reaction zone and active reaction zone based on pixel counts after manual classification using Adobe Photoshop 21.2.4 (Adobe Systems Inc.). In some cases, decayed sapwood was clearly divided into two consecu- . Inoculated spruce saplings that were subjected to drought suffered mortality sooner than those that received the well-watered treatment and the mortality rate accelerated after the dormancy period (30% vs. 90% of accumulated deaths before and after dormancy, respectively) . Spruce saplings subjected to drought alone suffered mortality later than saplings subjected to the other treatments, with the first deaths occurring 6 months after the onset of treatments and peaking in April 2018, coinciding with bud flush .
Drought influenced the capacity of trees to defend themselves from the pathogen. Two and three months after inoculation, droughtinoculated saplings showed larger necrotic lesions than well-watered inoculated saplings (Supporting Information: . In spruce, the survival of inoculated saplings at the end of the experiment was associated with the capacity to create a larger reaction zone in the sapwood to compartmentalize the pathogen and to protect the functional sapwood (31% vs. 3% of the cross-section in surviving and dead saplings, respectively). The size of the reaction zone was inversely correlated with the area colonized by the pathogen, and .
Drought reduced the Ψ md , A and g s of saplings and led to a higher PLC 3 months after treatments compared with that of control saplings (Supporting Information: . Inoculation alone did not cause any hydraulic impairment, with no increase observed in the PLC of xylem in inoculated-only spruce and all physiological variables were similar to those of controls (RWC, Ψ md , g s , A; and Supporting Information: and .
However, inoculation exacerbated the hydraulic stress seen in saplings subjected to drought given that lower Ψ md values were found in drought-inoculated saplings than in those only subjected to drought and Supporting Information: .
Gas exchange variables and the water content of needles were common predictors of mortality rates across the three stress treatments in spruce . Defoliation was significant only in the drought-inoculation treatment, where it was the second most F I G U R E 1 Predicted survival time from the accelerated failure time parametric model fit for each treatment and species. Treatments: W/NI, well-watered/non-inoculated; W/I, well-watered/inoculated with Heterobasidion annosum s.s.; D/I, drought/inoculated with H. annosum s.s.; D/NI, drought/non-inoculated. Different letters indicate statistically significant differences between treatments within a species at p = 0.05 when performing a multiple comparison procedure using Tukey. Thin lines indicate empirical data. Grey-shaded areas correspond to the dormancy period. NB: There are marginal differences between W/I and drought/inoculated treatments in spruce (p = 0.059).
## | drought or inoculation alone led to the mortality of scots pine saplings
Pines subjected to drought or pathogen infection died at a similar rate. The combined drought and inoculation treatment did not increase the mortality risk in pine. We found no differences in terms of survival among saplings that were either inoculated, subjected to drought or both and Supporting Information: .
Seventy-five percent of deaths of inoculated pine saplings occurred within the first 3 months of inoculation.
Pathogen infection caused hydraulic impairment in pine. The impact of inoculation on pine physiological variables was similar to that observed for drought and when the two stress treatments were combined. Saplings subjected to any of the three stress treatments showed a similar reduction in stomatal conductance and photosynthesis, as well as more negative midday water potentials than saplings that received the control treatment and Supporting Information: . Surprisingly, the starch concentration in needles of inoculated pine saplings was similar to that of noninoculated controls (despite decreased photosynthesis), but higher than that of the drought-only treatment and Supporting Information: . Like in spruce, inoculation under drought conditions did not increase PLC compared with that of pine saplings that were only subjected to drought (Supporting Information: .
Defoliation was the variable that most strongly increased the mortality risk in pine ,d,f). Before death, the youngest shoots of pine saplings displayed a conspicuous loss of turgor, coupled with a general shedding of older needles.
Like in spruce, variables related to water, the C economy and C uptake were significantly associated with mortality in all treatments, with needle RWC being the only variable that was significant across treatments . Mortality rates of inoculated-only pine were not associated with the NSC content of needles . By contrast, both inoculated and noninoculated pine saplings subjected to drought, which had lower starch concentrations in needles died faster .
## F i g u r e 3 soluble sugars and starch content (% dry weight) of picea abies (a, c) and
Pinus sylvestris needles (b, d). Different letters indicate statistically significant differences (p = 0.05) between groups when performing a multiple comparison procedure using Tukey. For spruce, mixed models include the three sampling dates. In pine, a linear model is fitted for the unique sampling date after treatments (refer to Section 2.5). Grey-shaded areas correspond to the dormancy period. [Color figure can be viewed at wileyonlinelibrary.com]
# | discussion
We studied the interactive effect of drought and pathogenic infection on the mortality process of two conifer species. We used H. annosum s.s., a necrotrophic fungus that attacks sapwood and causes internal decay and a reduction of functional sapwood. We only found an additive effect of drought and pathogen infection in spruce, where the combination of these two stressors accelerated death compared with that observed for each individual stressor (i.e., our results support Hypothesis [i] for one of the study species).
Spruce saplings suffered from decreased C reserves and increased hydraulic stress when subjected to both drought and pathogen infection. Specifically, pathogen infection decreased the concentration of starch in the needles of saplings subjected to drought, a variable linked to mortality in our survival analysis.
## | drought-pathogen interactions in spruce
Our results support two of our three hypothesized mechanisms by which the additive effect of drought and infection occurs in spruce : defence needs associated with pathogenic infection reduce the amount of C available to cope with drought, which eventually impairs the water status (mechanism 2); and pathogen infection affects hydraulic performance through a reduction in the sapwood cross-sectional area (mechanism 3). Firstly, we showed that building a reaction zone was needed to contain pathogen colonization and survive given that survivors had a larger reaction zone than dead spruce saplings. The larger the reaction zone size, the lower the SS concentration found at the site of infection. This, together with the reduced C content in needles of inoculated spruce, suggests that the rate of C fixation in the reaction zone was faster than the rate of water potential values than in noninoculated saplings subjected to drought. Finally, we did not find any direct drought effects on C reserves linked to spruce survival (mechanism 1, . However, the lowest photosynthetic rates were recorded in drought-inoculated spruce, which partly supports our first hypothesized mechanism (i.e., drought impairing C uptake, and hence, limiting the amount of C available to build up defence barriers against the pathogen).
Maintaining needle turgor to prevent desiccation is probably the highest priority of trees under drought stress (Martinez-Vilalta Reduced RWC resulted in an increased probability of death and was related to increased defoliation across treatments and species.
Although defoliation may be the result of an acclimation response to drought and pathogen infection to improve sapling performance [bib_ref] Hydraulic adjustment of Scots pine across Europe, Martínez-Vilalta [/bib_ref] [bib_ref] Physiological, biochemical, and anatomical responses of Araucaria araucana seedlings to controlled water..., Papú [/bib_ref] , severe defoliation signals extreme stress responses associated with an increased mortality risk [bib_ref] Drought-induced defoliation and long periods of near-zero gas exchange play a key..., Poyatos [/bib_ref]. In particular, defoliation implies lowered C assimilation (and eventually lower C reserves) due to reduced photosynthetically active tissue and may lead to an increase in energy costs to maintain cell turgor under more negative osmotic pressures [bib_ref] Carbon reserves and canopy defoliation determine the recovery of Scots pine 4..., Galiano [/bib_ref] [bib_ref] Balancing the risks of hydraulic failure and carbon starvation: atwig scale analysis..., Salmon [/bib_ref] [bib_ref] Plant water content integrates hydraulics and carbon depletion to predict drought-induced seedling..., Sapes [/bib_ref].
Purely pathogen-induced hydraulic impairment and defoliation was evident in pine; however, this was only apparent in combination with drought stress in spruce. Indeed, pathogen colonization of the sapwood, in the absence of drought, did not impose hydraulic constraints on spruce given that water potential, RWC and PLC in inoculated-only spruce saplings were similar to those of controls. This is consistent with previously reported sapwood redundancy, by which a 50% reduction in sapwood area caused relatively small reductions in leaf conductance and had no significant impact on shoot water potential in Norway spruce [bib_ref] Losing half the conductive area hardly impacts the water status of mature..., Dietrich [/bib_ref] see also [bib_ref] No need for pipes when the well is dry-a comment on hydraulic..., Körner [/bib_ref]. However, under drought conditions, F I G U R E 5 Time series of the physiological variables for each treatment and species: (a, b) relative water content in needles, (c, d) midday water potential, (e, f) stomatal conductance, and (g, h) net photosynthesis. Different letters indicate statistically significant differences (p = 0.05) between treatments within species when analysed by linear mixed models when performing a multiple comparison procedure using Tukey. The sample size (number of saplings) per sampling date is indicated in the top panels. Grey-shaded areas correspond to the dormancy period. [Color figure can be viewed at wileyonlinelibrary.com] pathogen-induced loss of sapwood had an impact on water potential, aggravating drought-induced hydraulic dysfunction.
We showed that a decrease in available C at the site of inoculation was associated with the saplings' defence response, particularly with the size of the reaction zone. In addition, C uptake was not reduced in inoculated-only spruce even though lower starch and SS concentrations in needles were associated with the mortality of inoculated saplings. This result suggests that the reaction zone formed to compartmentalize the pathogen increases the requirement for C, which needs to be fulfilled. This is in line with the fact that reaction zones are C-enriched compared to healthy sapwood (10% of C content increase reported for Ophiostoma brunneo ciliatum by [bib_ref] Do trees use reserve or newly assimilated carbon for their defense reactions?..., Guérard [/bib_ref] , with the activation of genes that allocate C to secondary metabolism [bib_ref] Transcriptional responses of Norway spruce (Picea abies) inner sapwood against Heterobasidion parviporum, Oliva [/bib_ref] , and with a density increment of ca. 15% of the reaction zone versus sapwood under Heterobasidion infection [bib_ref] Spread of Heterobasidion annosum s.s. and Heterobasidion parviporum in Picea abies 15..., Oliva [/bib_ref]. Spruce saplings surviving inoculation had both a higher NSC content in needles and twigs and a larger reaction zone than those that died, which implies that their C budget allowed for both an adequate defence response and the maintenance of C reserves. When infection coincides with drought, lower rates of photosynthesis and the requirement of NSC for osmoregulation in needles and shoots also contribute to the overall C demand. However, other mechanisms could also have limited the C supply to the site of infection. NSC transport could have been impaired by the necrosis of the phloem by the pathogen. Our study does not enable us to evaluate the impact of drought and pathogen infection on C transport in the phloem. However, lesions in the phloem were particularly large in spruce subjected to both drought and inoculation, as previously reported for other sapwood-and phloem-infecting fungi that cause larger phloem lesions under drought conditions [bib_ref] Defence reactions of mature Norway spruce (Picea abies) before and after inoculation..., Netherer [/bib_ref] [bib_ref] Predisposition, stress, and plant disease, Schoeneweiss [/bib_ref] [bib_ref] Drought predisposition to cytospora canker in blue spruce, Schoeneweiss [/bib_ref] [bib_ref] Low water availability increases necrosis in Picea abies after artificial inoculation with..., Terhonen [/bib_ref]. Drought has also been suggested to cause phloem transport failure [bib_ref] Phloem transport and drought, Sevanto [/bib_ref]. Thus, the possible impairment of C transport in the phloem under drought and pathogen infection warrants further research.
## | differences between species
Pathogen inoculation alone had more severe effects on pine than on spruce. The disease produced in our experiment corresponded well with how pine and spruce interact with the pathogen in nature, where spruce trees typically suffer from internal decay, whereas pines usually succumb as a result of cambial death and fungal growth in the outer sapwood [bib_ref] Biology of Heterobasidion annosum, Korhonen [/bib_ref]. Observed differences may be due to the capacity of spruce to create a thick reaction zone around the decay column, which was not seen in pine.
However, in our experiment, we deliberately introduced the pathogen into the sapwood, thereby eliminating bark and phloem defence barriers. These barriers might be more important for defending pine against Heterobasidion infection than they are for spruce; hence, the observed mortality differences between species may be partly due to our experimental setup. Although pine is the preferred host for H. annosum s.s., it is able to infect spruce [bib_ref] Biology, epidemiology, and control of heterobasidion species worldwide, Garbelotto [/bib_ref]. This may explain the high level of aggressivity observed in pine and the short time needed to cause mortality in our experiment. We argue that the rapid rate of mortality of inoculated pine did not allow for the expression of the interaction between Heterobasidion infection and drought. Lower inoculum loads of the pathogen in the field, compared with mycelial plugs inserted directly into the sapwood of saplings, may result in a slower mortality process due to infection in the field, which could be amplified by F I G U R E 6 Relationship between needle relative water content and the nonstructural carbohydrate (NSC) concentration of spruce needles (a). Model of beta-regression linking stem NSC concentration with relative water content in spruce needles (b). Linear model of the relative water content in needles and defoliation using average values at all sampling points; all treatments are included (c). The solid line corresponds to spruce and the dashed line to pine. Defoliation is graded from 0 to 4 (0, no defoliation; 1, 25% defoliated; 2, 50% defoliated; 3, 75% defoliated; 4, completely defoliated). reported previously as a host response to canker diseases, which mainly impact vascular cambium and phloem tissue [bib_ref] Eucalypt plants are physiologically and metabolically affected by infection with Ceratocystis fimbriata, Da Silva [/bib_ref] [bib_ref] Differential physiological and biochemical responses of Quercus infectoria and Q. libani to..., Ghanbary [/bib_ref] [bib_ref] Tree host-pathogen interactions as influenced by drought timing: linking physiological performance, biochemical..., Hossain [/bib_ref] [bib_ref] Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar..., Li [/bib_ref] [bib_ref] Monoterpene responses to interacting effects of drought stress and infection by the..., Madmony [/bib_ref] [bib_ref] Fungal pathogens of canker disease trigger canopy dieback in poplar saplings by..., Xing [/bib_ref]. The differential stomatal response to pathogenic infection between the two host species should be investigated in future work to clarify the relative role of the reduction in water potential, hormonal signals and potential hormonal crosstalk (defence and drought stress) in stomatal control [bib_ref] Compound stress response in stomatal closure: q mathematical model of ABA and..., Beguerisse-Diaz [/bib_ref]. Despite the differences in the physiological response to pathogen inoculation between the two species, they presented similar mortality rates when pathogen infection occurred under drought conditions.
Most of the spruce mortality events associated with the two inoculation treatments took place after the dormancy period. This was particularly evident for the well-watered inoculated spruce saplings: none died before dormancy, whereas 40% of deaths occurred after dormancy. A similar pattern was observed for spruce subjected to drought and inoculation, with 30% and 60% of the total deaths taking place before and after dormancy, respectively.
Hibernation conditions in our experiment resembled a mild winter (above-zero temperatures and a lack of light) in the boreal zone. We speculate that during dormancy, the fungus might have taken advantage of the saplings' inactive defence system and continued to grow in the sapwood, contributing to the reduction of functional sapwood. Heterobasidion annosum s.s. is able to grow at temperatures as low as 2°C (1.1 mm/day at 5°C;. The temperature in our experimental dormancy was never lower than 5°C, and therefore, likely permitted fungal growth in inoculated saplings. Thus, expansion of H. annosum might have happened during dormancy, leading to the high percentages of decayed sapwood (94% in inoculated saplings that died during the first month following dormancy) upon the reestablishment of light and vegetative activity.
The interaction between drought, the pathogen and mild winters may have important implications under ongoing climate change because a future warmer climate would increase the likelihood of these three factors co-occurring in many areas, including boreal forests.
However, we reached these conclusions based on our study of saplings and caution should be taken when extrapolating them to mature trees, whose larger dimensions may protect them from lethal reductions of functional sapwood.
# | conclusions
Our results suggest that mortality due to simultaneous drought and pathogen infection is linked to the limited availability of C, which is needed to respond to the two main C sinks created by the stresses: the defence response at the site of infection and osmoregulation to F I G U R E 7 Diagram of the mechanisms of tree mortality induced by the concurrence of drought and pathogen infection in spruce. Three mechanisms are highlighted: (1) drought affects carbon (C) reserves and impairs the capacity of trees to build up defence barriers to compartmentalize the pathogen; (2) pathogen infection triggers a defence response, which creates a C sink, reducing C availability for maintaining needle turgor; and (3) the pathogen reduces functional sapwood and disturbs water transport and storage in the presence of drought. A, net assimilation rate; g s , stomatal conductance; PLC, percentage loss of conductivity; NSC, nonstructural carbohydrates; RWC, needle relative water content; Ψ, water potential. [Color figure can be viewed at wileyonlinelibrary.com] maintain cell turgor in needles, leading to impaired water status. The additive effect of the two stressors was significant in spruce, which showed an accelerated death rate compared with saplings subjected to only one of the stressors. By contrast, pine saplings died at a similar rate when experiencing either drought or pathogen infection or both stressors. In our pathosystem, maintaining turgor was associated with a higher availability of NSC, presumably impairing the sapling's capacity to allocate NSC to build a reaction zone and contain the pathogen. Although this conclusion may differ in other pathogen-host trophic interactions affecting different plant organs, we argue that pathogens can exploit C allocation priorities under stress and increase their capacity to attack vital plant organs.
Similarly, our results show how pathogens can benefit from host dormancy to cause mortality.
We encourage empirical studies of trophic interactions other than necrotrophy to fully elucidate how co-occurring drought and pathogen infection can lead to tree mortality. The incorporation of both pathogen-induced and drought-induced mortality mechanisms in models has the potential to improve our capacity to forecast forest dynamics under climate change [bib_ref] Why is tree drought mortality so hard to predict?, Trugman [/bib_ref]. A way to capitalize on these results in tree mortality modelling could be the inclusion of an acceleration factor in death rates, if drought events coincide with the presence of necrotrophic pathogens infecting vascular tissues. However, we recognize that pathogen-drought interactions are complex and multidimensional. Drought can have simultaneous, non-linear impacts on both host and pathogen populations [bib_ref] Nonlinear shifts in infectious rust disease due to climate change, Dudney [/bib_ref] , and therefore, integrative research including all these impacts is needed to improve mechanistic tree mortality modelling. |
Harnessing adipose stem cell diversity in regenerative medicine
Since the first isolation of mesenchymal stem cells from lipoaspirate in the early 2000s, adipose tissue has been a darling of regenerative medicine. It is abundant, easy to access, and contains high concentrations of stem cells (ADSCs) exhibiting multipotency, proregenerative paracrine signaling, and immunomodulation-a winning combination for stem cell-based therapeutics. While basic science, preclinical and clinical findings back up the translational potential of ADSCs, the vast majority of these used cells from a single location-subcutaneous abdominal fat. New data highlight incredible diversity in the adipose morphology and function in different anatomical locations or depots. Even in isolation, ADSCs retain a memory of this diversity, suggesting that the optimal adipose source material for ADSC isolation may be application specific. This review discusses our current understanding of the heterogeneity in the adipose organ, how that heterogeneity translates into depot-specific ADSC characteristics, and how atypical ADSC populations might be harnessed for regenerative medicine applications. While our understanding of the breadth of ADSC heterogeneity is still in its infancy, clear trends are emerging for application-specific sourcing to improve regenerative outcomes.
# Introduction
Since its modern inception, the field of regenerative medicine has dedicated itself to harnessing the power of stem cells. Viewed broadly, the progress is staggering. From organ-in-a-dish models of disease to biomaterial scaffolds to direct stem cell fate in vivo, significant progress has been made both in understanding and exploiting the regenerative potential of stem cells. This is especially true of adult stem cells. These cells, which can be derived from the patient's own body and applied without the ethical challenges of embryonic stem cells (ESCs) or the technical hurdles of induced pluripotent stem cells (iPSCs), have pushed a new frontier of stem cell-based therapies. The hematopoietic and mesenchymal stem cells (MSCs) have been the most successful translationally, with over four thousand clinical trials and millions of patients treated (ClinicalTrials.gov). While hematopoietic stem cells are generally restricted to the myeloid and lymphoid lineages, MSCs exhibit plasticity toward a number of adult tissues such as bone, cartilage, adipose, muscle, and even nerve, making them promising candidates for treatment across a spectrum of injury and disease. MSCs are currently under development for the treatment of dozens of conditions from osteoarthritis to infertility.
Mesenchymal stem cells are now recognized to be a highly heterogeneous population. Not only are there variations in proliferative capacity and multipotency between clones of a single isolation 1 but also well documented differences between donors 2 and tissue of origin.Of course, heterogeneity is not all bad. Numerous studies suggest that MSCs from different sources possess preferential plasticity, 4,5 resilience in specialized environments,and unique immunomodulation,which suggest that the optimal MSC source may be application specific. While there is arguably more heterogeneity between tissue types (e.g., bone marrow vs adipose), there are likely also important differences between anatomical locations within a single type, which have only begun to be explored. Nowhere is this likely to have as big an impact on stem cell-based therapies than in adipose tissue: one because adipose-derived stem cells (ADSCs) show exceptional clinical promise in general and two because adipose tissue has extensive morphological and functional heterogeneity. Nearly all ADSCs in preclinical and clinical studies derive from abdominal subcutaneous fat, but adipose tissue also exists around internal organs, between muscles, and within joints. These other locations or "depots" are thought to be specialized to support adjacent tissues, and each has documented differences in tissue and ADSC function. While typically smaller and less accessible than subcutaneous belly fat, these depots are frequently accessible during surgeries on adjacent tissues and could offer improved regenerative potential as part of an ADSC adjuvant therapy.
In this review, we will discuss the morphological and functional heterogeneity among adipose depots and review the evidence for differential regenerative capacity among their resident ADSC populations. While heterogeneity in adipose depots has long been recognized to influence ADSC regenerative potential, this review moves beyond the traditional abdominal adipose sources to consider specific organand tissue-associated depots that could be harnessed to improve regeneration in specialized applications. We will discuss emerging data on the unique properties of ADSCs isolated from these depots and novel strategies to enhance their potential for clinical translation.
## Adipose-derived stem cells: fat in a new light
While stem cells have long been the holy grail of regenerative medicine as the body's natural source of regeneration, they are difficult to harness therapeutically. Since the discovery of hematopoietic stem cells in the 1960s and embryonic stem cells (ESCs) in the 1980s, scientists have devoted significant effort to utilize their therapeutic potential in tissue repair and regeneration. Three major issues currently challenge stem cell-based therapies: (1) accessing sufficient quantities of cells, (2) directing those cells to enhance regeneration, and (3) preventing rejection by the body. ESCs have the most regenerative potential in terms of differentiation capacity, but ethical issues concerning their sourcing limit their translational potential. Furthermore, because of their pluripotency and allogenic origin, ESCs have the potential to cause both immune rejection and tumor generation. The potency of ESCs can be regained without the ethical or immune issues in induced pluripotent stem cells (iPSCs) by genetic modification of a patient's own cells, but the expense and low rate of conversion currently limit the translational potential of these cells as well. Adult, or somatic, stem cells avoid the ethical and practical issues with ESCs and iPSCs but have faced other challenges.
Adult mesenchymal stem cells (MSCs) combine multipotency (adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic differentiation capacity) with low immunogenicity (because they can be derived from the patient's own tissues).Furthermore, they can be easily isolated by enzymatic digestion from nearly every tissue in the body and stably expanded in culture.The best characterized and most widely used MSCs for cell transplantations are derived from bone marrow. These were the first MSCs isolated for therapeutic use, targeted due to the proregenerative action of bone marrow on fracture healing.Despite their in vitro multipotency and low morbidity during cell culture, the painful harvesting process and relatively low yield limit their potential in clinical applications. Zuk et al. were the first to isolate and characterize a similar population of multipotent cells from lipoaspirate.These adipose-derived stem cells (ADSCs) circumvent some of the challenges associated with bone marrow-derived MSCs because of the abundance and accessibility of adipose tissue. For ADSC isolation, adipose tissue is typically harvested by liposuction, a popular cosmetic surgery yielding hundreds of milliliters of source material. ADSCs are conveniently isolated by enzymatic digestion and filtration and are typically defined as the plastic-adherent population remaining after passaging but may also be sorted by a panel of surface markers (CD31À, CD34þ, CD45À, CD90þ, CD105þ, and CD146À). 14 With yields as high as 400 000 cells per milliliter, ADSCs require minimal to no culture expansion for many applications.
Application of ADSCs in preclinical models of disease has shown significant regenerative benefits including improvements in cardiac ejection fraction post-infarction,immunosuppression in rheumatoid arthritis,learning and memory in Alzheimer's disease,dystrophin expression in Duchenne muscular dystrophy,and fibrosis in kidney injury.Because of their preclinical promise, extensive ADSC-based clinical trials for tissue regeneration and reconstruction are under way to assess their safety and efficiency. The number of relevant trials has risen from a total of nine in 2009 to 180 by June 2020 worldwide (ClinicalTrials.gov), investigating the efficacy in treating various diseases such as type I and type II diabetes, liver cirrhosis, fistulas, cardiovascular disease, limb ischemia, amyotrophic lateral sclerosis, and lipodystrophy (reviewed in Ref.. While most trials are still in phase I/II, some early promising results have been published. ADSC injection has shown promise in treating Crohn's disease with no adverse effects but a higher healing rate and a lower recurrence rate.In acute myocardial infarction, transplantation of ADSCs significantly reduced the infraction size and perfusion defect in phase I/II clinical trials.Furthermore, ADSCs are also investigated in clinical trials for a variety of musculoskeletal,immunological, 26,27 pulmonary,and reconstructiveapplications. However, not all preclinical findings have translated into clinically meaningful improvements (reviewed in Ref., highlighting a need to better define and refine the regenerative potential of ADSCs.
The mechanism of action of ADSCs (and MSCs in general) on tissue regeneration is not well characterized. In fact, substantial argument still exists regarding what these cells actually are (stem cells vs stromal cells vs pericytes vs connective tissue cells vs a heterogeneous combination).Based on the multipotency of MSCs in vitro and their role in maintaining tissue homeostasis in vivo, it is natural to conclude that they contribute to tissue regeneration by differentiating into target cells. However, few reports demonstrate direct contribution of injected MSCs to injured tissue, and they are now thought to elicit therapeutic effects primarily through paracrine signaling and immunomodulation (reviewed in Ref.. This is true of ADSCs as well, both in a therapeutic context and in their native tissue environment (reviewed in Ref.. ADSCs may alter the tissue microenvironment through secretion of growth factors, inflammatory cytokines, or extracellular vesicles (exosomes) loaded with proteins, lipids, DNA mRNA, micro-RNA, tRNA, and noncoding RNA. Exosomes in particular have emerged recently as modulators of inflammation and tissue regeneration in the brain, heart, liver, kidney, and skin (reviewed in Ref.. If, as studies suggest, native ADSCs contribute significantly to the paracrine and endocrine action of adipose tissueand this action varies by depot, tailored to specific tissue targets, it is reasonable to suggest that (1) depot-specific ADSC sourcing could improve regenerative outcomes for specific cell-based therapies and (2) an improved understanding of depot-specific signaling could inform the appropriate sourcing and application of ADSCs. Tailoring ADSC sourcing to enhance the efficacy of translational therapies is not a new concept and has been the subject of several reviews;however, emerging data on ADSCs isolated from nontraditional depots suggest that another look beyond abdominal subcutaneous and visceral adipose tissue (VAT) is warranted.
## Adipose tissue diversity in form and function wat, bat, vat, and sat
Originally considered an inert storage depot for energy, our understanding of the role of adipose tissue has greatly expanded to now being considered the largest endocrine organ of the body.In addition to its role in energy balance, adipose tissue secretes a host of cytokines, termed "adipokines," which regulate important biological processes and serve as markers for a multitude of cardiovascular, metabolic, and inflammatory diseases.Both the metabolic and endocrine functions of adipose tissue vary by depot.
Historically, adipose tissue is subdivided into two types: white adipose tissue (WAT) and brown adipose tissue (BAT). These two types are differentiated by gross appearance, cellular composition, gene expression signature, and developmental lineage (reviewed in Ref.. Specifically, BAT is brown in appearance, with mitochondriadense, multilocular adipocytes that express uncoupling protein 1 (UCP-1) and derive from a myogenic factor 5 (Myf5) positive progenitor, while WAT appears white or yellow, with large UCP-1 and Myf5 negative unilocular adipocytes. They are also functionally distinct, with WAT storing energy in the form of triglyceride and BAT burning energy through adaptive thermogenesis. WAT is further divided into subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). Unlike the classification of WAT vs BAT, which relies on fundamental differences within the adipose tissue, SAT and VAT are differentiated purely by anatomy, with SAT referring to all depots beneath the skin and VAT referring to all depots in the body cavity. Recent work has demonstrated considerable heterogeneity between depots within both the SAT and VAT categoriesand has even noted more similarity between some depots that cross categories than those within a category,calling into question the functional relevance of this anatomical classification.
Subcutaneous and visceral depots in the abdomen are the best characterized in humans. It was the subdivision of abdominal adiposity into these depots that launched the concept that not all WAT is created equal.Typically considered "good" fat, the deposition of abdominal SAT appears to have a cardio/metabolic-protective effect noted by increased antiatherogenic and antiinflammatory cytokines including interleukin 4 (IL-4) and transforming growth factor b (TGF-b). Opposingly, abdominal VAT as a "bad" fat is marked by heightened levels of lipolysis, immune cell infiltration, and proinflammatory adipokine release accompanied by metabolic detriments including higher risk of diabetes, dyslipidemia, and cardiovascular disease (reviewed in Ref..
Recently, more attention has been paid to smaller VAT depots including fat around the vasculature, heart, and kidneys. Intriguingly, these depots have features of both BAT and WAT, where they resemble WAT in appearance but are capable of elevated UCP-1 expression and adaptive thermogenesis in response to b-adrenergic stimulation.This subtype of adipose tissue has been coined "beige," "brite," or "inducible brown" (reviewed in Ref.. It is believed that beige fat contains mature adipocytes that possess a bi-directional phenotype and fluctuate between a "dormant" state capable of storing energy like typical WAT and transition to an "active" state with the enhanced BAT function upon external stimulation. This distinctive phenotype in conjunction with the anatomical juxtaposition of these depots with specific tissues and organs suggests a unique functionlikely distinct from classical abdominal SAT and VAT and potentially distinct from each other. The more that we uncover about adipose tissue, the more its inherent diversity defies the historical classification into WAT, BAT, VAT, and SAT. Thus, for the remainder of this review, individual depots will be named whenever possible.
## Adipose depots in rodents
The most extensive characterization of adipose depots has been performed in mice and rats.Exact definitions vary per researcher, but the following fundamental anatomical locations seem to be the most agreed upon. Murine SAT can be identified as four different depots: (1) inguinal fat is located subcutaneously posteriorly and runs dorsal along the hindlimb. It is the "classical" SAT and beige fat depot in rodents. (2) Anterior subcutaneous fat is a thin layer of WAT that runs on the sides of the ribcage. (3) Triceps-associated fat is a thin WAT depot located in the front legs and runs along the length of the triceps muscles. (4) Interscapular fat is located between the shoulder blades and is superior to the interscapular BAT fat pad. VAT can be identified as five depots in mice: (1) perigonadal/epididymal fat is considered prototypical visceral adipose in rodents and is located either attached to the epididymis and testis in males or near the ovaries in female mice. (2) Mesenteric fat lines the large intestines. (3) Perirenal fat is located posterior to the renal capsule of the kidneys. (4) Retroperitoneal fat is located anterior to the kidneys. (5) Cardiac fat is associated with the heart and can be further subdivided into epicardial adipose tissue, which is located between the myocardium and the visceral pericardium and pericardial fat that lies between the visceral and parietal pericardium. Classical BAT in rodents can be considered SAT or VAT depending on the anatomical location. Typically, it is defined as the four following depots: (1) Interscapular fat is considered the classical BAT depot and is present in two lobes between the scapulae.
(2) Cervical fat is two lobe-shaped fat pads located beneath the scapulae and head. (3) Perirenal fat in its brown form is distinct from perirenal VAT and is located on the hilum side of the kidney. (4) Axillary fat is located anterior to the anterior subcutaneous WAT. In addition to these, adipose tissue can be identified surrounding blood vessels (perivascular fat, reviewed in Ref., within synovial joints (articular fat pads, reviewed in Ref., around and within muscle (epimuscular and intramuscular adipose tissue, reviewed in Ref., and within bone marrow (bone marrow adipose tissue, reviewed in Ref.. Rather than existing as a single depot, this "auxiliary" adipose is composed of discrete small depots throughout the body. Although the small depots are frequently grouped under a single heading (e.g., perivascular), evidence suggests that physiology and phenotype vary with the anatomical location (e.g., peri-thoracic aortic vs peri-abdominal aortic).Comparative studies between depots demonstrate unique paracrine and endocrine functions, as well as marked differences in their contribution to health. As discussed above, by far, the most comparative data exist between the classical SAT and VAT depots. In mice, these are inguinal and epididymal fat. Extensive transcriptomic, proteomic, and secretomic studies reveal differences in the metabolic function and adipokine profiles both at the baselineand in response to stimuli (e.g., high fat feeding or exercise).However, perhaps the most convincing evidence for intrinsic differences in these depots comes from fat transplant studies. Stanford et al. transplanted inguinal and epididymal adipose from sedentary and exercise-trained mice into the subcutaneous space or the visceral cavity.Transplanting inguinal fat from exercised mice to either location of sedentary mice improved glucose tolerance. However, no improvements were found when transplanting exercised epididymal fat, suggesting that the source of the transplant, rather than its anatomical location, defines its physiological contribution.
In addition to the heterogeneous endocrine contributions of the major SAT and VAT depots, there appear to be specialized paracrine roles for many of the smaller auxiliary adipose depots. For example, the close proximity of perivascular adipose tissue and the blood vessel enables secreted adipokines to act locally either through the vasa vasorum or directly through the vessel wall (reviewed in Ref.. Indeed, mechanistic studies in rodents have demonstrated unique action of perivascular adipokines on vascular tone 69 and proliferation of smooth muscle cells.Similarly, paracrine adipokines are proposed to underlie the cardioprotective effects of epicardial fat (reviewed in Ref.and the cartilage protective effects of the infrapatellar fat pad (IPFP) (reviewed in Ref.. However, the vast majority of the literature concerning paracrine actions of these depots covers their negative role in disease states. Taken together, it suggests that paracrine adipose signaling, like endocrine adipose signaling, is plastic-promoting homeostasis in health and pathology in disease.
## Adipose depots in humans
Similar to rodents, adipose tissue in humans is a multidepot organ; however, specifics of the depots differ. In humans, abdominal VAT is classically subdivided into omental (anterior to the intestines), mesenteric (surrounding the intestines), and retroperitoneal (posterior to the abdominal organs) depots.Rodents mainly store VAT in the perigonadal depot, with the most analogous VAT in humans being mesenteric fat.On the flip side, while the omental VAT depot can be expansive in humans, it is not easily identifiable in mice. These differences do not allow for clear parallels between VAT in species. Human SAT is typically subdivided into upper-body and lower-body regions.Unlike in rodents where it exists in discrete depots (e.g., inguinal), in humans, SAT is distributed throughout the body, though the thickness of the fat varies by region and preferential storage varies by individual. Defining BAT depots in humans has been complicated by the fact that adipose in anatomical locations corresponding to rodent BAT more resembles rodent beige fat than classical BAT.Indeed, in humans, most of the classical BAT is thought to be lost in infancy and replaced with a beige spectrum of inducible thermogenesis.In addition to the typical paraspinal and supraclavicular depots, most of the auxiliary organ-and tissue-associated depots exhibit a beige signature. What data are available suggests functional overlap with rodent depots as well. Peri-thoracic aortic adipose tissue expresses transcriptional markers of beige/brown fat in miceand humans.The vasodilatory action of this fat has been confirmed in human explantsand appears to be lost in the context of obesity.Similarly, epicardial and rotator cuff epimuscular adipose tissues have confirmed beige signatures in humansand in mice 54 (our unpublished data). Epicardial adipose secretion of a number of adipokines identified in rodent studies, including adiponectin, adrenomedullin, and omentin-1, is confirmed to be correlated with the cardiac function in humans.Coculture models of human epimuscular adipose-muscle crosstalk also suggest that paracrine adipokines could regulate myogenesis.Interestingly, epicardial and epimuscular fat exhibit a concurrent decline in these "protective" effects and a transcriptional shift toward white fat in the presence of coronary artery disease and chronic rotator cuff disease, respectively,suggesting that phenotypic transitions could cause these fats to contribute negatively to disease states. Similarly, perirenal adipose tissue has a brown or beige phenotype in both humansand rodents,with a specific classification that varies by location and study. While the physiological role of perirenal adipose remains uncharacterized, it is responsive to obesity and type 2 diabetes where its expansion is linked to renal dysfunction in both species.Evidence in mice and humans suggests that bone marrow adipose tissue does not fully resemble brown, beige, or white fat and may have a unique phenotype.Paracrine actions of this adipose depot on bone remodeling are proposed in both mice and humans, but strong mechanistic supporting data are lacking in both species (reviewed in Ref.. Finally, while the infrapatellar fat pad is thought to be a white phenotype in both rodents and humans, it is also thought to be unique in its metabolic regulation.It has long been thought to be involved in the progression of osteoarthritis, likely through paracrine inflammatory adipokines (reviewed in Ref. 92) but intriguingly, ADSCs isolated from this depot have also been shown to ameliorate the condition (reviewed in Ref..
Amassed together, this substantial and growing body of evidence suggests that each adipose depot is unique in form and function. Most relevant to this review are notable differences in paracrine and endocrine secreted adipokines, which are likely to arise in part from the ADSC compartment.If ADSCs from different depots are tailored to uniquely regulate specific tissue functions and they maintain this purpose upon isolation, then selection of the source of ADSCs should be similarly tailored to maximize regenerative potential.
## Adsc regenerative potential across depots
Given the diversity in adipose tissue, it is not surprising that the characteristics of the ADSC population also vary by depot. However, before a comparative discussion can be undertaken, it must be noted that even within a single depot, the ADSC population is heterogeneous (reviewed in Ref.with 2-3 distinct subpopulations noted in mouse epididymal and inguinal depots. Therefore, differences between depots at the population level could arise from intrinsic differences in each ADSC, differences in specific subpopulations, or differences in the fractional composition of the depot. Only studies using single cell or clonal analyses can differentiate between these possibilities. Further complicating the generalization of ADSC characteristics, recent data indicate that lineage and transcriptional markers may identify different subpopulations in different depots.For instance, while the ADSCs that give rise to thermogenic adipocytes in BAT hail from a Myf-5 positive lineage,Myf-5 positive ADSCs in SAT are not the source of thermogenic beige adipocytes.Taken together, these data point to the need to individually characterize subpopulations in each depot to fully realize the translational potential of ADSCs. While most current translational work remains focused on the collective ADSC population of abdominal SAT, future identification of subpopulations with unique surface markers and regenerative potentials could hone the use of even the standard adipose source material, optimizing ADSC selection, and improving outcomes. However, as our understanding of inter-and intra-depot ADSC heterogeneity is still in its infancy, most comparative data exist at the population level.
In line with the characterization of adipose tissues, the most extensive comparison of ADSCs is between those derived from the classical SAT and VAT depots (inguinal and epididymal in mice and abdominal subcutaneous and omental in humans). Generally speaking, they have significant similarities: they both are stable in culture, exhibit similar degrees of pluripotency, and share a surface marker profile.However, a deeper comparison reveals subtle but significant differences (reviewed in detail in Ref.. Numerous studies in mice and humans confirm higher in vitro proliferation and adipogenic and chondrogenic differentiation rates in ADSCs derived from SAT compared with VAT, 96,97 while VAT ADSCs have higher osteogenic potential.Proliferative differences are supported by microarray analysis, indicating that VAT ADSCs have upregulated clusters of genes related to lipid synthesis and metabolism, while SAT ADSCs have highly expressed genes in DNA-dependent transcription.Ease of access and expansion certainly make SAT ADSCs an appealing therapeutic choice, and indeed, abdominal SAT is the primary source for clinical and preclinical ADSC based regenerative studies-but lineage preference during directed differentiation suggests that intrinsic differences might control the efficacy per quantity of transplanted cells. Secretome analyses support this hypothesis as, compared with abdominal SAT ADSCs, omental VAT ADSCs secrete elevated levels of inflammatory cytokines (IL-6 and IL-8),100 thought to be involved in immunomodulation of wound healing.However, the few studies that have investigated this directly have found both sources to equally promote cardiac and dermal regeneration in rodents.ADSCs can also be derived from BAT in humans and rodents and exhibit similar characteristics in culture to their WAT counterparts: fibroblastic morphology, stable expansion, multipotency, and mesenchymal surface marker expression,although the degree of potency and level of expression may differ.One notable exception is that BAT-derived ADSCs differentiate into adipocytes with high expression of thermogenic regulatory genes (Ucp1, Cidea, and Pgc1a) and elevated uncoupled respiration 76,105 compared with those derived from WAT. This suggests that the ADSCs retain a differentiation memory from their tissue of origin: BAT-derived and WAT-derived ADSCs want to differentiate into brown and white adipocytes, respectively, in the same culture conditions. A second notable exception is that BAT-derived ADSCs exhibit elevated potential for differentiation down the cardiomyocyte lineage, 106,108 which is currently being explored for its therapeutic potential. Aside from these studies, the vast majority of BAT ADSC-based work has focused on delineating factors that control the browning response or have used differentiated adipocytes as a model system for BAT. Comparisons of the transcriptional or secretory profiles between undifferentiated BAT and WAT ADSCs are lacking. However, adipocytes derived from BAT ADSCs maintain secretion of some BAT associated adipokines, and their secretome is responsive to adrenergic (browning) stimulation in vitro.Specifically, simulation with norepinephrine increases secretion of adipokines with antiinflammatory action in interscapular BAT ADSC-derived adipocytes but not in those derived from inguinal WAT.This suggests that even in isolation, the BAT ADSC secretome retains a unique responsiveness to environmental cues, which could be harnessed for regenerative applications.
A handful of studies have explored the unique features and regenerative potential of ADSCs isolated from depots outside the classical WAT and BAT described above. The most extensive characterization has been in the infrapatellar fat pad (IPFP)-an intraarticular fat pad that sits in the knee joint behind and just below the patella. Investigations in this small adipose depot were initially driven by studies suggesting that it played a role in joint pathologies such as post-arthroscopy fibrosis. As it is easily accessible during knee surgeries (and frequently removed as surgical waste), it has been well characterized in vitro. Generally, ADSCs isolated from the IPFP resemble those isolated from SAT in the surface marker profile, proliferative capacity, and potency-though reports vary as to which source exhibits higher proliferation and osteogenic capacity.However, there is a consensus that IPFP derived ADSCs have increased chondrogenic potential compared with other adipose-derived stromal cell (ADSC) sources, with numerous studies showing increased chondrogenic gene expression and production of cartilage matrix in directed differentiation.This finding combined with promising preclinical and clinical outcomes treating osteoarthritis with marrow derived MSCs and SAT derived ADSCs suggests that IPFP derived ADSCs could represent a unique therapeutic cell source. While no studies have yet pitted IPFP and SAT derived ADSCs against each other, injection of IPFP ADSCs into osteoarthritic animal models and human knees has improved radiological and functional outcomes, indicating that they can indeed impact degeneration.ADSCs derived from the cardiac-associated depots-epicardial and pericardial fat-have also been examined for their potential in cardiac regeneration. Epicardial fat is adjacent to the myocardium without a fascial division, while pericardial fat lies on the outer surface of the fibrous pericardium. ADSCs from both depots exhibit a typical Preclinical pyelonephritis model (rabbit); subscapular ADSC injection Improved interstitial fibrosis and cortical function compared with inguinal derived ADSCs 131
Preclinical ischemia-reperfusion liver injury model (mouse); delivery method not specified Reduced cellular necrosis and increased survival compared with untreated control 132 Preclinical muscle regeneration model (rabbit); intramuscular ADSC injection Increased muscle mass and contractile force compared with cell-free media mesenchymal morphology, surface marker profile, and potency,but, like BAT-derived ADSCs, they exhibit increased cardiomyocyte differentiation potential compared with abdominal SAT and VAT in both humans and rodents.A similar propensity for cardiomyocyte differentiation is found in perivascular fat around the aorta, which is also sometimes classified as cardiac adipose.In animal models of cardiac ischemia, injection of pericardial ADSCs increased the thickness of the infarcted wall with improved vascularization and function compared with injection of inguinal ADSCs,suggesting that cardiac ADSCs could elicit improved regenerative responses in the heart. Interestingly, despite their cardiomyogenic potential, the cells did not appear to contribute directly to the regenerating tissue but rather were thought to improve vascularization and myogenesis via secreted paracrine factors.A similar but less characterized depot of fat can also be found around some skeletal muscles-most notably the muscles of the rotator cuff. Rotator cuff epimuscular fat sits on the superior surface of the supraspinatus muscle, outside the fascial boundary of the muscle. Human ADSCs derived from this fat exhibit increased myogenic potency in vitro compared with subject-matched SAT ADSCs and are able to directly contribute to regenerating muscle in vivo.Interestingly, the progenitor cells that give rise to the other skeletal muscle associated adipose, intramuscular fat (adipocytes between myofibers within the fascial boundary of the muscle) do not exhibit myogenic potency either in vitro or in vivo, 122,123 highlighting the distinct characteristics of these two fats. Despite lacking myogenic potency, these muscle-resident fibro/adipogenic progenitor (FAP) cells are thought to facilitate regeneration, likely through paracrine signaling.While FAPs are less abundant and more difficult to isolate than ADSCs derived from traditional adipose depots, they are still being explored as a potential source for cell-based regenerative therapies in muscles.ADSCs derived from perirenal adipose tissue likewise exhibit an MSC-associated surface marker profile and are capable of trilineage differentiation in rodents and humans.Additionally, studies have demonstrated immunomodulation by these cells in both species.The degree of similarity between perirenal ADSCs and those from classical VAT and SAT depots depends on which portion of the fat is investigated.In rodents, perirenal fat is composed of a WAT and a BAT layer and in humans exhibits a graded regional variation in brown features, with the most prominent being close to the hilus.ADSCs derived from perirenal WAT are transcriptionally very similar to those derived from epididymal WAT in rats,while perirenal BAT ADSCs resemble human perithyroid BAT and mouse interscapular BAT.Only a few studies have compared ADSC differentiation potentials between perirenal and other adipose sources. To date, the only noted differences are higher proliferation, 47 adipogenic potential,and capacity to differentiate into Schwann-like cellscompared with epididymal ADSCs. Despite the lack of in vitro characterization, perirenal ADSCs have been applied therapeutically in preclinical models of disease with promising results. In a rabbit model of acute pyelonephritis, injection of autologous perirenal ADSCs enhanced histopathological outcomes and significantly improved the cortical function compared with neck subcutaneous ADSCs.Additionally, systemic injection of perirenal ADSCs improved liver regeneration in a mouse model of ischemia-reperfusion injuryand muscle regeneration in a rabbit model of toxin-induced injury.However, no comparison was made between ADSC sources in these studies.
The progenitor cells that give rise to the adipocytes in the bone marrow are a unique subpopulation within the MSCs derived from the bone marrow.They are closely associated with the bone marrow adipocytesand, in their niche, support neo-vascularization and bone regeneration through adipokine secretion.While bone marrow derived MSCs have been extensively characterized in vitro and evaluated in regenerative therapies in vivo, the contribution of this specific subpopulation has yet to be defined. Identification of unique surface markers could enable purification of these cells to harness their proregenerative paracrine signaling for regenerative therapies.
While extensive characterization is lacking and there may be a literature bias toward pairing ADSCs derived from specific auxiliary depots with regenerative therapies targeting the associated organ/tissue, what evidence exists suggests that selective ADSC sourcing could improve regenerative medicine strategies. Importantly, not only does there appear to be a propensity for ADSCs to support regeneration in the organ/tissue with which they are associated, but also these ADSCs are frequently accessible during the surgical treatment of that organ/ tissue. For example, the IPFP is accessible during knee osteotomy, pericardial fat is accessible during open heart surgery, and epimuscular fat is accessible during rotator cuff repair. In each of these scenarios, surgical success will depend on the degree of tissue regeneration and healing over subsequent months, and thus, post-surgical adjuvant treatment with ADSCs isolated during the surgery is an intuitive and feasible approach to improve outcomes. However, such strategies will face biological and technical challenges. Most significant among them is the limited source material. In comparison to abdominal SAT, these auxiliary depots are very small, which will likely necessitate culture expansion of ADSCs to generate sufficient numbers to achieve therapeutic benefits. Second, as mentioned previously, most of these depots undergo pathologic changes in response to disease in their associated organ/tissue and may even contribute to the organ/tissue pathology. It may not be beneficial to directly apply ADSCs isolated from the diseased fat to the diseased organ. However, part of the therapeutic potential of ADSCs lies in their plasticity and ability to be redirected by environmental cues. Significant advances have been made in tissue engineering to recreate healthy environmental cues in vitro and in vivo to maximize survival and senescence-free expansion and direct differentiation and adipokine secretion to maximize the pro-regenerative impact of stem cell-based therapies. Harnessing these strategies could make any adipose depot a realistic source for therapeutic ADSCs.
## Novel strategies to optimize adsc-based therapeutics
The field of tissue engineering has been working for over a decade to optimize ADSC-based therapeutics. The breadth of innovative strategies and designs has filled several reviews,but generally speaking and for the purpose of a brief introduction, engineering approaches can be broken down into four main categories: scaffold design, cytokine delivery, genetic engineering, and other culture environment modifications. Scaffold engineering takes advantage of the sensitivity of ADSCs to the stiffness, topography, and composition of the extracellular matrix (ECM). Scaffolds can be employed in vitro to promote quiescence or senescence-free expansion or to direct differentiation prior to in vivo applications. Alternatively, they can be applied with ADSCs to promote survival, reduce dispersion, and direct differentiation in vivo. Cytokine delivery approaches seek to direct ADSC behavior using soluble factors in the microenvironment and are frequently used in combination with engineered scaffolds to combine physical and chemical cues. Most of the current strategies either deliver growth factors to promote cell survival or deliver inflammatory cytokines to enhance the immunomodulatory effects of ADSCs. Genetic engineering can be used to elicit a stronger and more sustained survival and directed differentiation response by editing apoptotic genes and the transcription factors that control pluripotency.
Finally, culture conditions are continually being optimized to improve ADSC survival and performance in diseased microenvironments. Two major strategies are hypoxic preconditioning and 3D spheroid culture. Both strategies are designed to better mimic the ADSC native microenvironment and have been shown to improve the viability and survival of transplanted ADSCs.
These strategies were developed and optimized with ADSCs derived from abdominal SAT, but they have clear potential for improving the translational potential of ADSCs derived from other depots. The limited quantity of source material limits the. Similarly, when native ECM proteins were used as a culture surface (rather than tissue culture plastic), proliferation and chondrogenic and adipogenic potency were enhanced in IPFP ADSCs.Proliferation of perirenal ADSCs was also increased by culture on the decellularized ECM.While these coatings provide some of the native cell-protein interactions, they lack the native topography. 3D printing and electrospinning of ECM proteins can combine structural and biochemical cues to further guide cellular behavior. In vitro, induced osteogenic differentiation of epicardial ADSCs is improved with culture on electrospun collagen.Electrospun collagen matricesand bioprinted ECMhave also been used to develop tissue engineered constructs from IPFP ADSCs. These constructs are designed for implantation to improve the survival and efficacy of transplanted cells in vivo, thereby requiring fewer ADSCs to elicit a therapeutic effect. Chronic diseases such as aging and diabetes impair both the potency and immunomodulatory functions of SAT ADSCs.Given the pathological changes to many of the tissue/organ associated fat depots in the conditions in which they would be accessible (e.g., major surgery), disease and aging are likely to affect the translational potential of autologous ADSCs from these depots as well. Indeed, obesity impairs the chondrogenic potential of IPFP ADSCs 146 as likely does osteoarthritis.However, IPFP ADSCs isolated from arthritic knees can still be conditioned to generate tissue engineered cartilaginous grafts in vitro using a combination of hypoxic preconditioning, biochemical cues, and biophysical cues.Similarly, the angiogenic function of epicardial ADSCs isolated from diabetic rats can be rescued by pretreatment with a soluble Notch pathway inhibitor.While deficits have not been investigated in perirenal ADSCs in the context of aging or metabolic derangement, inflammatory priming has been shown to enhance their immunosuppressive capacity.These strategies to "recondition" ADSCs isolated from aged tissue or disease states (hypoxic and inflammatory priming in particular) have been very effective in restoring the regenerative potential of SAT derived ADSCs (reviewed in Ref.. Interestingly, pericardial ADSCs were shown to respond to myocardial infarction by switching to a pro-repair phenotype,suggesting that sourcing ADSCs from some injury/disease states could confer a regenerative benefit. In this study, Tang et al. were able to identify a surface marker to sort the proregenerative ADSCs to maximize the therapeutic benefit of cell injection.Alternatively, it is possible to isolate ADSCs from these depots in healthy adults in situations of organ and tissue donation. In this case, cell-free strategies harnessing paracrine signals present in exosomes could overcome both the issue of autologous ADSC dysfunction and allogenic ADSC rejection.Exosomes derived from human IPFP ADSCs were recently shown to protect articular cartilage in a mouse model of osteoarthritis, demonstrating that the regenerative power of exosomes can be harnessed outside of traditional abdominal adipose depots.ADSC based therapies sourcing from these nontraditional depots may also be able to capitalize on some new engineering strategies targeting beige and brown fat. While studies have yet to fully differentiate the regenerative potential-and heterogeneity-of ADSCs derived from the nonwhite fats, many have hypothesized that their paracrine effects are modifiable by adrenergic (browning) stimuli. Positive selection of progenitors that are sensitive to browning stimuli, priming with browning stimuli prior to cell delivery and delivery of cells in conjunction with browning stimuli all have the potential to further direct the regenerative action of beige fat derived ADSCs. While current engineering strategies are focused on generating and transplanting mature BAT,they have demonstrated the value of optimizing engineering strategies to brown and beige progenitors.
As our understanding of the diversity of ADSCs expands, population-specific engineering will likely play a significant role in expanding the use of ADSCs in regenerative medicine. However, a word of caution is warranted as much remains to be done at the preclinical and clinical levels before ADSCs can be effectively used in therapeutically. A heterogeneous stromal vascular fraction (SVF) containing ADSCs has become a popular therapy, sometimes discussed as a "cure-all" despite a lack of careful characterization and consistent documentation of clinical efficacy. Variability in outcomes across clinical trials, and in clinics, no doubt reflects a lack of uniformity in isolation and delivery, as well as a limited understanding of the factors that influence the transplanted ADSC behavior. A step back to fundamentally understand these cells and their heterogeneity will enable guided, targeted ADSC-based therapeutics with consistent and positive outcomes.
# Conclusions
There is incredible diversity in function across adipose depots, and data support the concept that each depot is specialized to play a specific role in homeostasis and disease. At least some of this specialization is retained in isolated ADSCs. Preferential potencies, different cytokine secretion profiles, and varied immunomodulatory capacities suggest that ADSCs from specific sources may be optimal for specific applications. While much more characterization and comparative data are needed, work to date strongly suggests that improving methodologies to utilize ADSCs from multiple depots could significantly improve regenerative medicine outcomes.
AUTHORS' CONTRIBUTIONS C.G. and J.P. contributed equally to this work.
# Acknowledgments
This work was supported by the National Institutes of Health (NIH) under Grant Nos. R21AR071582 and R01AR075773.
## Data availability
Data sharing is not applicable to this article as no new data were created or analyzed in this study.
## Apl bioengineering
## Review
scitation.org/journal/apb |
The Florence Statement on Triclosan and Triclocarban
The Florence Statement on Triclosan and Triclocarban documents a consensus of more than 200 scientists and medical professionals on the hazards of and lack of demonstrated benefit from common uses of triclosan and triclocarban. These chemicals may be used in thousands of personal care and consumer products as well as in building materials. Based on extensive peer-reviewed research, this statement concludes that triclosan and triclocarban are environmentally persistent endocrine disruptors that bioaccumulate in and are toxic to aquatic and other organisms. Evidence of other hazards to humans and ecosystems from triclosan and triclocarban is presented along with recommendations intended to prevent future harm from triclosan, triclocarban, and antimicrobial substances with similar properties and effects. Because antimicrobials can have unintended adverse health and environmental impacts, they should only be used when they provide an evidence-based health benefit. Greater transparency is needed in product formulations, and before an antimicrobial is incorporated into a product, the long-term health and ecological impacts should be evaluated.
# Introduction
In September 2016, the U.S. Food and Drug Administration (FDA) banned nineteen antimicrobial ingredients, including triclosan and triclocarban, in over-the-counter consumer antiseptic wash products based on insufficient evidence demonstrating their safety for long-term daily use and that they reduce the spread of illness and infection. Many of those 19 chemicals have been in widespread use for decades, and many are still allowed in a number of other over-the-counter personal care products as well as in consumer and building products. The FDA first indicated in a 1974 Tentative Final Monograph that there was insufficient evidence to show that triclosan was effective and safe for long-term use [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref]. The FDA's decades-long path to issuing a final rule, and the narrow scope of the September 2016 Final , indicate that existing regulatory practices are not sufficient to protect human and ecosystem health from adverse impacts of antimicrobial chemicals. Scientists from both academia and nonprofit organizations coauthored The Florence Statement in 2016 to share current scientific research on two widely used antimicrobial chemicals and to motivate broader consideration of the long-term impacts of antimicrobial use (see Appendix I). The Statement was introduced at DIOXIN 2016, the 36th International Symposium on Halogenated Persistent Organic Pollutants in Florence, Italy, and has been signed by more than 200 international scientists and medical professionals (see Appendix II).
## The florence statement on triclosan and triclocarban
As scientists, medical doctors, and public health professionals, we are concerned about the continued widespread use of the chlorinated antimicrobials triclosan and triclocarban for the following reasons:
1. Triclosan and triclocarban are used as antimicrobials, a class of chemicals present in >2,000 products including soaps, toothpastes, detergents, clothing, toys, carpets, plastics, and paints. In personal care products like hand soap, there is no evidence that use of triclosan or triclocarban improves consumer or patient health or prevents disease. 2. Triclosan and triclocarban used in consumer products end up in the environment and have been detected in a wide variety of matrices worldwide. 3. Triclosan and triclocarban persist in the environment and are a source of toxic and carcinogenic compounds including dioxins, chloroform, and chlorinated anilines. 4. Triclosan, triclocarban, and their transformation products and byproducts bioaccumulate in aquatic plants and animals, and triclosan partitions into human blood and breast milk. 5. Triclosan and triclocarban have detrimental effects on aquatic organisms. 6. Humans are exposed to triclosan and triclocarban through direct contact with personal care products and from other sources including food, drinking water, and dust. Triclosan has been detected in the urine of a majority of humans tested. 7. Triclosan and triclocarban are endocrine disruptors and are associated with reproductive and developmental impacts in animal and in vitro studies. Potential implications for human reproduction and development are of concern and merit further study. 8. Human epidemiology and animal studies suggest triclosan exposure can increase sensitivity to allergens. 9. Overuse of triclosan may contribute to antibiotic/antimicrobial resistance and may modify the microbiome. 10. A number of authorities, including the FDA, have restricted the use of triclosan and triclocarban in certain types of soaps. These and other antimicrobial chemicals are generally not restricted from use in other products. We therefore call on the international community to limit the production and use of triclosan and triclocarban and to question the use of other antimicrobials. We urge scientists, governments, chemical and product manufacturers, purchasing organizations, retailers, and consumers to take the actions recommended below.
Recommendations 1. Avoid the use of triclosan, triclocarban, and other antimicrobial chemicals except where they provide an evidencebased health benefit (e.g., physician-prescribed toothpaste for treating gum disease) and there is adequate evidence demonstrating they are safe. 2. Where antimicrobials are necessary, use safer alternatives that are not persistent and pose no risk to humans or ecosystems. 3. Label all products containing triclosan, triclocarban, and other antimicrobials, even in cases where no health claims are made. 4. Evaluate the safety of antimicrobials and their transformation products throughout the entire product life cycle, including manufacture, long-term use, disposal, and environmental release.
Appendix I: Supporting Information 1. Triclosan and triclocarban are used as antimicrobials, a class of chemicals present in >2,000 products including soaps, toothpastes, detergents, clothing, toys, carpets, plastics, and paints [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref]. In personal care products like hand soap, there is no evidence that use of triclosan and triclocarban improves consumer or patient health or prevents disease [Centers for Disease Control and Prevention (CDC) 2003; FDA 2016). Triclosan and triclocarban are not well regulated and may be found in >2,000 consumer and building products [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref]. In 1998, the worldwide annual production of triclosan was approximately 1,500 tons, with a majority produced in Europe (350 tons) and the United States (450 tons) [bib_ref] Triclosan: Current status, occurrence, environmental risks and bioaccumulation potential, Dhillon [/bib_ref]. In 2006, an estimated 450 tons of triclosan was used within the European Union (EU) [Scientific Committee on Consumer Safety (SCCS) 2010]. In 2007, an estimated 85% of the total volume of triclosan in the EU was used in personal care and cosmetic products (SCCS 2010). Triclocarban has been primarily used in bar soaps at concentrations ranging from approximately 0.5% to 2% by weight [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref] [bib_ref] Urinary concentrations of the antibacterial agent triclocarban in United States residents: 2013-2014..., Ye [/bib_ref].
Epidemiological studies indicate that the use of triclosan and triclocarban by the general population has no significant health benefits for reducing common respiratory and gastrointestinal infections [bib_ref] Consumer antibacterial soaps: effective or just risky?, Aiello [/bib_ref] [bib_ref] Effect of hand hygiene on infectious diseases risk in the community setting:..., Aiello [/bib_ref]. A 2003 report by the U.S. Centers for Disease Control and Prevention Healthcare Infection Control Practices Advisory Committee concluded, "No evidence is available to suggest that use of [antimicrobial-impregnated articles and consumer items bearing antimicrobial labeling] will make consumers and patients healthier or prevent disease" .
According to the FDA, which is responsible for regulation of foods, drugs, cosmetics, medical devices, and similar products, there is no evidence that antibacterial soaps are more effective than nonantibacterial soap and water . This is likely because the contact time during typical hand washing (an average of 6 s) is too short to deliver measurable benefits [bib_ref] Hand washing practices in a college town environment, Borchgrevink [/bib_ref] and because the antibacterial ingredient is highly diluted during the washing process.
2. Triclosan and triclocarban used in consumer products end up in the environment [bib_ref] Fate of organohalogens in US wastewater treatment plants and estimated chemical releases..., Heidler [/bib_ref] and have been detected in a wide variety of matrices worldwide [bib_ref] Co-occurrence of triclocarban and triclosan in U.S. water resources, Halden [/bib_ref] [bib_ref] Triclosan: Occurrence and fate of a widely used biocide in the aquatic..., Singer [/bib_ref]. Triclosan and triclocarban are commonly used in products intended for washing [e.g., an estimated 96% of triclosan is used in products that are intentionally disposed of down the drain, such as soaps and detergents [bib_ref] An ecological risk assessment for triclosan in lotic systems following discharge from..., Reiss [/bib_ref] ]. These substances are also used in products that may be frequently washed (e.g., textiles, food contact materials, plastic surfaces). A large amount of triclosan and triclocarban is therefore discharged directly to conventional wastewater treatment plants [bib_ref] Fate of triclosan and triclosan-methyl in sewage treatment plants and surface waters, Bester [/bib_ref] [bib_ref] Co-occurrence of triclocarban and triclosan in U.S. water resources, Halden [/bib_ref]. During wastewater treatment, these chemicals partition preferentially into sewage sludge [bib_ref] Triclosan in a sewage treatment process -Balances and monitoring data, Bester [/bib_ref] [bib_ref] Fate of triclosan and triclosan-methyl in sewage treatment plants and surface waters, Bester [/bib_ref] [bib_ref] Partitioning, persistence, and accumulation in digested sludge of the topical antiseptic triclocarban..., Heidler [/bib_ref].
An analysis of U.S. sewage sludge found triclosan and triclocarban at high levels, on average in the tens of milligrams per kilogram dry weight [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref] ; U.S. Environmental Protection Agency (EPA) 2009]. In the United States, ∼ 15% of sewage sludge is incinerated, 30% is deposited in landfills, and 55% is deposited on land where the antimicrobial compounds and their transformation products may enter adjacent surface waters [bib_ref] Removal and formation of chlorinated triclosan derivatives in wastewater treatment plants using..., Buth [/bib_ref]. Through land application of biosolids, antimicrobials can also end up in livestock feed and in crops destined for human consumption [bib_ref] Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to..., Aryal [/bib_ref] [bib_ref] Bioaccumulation of triclosan and triclocarban in plants grown in soils amended with..., Prosser [/bib_ref].
Persisting fractions of triclosan and triclocarban that do not partition into the sludge are discharged to surface waters via effluent, where they can reach levels of thousands of nanograms per liter [bib_ref] Fate of triclosan and triclosan-methyl in sewage treatment plants and surface waters, Bester [/bib_ref] [bib_ref] Removal and formation of chlorinated triclosan derivatives in wastewater treatment plants using..., Buth [/bib_ref] [bib_ref] Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater..., Coogan [/bib_ref] [bib_ref] Measurement of triclosan in wastewater treatment systems, Mcavoy [/bib_ref] [bib_ref] Triclosan: Occurrence and fate of a widely used biocide in the aquatic..., Singer [/bib_ref].
Triclosan and triclocarban have been detected in the environment throughout the world. Triclosan has been detected in both raw and finished drinking water [bib_ref] Seasonal variations in concentrations of pharmaceuticals and personal care products in drinking..., Loraine [/bib_ref] , in ocean water [bib_ref] Occurrence and distribution of triclosan in the German Bight (North Sea), Xie [/bib_ref] , and in fresh water [bib_ref] Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999 -2000:..., Kolpin [/bib_ref]. A nationwide survey detected triclosan in ∼ 60% of U.S. streams [bib_ref] Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999 -2000:..., Kolpin [/bib_ref]. Triclocarban is expected to be similarly prevalent [bib_ref] Co-occurrence of triclocarban and triclosan in U.S. water resources, Halden [/bib_ref]. In surface waters, even when discharged at nanograms per liter concentrations, triclosan and triclocarban can concentrate and accumulate in sediments [bib_ref] Quantification of triclosan, chlorinated triclosan derivatives, and their dioxin photoproducts in lacustrine..., Anger [/bib_ref] [bib_ref] Dioxin photoproducts of triclosan and its chlorinated derivatives in sediment cores, Buth [/bib_ref] [bib_ref] Temporal trends of triclosan contamination in dated sediment cores from four urbanized..., Cantwell [/bib_ref] [bib_ref] Bioaccumulation of triclocarban in Lumbriculus Variegatus, Higgins [/bib_ref] [bib_ref] Quantification of hydroxylated polybrominated diphenyl ethers (OH-BDEs), triclosan, and related compounds in..., Kerrigan [/bib_ref] [bib_ref] Fate of triclosan and evidence for reductive dechlorination of triclocarban in estuarine..., Miller [/bib_ref] [bib_ref] Occurrence of triclosan, triclocarban, and its lesser chlorinated congeners in Minnesota freshwater..., Venkatesan [/bib_ref].
3. Triclosan and triclocarban persist in the environment and are a source of toxic and carcinogenic compounds including dioxins, chloroform, and chlorinated anilines [bib_ref] Dioxin photoproducts of triclosan and its chlorinated derivatives in sediment cores, Buth [/bib_ref] [bib_ref] Photodegradation of the antimicrobial triclocarban in aqueous systems under ultraviolet radiation, Ding [/bib_ref] [bib_ref] Formation of chloroform and other chlorinated byproducts by chlorination of triclosan-containing antibacterial..., Fiss [/bib_ref]. Triclosan and triclocarban are persistent in the environment. Both compounds are predicted to have half-lives on the order of 60d in water, 120d in soil, and 540d in sediment [bib_ref] Co-occurrence of triclocarban and triclosan in U.S. water resources, Halden [/bib_ref].
Sediment cores indicate long-term preservation of triclosan and triclocarban dating to approximately 1964 (when triclosan was patented) [bib_ref] Quantification of triclosan, chlorinated triclosan derivatives, and their dioxin photoproducts in lacustrine..., Anger [/bib_ref] [bib_ref] Occurrence and toxicity of antimicrobial triclosan and by-products in the environment, Bedoux [/bib_ref] [bib_ref] Temporal trends of triclosan contamination in dated sediment cores from four urbanized..., Cantwell [/bib_ref] [bib_ref] Quantification of hydroxylated polybrominated diphenyl ethers (OH-BDEs), triclosan, and related compounds in..., Kerrigan [/bib_ref] [bib_ref] Fate of triclosan and evidence for reductive dechlorination of triclocarban in estuarine..., Miller [/bib_ref] [bib_ref] Triclosan: Occurrence and fate of a widely used biocide in the aquatic..., Singer [/bib_ref]. In biosolids-amended soils, triclocarban and triclosan can persist for extended periods of time while exhibiting very slow or no measurable degradation [bib_ref] Field dissipation of 4-nonylphenol, 4-t-octylphenol, triclosan and bisphenol A following land application..., Langdon [/bib_ref] [bib_ref] Occurrence and loss over three years of 72 pharmaceuticals and personal care..., Walters [/bib_ref]. Triclosan may also be transformed to methyl triclosan or to other products [bib_ref] Fate of flame retardants and the antimicrobial agent triclosan in planted and..., Davis [/bib_ref] [bib_ref] Field dissipation of 4-nonylphenol, 4-t-octylphenol, triclosan and bisphenol A following land application..., Langdon [/bib_ref] [bib_ref] Occurrence and loss over three years of 72 pharmaceuticals and personal care..., Walters [/bib_ref]. Methyl triclosan may be more persistent than triclosan [bib_ref] Occurrence of methyl triclosan, a transformation product of the bactericide triclosan, in..., Balmer [/bib_ref] [bib_ref] Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater..., Coogan [/bib_ref] , and it has been consistently detected in surface waters and sediments [bib_ref] Fate of triclosan and triclosan-methyl in sewage treatment plants and surface waters, Bester [/bib_ref] [bib_ref] Development and use of polyethylene passive samplers to detect triclosans and alkylphenols..., Sacks [/bib_ref].
Triclosan is a "pre-dioxin" and is associated with formation of polychlorinated dioxins and furans (PCDDs/Fs) throughout its life cycle. Triclosan contains detectable contaminant levels of polychlorinated dioxins and furans, including toxic and carcinogenic 2,3,7,8-substituted PCDD/Fs, which are formed in amounts that vary with the quality of production technology [bib_ref] Testing for dioxin and furan contamination in triclosan, Menoutis [/bib_ref] ; United Nations Environment Programme (UNEP) 2013; [bib_ref] Polychlorinated dibenzo-p-dioxins and dibenzofurans pollution in China: Sources, environmental levels and potential..., Zheng [/bib_ref] ; International Agency for Research on Cancer (IARC) 2012]. The high persistence, bioaccumulation, and toxicity of these dioxins and furans in the environment is wellestablished [bib_ref] Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling, Sinkkonen [/bib_ref]. Furthermore, triclosan undergoes conversion to 2,8-dibenzodichloro-p-dioxin (2,8-DCDD) in water when exposed to natural sunlight [bib_ref] Photolytic degradation of triclosan in freshwater and seawater, Aranami [/bib_ref] [bib_ref] Photochemical conversion of triclosan to 2,8-dichlorodibenzo-p-dioxin in aqueous solution, Latch [/bib_ref] and during heating and combustion [bib_ref] Chlorination of Irgasan DP300 and formation from its chlorinated derivatives, Kanetoshi [/bib_ref] [bib_ref] Formation of polychlorinated dibenzo-p-dioxins upon combustion of commercial textile products containing 2,4,4..., Kanetoshi [/bib_ref]. In a recent study using an artificial skin model, topically applied triclosan transformed into 2,8-DCDD under ultraviolet irradiation [bib_ref] Pressurized liquid extraction-gas chromatography-mass spectrometry for confirming the photoinduced generation of dioxin-like..., Alvarez-Rivera [/bib_ref]. Chlorinated triclosan derivatives (formed during chlorine disinfection of wastewater and drinking water) transform into tri-and tetra-chlorinated dibenzo-p-dioxins in sunlight-exposed surface waters [bib_ref] Aquatic photochemistry of chlorinated triclosan derivatives: potential source of polychlorodibenzo-p-dioxins, Buth [/bib_ref] [bib_ref] Dioxin photoproducts of triclosan and its chlorinated derivatives in sediment cores, Buth [/bib_ref] and upon heating and combustion [bib_ref] Chlorination of Irgasan DP300 and formation from its chlorinated derivatives, Kanetoshi [/bib_ref] [bib_ref] Formation of polychlorinated dibenzo-p-dioxins upon combustion of commercial textile products containing 2,4,4..., Kanetoshi [/bib_ref]. Calculations suggest that incineration of sewage sludge containing triclosan and chlorinated triclosan derivatives contributes significantly to total dioxin emissions in the United States [bib_ref] Assessment of the contribution of triclosan to dioxin emissions from sludge incineration..., Doudrick [/bib_ref].
In water disinfection processes, triclosan can react with free chlorine to produce chloroform [bib_ref] Formation of chloroform and chlorinated organics by free-chlorine-mediated oxidation of triclosan, Rule [/bib_ref] , a probable human carcinogen (U.S. EPA 2001) that is also recognized by the State of California as a developmental toxicant [State of California Environmental Protection Agency (CalEPA) 2017]. In a study testing household dishwashing soaps, lotions, and body washes in chlorinated water under simulated normal household use conditions, all of the products containing triclosan produced either chloroform or other chlorinated byproducts [bib_ref] Formation of chloroform and other chlorinated byproducts by chlorination of triclosan-containing antibacterial..., Fiss [/bib_ref]. The results suggest that under some conditions, the use of triclosan in such products could potentially increase chloroform exposure to nearly double the background levels in tap water.
Triclocarban degrades via aerobic biodegradation and photolysis into 4-chloroaniline and 3,4-dichloroaniline [bib_ref] Photodegradation of the antimicrobial triclocarban in aqueous systems under ultraviolet radiation, Ding [/bib_ref] [bib_ref] Identification of wastewater bacteria involved in the degradation of triclocarban and its..., Miller [/bib_ref]. 4-Chloroaniline is recognized by the State of California as known to cause cancer (CalEPA 2017).
4. Triclosan, triclocarban, and their transformation products and byproducts bioaccumulate in aquatic plants [bib_ref] Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater..., Coogan [/bib_ref] and animals [bib_ref] Snail bioaccumulation of triclocarban, triclosan, and methyltriclosan in a North Texas, USA,..., Coogan [/bib_ref] [bib_ref] Occurrence of triclosan in plasma of wild Atlantic bottlenose dolphins (Tursiops truncatus)..., Fair [/bib_ref] , and triclosan partitions into human blood and breast milk [bib_ref] Triclosan in plasma and milk from Swedish nursing mothers and their exposure..., Allmyr [/bib_ref]. Triclosan and triclocarban are highly hydrophobic and bioaccumulate in organisms living in aquatic systems exposed to effluent from wastewater treatment plants. Triclosan has been detected in wild bottlenose dolphins at levels similar to those in humans [bib_ref] Occurrence of triclosan in plasma of wild Atlantic bottlenose dolphins (Tursiops truncatus)..., Fair [/bib_ref] , and it has also been detected at high levels in fish [bib_ref] Triclosan, a commonly used bactericide found in human milk and in the..., Adolfsson-Erici [/bib_ref] [bib_ref] Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in..., Valters [/bib_ref]. These levels are potentially high enough to cause harm [bib_ref] Contaminants of emerging concern in a large temperate estuary, Meador [/bib_ref]. Triclosan was recently detected in the eggs of skimmers, seabirds that serve as sensitive indicators of coastal health and of contaminant threats to fish-eating birds and animals [bib_ref] Identifying bioaccumulative halogenated organic compounds using a nontargeted analytical approach: Seabirds as..., Millow [/bib_ref]. Methyl triclosan, an even more lipophilic and stable bacterial transformation product of triclosan, has been detected in fish at levels considerably higher than in the surrounding water [bib_ref] Occurrence of methyl triclosan, a transformation product of the bactericide triclosan, in..., Balmer [/bib_ref] [bib_ref] Identification of methyl triclosan and halogenated analogues in male common carp (Cyprinus..., Leiker [/bib_ref]. The bioaccumulation and slow conversion of methyl triclosan in lower-level consumers such as catfish could transfer environmental triclosan to higher-level consumers in the food chain, including humans [bib_ref] Slow O-demethylation of methyl triclosan to triclosan, which is rapidly glucuronidated and..., James [/bib_ref]. Triclocarban bioaccumulates in freshwater worms [bib_ref] Bioaccumulation of triclocarban in Lumbriculus Variegatus, Higgins [/bib_ref] and fish [bib_ref] Bioconcentration, metabolism and excretion of triclocarban in larval Qurt medaka (Oryzias latipes), Schebb [/bib_ref]. Triclosan, methyl triclosan, and triclocarban all bioaccumulate rapidly in algae and snails exposed to wastewater treatment effluent with calculated bioaccumulation factors in the thousands [bib_ref] Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater..., Coogan [/bib_ref] [bib_ref] Snail bioaccumulation of triclocarban, triclosan, and methyltriclosan in a North Texas, USA,..., Coogan [/bib_ref].
In biosolids-amended soil ecosystems, triclosan, methyl triclosan, and triclocarban bioaccumulate in earthworms [bib_ref] Persistence of triclocarban and triclosan in soils after land application of biosolids..., Higgins [/bib_ref] [bib_ref] Bioaccumulation of pharmaceuticals and other anthropogenic waste indicators in earthworms from agricultural..., Kinney [/bib_ref] [bib_ref] Triclocarban, triclosan and its transformation product methyl triclosan in native earthworm species..., Macherius [/bib_ref] , the basis of many terrestrial food webs. Phytoaccumulation of triclosan and triclocarban has been observed in certain vegetable crops grown in biosolids-amended soils. Calculations suggest that potential human exposure from contaminated vegetable consumption is less than exposure from personal care product use but greater than exposure from consumption of drinking water [bib_ref] Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to..., Aryal [/bib_ref] [bib_ref] Uptake and accumulation of antimicrobials, triclocarban and triclosan, by food crops in..., Mathews [/bib_ref].
Upon human exposure and uptake, triclosan and triclocarban are metabolized and excreted by the body within 36-72h [bib_ref] Investigation of human exposure to triclocarban after showering and preliminary evaluation of..., Schebb [/bib_ref] [bib_ref] Whole blood is the sample matrix of choice for monitoring systemic triclocarban..., Schebb [/bib_ref]. One study calculated a terminal plasma half-life of 21h for triclosan ). Blood-borne triclosan and triclocarban can cross the placenta, and triclosan and its metabolites have been detected in umbilical cord blood at birth [bib_ref] Triclosan in plasma and milk from Swedish nursing mothers and their exposure..., Allmyr [/bib_ref] [bib_ref] Human fetal exposure to triclosan and triclocarban in an urban population from..., Pycke [/bib_ref] [bib_ref] Detection of phenolic endocrine disrupting chemicals (EDCs) from maternal blood plasma and..., Shekhar [/bib_ref] , raising concerns about prenatal exposure to the developing fetus. Triclosan, triclocarban, and their metabolites have also been detected in human milk samples [bib_ref] Triclosan, a commonly used bactericide found in human milk and in the..., Adolfsson-Erici [/bib_ref] [bib_ref] Triclosan in plasma and milk from Swedish nursing mothers and their exposure..., Allmyr [/bib_ref] [bib_ref] Risk assessment of triclosan [Irgasan ® ] in human breast milk, Dayan [/bib_ref] [bib_ref] Triclosan in individual human milk samples from Australia, Toms [/bib_ref]. For example, in one population sample (n = 151), triclosan levels were detected in >93% of milk samples over a wide range of concentrations [bib_ref] Triclosan in individual human milk samples from Australia, Toms [/bib_ref]. The ability of triclosan to partition into human milk raises concerns about impacts from exposure on nursing infants. 5. Triclosan and triclocarban have detrimental effects on aquatic organisms [bib_ref] Ecotoxicity and screening level ecotoxicological risk assessment of five antimicrobial agents: Triclosan,..., Tamura [/bib_ref]. The continuous exposure of aquatic organisms to triclosan and triclocarban, coupled with their bioaccumulation potential, have led to detectable levels of triclosan and triclocarban throughout aquatic food chains in species such as algae, crustaceans, fish, and marine mammals [bib_ref] Triclosan, a commonly used bactericide found in human milk and in the..., Adolfsson-Erici [/bib_ref] [bib_ref] Environmental exposure of aquatic and terrestrial biota to triclosan and triclocarban, Chalew [/bib_ref] [bib_ref] Occurrence of triclosan in plasma of wild Atlantic bottlenose dolphins (Tursiops truncatus)..., Fair [/bib_ref] [bib_ref] Contaminants of emerging concern in a large temperate estuary, Meador [/bib_ref]. Highly sensitive indicator organisms, such as algae and crustaceans, experience potentially harmful exposures to triclosan and triclocarban in surface waters receiving raw and treated sewage ). Benthic organisms such as worms, crabs, and shellfish can be exposed to triclosan and triclocarban via particulate matter and sediments .
In laboratory studies of algae, crustaceans, and fish, both triclosan and triclocarban have been shown to exhibit acute and subchronic toxicity at concentrations found in the environment [bib_ref] Ecotoxicity and screening level ecotoxicological risk assessment of five antimicrobial agents: Triclosan,..., Tamura [/bib_ref] [bib_ref] Toxic assessment of triclosan and triclocarban on Artemia salina, Xu [/bib_ref]. Triclosan exposure inhibits algal growth [bib_ref] Aquatic toxicity of triclosan, Orvos [/bib_ref] , which can alter aquatic ecosystem dynamics. Triclosan is acutely toxic to aquatic macrobiota at microgram per liter (lg=L) concentrations [bib_ref] What contributes to the sensitivity of microalgae to triclosan?, Franz [/bib_ref] [bib_ref] Effects of triclosan on the early life stages and reproduction of medaka..., Ishibashi [/bib_ref] [bib_ref] Triclosan persistence through wastewater treatment plants and its potential: Toxic effects on..., Ricart [/bib_ref] , with acute toxicity values ranging from 1:4 lg=L to 3,000 lg=L (von der Ohe et al. 2012).
Triclosan affects reproduction and development in some fish [bib_ref] Triclosan: Environmental exposure, toxicity and mechanisms of action, Dann [/bib_ref] and may interfere with the action of thyroid hormones in amphibians at environmentally relevant concentrations [bib_ref] The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic..., Veldhoen [/bib_ref]. Triclosan and triclocarban can also affect reproduction in snails at environmentally relevant concentrations [bib_ref] The antimicrobial agents triclocarban and triclosan as potent modulators of reproduction in..., Geiß [/bib_ref] [bib_ref] The antimicrobial triclocarban stimulates embryo production in the freshwater mudsnail Potamopyrgus antipodarum, Giudice [/bib_ref].
6. Humans are exposed to triclosan and triclocarban through direct contact with personal care products [bib_ref] Absorption, pharmacokinetics, and safety of triclosan after dermal administration, Queckenberg [/bib_ref] [bib_ref] Investigation of human exposure to triclocarban after showering and preliminary evaluation of..., Schebb [/bib_ref] and from other sources including food, drinking water, and dust [bib_ref] Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to..., Aryal [/bib_ref]. Triclosan has been detected in the urine of a majority of humans tested [bib_ref] Urinary concentrations of triclosan in the, Calafat [/bib_ref]. Human exposure to triclosan occurs primarily from the topical application and use of personal care products such as lotions, soaps, toothpastes, and mouthwashes [bib_ref] Triclosan: Applications and safety, Bhargava [/bib_ref] [bib_ref] Percutaneous penetration and dermal metabolism of triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether), Moss [/bib_ref] [bib_ref] Absorption, pharmacokinetics, and safety of triclosan after dermal administration, Queckenberg [/bib_ref]. Minor routes of exposure could include contaminated food and drinking water [bib_ref] Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to..., Aryal [/bib_ref] [bib_ref] Uptake of human pharmaceuticals and personal care products by cabbage (Brassica campestris)..., Holling [/bib_ref] [bib_ref] Simultaneous determination and assessment of 4-nonylphenol, bisphenol A and triclosan in tap..., Li [/bib_ref] [bib_ref] Seasonal variations in concentrations of pharmaceuticals and personal care products in drinking..., Loraine [/bib_ref] [bib_ref] Metabolization of the bacteriostatic agent triclosan in edible plants and its consequences..., Macherius [/bib_ref] [bib_ref] Uptake of pharmaceutical and personal care products by soybean plants from soils..., Wu [/bib_ref] [bib_ref] Comparative uptake and translocation of pharmaceutical and personal care products (PPCPs) by..., Wu [/bib_ref] and indoor dust [bib_ref] Simultaneous quantitation of parabens, triclosan, and methyl triclosan in indoor house dust..., Fan [/bib_ref] [bib_ref] Assessment of human exposure to bisphenol-A, triclosan and tetrabromobisphenol-A through indoor dust..., Geens [/bib_ref].
A large U.S. national survey found triclosan in the urine of the majority of people tested [bib_ref] Urinary concentrations of triclosan in the, Calafat [/bib_ref]. Other studies have measured triclosan in the urine of pregnant women [bib_ref] Distribution, variability, and predictors of urinary concentrations of phenols and parabens among..., Meeker [/bib_ref] [bib_ref] Urinary concentrations of environmental phenols in pregnant women in a pilot study..., Mortensen [/bib_ref] [bib_ref] Human fetal exposure to triclosan and triclocarban in an urban population from..., Pycke [/bib_ref] , children [bib_ref] Pilot study of urinary biomarkers of phytoestrogens, phthalates, and phenols in girls, Wolff [/bib_ref] , and a large sampling of people in Denmark [bib_ref] Human urinary excretion of non-persistent environmental chemicals: An overview of Danish data..., Frederiksen [/bib_ref]. Triclosan has been detected in breast milk [bib_ref] Risk assessment of triclosan [Irgasan ® ] in human breast milk, Dayan [/bib_ref] [bib_ref] Triclosan in individual human milk samples from Australia, Toms [/bib_ref] [bib_ref] Triclosan in plasma and milk from Swedish nursing mothers and their exposure..., Allmyr [/bib_ref] , serum and plasma [bib_ref] Triclosan in plasma and milk from Swedish nursing mothers and their exposure..., Allmyr [/bib_ref] [bib_ref] The influence of age and gender on triclosan concentrations in Australian human..., Allmyr [/bib_ref] , cord blood [bib_ref] Human fetal exposure to triclosan and triclocarban in an urban population from..., Pycke [/bib_ref] , amniotic fluid [bib_ref] Prenatal exposure to environmental phenols: Concentrations in amniotic fluid and variability in..., Philippat [/bib_ref] [bib_ref] Detection of phenolic endocrine disrupting chemicals (EDCs) from maternal blood plasma and..., Shekhar [/bib_ref] , and fingernails and toenails [bib_ref] Chinese population exposure to triclosan and triclocarban as measured via human urine..., Yin [/bib_ref].
Dermal exposure from personal care products is believed to be the main route of human exposure to triclocarban [bib_ref] Biomarkers of exposure to triclocarban in urine and serum, Ye [/bib_ref]. A human study showed a small but significant amount of triclocarban was absorbed during showering for 15 min with triclocarban-containing antibacterial soap [bib_ref] Investigation of human exposure to triclocarban after showering and preliminary evaluation of..., Schebb [/bib_ref]. In addition, minor routes of triclocarban exposure may include contaminated food [bib_ref] Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to..., Aryal [/bib_ref] [bib_ref] Metabolization of the bacteriostatic agent triclosan in edible plants and its consequences..., Macherius [/bib_ref] [bib_ref] Uptake of pharmaceutical and personal care products by soybean plants from soils..., Wu [/bib_ref] [bib_ref] Comparative uptake and translocation of pharmaceutical and personal care products (PPCPs) by..., Wu [/bib_ref]. In a recent study of 209 adults living in China, triclocarban was detected in the urine and in the nails of 99% and 100% of study participants, respectively [bib_ref] Chinese population exposure to triclosan and triclocarban as measured via human urine..., Yin [/bib_ref]. Triclocarban was detected in 86% of urine samples and in 23% of cord blood samples from 181 pregnant U.S. women between 2007 and 2009 [bib_ref] Human fetal exposure to triclosan and triclocarban in an urban population from..., Pycke [/bib_ref]. In a 2012 study of 158 U.S. adults with no known exposure to triclocarban, the compound was detected in 35% of urine samples [bib_ref] Automated on-line column-switching HPLC-MS/ MS method for the quantification of triclocarban and..., Zhou [/bib_ref]. In a smaller 2011 study, triclocarban was detected in 50% of serum samples and in 28% of urine samples from U.S. adults [bib_ref] Biomarkers of exposure to triclocarban in urine and serum, Ye [/bib_ref].
Monitoring and explorative studies of other potential sources of triclosan and triclocarban exposure are warranted [bib_ref] Consumer products as sources of chemical exposures to children: Case study of..., Ginsberg [/bib_ref].
7. Triclosan and triclocarban are endocrine disruptors and are associated with reproductive and developmental impacts in animal and in vitro studies [bib_ref] Application of the navigation guide systematic review methodology to the evidence for..., Johnson [/bib_ref] [bib_ref] Reproductive endocrine-disrupting effects of triclosan: Population exposure, present evidence and potential mechanisms, Wang [/bib_ref]. Potential implications for human reproduction and development are of concern and merit further study.
Triclosan and triclocarban have been shown to interfere with estrogen and androgen systems in mammalian models [bib_ref] Effects of triclocarban on intact immature male rat: Augmentation of androgen action, Duleba [/bib_ref] [bib_ref] Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats..., Kumar [/bib_ref] [bib_ref] Triclosan exposure modulates estrogendependent responses in the female Wistar rat, Stoker [/bib_ref] and in vitro [bib_ref] Oestrogenic and androgenic activity of triclosan in breast cancer cells, Gee [/bib_ref] [bib_ref] Comparison of in vitro cytotoxicity, estrogenicity, and anti-estrogenicity of triclosan, perfluorooctane sulfonate..., Henry [/bib_ref] [bib_ref] The in vitro estrogenic activities of triclosan and triclocarban, Huang [/bib_ref]. In vitro screening assays suggest that triclosan can interact with the estrogen receptor (ER) in certain cell types at relatively low (nanomolar) concentrations . In vitro studies have shown a weak estrogenic effect of triclosan and triclocarban in the ER reporter gene assay [bib_ref] The in vitro estrogenic activities of triclosan and triclocarban, Huang [/bib_ref] and in MCF7-BOS breast cancer cells [bib_ref] Comparison of in vitro cytotoxicity, estrogenicity, and anti-estrogenicity of triclosan, perfluorooctane sulfonate..., Henry [/bib_ref]. Triclosan has also shown estrogenic and androgenic activity in vitro in breast cancer cells at environmentally relevant concentrations ). However, in vivo studies suggest that the estrogenic effects of triclosan may not be a result of direct binding with the estrogen receptor. Triclosan has been shown to enhance the estrogenic activity of synthetic estrogenic compounds [bib_ref] The effect of triclosan on the uterotrophic response to extended doses of..., Louis [/bib_ref] and to increase estradiol [bib_ref] Triclosan elevates estradiol levels in serum and tissues of cycling and peri-implantation..., Pollock [/bib_ref] and bisphenol Auptake in certain tissues in adult mice. In male roaches, coexposure to triclosan and to other anti-androgenic chemicals enhanced the feminizing effect of the estrogen 17a-ethinylestradiol on reproductive duct development [bib_ref] Environmental chemicals active as human antiandrogens do not activate a stickleback androgen..., Lange [/bib_ref]. These studies suggest that the estrogenic effect of triclosan in vivo may be due to inhibition of estrogen metabolism. An in vitro study with sheep placental tissue also showed that triclosan is a potent inhibitor of estrogen sulfotransferase [bib_ref] Triclosan is a potent inhibitor of estradiol and estrone sulfonation in sheep..., James [/bib_ref].
In rodent studies, triclosan exposure has been associated with reduced testosterone, luteinizing hormone, follicle stimulating hormone, and sperm production [bib_ref] Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats..., Kumar [/bib_ref] , as well as with implantation failure [bib_ref] Disruption of blastocyst implantation by triclosan in mice: Impacts of repeated and..., Crawford [/bib_ref] and spontaneous abortion .The varying results of in vivo studies to date may result from the use of different rodent strains and experimental procedures [bib_ref] Reproductive endocrine-disrupting effects of triclosan: Population exposure, present evidence and potential mechanisms, Wang [/bib_ref].
The possible effects of triclosan and triclocarban on human endocrine and reproductive systems have not been sufficiently studied. There is emerging evidence of associations between triclosan exposure and reduced semen quality [bib_ref] Environmental exposure to triclosan and semen quality, Zhu [/bib_ref] and reduced inhibin B and luteinizing hormones in men [bib_ref] Human exposure to endocrine disrupting chemicals and fertility: A casecontrol study in..., Hond [/bib_ref] and with longer time-to-pregnancy in a large retrospective study of pregnant women [bib_ref] Female exposure to phenols and phthalates and time to pregnancy: The Maternal-Infant..., Velez [/bib_ref].
Triclosan can disrupt the thyroid hormone system in animal models [bib_ref] Short-term exposure to triclosan decreases thyroxine in vivo via upregulation of hepatic..., Paul [/bib_ref] [bib_ref] Triclosan exposure modulates estrogendependent responses in the female Wistar rat, Stoker [/bib_ref] [bib_ref] The effects of triclosan on puberty and thyroid hormones in male Wistar..., Zorrilla [/bib_ref]. A meta-analysis of rodent data found significant and dose-dependent reductions in serum thyroxine after early postnatal administration of triclosan [bib_ref] Application of the navigation guide systematic review methodology to the evidence for..., Johnson [/bib_ref]. Perinatal triclosan exposure can reduce blood levels of maternal, fetal, and neonatal thyroxine levels in rodents [bib_ref] Triclosan exposure reduces thyroxine levels in pregnant and lactating rat dams and..., Axelstad [/bib_ref] [bib_ref] Developmental triclosan exposure decreases maternal, fetal, and early neonatal thyroxine: Dynamic and..., Paul [/bib_ref]. Potential effects of prenatal exposure on thyroxine levels should be carefully considered because even small reductions in thyroxine in pregnant women can have adverse effects on the neurodevelopment of children [bib_ref] Downstream effects of maternal hypothyroxinemia in early pregnancy: Nonverbal IQ and brain..., Ghassabian [/bib_ref] [bib_ref] Maternal hypothyroxinemia and effects on cognitive functioning in childhood: How and why, Henrichs [/bib_ref] [bib_ref] Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes, Miller [/bib_ref] [bib_ref] Upstream adverse effects in risk assessment: A model of polychlorinated biphenyls, thyroid..., Wise [/bib_ref] [bib_ref] Meeting report: Moving upstream-Evaluating adverse upstream end points for improved risk assessment..., Woodruff [/bib_ref].
Few human studies have examined the potential impacts of prenatal triclosan and triclocarban exposure on fetal growth and development. However, there is suggestive evidence that prenatal triclosan exposure is associated with reduced fetal growth late in pregnancy [bib_ref] Prenatal exposure to phenols and growth in boys, Philippat [/bib_ref] and with smaller head circumference at birth in boys [bib_ref] Prenatal triclosan exposure and anthropometric measures including anogenital distance in Danish infants, Lassen [/bib_ref] [bib_ref] Prenatal exposure to phenols and growth in boys, Philippat [/bib_ref] and that prenatal triclocarban exposure is associated with decreased gestational age at birth [bib_ref] Association of birth outcomes with fetal exposure to parabens, triclosan and triclocarban..., Geer [/bib_ref].
8. Human epidemiology [bib_ref] The associations of triclosan and paraben exposure with allergen sensitization and wheeze..., Spanier [/bib_ref] and animal studies [bib_ref] Exposure to triclosan augments the allergic response to ovalbumin in a mouse..., Anderson [/bib_ref] suggest triclosan exposure can increase sensitivity to allergens.
Large cross-sectional analyses of U.S. National Health and Nutrition Examination Survey (NHANES) participants have found positive associations between urinary triclosan concentrations in children and aeroallergen sensitization [bib_ref] Urinary levels of triclosan and parabens are associated with aeroallergen and food..., Savage [/bib_ref] [bib_ref] The associations of triclosan and paraben exposure with allergen sensitization and wheeze..., Spanier [/bib_ref] , atopic asthma [bib_ref] The associations of triclosan and paraben exposure with allergen sensitization and wheeze..., Spanier [/bib_ref] , diagnosis of allergic rhinitis or other allergies in those 18 y old [bib_ref] The impact of bisphenol A and triclosan on immune parameters in the..., Clayton [/bib_ref] , and food sensitization [bib_ref] Urinary levels of triclosan and parabens are associated with aeroallergen and food..., Savage [/bib_ref]. Similarly, a large cross-sectional analysis of Norwegian children found an association between urinary triclosan concentrations and allergic sensitization and rhinitis [bib_ref] Triclosan exposure and allergic sensitization in Norwegian children, Bertelsen [/bib_ref]. Among both child and adult NHANES participants with asthma, urinary triclosan concentration was associated with increased risk of asthma exacerbation in the previous year .
Animal studies support these findings and suggest that although triclosan may not be an allergen itself, it may act as an adjuvant and enhance allergic responses to a known allergen [bib_ref] Exposure to triclosan augments the allergic response to ovalbumin in a mouse..., Anderson [/bib_ref]. In mouse models, dermal exposure to triclosan at concentrations similar to those used in consumer products enhanced the hypersensitivity response to the egg-white allergen ovalbumin [bib_ref] Exposure to triclosan augments the allergic response to ovalbumin in a mouse..., Anderson [/bib_ref] , promoted sensitization and anaphylaxis to peanut [bib_ref] Triclosan promotes epicutaneous sensitization to peanut in mice, Tobar [/bib_ref] , promoted sensitization to the milk allergen alpha-lactalbumin [bib_ref] Triclosan promotes epicutaneous sensitization to peanut in mice, Tobar [/bib_ref] , and induced stimulation of the immune system [bib_ref] Investigations of immunotoxicity and allergic potential induced by topical application of triclosan..., Anderson [/bib_ref]. Demonstrating a potential mechanism for this immune alteration, dermal triclosan exposure changed gene expression and cytokine levels promoting a food sensitization phenotype in mice and in a human skin model [bib_ref] Triclosan induces thymic stromal lymphopoietin in skin promoting Th2 allergic responses, Marshall [/bib_ref].
9. Overuse of triclosan may contribute to antibiotic/antimicrobial resistance [bib_ref] Efficacy of triclosan as an antimicrobial hand soap and its potential impact..., Giuliano [/bib_ref] and may modify the microbiome. Concerns about triclosan-induced cross-resistance to antibiotics used in human medicine were voiced as early as 2001, although the extent to which triclosan and triclocarban contribute to antibiotic resistance is not yet clear [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref] [bib_ref] Antimicrobial chemicals are associated with elevated antibiotic resistance genes in the indoor..., Hartmann [/bib_ref] [bib_ref] Triclosan and antimicrobial resistance in bacteria: An overview, Yazdankhah [/bib_ref]. One large randomized controlled trial that examined bacterial flora isolated from hands showed decreased susceptibility over time to triclosan in the studied community [bib_ref] Relationship between triclosan and susceptibilities of bacteria isolated from hands in the..., Aiello [/bib_ref]. There is evidence that bacteria that develop resistance to triclosan can also exhibit lowered susceptibilities to other antimicrobial agents [bib_ref] Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and..., Braoudaki [/bib_ref]. Triclosan in stream sediments has been shown to trigger increases in triclosan resistance and changes in benthic bacterial community composition [bib_ref] Triclosan exposure increases triclosan resistance and influences taxonomic composition of benthic bacterial..., Drury [/bib_ref]. The clinical significance of these observations is unclear, but a legitimate concern remains: antimicrobials may exacerbate the problem of bacterial resistance to antibiotics [bib_ref] The impact of triclosan on the spread of antibiotic resistance in the..., Carey [/bib_ref] [bib_ref] Antimicrobial chemicals are associated with elevated antibiotic resistance genes in the indoor..., Hartmann [/bib_ref] [bib_ref] Characterization of triclosan-resistant mutants reveals multiple antimicrobial resistance mechanisms in Rhodospirillum rubrum..., Pycke [/bib_ref].
Recently, several animal studies have suggested that exposure to triclosan modifies the microbiome, including in the gut and intranasally [bib_ref] Triclosan exposure is associated with rapid restructuring of the microbiome in adult..., Gaulke [/bib_ref] [bib_ref] Triclosan promotes Staphylococcus aureus nasal colonization, Syed [/bib_ref]. However, longer-term human studies are needed to identify the impact of triclosan and other antimicrobial substances on the human microbiome both on the skin and in the gut.
10. A number of authorities, including the U.S. Food and Drug Administration, have restricted the use of triclosan and triclocarban in certain types of soaps [European Commission (EC) 2016; FDA 2016]. These and other antimicrobial chemicals are generally not restricted from use in other products. Several jurisdictions have recognized the risks from triclosan and triclocarban and have taken steps to reduce their use. Following an evaluation of triclosan by the Biocidal Products Committee of the European Chemicals Agency (ECHA), the European Commission (EC) decided in 2016 that triclosan is not approved for use in human hygiene biocidal products . Beginning in February 2017, triclosan will no longer be available in such products in the EU. Triclosan has also been banned from use in consumer sanitizing and cleansing products by the state of Minnesota, effective January 2017 (State of Minnesota 2016). In September 2016, the FDA issued a final rule, effective in 2017, that over-the-counter consumer antiseptic wash products containing the antibacterial active ingredients triclosan and triclocarban, or any of seventeen other antimicrobial ingredients, can no longer be marketed because they "are not generally recognized as safe and effective" (FDA 2016). In the United States, the FDA regulates the use of antimicrobials in personal care products and medical devices, whereas the U.S. EPA regulates the pesticidal uses of antimicrobials in other products [bib_ref] Application of the navigation guide systematic review methodology to the evidence for..., Johnson [/bib_ref].
Triclosan is being phased out of certain products by Procter & Gamble, Johnson & Johnson, and other manufacturers. The use of triclosan and triclocarban may continue in household, building, and other products not covered under existing restrictions.
Despite regulatory restrictions on triclosan, triclocarban, and certain other antimicrobials, the overall market for antimicrobial products has been predicted to grow [bib_ref] On the need and speed of regulating triclosan and triclocarban in the..., Halden [/bib_ref]. It is not yet clear what impact the 2016 EC decision, the FDA Final Rule, and other authoritative actions may have on market growth. Alternative antimicrobial substances may be used in place of triclosan and triclocarban in personal care, consumer, and building products. These replacement substances may have little to no publicly available safety information.
# Appendix ii: signatories
Institutional affiliations are provided for identification purposes only.
AcknowledgmentsThe content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of their organizations or funding sources. R.U.H.'s contribution to this project was supported in part by grant number R01ES020889 and its supplements from the National Institute of Environmental Health Sciences (NIEHS) and by grant number LTR 05/01/12 from the Virginia G. Piper Charitable Trust. A.E.A. received an unrestricted research grant from Gojo; Gojo had no role in the support of this research or any of A.E.A.'s research related to triclosan. W.A.A. received a grant from the National Science Foundation [CBET 0,967,163 (Using triclosan and polyhalogenated dibenzo-p-dioxins to elucidate the importance of natural and anthropogenic sources of OH-PBDEs in fresh and estuarine waters)] that ended in 2014. The Green Science Policy Institute [a 501(c)(3) nonprofit organization] received funding from New York Community Trust that was used to support the contributions of A. E.L., R.E.F., V.P.S., and A.B. to this project. Green Science Policy Institute has no actual or potential competing financial interests relating to this publication. D.A. is employed by Environmental Working Group and has no actual or potential competing financial interests to declare. T.S. works with Science and Environmental Health Network and has no actual or potential competing financial interests to declare. All other authors have no actual or potential competing financial interests to declare. |
R-spondin2 promotes hematopoietic differentiation of human pluripotent stem cells by activating TGF beta signaling
Background: Human pluripotent stem cells (hPSCs) provide supplies of potential functional blood cells to suffice the clinical needs. However, the underlying mechanism of generating genuine hematopoietic stem cells (HSCs) and functional blood cells from hPSCs remains largely elusive. Method: In this study, we supplied R-spondin2 exogenously during hematopoietic differentiation of hPSCs under various culture conditions and analyzed the production of hematopoietic progenitor cells (HPCs). We further added R-spondin2 at different temporal window to pin down the stage at which R-spondin2 conferred its effects. RNA-SEQ-based gene profiling was applied to analyze genes with significantly altered expression and altered signaling pathways. Finally, megakaryocytic differentiation and platelet generation were determined using HPCs with R-spondin2 treatment.Results: We found that R-spondin2 generated by hematopoiesis-supporting stromal cells significantly enhances hematopoietic differentiation of hPSCs. Supply of R-spondin2 exogenously at the early stage of mesoderm differentiation elevates the generation of APLNR + cells. Furthermore, early treatment of cells with R-spondin2 enables us to increase the output of hPSC-derived platelet-like particles (PLPs) with intact function. At the mechanistic level, R-spondin2 activates TGF-β signaling to promote the hematopoietic differentiation. Conclusions: Our results demonstrate that a transient supply of R-spondin2 can efficiently promote hematopoietic development by activating both WNT and TGF-β signaling. R-spondin2 can be therefore used as a powerful tool for large-scale generation of functional hematopoietic progenitors and platelets for translational medicine.
# Background
Hematopoietic stem cell transplantation and blood cell transfusion such as the use of red blood cells and platelet have been widely applied as potent treatments of multiple blood disorders. The limited availability of suitable HLA-matched donors and blood donors, especially in China, the modest expansion capability of LT-HSCs after chemotherapy treatment during allogenic transplantation, incidences of graft failure and GVHD (graft-versus-host disease) in haploidentical HSC transplantation, a short shelf life of platelets from blood donors, and potential infections or associated complications are currently limiting the application of these strategies, calling for a new and abundant source for blood cells. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), serve as a potential source of various blood cells due to their capability of long-term self-renewal and multipotent differentiation. This feature provides a promising alternative to the production of hematopoietic cells, such as hematopoietic stem cells (HSCs), platelets, and red blood cells. However, despite of numerous attempts to recapture the hematopoietic developmental process in vivo, the generation of functional blood cells remains at a low level of efficacy and quality, thereby calling for a more thorough understanding of the regulatory mechanism of hematopoietic differentiation and the development of more robust strategies for blood cell generation.
During embryonic development in vivo, hematopoietic development occurs from the mesoderm, which gives rise to both hematopoietic and vascular lineages. Hemogenic endothelium (HE) has been identified as the direct precursor of HSCs following an endothelium-hematopoietic transition (EHT). hPSC hematopoietic differentiation in vitro goes through similar stages, mimicking the in vivo progress of hematopoietic development. Multiple studies have shown that every stage of hematopoietic differentiation is under precise regulation by several key signaling pathways. For example, BMP4 is crucial for induction of primitive streak from hESCs, while formation of mesodermal cells also depends on synergistic regulation of the canonical WNT/β and Activin/Nodal signaling pathways. VEGF is necessary and sufficient for specifying HE cells, and bFGF acts synergistically with VEGF. In addition, the NOTCH signaling pathway plays a vital role in specifying arterial-type definitive HE from hPSCs. In the subsequent process of EHT, inhibition of the TGFβ signaling pathway is required for generating hematopoietic progenitors. Because of the pivotal roles of these signaling pathways, identification of novel components of the pathways and manipulation of the factors should benefit the efficiency of hematopoietic differentiation.
In addition to the intracellular signaling pathways, accumulating evidence has revealed the significance of extracellular factors in hematopoietic differentiation. During embryonic development and in adulthood, a wide range of soluble factors secreted from various cells exerts influences on development and maintenance of HSCs. Recent studies also emphasized the importance of extracellular paracrine factors in hematopoietic development in vitro. Menendez et al. reported that mesenchymal stem cell (MSC)-conditioned media augments hematopoietic specification from hESCs, implying an important role of secreted microenvironmental factors. Tenascin C, an extracellular matrix protein identified from OP9 feeder expression profiling, has been used in an hPSC hematopoietic differentiation system to enhance HEP generation and definitive hematopoiesis. Extracellular CXCL12/CXCR4 signaling can further confer hPSC-derived hematopoietic progenitor cells function of in vivo transplantation. To date, the mechanisms by which these extracellular factors function have not been well defined. Thus, exploring novel extracellular factors and unraveling their connection to key intracellular pathways should prove helpful in revealing the mechanisms of hematopoietic development and ameliorate the strategy of blood cell production in vitro.
We recently conducted RNA-SEQ screening and successfully identified new factors in controlling hPSC hematopoietic differentiation, including MEIS1 (Myeloid Ectopic Viral Integration Site 1 homolog) and MEIS2 (Myeloid Ectopic Viral Integration Site 2 homolog), which regulate HEP generation and EHT, respectively. In the current study, we have further revealed R-spondin2 as a key modulator of early hematopoietic differentiation of hPSCs, which augments APLNR + mesoderm cell generation. We have also identified TGFβ signaling as a novel downstream target of R-spondin2, which works in parallel to WNT signaling to mediate the effects of R-spondin2 on hPSC hematopoietic differentiation., and Z-15 hiPSCs (from Dr. Zhijian Xiao)were used in this study. BC1 cells were derived from BM CD34 + cells reprogrammed by OCT4, SOX2, KLF4, c-MYC, and LIN28 and characterized with pluripotency markers, karyotyping, in vitro pluripotency assay of embyroid body formation, and in vivo pluripotency assay of teratoma formation. Z-15 cells were derived from peripheral blood mononuclear cells reprogrammed by OCT4, SOX2, KLF4, c-MYC, and BCL-XL and characterized with pluripotency markers, karyotyping, and in vivo pluripotency assay of teratoma formation. hPSCs were cultured on Matrigel-coated plates (Corning) in mTeSR1 (Stem cell Technology) to maintain a pluripotent state. Medium was changed daily, and colonies were passaged every 4 days with 2 U/mL Dispase (Sigma) according to the manufacturer's instructions.
# Material and methods
Hematopoietic differentiation from hPSCs in mAGM-S3 co-culture Hematopoietic differentiation from hPSCs in mAGM-S3 co-culture system was performed as described earlier. hPSCs were first dissociated into single cells with Accutase (Sigma) and seeded at the density of 6 × 10 4 / mL with 10 μM ROCK inhibitor Y-27632. After 48 h of culture, when cells grew into small colonies with similar sizes, the hPSC colonies were passaged and plated onto over-confluent mAGM-S3 feeder cells. Forty-eight hours later, medium was replaced with IMDM hematopoietic differentiation medium containing 10% fetal bovine serum. Media were changed every day for up to 14 days. Re-constituted R-spondin2 was obtained from Peprotech. R-spondin2 was added directly to the culture medium at 20 ng/mL at day 0 of hematopoietic differentiation.
## Hematopoietic differentiation from hpscs in chemical defined system
Hematopoietic differentiation from hPSCs in chemically defined system was performed as described before. hPSCs were dissociated into single cells with Accutase and seeded with Y-27632 onto 12-well plates coated with growth factor-reduced Matrigel (Corning) at the density of 3.5 × 10 4 cells/mL. After 24 h, hematopoietic differentiation was performed using a stepwise induction procedure. Medium was replaced with Custom mTeSR1 (Stem cell Technology) supplemented with BMP4 (5 ng/mL) and Activin A (5 ng/mL) for days 0-2, VEGF (40 ng/mL) and bFGF (50 ng/mL) for days 2-4, and VEGF, bFGF, and SB431542 (20uM)(STEMGENT) for days 4-7. All cytokines used were from PeproTech. R-spondin2 was added directly to the culture medium at 20 ng/mL at day 0 of hematopoietic differentiation.
## Flow cytometry
To determine the proportion of defined populations in aforementioned differentiation culture conditions, differentiated cells were disassociated with 0.25% trypsin-EDTA, and cell suspension was stained with fluorescein-conjugated antibodies as follows: TRA-1-85-APC, hAPLNR-APC, hCD31-PE, hCD34-APC, hCD43-APC, and hCD45-PE. Detailed information for the antibodies is listed in Additional file 1: . Before analysis, cells were filtered through 70 μm cell strainer to obtain single cell suspension and stained with DAPI to exclude dead cells. Flow cytometry analysis was performed using a FACS CantoII flow cytometer, and cell sorting was performed using FACS Aria III sorter (BD Biosciences).
## Immunofluorescence
Cultured cells were fixed with 4% PFA in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 20 min, and blocked with 1-5% BSA in PBS for 1 h. Cells were then incubated with hCD43 and hCD45 primary antibodies at 4°C overnight. After washes of PBS, cells were incubated with fluorescent anti-mouse secondary antibody conjugated with Alexa Fluor 488 or 594 at room temperature (RT) for 1 h. The nuclei were stained with DAPI for 10 min before experimentation. The cells were examined and recorded using a fluorescence microscope. Information and dilution for the antibodies are listed in Additional file 1: .
Colony-forming unit (CFU) assay CFU assays were performed by plating 5 × 10 3 cells/ well from single cells obtained from day 12 of co-culture into methylcellulose H4435 in a 24-well plate. Cells were incubated at 37°C for 14 days before the colonies were counted based on standard morphological criteria. BFU-E (burst-forming unit-erythroid), CFU-E (colony-forming unit-erythrocyte), CFU-GM (colony-forming unit-granulocyte/macrophage), and CFU-GEMM (colony-forming unit-granulocyte/erythroid/macrophage/ monocyte) were classified and enumerated based on morphological recognition.
## Western blotting
Western blotting analysis was performed as described previously. hPSCs were lysed directly on ice in Laemmli sample buffer (BioRad) supplemented with phosphatase inhibitor cocktail. The cell lysates were electrophoresed on 10% SDS-PAGE gel and transferred onto PVEF membranes. The membranes were blocked at RT with 5% nonfat milk in 0.1% Tween-20 in PBS (PBST) for 1 h and then incubated with primary antibodies overnight at 4°C. After being washed with TBST, membranes were then incubated with HRP-coupled secondary antibodies for 2 h at room temperature and detected using the Super-Signal West Pico Chemiluminescent Substrate (Thermo) in ImageQuant LAS-4010 (GE). GAPDH was used as a loading control. Dilutions for various antibodies are shown in Additional file 1: . Densitometric quantitation of Western blotting results was performed with ImageJ software. Western blotting images were quantified by analyzing the intensities of greyscales of each band.
## Quantitative real-time pcr
RNA was extracted with the Trizol according to the manufacturer's instructions. RNA was transcribed into cDNA using random primers. Real-time PCR was carried out with SYBR green detection on an ABI 7900HT Fast Real-Time PCR cycler. Relative quantification of transcript levels was calculated using CT values normalized to ACTIN. Sequences for various primer pairs are shown in Additional file 1: .
## Rna-seq
RNA-SEQ analysis was performed as previously described. All human cells sorted with hTRA-1-85 + from day 2 of co-culture were enriched and analyzed by RNA-SEQ. The expression levels were visualized using a heatmap built based on the value of log10 (FPKM+1). GO enrichment and GSE analyses were performed. The data are available at Gene Expression Omnibus (GEO) (Accession number GEO: GSE118596).
# Statistical analysis
At least three independent experiments were performed for each analysis. The number of biological replicates is indicated by the n value. All graphs depict mean ± SD. Statistical analysis was performed using a two-tailed unpaired Student's t test, and the results were considered statistically significant at P value < 0.05 and were denoted as NS, not significant; *P < 0.05; ** P < 0.01; ***P < 0.001. The graphs and statistical evaluation were performed using GraphPad Prism (GraphPad Software).
# Results
## R-spondin2 promotes generation of hematopoietic progenitors from hescs
To discover novel regulators of hPSC early hematopoietic differentiation, we recently conducted RNA-SEQ screening and identified the role of MEIS1 and MEIS2 in modulating formation of HEP from mesoderm cells and in EHT, respectively. In the current study, we focused on the identification of potential extracellular regulators. We initially speculated that cytokines or growth factors may be produced by hematopoietic differentiation supporting stromal cells including mAGM-S3 and OP9-two cell lines extensively used for hematopoietic differentiation of hPSCs in a variety of studies including ours. Interestingly, from the published RNA-seq results, we discovered high expression of members of R-spondin family that are well-known WNT signaling agonists. R-spondin family includes four members: R-spondin1 to R-spondin4. Their expression was measured in mAGM-S3 cells, enabling us to find that R-spondin2 exhibited the highest expression among four members. Thus, we chose R-spondin2 for further functional studies.
To test the potential role of R-spondin2 in hESC hematopoietic differentiation, we added human R-spondin2 (20 ng/mL) to hESCs induced to undergo hematopoietic differentiation by mAGM-S3 co-culture. Indeed, we found that the addition of R-spondin2 significantly enhanced the appearance of cobble-stone-like hematopoietic progenitors from H1 hESCs (Additional file 2:. Immunofluorescence and flow cytometry analyses showed that much more CD43 + HPCs were generated upon R-spondin2 treatment in H1 cells (Additional file 2:, d; control 4.50% ± 0.12% vs R-spondin2 7.78% ± 0.07%, P < 0.001). This stimulatory effect was dose-dependent (Additional file 2:. In addition, a greater number of CD45 + HPCs were derived upon R-spondin2 treatment, f; control 4.44% ± 0.14% vs R-spondin2 7.03% ± 0.29%, P < 0.01; Additional file 2:, S1E). These results suggest that R-spondin2 is capable of enhancing hematopoietic differentiation of hESCs in a dosagedependent manner. To further assess the differentiation potential of generated hematopoietic progenitors, we utilized the colony-forming unit (CFU) assay and found significantly elevated number of total colonies after R-spondin2 treatment, upper panel; control 23.7 ± 1.9 vs R-spondin2 43 ± 4.2, P < 0.05). However, there was minimal change in the percentage of various types of colonies, lower panel, Additional file 2:. Thus, R-spondin2 enhances the generation of hematopoietic progenitors from hPSCs without changing the colony-forming capacity.
## R-spondin2 enhances hematopoietic differentiation of hpscs independently of culture conditions and cell lines
To exclude the possibility that the elevated hematopoietic differentiation conferred by R-spondin2 is cell line specific, we measured the effect of R-spondin2 on a human iPSC line BC1. Similar to the results from H1 cells, immunofluorescence and flow cytometry analyses also showed that much more CD43 + HPCs were generated with R-spondin2 treatment of BC1 cells ; control 1.72% ± 0.10% vs R-spondin2 2.48% ± 0.16%, P < 0.05). In addition, the proportion of CD45 + HPCs was elevated upon R-spondin2 treatment in BC1 cells (Additional file 3: -S2B; control 1.67% ± 0.38% vs R-spondin2 3.27% ± 0.46%, P < 0.05). Therefore, R-spondin2 also promotes hematopoietic differentiation from BC1 cells. In addition, we tested R-spondin2 function in two more hPS cell lines, H9 hES and Z-15 hiPS cells. In line with our observations for H1 and BC1 cells, R-spondin2 treatment enhanced the generation of CD43 + HPCs from both H9 and Z-15 cells ; H9 control 9.73 ± 0.47% vs R-spondin2 13.93 ± 0.38%, P < 0.01; Z-15 control 5.12 ± 0.27% vs R-spondin2 8.00 ± 0.36%, P < 0.01). These results strongly suggest that R-spondin2 promotes hematopoietic differentiation independently of cell lines.
Furthermore, to exclude the possibility that the elevated differentiation solely depended upon the mAGM-S3 culture system, we tested the effect of R-spondin2 in a chemically defined system described previously by us. Consistently, a similar increase of CD43 + HPCs was also observed for H1 cells with R-spondin2 treatment under this condition , h; control 11.77% ± 0.92% vs R-spondin2 22.33% ± 1.24%, P < 0.01) and BC1 cells , j; control 4.42% ± 0.16% vs R-spondin2 8.58% ± 0.72%, P < 0.01). In addition, we also verified the results in H9 and Z-15 cells (Additional file 3: -F; H9 control 4.57 ± 0.52% vs R-spondin2 7.59 ± 0.36%, P < 0.01; Z-15 control 7.44 ± 0.22% vs R-spondin2 11.4 ± 0.44%, P < 0.01). Besides, we analyzed the number of CD43 + HPCs generated per hPSC from all the above four cell lines in both culture systems and observed a similar increase of CD43 + HPC number generated from each hPSC in both differentiation system (Additional file 3: . By using four hPS cell lines including H1 and H9 hESCs, BC1, and Z-15 hiPSCs, we showed that R-spondin2 promotes hematopoietic differentiation of these cells in both the co-culture and chemical defined differentiation systems.
R-spondin2 treatment during early mesoderm differentiation suffices to promote hPSC hematopoietic differentiation As described in our previous studies, hematopoietic differentiation from hPSCs goes through a stepwise process including three stages-mesoderm induction (days 0-3), HE progenitor emergence (days 3-5), and HPC generation (days 5-7). Accordingly, we added R-spondin2 at these different stages to identify the stage(s) at which R-spondin2 exerts its functions (Additional file 4: . Interestingly, R-spondin-2 addition during days 0-3 led to the generation of the highest percentage of CD43 + and CD45 + HPCs compared to the treatment during other stages, as revealed by immunofluorescence studies . Little enhancement of differentiation was observed when R-spondin2 was added during the other two stages. Quantification with flow cytometry analysis further confirmed the observations .
Similar results were also observed in BC1 cells (Additional file 4: . To further narrow down the temporal window of R-spondin2 function, we included R-spondin2 at different time points of mesoderm for duration of 1 day (Additional file 4: . Surprisingly, we found that the addition of R-spondin2 at the first day of differentiation was capable of significantly enhancing CD43 + HPC generation in both H1 and BC1 cells. The effect was comparable to that of consecutive R-spondin2 treatment from day 0 to day 3 , d, Additional file 4: . In contrast, the addition of R-spondin2 at the second day or third day after induction of differentiation only induced minor or no . Together, our results showed that transient treatment of R-spondin2 at the early stage of mesoderm induction is sufficient to achieve maximal enhancement of hPSC hematopoietic differentiation.
## R-spondin2 augments aplnr + mesodermal cells
The ability of R-spondin2 to stimulate differentiation at the early stage led us to speculate that R-spondin2 might enhance the generation of mesoderm cells. Indeed, we found that the proportion of APLNR + cells, a subpopulation of lateral-plate mesodermal cells that can be eventually converted into CD31 + CD34 + HEPs, was elevated after R-spondin2 treatment in both H1 and BC1 cells (Additional file 5:; control 28.30% ± 4% vs R-spondin2 53.93% ± 1.64% P < 0.01, Additional file 5:. Consistently, the subsequent production of CD31 + CD34 + HEPs was also enhanced (Additional file 5:; control 1.35% ± 0.04% vs R-spondin2 4.27% ± 0.25%, P < 0.01, Additional file 5:. Similar results were also obtained for the chemically defined differentiation system, therefore ruling out culture system dependency (Additional file 5:R-spondin2 enhances hematopoiesis by augmenting APLNR + mesodermal cells.
To further confirm the role of R-spondin2 in mesoderm formation, we sorted TRA-1-85 + cells from R-spondin2-treated H1 cells at day 2 of mAGM-S3 co-culture differentiation and analyzed gene expression profiles by performing RNA-SEQ. In accordance with enhanced mesodermal differentiation, R-spondin2 treatment caused significant enrichment of mesodermassociated genes. In contrast, no significant difference was observed with respect to genes associated with other germ layers. Several cellular responses associated with mesoderm development such as mesoderm morphogenesis and mesoderm formation were enriched, as revealed by gene ontology (GO) of the upregulated genes in cells upon R-spondin2 treatment. Gene set enrichment analysis (GSEA) assay further confirmed significant changes (P < 0.05) in mesoderm-associated gene sets. We also validated these results by utilizing real-time RT-PCR. Consistently, representative genes associated with mesoderm development, such as BRACHYURY, MIXL1, APLNR, and KDR, were markedly upregulated from day 1 of differentiation. Together, these results suggest that R-spondin2 promotes hematopoietic differentiation of hPSCs by augmenting APLNR + mesoderm cells.
## Activation of tgf-β signaling by r-spondin2
To identify the downstream signaling pathways controlled by R-spondin2, we analyzed the RNA-SEQ results from cells with R-spondin2 treatment at day 2 of differentiation. KEGG analysis showed the enrichment of the WNT signaling pathway after R-spondin2 treatment, consistent with previous findings . Interestingly, among the significantly altered signaling pathways, enrichment of TGF-β signaling was even more significant than that of WNT signaling . GSEA confirmed that the enrichment of TGF-β signaling-associated gene sets in R-spondin2-treated cells was statistically significant (P = 0.02) . Consistently, as shown in the heatmap, a large number of genes associated with TGF-β signaling were upregulated after R-spondin2 treatment, including BMPs, TGFB1, ACVR1, and SMADs . Because R-spondins have been widely shown as WNT agonists, here we mainly focused on investigating the function of TGF-β signaling. We found that R-spondin2 treatment caused a dose-dependent elevation of phosphorylated SMAD2/3, which is widely used as an indicator of TGF-β signaling activity . These results are in agreement with the bioinformatics analyses. To further determine whether activation of TGF-β signaling has functional significance, we tested whether SB-431542, an inhibitor of TGF-β signaling pathway, could block the enhancement of mesoderm development triggered by R-spondin2. Indeed, addition of SB-431542 inhibited the elevated generation of APLNR + cells caused by R-spondin2 in a dose-dependent manner . When its dose reached 2 μM, SB-431542 nearly completely abolished the increase of APLNR + cells induced by R-spondin2 treatment . Together, both the bioinformatics analyses and functional studies demonstrated that the effect of R-spondin2 on hematopoietic differentiation of hPSCs is mediated, at least partially, by the activation of TGF-β signaling.
## R-spondin2 ultimately augments the production of functional platelets
We recently reported a three-step strategy for robust derivation of functional PLPs from hPSCs. To test whether the HPCs generated with R-spondin2 treatment ultimately increased the final production of PLPs from hPSCs, we harvested the cobblestone-like HPCs from day 12 of hematopoietic differentiation with or without R-spondin2 treatment and induced them to form megakaryocytes (MKs) and gradually PLPs. Consistent with previous observations, upon R-spondin2 treatment, the co-culture procedure yielded a significant larger number of cobblestone-like HPCs per dish . When induced to undergo further MK differentiation, the HPCs from both the control and R-spondin2-treated cells are capable of generating large MK cells at day 6 of differentiation . Quantitative analysis of CD41a + CD42b + MKs showed that R-spondin2 indeed increased the production of MK cells from H1 cells , while the MKs from both cultures showed high degree of polyploidy . Quantification of PLPs with flow cytometry analysis also showed increased PLP generation from H1 cells after R-spondin2 treatment (shown as per H1 cell; . Furthermore, we measured the in vitro function of PLPs derived from this culture condition. We found that the derived PLPs from cells with or without R-spondin2 treatment were capable of adhering to immobilized fibrinogen in the presence of thrombin, indicating normal function of adhesion and spreading . In addition, the PLPs derived from R-spondin2-treated cells could aggregate upon thrombin stimulation . Analysis of P-selectin (CD62P) expression revealed little difference in the function of α-granule release for PLPs derived from cells with or without R-spondin2 treatment . Thus, enhanced HPC generation by early stimulation of R-spondin2 led to an ultimate increase in the final production of functionally intact PLPs. As such, this approach might be used as a powerful tool for in vitro large-scale generation of PLPs for future transfusion purposes.
# Discussion
In this study, we found that R-spondin2 can be produced by hematopoietic differentiation-supporting stromal cells and is capable of strongly promoting hematopoietic differentiation of hPSCs independently of culture conditions and cell lines. A short-term treatment during the early window of mesoderm induction with R-spondin2 enhances the generation of a subpopulation of APLNR + cells more potent for hematopoietic differentiation, thus increasing the efficiency of HPC derivation. HPCs generated with R-spondin2 treatment are fully potent in further production of functional intact PLPs. Because of its potent effects on hematopoietic differentiation, we propose that R-spondin2 might potentially be used as a powerful tool for derivation of transplantable functional PLPs for translational medicine in the future. R-spondin2 has been shown to play a pivotal role in embryogenesis. Rspo2-deficient mice display distal limb loss and lung hypoplasia in embryos and die immediately after birth due to respiratory failure. Nevertheless, the role of R-spondin2 in hematopoiesis has not been reported to date. While searching potential extracellular regulators of hPSC hematopoietic differentiation, we found from previously published sequencing results that R-spondin2 is highly expressed in active stromal cells, while this expression is validated. Interestingly, in this study we found that exogenous R-spondin2 can robustly promote hematopoietic differentiation and that this effect is independent of cell lines and culture conditions. Functionally intact PLPs can be further generated using the HPCs derived with R-spondin2 treatment. Therefore, our study identified, for the first time, the role of R-spondin2 in hematopoietic differentiation of hPSCs. Thus, R-spondin2 can be potentially applied as a powerful tool for large-scale generation of functional HPCs and mature blood cells, including platelets, for regeneration medicine.
hPSC hematopoietic differentiation goes through the process of mesoderm induction, HE generation, and EHT, mimicking the embryonic hematopoietic development in vivo. We recently identified the role of. In this study, we further improved the understanding of the regulatory process by identifying a novel factor, R-spondin2, which confers its effect during the early window of mesoderm induction. As the origin of hematopoietic lineage, the mesoderm development in vivo and in vitro is under the regulation of several key signaling pathways. Among them, WNT signaling is one of the most critical factors as described previously. For example, Wnt3 deficiency causes gastrulation defects and loss of primitive streak in mouse embryos. During hPSC differentiation, activation of WNT signaling promotes mesoderm specification and consequently its derivative lineages, while WNT inhibition blocks this process. Consistent with these observations, we found that a well-established WNT agonist, R-spondin2, is sufficient to significantly promote hematopoietic differentiation and does so by augmenting APLNR + mesoderm subsets at the early stage of mesoderm induction. However, how R-spondin2 functions to stimulate this early population remains elusive. Therefore, it will be of great interest to explore the molecule mechanism underlying R-spondin2 function in mesoderm induction in future studies.
Interestingly, in addition to WNT signaling, we found that R-spondin2 can also activate TGFβ signaling to further regulate mesoderm development. To our knowledge, ours is the first study to reveal the functional link between R-spondin2 and TGFβ signaling. TGFβ signaling has been implicated in mesoderm specification both in vitro and in vivo. For example, Smad2, one of the key signaling mediators of TGFβ signaling, is essential for early embryonic development. hPSC mesoderm differentiation requires activation of TGFβ signaling while treatment with chemical inhibitors of TGFβ signaling severely impairs hPSC hematopoietic differentiation. Our study reveals a functional connection between R-spondin2 and TGFβ signaling, which contributes to mesoderm differentiation, providing a mechanistic explanation of how R-spondin2 exerts its effects on mesoderm induction beyond the activation of WNT signaling. However, it remains to be determined how R-spondin2 modulates TGFβ signaling in hPSC hematopoiesis. A comprehensive delineation of the detailed mechanisms underlying the crosstalk between R-spondin2 and TGFβ awaits future studies. |
Defense Mechanisms of Cotton Fusarium and Verticillium Wilt and Comparison of Pathogenic Response in Cotton and Humans
Citation: Man, M.; Zhu, Y.; Liu, L.; Luo, L.; Han, X.; Qiu, L.; Li, F.; Ren, M.; Xing, Y. Defense Mechanisms of Cotton Fusarium and Verticillium Wilt and Comparison of Pathogenic Response in Cotton and Humans. Int.
# Introduction
Cotton (Gossypium spp.) is an important cash crop as it yields grain, fiber, and oil [bib_ref] Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic..., Sunilkumar [/bib_ref]. Cotton is the world's most important source of natural fiber, providing approximately 35% of the world's total fiber [bib_ref] Regulatory Network of Cotton Genes in Response to Salt, Drought and Wilt..., Billah [/bib_ref]. Compared with synthetic fiber, cotton is a renewable resource and cotton plantations generate a higher number of jobs that involve planting, processing, and textile manufacturing. Cotton has important environmental and social benefits, and cottonseed can be used as animal feed after oil extraction. Cotton is grown in more than 80 countries, of which approximately 30 regard cotton as a major crop [bib_ref] Progress and perspective on drought and salt stress tolerance in cotton, Abdelraheem [/bib_ref]. China is the world's largest producer of cotton fiber. However, cotton has been affected by various pests and pathogens, among which Fusarium and Verticillium wilt cause serious global cotton economic losses [bib_ref] Fusarium Wilt of Cotton: Population Diversity and Implications for Management, Davis [/bib_ref] [bib_ref] The advances in cotton breeding resistance to Fusarium and Verticillium wilts in..., Cun [/bib_ref] , thus leading to them being considered as the main obstacle to sustainable high-quality cotton production in China [bib_ref] Temporal patterns of cotton Fusarium and Verticillium wilt in Jiangsu coastal areas..., Li [/bib_ref]. Therefore, this review describes the relationship between cotton and pathogenic fungi, using Fusarium and Verticillium as examples. Fusarium oxysporum (FOV) is the cause of Fusarium cotton wilt and is classified into eight physiological types, of which three exist in China [bib_ref] Identification of races of cotton Fusarium wilt in China, Qiying [/bib_ref]. Based on their symptoms, there are two species of Verticillium dahliae that infect cotton, namely defoliating and nondefoliating strains [bib_ref] Nondefoliating and defoliating strains from cotton correlate with races 1 and 2..., Hu [/bib_ref]. These pathogens can exist in soil and plant debris in the form of mycelium, chlamydia spores, and microsclerotia and survive in soil for a long time (until the beginning of a new infection cycle) [bib_ref] Identification of races of cotton Fusarium wilt in China, Qiying [/bib_ref]. In addition, they can survive saprophytically on other crops and weeds [bib_ref] Temporal patterns of cotton Fusarium and Verticillium wilt in Jiangsu coastal areas..., Li [/bib_ref]. Diseases caused by Fusarium typically manifest following the seedling stage and are most severe during the budding stage. The onset of Fusarium wilt is characterized by plaques between the main veins, while the rest of the leaves remain green; chlorotic plaques gradually cause the leaves to become necrotic and fall off the stem [bib_ref] Identification of races of cotton Fusarium wilt in China, Qiying [/bib_ref]. Verticillium wilt occurs before the budding stage, manifesting as yellow mottled or fallen leaves during the boll setting stage, when the disease is also the most serious [bib_ref] Nondefoliating and defoliating strains from cotton correlate with races 1 and 2..., Hu [/bib_ref]. However, the determinants that lead to the development of these diseases remain unclear [bib_ref] Temporal patterns of cotton Fusarium and Verticillium wilt in Jiangsu coastal areas..., Li [/bib_ref].
Humans are warm-blooded, their innate and adaptive immune system is complex, and they are naturally resistant to most invasive fungal infections. For fungi to successfully infect humans they must: (1) grow at or above human body temperature (37 - C), (2) bypass or penetrate host surface barriers to reach internal tissues, (3) digest and absorb human tissues, and (4) resist against the human immune system. Thus, the number of fungi capable of infecting a healthy human host is very limited [bib_ref] Comparative pathobiology of fungal pathogens of plants and animals, Dickman [/bib_ref] [bib_ref] Fungi infecting plants and animals: Killers, non-killers, and cell death, Sharon [/bib_ref]. However, the pathogenic fungi Fusarium and Aspergillus have these abilities and can therefore infect humans. Although there are limited data on the role of host defenses against these fungi, fusariosis, aspergillosis, and other fungal infections share many features [bib_ref] Fusarium infections in immunocompromised patients, Nucci [/bib_ref]. Hence, this review discusses Fusarium to illustrate the relationship between pathogenic fungi and humans. Fusarium can produce mycotoxins that pose a significant health risk to humans and animals. There has been a gradual increase in infections caused by Fusarium in immunosuppressed patients that have undergone organ transplantation and chemotherapy in recent years [bib_ref] Fusarium infection in hematopoietic stem cell transplant recipients, Nucci [/bib_ref]. Moreover, Fusarium is second only to Candida albicans and Aspergillus in terms of morbidity and mortality in immunodeficient individuals and is internationally classified as an important infectious disease [bib_ref] Chronic leg ulcer caused by Fusarium sonali: A case report and super-microstructure..., Ran [/bib_ref].
To eliminate, or significantly mitigate, the harm of Fusarium and Verticillium wilt on cotton so as to improve yield, and simultaneously seek a method to treat patients suffering from Fusarium spp. infectious diseases, we review the studies on cotton genes related to resistance and susceptibility to Fusarium and Verticillium wilt in recent years, summarize the molecular mechanisms by which humans resist Fusarium disease, compare resistance strategies in humans and plants, and provide novel insights into the improvements in the resistance of plants and humans against Fusarium.
## Molecular mechanisms of plant resistance to fusarium and verticillium wilt
Plants can use various strategies to counter invasion by Fusarium and Verticillium by activating complex cellular and molecular regulatory networks in vivo. This includes using pattern recognition receptors (PRRs) to induce immune responses, modifying cell walls to block pathogens, producing extracellular enzymes to directly degrade pathogens, or activating the expression of specific genes through various signaling pathways and transcription factors [bib_ref] An Overview of the Molecular Genetics of Plant Resistance to the Verticillium..., Song [/bib_ref].
In recent years, considerable research has been carried out on the physiological and molecular mechanisms of cotton Fusarium wilt caused by FOV and Verticillium wilt caused by V. dahliae [bib_ref] Regulatory Network of Cotton Genes in Response to Salt, Drought and Wilt..., Billah [/bib_ref]. This review highlights the relatively recent discovery of genes involved in defenses against Fusarium and Verticillium and classifies them according to whether they act in signaling cascades and transcriptional regulation or are directly involved in cell wall and protein defense [fig_ref] Figure 1: Functional schematic diagram of resistance genes in cotton [/fig_ref]. This provides a theoretical basis to better understand the molecular genetic mechanisms of plant resistance to Fusarium and Verticillium wilt and provides a reference for future studies on genetic resistance mechanisms to fungal wilt. Pink boxes represent receptorlike proteins that recognize and bind to extracellular pathogens or damaged plant tissue before then transmitting the signal downstream. Blue boxes represent genes involved in signal transduction, which are important transmitters of signals from the cell surface to the nucleus and ultimately to downstream reaction substrates. Yellow boxes represent transcription factors that interact with RNA polymerase to influence the initiation of the transcription of genes related to disease resistance. Grey boxes are defense-related proteins that both strengthen the defenses of the cell wall, keeping pathogenic bacteria out of the cell, and accumulate ROS levels, thereby activating the cell's hypersensitivity response, etc. Parts of the figure were drawn using pictures from Servier Medical Art (https://creativecommons.org/licenses/by/3.0/ accessed on 15 July 2022).
## Genes involved in signaling cascades and transcriptional regulation
During the long-term evolution of plants, an immune system directly controlled by a series of cross-linked signal transduction pathways and various plant hormones has been developed to rapidly regulate gene expression, thereby adapting, resisting, and tolerating various biotic stresses. The reaction substrates of these intricate signaling networks are usually transcription factors (TFs). TFs can be regulated via multiple signals and can also regulate the expression of multiple genes related to disease resistance. They also play an important regulatory role in the process of plant stress signal transmission.
Within this intricate signaling network, the mitogen-activated protein kinase (MAPK) cascade response is the primary pathway for sorting and amplifying external signals into intracellular signals, including three kinases-MAPK kinase kinases (MAPKKKs), MAPK kinases (MKKs), and MAPKs-which are important in response to biotic and abiotic stresses [bib_ref] MAPK cascades in plant disease resistance signaling, Meng [/bib_ref] [bib_ref] GhMPK16, a novel stress-responsive group D MAPK gene from cotton, is involved..., Shi [/bib_ref]. Infection of cotton by FOV significantly induced GhMPK20 expression, whereas silencing GhMPK20 enhanced resistance to Fusarium cotton wilt. The WRKY family of TFs regulate various biotic stress responses and physiological processes. GhWRKY40 is a substrate of GhMPK20; FOV induces GhWRKY40 expression through the GhMKK4-GhMPK20 cascade reaction, whereas silencing of GhMKK4 and GhWRKY40 enhances resistance to Fusarium cotton wilt [bib_ref] The cotton MAPK kinase GhMPK20 negatively regulates resistance to Fusarium oxysporum by..., Wang [/bib_ref]. However, GhWRKY53 silencing activates the jasmonic acid (JA) signaling pathway and inhibits the salicylic acid (SA) signaling pathway, which reduces resistance to Verticillium cotton wilt. Other members of the WRKY family are regulated by other hormones. GbWRKY1 is [fig_ref] Figure 1: Functional schematic diagram of resistance genes in cotton [/fig_ref]. Functional schematic diagram of resistance genes in cotton. Pink boxes represent receptorlike proteins that recognize and bind to extracellular pathogens or damaged plant tissue before then transmitting the signal downstream. Blue boxes represent genes involved in signal transduction, which are important transmitters of signals from the cell surface to the nucleus and ultimately to downstream reaction substrates. Yellow boxes represent transcription factors that interact with RNA polymerase to influence the initiation of the transcription of genes related to disease resistance. Grey boxes are defense-related proteins that both strengthen the defenses of the cell wall, keeping pathogenic bacteria out of the cell, and accumulate ROS levels, thereby activating the cell's hypersensitivity response, etc. Parts of the figure were drawn using pictures from Servier Medical Art (https://creativecommons.org/licenses/by/3.0/ accessed on 15 July 2022).
## Genes involved in signaling cascades and transcriptional regulation
During the long-term evolution of plants, an immune system directly controlled by a series of cross-linked signal transduction pathways and various plant hormones has been developed to rapidly regulate gene expression, thereby adapting, resisting, and tolerating various biotic stresses. The reaction substrates of these intricate signaling networks are usually transcription factors (TFs). TFs can be regulated via multiple signals and can also regulate the expression of multiple genes related to disease resistance. They also play an important regulatory role in the process of plant stress signal transmission.
Within this intricate signaling network, the mitogen-activated protein kinase (MAPK) cascade response is the primary pathway for sorting and amplifying external signals into intracellular signals, including three kinases-MAPK kinase kinases (MAPKKKs), MAPK kinases (MKKs), and MAPKs-which are important in response to biotic and abiotic stresses [bib_ref] MAPK cascades in plant disease resistance signaling, Meng [/bib_ref] [bib_ref] GhMPK16, a novel stress-responsive group D MAPK gene from cotton, is involved..., Shi [/bib_ref]. Infection of cotton by FOV significantly induced GhMPK20 expression, whereas silencing GhMPK20 enhanced resistance to Fusarium cotton wilt. The WRKY family of TFs regulate various biotic stress responses and physiological processes. Gh-WRKY40 is a substrate of GhMPK20; FOV induces GhWRKY40 expression through the GhMKK4-GhMPK20 cascade reaction, whereas silencing of GhMKK4 and GhWRKY40 enhances resistance to Fusarium cotton wilt [bib_ref] The cotton MAPK kinase GhMPK20 negatively regulates resistance to Fusarium oxysporum by..., Wang [/bib_ref]. However, GhWRKY53 silencing activates the jasmonic acid (JA) signaling pathway and inhibits the salicylic acid (SA) signaling pathway, which reduces resistance to Verticillium cotton wilt. Other members of the WRKY family are regulated by other hormones. GbWRKY1 is a negative regulator of the JA-mediated defense pathway involved in plant resistance to V. dahliae and mediates the transition from defense to development by activating JAZ1 expression during infection by V. dahliae [bib_ref] Cotton WRKY1 mediates the plant defense-to-development transition during infection of cotton by..., Li [/bib_ref]. GhWRKY48 expression in roots, stems, and leaves is induced by V. dahliae infestation, JA, and SA; whereas its silencing enhances resistance to Verticillium cotton wilt and may act as a negative regulatory transcription factor to repress the expression of downstream disease resistance genes, thereby affecting resistance to Verticillium cotton wilt [bib_ref] Resistance of GhWRKY48 Negatively Regulated Cotton Against Verticillium dahlia, Liu [/bib_ref]. MKK family members play a dual role in regulating resistance to fungal pathogens in cotton; GhMKK4, GhMKK6, and GhMKK9 positively regulate resistance to Verticillium wilt, whereas GhMKK10 negatively regulates resistance to Verticillium wilt [bib_ref] Subtle regulation of cotton resistance to Verticillium wilt mediated by MAPKK family..., Meng [/bib_ref]. MAPK scaffold protein (GhMORG1) interacts with GhMKK6 and GhMPK4, and its overexpression significantly enhances the activity of the GhMKK6-GhMPK4 cascade response and positively regulates resistance to Fusarium cotton wilt [bib_ref] Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by..., Wang [/bib_ref]. GhAP2C1 is a negative regulator of the MAPK cascade that interacts with GhMPK4 to negatively regulate the immune response to Fusarium cotton wilt [bib_ref] The Protein Phosphatase GhAP2C1 Interacts Together with GhMPK4 to Synergistically Regulate the..., Guo [/bib_ref]. Gene silencing experiments demonstrated that GhNDR1 and GhMKK2 are essential for cotton resistance to Verticillium wilt [bib_ref] Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt, Gao [/bib_ref].
Glutathione transferases (GSTs) catalyze their binding to glutathione for various physiological functions, including disarming endogenous and exogenous toxins, participating in the translocation of flavonoids, and affecting disease resistance in plants. GaGSTF9 overexpression positively regulates resistance to Verticillium wilt in Arabidopsis [bib_ref] A Phi-Class Glutathione S-Transferase Gene for Verticillium Wilt Resistance in Gossypium arboreum..., Gong [/bib_ref]. Similarly, GbGSTU7 positively regulates resistance to Fusarium wilt in Gossypium barbadense, whereas its silencing significantly reduces glutathione peroxidase activity in vivo and increases the incidence of Fusarium cotton wilt [bib_ref] Effect of Silencing GbGSTU7 Gene on the Resistance to Fusarium Wilt of..., Deng [/bib_ref].
Expression of ribosomal protein GaRPL18 is induced by SA treatment, suggesting that GaRPL18 is associated with SA-mediated signaling pathways. Silencing GaRPL18 significantly reduces the number of immune molecules induced by the SA signaling pathway and reduces Verticillium wilt resistance in cotton, whereas GaRPL18 overexpression increases plant resistance to Verticillium wilt [bib_ref] Salicylic acid-related cotton (Gossypium arboreum) ribosomal protein GaRPL18 contributes to resistance to..., Gong [/bib_ref]. Expression of cotton cyclin-dependent kinase E (GhCDKE) is induced by Verticillium wilt infestation and methyl jasmonate (MeJA); GhCDKE silencing in cotton increases vulnerability to Verticillium wilt, whereas its overexpression in Arabidopsis enhances resistance [bib_ref] A Cotton Cyclin-Dependent Kinase E Confers Resistance to Verticillium dahliae Mediated by..., Li [/bib_ref]. The JA-induced calcium-dependent protein kinase GhCPK33 negatively regulates resistance to Verticillium cotton wilt, and knockdown of GhCPK33 enhances resistance [bib_ref] Dual-targeting Wnt and uPA receptors using peptide conjugated ultra-small nanoparticle drug carriers..., Miller-Kleinhenz [/bib_ref]. The expression of GhIQM1 Ca 2+ -independent calmodulin-binding protein is induced by V. dahliae and SA, which may negatively regulate resistance to Verticillium cotton wilt by inhibiting the SA pathway [bib_ref] Cloning and functional verification of GhIQM1 gene of cotton in response to..., Li [/bib_ref]. GhBIN2 interacts with JAZ (jasmonate-ZIM domain) family proteins and phosphorylates JAZ proteins in response to infestation by V. dahliae. This may negatively regulate resistance to Verticillium cotton wilt by regulating the JA signaling pathway, since GhBIN2 overexpression in cotton increases sensitivity to Verticillium wilt [bib_ref] BIN2 negatively regulates plant defence against Verticillium dahliae in Arabidopsis and cotton, Song [/bib_ref].
Several stress factors induce the production of large amounts of reactive oxygen species (ROS) in plants, which act as direct toxic agents against pathogens, and may act as signaling molecules for defense; ROS levels are precisely regulated by the plant [bib_ref] Plant genes for abiotic stress, Ciarmiello [/bib_ref] [bib_ref] Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes, Mou [/bib_ref]. Ascorbic acid (AsA) is an important cellular antioxidant that oxidizes ROS and affects signaling pathways involving ROS, while oxidized inositol can be involved in AsA synthesis [bib_ref] myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis, Lorence [/bib_ref]. Inhibition of myo-inositol oxygenase GbMIOX5 causes H 2 O 2 accumulation, and a reduction in AsA content in cotton cells significantly reduces resistance to Verticillium cotton wilt [bib_ref] Suppressed expression of GbMIOX5 gene reduced the Verticillium dahlia resistance in Gossypium..., Tu [/bib_ref].
Lipids are essential plant cell components and can act as signaling molecules to regulate plant pathogen resistance [bib_ref] Fatty Acid-and Lipid-Mediated Signaling in Plant Defense, Lim [/bib_ref]. Silencing of stearoyl-ACP desaturase GhSSI2 enhances resistance to Fusarium and Verticillium cotton wilt. Inhibition of GhSSI2 expression reduces oleic acid content, which adversely affects cell membrane function and activates the immune response, leading to increased SA content. SA triggers a hypersensitive response (HR) in plants, with rapid accumulation of ROS and induction of high levels of PR (pathogenesis-related) gene expression, which seals the pathogen in the tomb of dead cells [bib_ref] Programmed cell death in the plant immune system, Coll [/bib_ref]. Oleic acid is also a defense signal which, at low levels, can directly upregulate the expression of different disease resistance genes in a pathway independent of SA and JA, whereas activating the second messenger, NO (nitric oxide), upregulates a wide range of intranuclear disease resistance genes [bib_ref] Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase..., Mo [/bib_ref].
Knockout of the SA-related spermine (Spm) protein, Spm synthase (GhSPMS), and S-adenosylmethionine decarboxylase (GhSAMDC) genes increases the susceptibility to Verticillium cotton wilt. Conversely, transfection of Arabidopsis with these genes enhances its resistance to Verticillium wilt, indicating that the SA signaling pathway and Spm biosynthesis are associated with plant resistance to Verticillium wilt [bib_ref] Cotton S-adenosylmethionine decarboxylasemediated spermine biosynthesis is required for salicylic acid-and leucine-correlated signaling..., Mo [/bib_ref]. Expression of cotton polyamine oxidase (GhPAO) in Arabidopsis increases its resistance to Verticillium wilt and causes significant accumulation of H 2 O 2 , SA, and camalexin (a plant antitoxin), implying that GhPAO promotes plant resistance to Verticillium wilt by activating signaling pathways associated with Spm and camalexin [bib_ref] Cotton polyamine oxidase is required for spermine and camalexin signalling in the..., Mo [/bib_ref].
The MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor family is involved in resistance to pathogen infection, and knockout of GhMYB108 makes cotton more susceptible to Verticillium wilt, whereas GhMYB108 overexpression improves resistance to Verticillium wilt in Arabidopsis [bib_ref] The cotton MYB108 forms a positive feedback regulation loop with CML11 and..., Cheng [/bib_ref]. Silencing the homeodomain transcription factor (HDTF1) activates the JA signaling pathway and further improves Verticillium cotton wilt resistance [bib_ref] Suppression of the homeobox gene HDTF1 enhances resistance to Verticillium dahliae and..., Gao [/bib_ref]. Both JA and V. dahliae can induce the expression of HD-ZIP transcription factor GhHB12, with its overexpression reducing the resistance of cotton to V. dahliae [bib_ref] HD-ZIP I Transcription Factor, Negatively Regulates the Cotton Resistance to Verticillium dahliae, He [/bib_ref]. GbbHLH171 interacts with, and is phosphorylated by, a defense-associated receptor-like kinase (GbSOBIR1) and positively regulates resistance to Verticillium cotton wilt [bib_ref] GbSOBIR1 confers Verticillium wilt resistance by phosphorylating the transcriptional factor GbbHLH171 in..., Zhou [/bib_ref].
Ethylene (ET) response factors (ERFs) are required for pathogen defense responses, and genes from different hormone signaling pathways all play important roles in response to infection by fungal pathogens. GbABR1 of the apetala 2 (AP2) family positively regulates plant resistance to Verticillium wilt, whereas its silencing increases disease rates in cotton [bib_ref] GbABR1 is associated with Verticillium wilt resistance in cotton, Liu [/bib_ref]. GbERFb is an AP2/ERF type TF which increases disease resistance in cotton [bib_ref] A novel Gossypium barbadense ERF transcription factor, GbERFb, regulation host response and..., Liu [/bib_ref]. GbERF1-like ERFs enhance plant resistance to Verticillium wilt by positively regulating lignin synthesis [bib_ref] An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium..., Guo [/bib_ref]. V. dahliae and phytohormones (SA, JA, and ET) induce the expression of the nucleotide binding site-leucine-rich repeat (NBS-LRR) gene GbaNA1 in cotton, which is involved in its response to Verticillium wilt [bib_ref] The island cotton NBS-LRR gene GbaNA1 confers resistance to the non-race 1..., Li [/bib_ref] ; GbaNA1 overexpression increases ROS levels in Arabidopsis and promotes the expression of genes related to the ET signaling pathway [bib_ref] Heterologous Expression of the Cotton NBS-LRR Gene GbaNA1 Enhances Verticillium Wilt Resistance..., Li [/bib_ref]. The CC-NBS-LRR gene GbCNL130 activates the SA-mediated signaling pathway, increases ROS accumulation, and promotes the expression of downstream PR genes, thereby enhancing resistance to Verticillium cotton wilt [bib_ref] Cotton CC-NBS-LRR Gene GbCNL130 Confers Resistance to Verticillium Wilt Across Different Species...., Li [/bib_ref]. In addition, knockdown of positively regulated genes GhJAZ10, GhbHLH18 (JA), GhPUB17 (SA), and GhEBF1 (ET) significantly increases susceptibility to Verticillium wilt in resistant varieties, whereas silencing of negatively regulated genes GhTGA7 (SA) and GhBZR1 (Brassinolide, BR) significantly increases resistance to Verticillium wilt in susceptible varieties [bib_ref] The genes involved in the protective effects of phytohormones in response to..., Zhang [/bib_ref].
The bZIP transcription factor GbVIP1 (VirE2 interacting protein 1) is induced by V. dahliae and ET, and its silencing reduces Verticillium cotton wilt resistance, whereas its ectopic expression in tobacco enhances resistance to Verticillium wilt in tobacco by positively regulating the expression of PR1, PR1-like, and HSP70 genes [bib_ref] Isolation and characterization of the GbVIP1 gene and response to Verticillium wilt..., Zhang [/bib_ref]. JA induces the expression of BEL1-like transcription factor GhBLH7-D06 in vascular tissues, acting on secondary cell formation and negatively regulating resistance to Verticillium cotton wilt [bib_ref] The Cotton BEL1-Like Transcription Factor GhBLH7-D06 Negatively Regulates the Defense Response against..., Ma [/bib_ref]. In addition, knockdown of transcription factor GbNAC1 reduces resistance to Verticillium cotton wilt, whereas its overexpression increases Verticillium wilt resistance in Arabidopsis [bib_ref] Characterization, Expression, and Functional Analysis of a Novel NAC Gene Associated with..., Wang [/bib_ref].
## Genes directly involved in cell wall and protein defense
Plant resistance to fungal pathogens is largely influenced by defense-related proteins. Plants have evolved sophisticated sensory mechanisms to detect microbial invasion and overcome the negative effects caused by microbes in terms of growth, yield, and survival [bib_ref] Plant Defense Responses to Biotic Stress and Its Interplay with Fluctuating Dark/Light..., Iqbal [/bib_ref].
V. dahliae invades host plants mainly through plant cell wall degradation; therefore, the cell wall is the first barrier for plants that helps prevent pathogen entry, often effectively stopping pathogen invasion [bib_ref] The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis..., Cantu [/bib_ref] [bib_ref] Characterization of the Verticillium dahliae Exoproteome Involves in Pathogenicity from Cotton-Containing Medium, Chen [/bib_ref]. Pectin is one of the main components of the plant cell wall, and its structure and function are closely related to the degree of esterification. Pectin methylesterases (PMEs) protect plant cell walls by catalyzing the dimethyl esterification of pectin polygalacturonate. GhPMEI3 regulates resistance to V. dahliae in cotton since its silencing increases susceptibility to V. dahliae infestation, while ectopic expression of GhP-MEI3 increases pectin methyl esterification and limits fungal infection by regulating root growth [bib_ref] A Pectin Methylesterase Inhibitor Enhances Resistance to Verticillium Wilt, Liu [/bib_ref]. Knockout of pectin lyase genes VdPL3.1 and VdPL3.3 reduces pathogen virulence to cotton [bib_ref] Characterization of the Verticillium dahliae Exoproteome Involves in Pathogenicity from Cotton-Containing Medium, Chen [/bib_ref]. Cell wall polysaccharides are mainly modified by acetylation; the level and location of this modification directly affects the structure, physicochemical properties, and ability of the cell wall to resist pathogens [bib_ref] O-acetylation of plant cell wall polysaccharides. Front, Gille [/bib_ref]. Acetyltransferase GhTBL34 (trichome birefringence-like) expression is induced by V. dahliae, ET, SA, and JA and mediates cell wall polysaccharide acetylation to block pathogen invasion and regulate Verticillium wilt resistance [bib_ref] The role of the plant-specific ALTERED XYLOGLUCAN9 protein in Arabidopsis cell wall..., Schultink [/bib_ref] [bib_ref] GhTBL34 Is Associated with Verticillium Wilt Resistance in Cotton, Zhao [/bib_ref]. Resistance of cotton to wild-type fungal diseases is largely dependent on lignin content, and its levels can be used as an indicator of resistance against Fusarium wilt [bib_ref] Lignin metabolism has a central role in the resistance of cotton to..., Xu [/bib_ref]. Transcriptome analysis suggests that caffeic acid 3-O-methyltransferase (COMT) and peroxidase 2 (POD2) may be involved in the synthesis and accumulation of lignin in response to Fusarium cotton wilt [bib_ref] Lignin synthesis related genes with potential significance in the response of upland..., Hou [/bib_ref]. Patatin-like proteins (PLPs) are defense proteins with non-specific lipid acyl hydrolytic activity that hydrolyze cell membranes into fatty acids and lysophospholipids. GhPLP2, which is induced by FOV, V. dahliae, ET, and JA, plays an important role in reducing fungal pathogenicity and regulating plant resistance to Verticillium wilt [bib_ref] GhPLP2 Positively Regulates Cotton Resistance to Verticillium Wilt by Modulating Fatty Acid..., Zhu [/bib_ref]. Overexpression of polygalacturonase inhibitor proteins CkPGIP1 and GhPGIP1 from Cynanchum komarovii and G. hirsutum enhances cotton resistance to Verticillium wilt by promoting xylem lignification in cotton. The apoplastic thioredoxin protein GbNRX1 enhances the immune response of cotton to V. dahliae; GbNRX1 silencing causes a large accumulation of ROS and increases susceptibility to V. dahliae infection [bib_ref] The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive..., Li [/bib_ref].
BLADE-ON-PETIOLE (BOP) 1 and BOP2 are two BTB-ankyrin proteins specifically expressed at lateral-organ boundaries (LOBs). GhBOP1 acts synergistically with GhBP1 to co-regulate lignin biosynthesis and enhance resistance to Verticillium cotton wilt [bib_ref] Cotton plant defence against a fungal pathogen is enhanced by expanding BLADE-ON-PETIOLE1..., Zhang [/bib_ref]. GbHyPRP1 encodes a proline-rich protein with a POLE1 structural domain that negatively regulates resistance to Verticillium cotton wilt. Expression of GbHyPRP1 is inhibited by the SA signaling pathway when cotton is infected with V. dahliae, and GbHyPRP1 silencing enhances resistance to V. dahliae by thickening the cell wall and accumulating ROS; in contrast, HyPRP1 overexpression reduces resistance to Verticillium wilt in Arabidopsis [bib_ref] HyPRP1 performs a role in negatively regulating cotton resistance to V. dahliae..., Yang [/bib_ref].
Latex protein GhMLP28 is induced by V. dahliae, JA, SA, or ET to produce and regulate plant disease resistance [bib_ref] Regulatory Network of Cotton Genes in Response to Salt, Drought and Wilt..., Billah [/bib_ref]. GhDIR1 overexpression increases cotton lignin content and enhances resistance to Verticillium cotton wilt [bib_ref] Overexpression of cotton (Gossypium hirsutum) dirigent1 gene enhances lignification that blocks the..., Shi [/bib_ref]. Defense regulator EDS1 encodes a lipase-like protein that is induced by SA and regulates plant resistance to Verticillium wilt. GbEDS1 overexpression promotes the production of SA and H 2 O 2 and enhances Arabidopsis resistance to Verticillium wilt, whereas GbEDS1 silencing reduces cotton resistance to Verticillium wilt [bib_ref] Island Cotton Enhanced Disease Susceptibility 1 Gene Encoding a Lipase-Like Protein Plays..., Yan [/bib_ref].
Papain-like cysteine proteases (PLCPs) play an important role in plant defense against pathogen invasion. GhRD21-7 substantially promotes the resistance to Verticillium cotton wilt and its overexpression further enhances this resistance [bib_ref] CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance, Li [/bib_ref]. Germin-like proteins (GLPs) are a family of glycoproteins involved in plant resistance to various stresses. GhGLP2 silencing increases susceptibility to FOV and V. dahliae infection in cotton, whereas GhGLP2 overexpression attenuates mycelial growth of these fungi, increases callus formation at leaf infection sites, enhances cell wall lignification, and improves resistance to Fusarium and Verticillium wilt in Arabidopsis. Thaumatin-like proteins (TLPs) are a family of multimember defense-related proteins; GhTLP19 silencing increases malondialdehyde levels and decreases catalase levels in cotton, which increases its susceptibility to Verticillium wilt [bib_ref] Comprehensive Genome-Wide Analysis of Thaumatin-Like Gene Family in Four Cotton Species and..., Li [/bib_ref].
Extracellular enzymes help plants to resist fungal pathogen infection. For example, chitinase (Chi) hydrolyzes chitin (a main component of the fungal cell wall) into N-ethoxylated oligosaccharides and glucose to inhibit spore germination and mycelial growth [bib_ref] Discovery and identification of candidate genes from the chitinase gene family for..., Xu [/bib_ref]. There are low levels of Chi (with low activity) in all higher plant organs; however, its levels rapidly increase following pathogen infestation [bib_ref] The endochitinase VDECH from Verticillium dahliae inhibits spore germination and activates plant..., Cheng [/bib_ref]. Chi23, Chi32, or Chi47 positively regulate Verticillium wilt resistance; their knockout reduces resistance to Verticillium cotton wilt [bib_ref] Discovery and identification of candidate genes from the chitinase gene family for..., Xu [/bib_ref]. Cotton infestation by V. dahliae triggers the secretion of Chi28 and cysteine protein CRR1 in order to degrade the fungal cell wall and protect Chi28 from degradation by serine protease secreted by V. dahlia, respectively. Thus, silencing either Chi28 or CRR1 reduces resistance to Verticillium cotton wilt, whereas CRR1 overexpression enhances its resistance [bib_ref] The Cotton Apoplastic Protein CRR1 Stabilizes Chitinase 28 to Facilitate Defense against..., Han [/bib_ref]. Plant lysine motif-containing (LysM) proteins recognize chitin in the cell wall of fungal pathogens and initiate the corresponding defense response. Silencing Lyp1, Lyk7, and LysMe3 significantly reduces the production of SA, JA, and ROS, decreases the activity of defense-related genes, and ultimately reduces resistance to Verticillium cotton wilt [bib_ref] The lysin motif-containing proteins, Lyp1, Lyk7 and LysMe3, play important roles in..., Xu [/bib_ref].
Verticillium highly induces laccase GhLAC15 expression, and the cell walls of overexpressing plants enhance resistance to Verticillium wilt through a defense-mediated response that enhances lignification and increases arabinose and xylose accumulation [bib_ref] The cotton laccase gene GhLAC15 enhances Verticillium wilt resistance via an increase..., Zhang [/bib_ref]. GhUMC1, a divalent copper-binding protein, is an umecyanin-like gene involved in resistance to Verticillium cotton wilt through regulation of the JA signaling pathway and lignin metabolism [bib_ref] GhUMC1, a blue copper-binding protein, regulates lignin synthesis and cotton immune response, Zhu [/bib_ref]. Walls are thin (WAT) genes regulate SA metabolism and signaling by affecting polar transport of growth hormones to further enhance plant resistance to a variety of pathogens [bib_ref] Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia..., Denancé [/bib_ref]. Knockout of GhWATs increases SA accumulation, activates the expression of SA pathway-related genes, and increases lignin accumulation in the xylem, each of which promotes plant resistance to Verticillium wilt [bib_ref] Cotton WATs Modulate SA Biosynthesis and Local Lignin Deposition Participating in Plant..., Tang [/bib_ref]. Simultaneous silencing of GhWAT1, GhWAT2, and GhWAT3 increases lignin accumulation in the xylem and enhances plant resistance to Verticillium wilt but inhibits plant growth [bib_ref] Cotton WATs Modulate SA Biosynthesis and Local Lignin Deposition Participating in Plant..., Tang [/bib_ref]. Knockdown of the Gh4CL30 lignin synthesis gene in cotton decreases flavonoid, lignin, and butyryl content but increases guaiacyl, caffeic acid, and ferulic acid content, which provides a new idea for resistance to Verticillium cotton wilt [bib_ref] The Cotton Lignin Biosynthetic Gene Gh4CL30 Regulates Lignification and Phenolic Content and..., Xiong [/bib_ref].
Enoyl-CoA reductase (GhECR) is involved in the formation of long-chain fatty acids, and its silencing increases the susceptibility of cotton to FOV and V. dahliae infection [bib_ref] Tobacco Rattle Virus-Based Silencing of Enoyl-CoA Reductase Gene and Its Role in..., Mustafa [/bib_ref]. The subtilase-like protein GbSBT1 is highly induced following infection with V. dahliae, or by JA and ET treatment. The protein acts at the cell membrane and regulates plant resistance to Fusarium and Verticillium wilt [bib_ref] Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular..., Duan [/bib_ref].
Plants infested with pathogens over a long period of time have evolved various innate immune mechanisms [bib_ref] Plant innate immunity-direct and indirect recognition of general and specific pathogen-associated molecules, Jones [/bib_ref]. In general, plants employ two sets of defense strategies: microbial (or pathogen)-associated molecular pattern (MAMP/PAMP)-triggered immunity (MTI/PTI) and effector-triggered immunity (ETI) [bib_ref] The plant immune system, Jones [/bib_ref]. Some pathogenic bacteria disrupt plant tissue structure and produce damage-associated molecular patterns (DAMPs) such as cell wall fragments, which induce innate immunity similar to that of MAMPs [bib_ref] Membrane receptors recognizing MAMP/PAMP and DAMP molecules that activate first line of..., Hetmann [/bib_ref].
Activation of plant innate immunity requires receptor recognition of pathogens. MTI/PTI is triggered by PRRs located across the cell surface, whereas ETI is caused by NBS-LRR receptor proteins located intracellularly [bib_ref] Plant Immune Mechanisms: From Reductionistic to Holistic Points of View, Zhang [/bib_ref]. PRRs include receptor-like proteins (RLPs) and receptor-like kinases (RLKs) that are located on plant cell membranes and which are responsible for detecting molecular markers of invading pathogens or damaged plant tissues and activating defense responses, thereby preventing infection before pathogen invasion of the plant cell [bib_ref] Responses to Plant Pathogens, Hammond-Kosack [/bib_ref] [bib_ref] Receptor-like kinases and receptor-like proteins: Keys to pathogen recognition and defense signaling..., Yang [/bib_ref]. The GhRLPGSO1-like gene, GhRLP44, GhRLP6, and GhRLP34 may play a role in resistance to Fusarium cotton wilt. Verticillium wilt infestation of disease-resistant G. barbadense cotton induces GbRLK; transfection of cotton and Arabidopsis with this gene increases resistance to Verticillium wilt [bib_ref] Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to..., Jun [/bib_ref]. The TIR-NBS-LRR protein encoded by CG02 is likely to be a key gene for resistance to Verticillium cotton wilt since it specifically promotes the expression of disease resistance genes, while silencing this gene increases its susceptibility to Verticillium wilt [bib_ref] Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland..., Li [/bib_ref].
Invasion of cotton cells by FOV causes the release of large amounts of intracellular Ca 2+ , which acts as a second messenger to initiate the expression of various downstream disease resistance genes [bib_ref] Structural insights into long-distance signal transduction pathways mediated by plant glutamate receptor-like..., Grenzi [/bib_ref]. GhGLR4.8 encoding glutamate-like receptor (GLR) protein plays an active role in resistance to Fusarium cotton wilt and it is hypothesized that it may be the ion channel required for Ca 2+ to flood into the cell and enhance the disease resistance response [bib_ref] Battle for Cotton against Fusarium, Wang [/bib_ref].
In addition, the Ve R-gene enhances plant resistance to Verticillium wilt by encoding RLPs with LRR structural domains [bib_ref] Antagonistic function of the Ve R-genes in tomato, Nazar [/bib_ref]. The transcription products of GhlncNAT-ANX2 and GhlncNAT-RLP7 are long non-coding RNAs whose silencing enhances resistance to Verticillium cotton wilt, possibly by promoting lipoxygenase 1 and lipoxygenase 2 expression [bib_ref] Long noncoding RNAs involve in resistance to Verticillium dahliae, a fungal disease..., Zhang [/bib_ref]. GbAt11 (AXMN toxin-induced protein-11) is highly resistant to Verticillium wilt, and its overexpression stimulates the expression of disease resistance genes, including FLS2 and BAK1, to increase resistance [bib_ref] GbAt11 gene cloned from Gossypium barbadense mediates resistance to Verticillium wilt in..., Qiu [/bib_ref]. GhPUB17 is a U-box ubiquitin ligase E3 that can be inhibited by antifungal protein GhCyP3 to negatively regulate Verticillium cotton wilt resistance [bib_ref] GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the..., Qin [/bib_ref]. GbANS is involved in the biosynthesis of anthocyanins in cotton and its silencing significantly reduces anthocyanin production and resistance to Verticillium cotton wilt [bib_ref] Silencing of GbANS reduces cotton resistance to Verticillium dahliae through decreased ROS..., Long [/bib_ref]. Similarly, GbCAD1 is involved in cotton phenol synthesis, and its silencing also reduces resistance to Verticillium cotton wilt [bib_ref] Proteomic and virus-induced gene silencing (VIGS) Analyses reveal that gossypol, brassinosteroids, and..., Gao [/bib_ref].
Taken together, these findings suggest that defense genes induced during pathogen attack provide potent protection to the plant by their respective defense mechanisms [fig_ref] Figure 2: Regulatory network of disease resistance genes in cotton [/fig_ref]. However, more studies are required to understand the mechanisms of action of these genes.
to Verticillium cotton wilt since it specifically promotes the expression of disease resistance genes, while silencing this gene increases its susceptibility to Verticillium wilt [bib_ref] Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland..., Li [/bib_ref].
Invasion of cotton cells by FOV causes the release of large amounts of intracellular Ca 2+ , which acts as a second messenger to initiate the expression of various downstream disease resistance genes [bib_ref] Structural insights into long-distance signal transduction pathways mediated by plant glutamate receptor-like..., Grenzi [/bib_ref]. GhGLR4.8 encoding glutamate-like receptor (GLR) protein plays an active role in resistance to Fusarium cotton wilt and it is hypothesized that it may be the ion channel required for Ca 2+ to flood into the cell and enhance the disease resistance response [bib_ref] Battle for Cotton against Fusarium, Wang [/bib_ref].
In addition, the Ve R-gene enhances plant resistance to Verticillium wilt by encoding RLPs with LRR structural domains [bib_ref] Antagonistic function of the Ve R-genes in tomato, Nazar [/bib_ref]. The transcription products of GhlncNAT-ANX2 and GhlncNAT-RLP7 are long non-coding RNAs whose silencing enhances resistance to Verticillium cotton wilt, possibly by promoting lipoxygenase 1 and lipoxygenase 2 expression [bib_ref] Long noncoding RNAs involve in resistance to Verticillium dahliae, a fungal disease..., Zhang [/bib_ref]. GbAt11 (AXMN toxin-induced protein-11) is highly resistant to Verticillium wilt, and its overexpression stimulates the expression of disease resistance genes, including FLS2 and BAK1, to increase resistance [bib_ref] GbAt11 gene cloned from Gossypium barbadense mediates resistance to Verticillium wilt in..., Qiu [/bib_ref]. GhPUB17 is a U-box ubiquitin ligase E3 that can be inhibited by antifungal protein GhCyP3 to negatively regulate Verticillium cotton wilt resistance [bib_ref] GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the..., Qin [/bib_ref]. GbANS is involved in the biosynthesis of anthocyanins in cotton and its silencing significantly reduces anthocyanin production and resistance to Verticillium cotton wilt [bib_ref] Silencing of GbANS reduces cotton resistance to Verticillium dahliae through decreased ROS..., Long [/bib_ref]. Similarly, GbCAD1 is involved in cotton phenol synthesis, and its silencing also reduces resistance to Verticillium cotton wilt [bib_ref] Proteomic and virus-induced gene silencing (VIGS) Analyses reveal that gossypol, brassinosteroids, and..., Gao [/bib_ref].
Taken together, these findings suggest that defense genes induced during pathogen attack provide potent protection to the plant by their respective defense mechanisms [fig_ref] Figure 2: Regulatory network of disease resistance genes in cotton [/fig_ref]. However, more studies are required to understand the mechanisms of action of these genes.
## Mechanism of human resistance to fusarium and its pathogenic features
Fusarium is a naturally widespread, highly adaptable saprophytic fungus that can survive in a wide range of extreme environments [bib_ref] Fusarium infection in hematopoietic stem cell transplant recipients, Nucci [/bib_ref]. Some Fusarium species are nonpathogenic, while others are extremely harmful. Common pathogenic Fusarium species include F. moniliforme, F. solani, and FOV [bib_ref] The epidemiology of Fusarium infection and advances in its diagnosis and treatment, Shi [/bib_ref]. They infect a wide range of crops (cotton, bananas, aubergines, etc.), cause the worldwide spread of many plant diseases, and infect animals such as fish and shrimps [bib_ref] A Case Report of Fusariomycosis, Liu [/bib_ref]. They may even cause skin, corneal, nail, and systemic damage in humans [bib_ref] Three cases of corneal, perioral and genital infections with Fusarium solani, Yang [/bib_ref].
## Fungal toxins
Fungi are often found in various types of grain in fields and the grain stored in warehouses. This is accompanied by the production of mycotoxins which affect plant regeneration, cause programmed cell death, and lead to disease in humans and animals following accidental consumption of grain contaminated with mycotoxins or through fungi-contaminated grain coming into contact with wounds or eyes [bib_ref] A Review on Mycotoxins in Food and Feed: Malaysia Case Study, Afsah-Hejri [/bib_ref].
The gastrointestinal tract is a barrier to harmful substances such as pathogens, toxins, and exogenous antigens. It is a habitat for intestinal flora that synergistically protects the host from fungi and plays a key role in regulating the body's immune system [bib_ref] The Immunology of the Gastrointestinal System, Staples [/bib_ref]. Mycotoxins that enter the body through food initially interact with the gastrointestinal tract, break through its defenses, disrupt its homeostasis, and alter its histomorphology, nutrient absorption, and barrier function, which results in intestinal dysfunction and local immune compromise and may eventually lead to serious illnesses such as systemic chronic mycotoxicosis [bib_ref] Patulin and ochratoxin A co-occurrence and their bioaccessibility in processed cereal-based foods:..., Assunção [/bib_ref]. Mycotoxins can also affect the susceptibility of animals to infectious diseases, such as bacteria, viruses, and parasites, by affecting the innate and acquired immune systems of healthy animals. Thus, exposure of animals to mycotoxins increases their susceptibility to these infectious diseases.
Trichothecenes are mainly produced by F. graminearum and include two major toxins: T-2 toxin and deoxynivalenol (DON). Their oral ingestion causes toxicity in animals and humans, results in a variety of symptoms (such as vomiting and diarrhea), and may poison white blood cells and affect the host's immune system [bib_ref] Deoxynivalenol: Toxicology and potential effects on humans, Pestka [/bib_ref] [bib_ref] Metabolism of the Fusarium Mycotoxins T-2 Toxin and HT-2 Toxin in Wheat, Nathanail [/bib_ref]. The toxicity of T-2 and DON is generally mediated by oxidative stress-mediated DNA damage and apoptosis, although they can also bind to the large subunit of the ribosome and interfere with its peptidyl transferase activity to inhibit protein synthesis [bib_ref] Oxidative stress-mediated cytotoxicity and metabolism of T-2 toxin and deoxynivalenol in animals..., Wu [/bib_ref].
Fumonisins are mainly produced by F. verticillioides; fumonisin B1 (FB1) is the most abundant type found in nature [bib_ref] Fumonisins in foods from Cordoba (Argentina), presence: Mini review, Lerda [/bib_ref]. Epidemiological studies have shown that esophageal cancer in humans and liver cancer in animals are associated with the ingestion of foods containing fumonisins [bib_ref] The Status of Fusarium Mycotoxins in Sub-Saharan Africa: A Review of Emerging..., Chilaka [/bib_ref] [bib_ref] Mycotoxins and their effects on human and animal health, Bezerra Da Rocha [/bib_ref]. FB1 has a similar structure to that of sphingolipids and can affect sphingolipid metabolism by interfering with ceramide synthase, leading to increased intracellular sphingomyelin levels that ultimately lead to oxidative stress and apoptosis [bib_ref] Gastrointestinal Degradation of Fumonisin B 1 by Carboxylesterase FumD Prevents Fumonisin Induced..., Masching [/bib_ref] [bib_ref] Effects of orally administered fumonisin B 1 (FB 1 ), partially hydrolysed..., Hahn [/bib_ref]. In addition, FB1 may weaken the intestinal barrier by affecting the synthesis of related proteins and lead to immune dysfunction [bib_ref] Toxic mechanisms induced by fumonisin b1 mycotoxin on human intestinal cell line, Minervini [/bib_ref] [bib_ref] Mycotoxins modify the barrier function of Caco-2 cells through differential gene expression..., Romero [/bib_ref].
Zearalenone (ZEA) is a fungal toxin mainly produced in grain by F. graminearum and F. culmorum which may act synergistically with FBs and DON. It is structurally similar to estrogen; therefore, ZEA competes with natural estrogens for estrogen receptor binding and has a major impact on the reproductive system [bib_ref] Zearalenone, an estrogenic mycotoxin, is an immunotoxic compound, Hueza [/bib_ref]. In addition, ZEA can downregulate tumor suppressor genes in intestinal cells, which can cause cancer [bib_ref] Exposure to zearalenone mycotoxin alters in vitro porcine intestinal epithelial cells by..., Taranu [/bib_ref].
## Fusariosis-fungal keratitis
Fusariosis is an infectious disease of the skin, eyes, and internal organs mainly caused by Fusarium infections [bib_ref] Chronic leg ulcer caused by Fusarium sonali: A case report and super-microstructure..., Ran [/bib_ref]. Fusarium is an opportunistic pathogen that does not normally cause disease; however, at times it causes local infections in immunocompetent individuals and invasive infections when the body's immunity is compromised or when there is trauma. Therefore, neutrophils and macrophages play an important role in the immune defense against Fusarium, which infects immunocompromised patients in two main ways: (1) through trauma or foreign bodies that cause infection of the patient's soft tissues or mucous membranes, resulting in keratitis and nail infection, among others [bib_ref] Human fusariosis, Dignani [/bib_ref] ; and (2) through rhinitis and pneumonia caused by inhalation or ingestion of Fusarium and its toxins [bib_ref] The epidemiology of Fusarium infection and advances in its diagnosis and treatment, Shi [/bib_ref].
Fungal keratitis is the most common type of fusariosis in normal individuals and it has a significant medical and socioeconomic impact [bib_ref] Fungal infections of the cornea, Thomas [/bib_ref] [bib_ref] Pathogenic Aspergillus and Fusarium as important causes of blinding corneal infections-the role..., Ratitong [/bib_ref]. Most patients have an infected corneal stroma due to trauma related to agricultural work, particularly in hot and humid areas that lead to an increase in the growth rate of Fusarium and mycotoxin levels [bib_ref] Fungal keratitis, Srinivasan [/bib_ref] [bib_ref] A mini review on aflatoxin exposure in Malaysia: Past, present and future, Mohd-Redzwan [/bib_ref]. The next section presents the pathogenic mechanisms of Fusarium and the host defense response using the example of fungal keratitis to help advance the prevention and control of fungal keratitis.
The severity of a fungal infection is determined by the strength of the pathogen and the immunity of the host [bib_ref] Pathogenic Aspergillus and Fusarium as important causes of blinding corneal infections-the role..., Ratitong [/bib_ref]. The innate immune response against fungal keratitis involves detection of the fungus by macrophages residing in the cornea followed by chemokine secretion by the macrophages, which recruits neutrophils to the infected corneal stroma [bib_ref] Distinct roles for Dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus..., Leal [/bib_ref]. The neutrophils act as killer cells and remove the invading fungus. However, insufficient levels of chemokines restrict the number of neutrophils infiltrating the corneal stroma, resulting in a failure to clear the fungus in time for the early stages of infection.
Recognition of fungal invasion by immune cells through the C-type lectin receptor (CLR) and toll-like receptor (TLR) is the first step in the defense response [bib_ref] The first line of defense: Effector pathways of anti-fungal innate immunity, Ward [/bib_ref]. The CLR and TLR of macrophages recognize intrinsic components of the fungal cell wall, while neutrophils use different oxidative and non-oxidative mechanisms to inhibit mycelial growth and regulate the host immune response to the fungus [bib_ref] Expression of innate and adaptive immune mediators in human corneal tissue infected..., Karthikeyan [/bib_ref] [bib_ref] C-type lectin receptors orchestrate antifungal immunity, Hardison [/bib_ref]. However, Fusarium also expresses a variety of proteins and small molecule virulence factors that inhibit the host's defense response. For example, conidia secrete the hydrophobic protein RodA, which provides a protective layer for Fusarium and prevents recognition by the receptor, thus delaying initiation of the immune response and neutrophil recruitment and allowing the fungus to survive and cause disease [bib_ref] The RodA hydrophobin on Aspergillus fumigatus spores masks dectin-1-and dectin-2-dependent responses and..., Carrion Sde [/bib_ref].
Neutrophils can destroy fungi by producing ROS such as O 2− , H 2 O 2 , and HClO through the NADPH oxidase (NOX) system [bib_ref] Eradicating, retaining, balancing, swarming, shuttling and dumping: A myriad of tasks for..., Urban [/bib_ref]. Neutrophils in mice with mutations in the NOX subunit have impaired ability to produce ROS, leading to a marked increase in mycelial growth in the cornea and more severe disease progression [bib_ref] Fungal antioxidant pathways promote survival against neutrophils during infection, Leal [/bib_ref]. Fungi produce antioxidant factors including thioredoxin and superoxide dismutase (regulated by the Yap1 transcription factor) to counteract neutrophil ROS production. Although most fungal secondary metabolites and catalases have antioxidant activity, they do not enhance fungal resistance to ROS [bib_ref] Fungal antioxidant pathways promote survival against neutrophils during infection, Leal [/bib_ref]. Protein phosphatase Z (Ppz) also plays an important role in oxidative stress tolerance in filamentous fungi [bib_ref] Farkas, I. Protein phosphatase Z modulates oxidative stress response in fungi, Leiter [/bib_ref].
Neutrophils can use various non-oxidative mechanisms to limit microbial growth, such as the production of proteins that compete with microbes for metal ions necessary for growth (a process also known as nutritional immunity) [bib_ref] Nutritional immunity: Transition metals at the pathogen-host interface, Hood [/bib_ref]. Iron is essential for thioredoxin production by fungi; neutrophils secrete iron-binding proteins, such as transferrin and lactoferrin, to compete with fungi for iron ions. Zinc is also essential for mycelial growth since it is an important component of many enzymes and TFs, and neutrophils secrete calprotectin, which binds zinc and manganese to compete with fungi [bib_ref] Nutritional Immunity: S100 Proteins at the Host-Pathogen Interface, Zackular [/bib_ref]. Neutrophils more efficiently kill Fusarium species with mutated ion transport carriers, and the growth of these mutant fungi on the cornea itself is inhibited [bib_ref] Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and..., Leal [/bib_ref]. New mobile, germline-specific chromosomes were identified in FOV that can transmit pathogenicity by transfer in plants. These germline-specific chromosomes have numerous genes that encode metal ion transport proteins that may play an important role in the evasion of nutritional immunity by Fusarium; these genes are also present in cancer patients with systemic Fusarium infections and in those with fungal keratitis [bib_ref] The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set..., Zhang [/bib_ref] [bib_ref] Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium, Ma [/bib_ref].
Macrophages and neutrophils can secrete acidic mammalian chitinase (AMCase) to inhibit fungal growth [bib_ref] Neutrophils as a Source of Chitinases and Chitinase-Like Proteins in Type 2..., Żurawska-Płaksej [/bib_ref] [bib_ref] Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation, Zhu [/bib_ref]. In vitro experiments showed that neutrophils lacking AMCase are less potent against fungi, while fungi survival is higher in mice lacking AM-Case [bib_ref] Aspergillus fumigatus corneal infection is regulated by chitin synthases and by neutrophil-derived..., De Jesus Carrion [/bib_ref]. Neutrophils are an important source of cytokines during infection, producing large amounts of pro-inflammatory and chemotactic cytokines. The expression of IL-1α and IL-1β cytokines is significantly higher in diseased corneas compared to healthy corneas [bib_ref] Pathogenic Aspergillus and Fusarium as important causes of blinding corneal infections-the role..., Ratitong [/bib_ref] [bib_ref] Transcriptome Analysis of the Gene Expression Profiles Associated with Fungal Keratitis in..., Zhang [/bib_ref] [bib_ref] Neutrophil-derived cytokines: Facts beyond expression, Tecchio [/bib_ref].
Neutrophils are bactericidal in two main ways: direct phagocytosis of pathogens or by the release of a specific structure, namely neutrophil extracellular traps (NETs) consisting of depolymerized chromatin and intracellular granule proteins that cause death to the outside of the cell. This process involves the generation of ROS and the depolymerization of chromatin by neutrophil elastase (NE) to trap and kill pathogenic bacteria [bib_ref] Neutrophil extracellular traps kill bacteria, Brinkmann [/bib_ref]. In addition, peptidyl arginine deiminase 4 (PAD4) guanylates histones, which changes the chromatin charge and promotes chromatin depolymerization and nucleus expansion [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref]. NETosis is induced by fungi such as Fusarium, leading to the release of histones, calprotectin, and other particles. Therefore, NETs kill pathogenic fungi and can also cause damage to their own tissues [bib_ref] Neutrophil extracellular traps in fungal infection, Urban [/bib_ref].
## Comparison of pathogenic mechanisms against fusarium in cotton and humans
Plants and humans possess conserved innate immune systems to respond to microbial invasion [fig_ref] Table 1: Comparison between the pathogenic response in humans and cotton [/fig_ref]. They use specialized receptors to sense PAMPs and endogenous DAMPs, which then activates the appropriate defense response. The immune response includes transcriptional reprogramming, production of antimicrobial substances, programmed cell death of infected cells, and the release of soluble factors (including cytokines and phytohormones) that produce a signal from the site of infection to alert the host of the arrival of danger [bib_ref] Cross Kingdom Immunity: The Role of Immune Receptors and Downstream Signaling in, Roudaire [/bib_ref]. In vertebrates, membrane-bound immune receptors belonging to the TLR family are located on the cell surface or in endosomal compartments, while the CLR is located on the cell surface. Plant RLKs and RLPs are mainly located on the plasma membrane, with RLPs being structurally similar to TLRs in animal cells [bib_ref] Recognition of microbial infection by Toll-like receptors, Kopp [/bib_ref]. Both plants and vertebrates use membrane PRRs to recognize PAMPs/DAMPs to then trigger activation of transcriptional programs that ultimately produce antimicrobial molecules and enable host adaptive responses to resist pathogen attack.
Activating programmed cell death at the site of pathogen infection is a strategy shared by plants and humans that protects the organism from further invasion by pathogens through directly disrupting the environment in which they live. Cell death also releases intracellular components that can activate or enhance antimicrobial responses and signal alarms to neighboring cells. Apoptosis, pyroptosis, and necroptosis limit pathogen development in humans [bib_ref] Pattern Recognition Receptors and the Host Cell Death Molecular Machinery, Amarante-Mendes [/bib_ref]. Recognition of invading microorganisms by plants also triggers an HR which prevents their spread in healthy tissues by limiting the uptake of plant metabolites by living trophic pathogens [bib_ref] Programmed Cell Death in Plants: An Overview, Locato [/bib_ref]. The characteristics of HR-associated cell death are remarkably similar to programmed cell death in humans, and leakage of cell contents can constitute an alarm signal to neighboring cells and prepare them to respond to infection, ensuring a comprehensive and effective defense response [bib_ref] Programmed cell death in C. elegans, mammals and plants, Lord [/bib_ref] [bib_ref] Cell Death in Plant Immunity, Pitsili [/bib_ref]. In addition, humans and plants secrete chitinase and ROS to inhibit fungal growth [bib_ref] Plant genes for abiotic stress, Ciarmiello [/bib_ref] [bib_ref] Discovery and identification of candidate genes from the chitinase gene family for..., Xu [/bib_ref] [bib_ref] Pathogenic Aspergillus and Fusarium as important causes of blinding corneal infections-the role..., Ratitong [/bib_ref] [bib_ref] Eradicating, retaining, balancing, swarming, shuttling and dumping: A myriad of tasks for..., Urban [/bib_ref].
Plants and humans have innate immune memory and immune memory, which enables rapid mobilization of large amounts of resources for the synthesis of disease-resistant proteins that protect them from injury when reinfested by pathogenic bacteria. However, this depends on the plant life cycle, with long-lived perennials exhibiting better immune memory than annuals [bib_ref] Immunological memory: What's in a name?, Pradeu [/bib_ref] [bib_ref] Ecological costs of induced resistance, Heil [/bib_ref].
The biggest difference between animals and plants in the defense against Fusarium infestation is that animals have an innate immune system, which is triggered by allergenic proteins such as Fusarium's beta-1,3 glucan and enzymes released during budding. Furthermore, allergen proteins are recognized by phagocytes, neutrophils, and dendritic cells of the host through receptors such as CLR, thereby initiating the allergic process [bib_ref] Mycotoxins and a Dysregulated Immune System: A Combination of Concern?, Kraft [/bib_ref] [bib_ref] Surface availability of beta-glucans is critical determinant of host immune response to..., Mintz-Cole [/bib_ref]. In addition, these receptors activate intracellular signaling pathways that lead to inflammatory responses. Macrophages and neutrophils also inhibit Fusarium infection through the presence of interferon-gamma, chemokines, tumor necrosis factor-alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and various interleukins (IL-1β, IL-6, IL-15, IL-23) [bib_ref] Fusarium infections in immunocompromised patients, Nucci [/bib_ref] [bib_ref] The role of Syk/CARD9 coupled C-type lectins in antifungal immunity, Drummond [/bib_ref]. After the innate immune cells are activated, antigen-presenting cells such as macrophages can engulf pathogens and produce antigens, which are then presented to two types of T cells. Helper T cells mainly help B cells to generate antibody-labeled antigens, whereas cytotoxic T cells directly kill pathogen-infected somatic cells [bib_ref] The immune system, Nicholson [/bib_ref]. This is centered on neutrophils and is key to an animal's defense against Fusarium. The prognosis for invasive fusariosis in patients with neutropenia or organ transplantation is poor and up to 100% lethal in the absence of recovery of neutrophil function. At the onset of inflammation, killer cells such as neutrophils are attracted by chemotactic substances and rapidly accumulate through blood vessels to the site of disease to surround and destroy the invading Fusarium in a single locality and prevent its spread in the body in immunocompetent individuals.
Unlike animals, plants have not evolved motile immune cells or an adaptive immune system [bib_ref] Innate immunological memory: From plants to animals, Sharrock [/bib_ref] ; they also lack a well-developed cellular circulation system similar to that of blood vessels and lymphatic vessels that can produce a direct immune response in the bloodstream. However, every cell in a plant has the ability to sense and resist pathogens [bib_ref] The plant immune system, Jones [/bib_ref]. After sensing pathogen signals, plants activate or inhibit TFs through hormone signals such as SA, JA, and ET, thereby affecting the expression of downstream PR genes, the thickening of cell walls, and the production of phytoalexins [bib_ref] Physiological and molecular mechanism of defense in cotton against Verticillium dahliae, Shaban [/bib_ref]. Initial animal defenses against pathogens mainly involve the mucous membranes and cuticles. Plant structures also provide physical defense strategies against pathogenic bacteria, such as epicuticular waxes (EWs) and the closure of stomata in many land plants [bib_ref] Epicuticular Wax Rice Mutants Show Reduced Resistance to Rice Water Weevil (Coleoptera:..., Bernaola [/bib_ref] [bib_ref] Function of ABA in Stomatal Defense against Biotic and Drought Stresses, Lim [/bib_ref].
In animals, immune cells such as effector T cells are directed to move flexibly and specifically throughout the body through endogenous cellular factors, such as chemokines or exogenous cellular signals generated by the inflammatory microenvironment, so that resistance is acquired throughout the body [bib_ref] The spatio-temporal control of effector T cell migration, Fowell [/bib_ref]. In contrast, plants send a systemic signal at the site of pathogen infestation, such as methyl salicylate (MeSA) which transmits the pathogen signal from the infected site to healthy tissues distal to the plant via intercellular filaments or phloem [bib_ref] Methyl salicylate is a critical mobile signal for plant systemic acquired resistance, Park [/bib_ref]. This allows the whole plant to acquire resistance to the disease; this process is also known as systemic acquired resistance (SAR) [bib_ref] Systemic acquired resistance, Durrant [/bib_ref]. Systemic resistance in plants is temporarily built up to keep out soon-to-be invading microbes [bib_ref] Systemic Acquired Resistance and Salicylic Acid: Past, Present, and Future, Klessig [/bib_ref]. Conversely, vertebrates have evolved an adaptive immune system with specialized immune cells dedicated to producing a stronger and more specific immune response while ensuring long-term protection of the organism [fig_ref] Figure 3: Similarities and differences in the resistance of plants [/fig_ref].
Furthermore, in addition to their highly complex adaptive immune system, humans are warm-blooded. Higher body temperature makes the body resistant to most invasive fungal infections, and humans can further raise their body temperature during immune defense; thus, only a few fungi that can grow at or above 37 - C can infect humans.
invading microbes [bib_ref] Systemic Acquired Resistance and Salicylic Acid: Past, Present, and Future, Klessig [/bib_ref]. Conversely, vertebrates have evolved an adaptive immune system with specialized immune cells dedicated to producing a stronger and more specific immune response while ensuring long-term protection of the organism [fig_ref] Figure 3: Similarities and differences in the resistance of plants [/fig_ref].
Furthermore, in addition to their highly complex adaptive immune system, humans are warm-blooded. Higher body temperature makes the body resistant to most invasive fungal infections, and humans can further raise their body temperature during immune defense; thus, only a few fungi that can grow at or above 37°C can infect humans. Blue represents the similarities between the defense mechanisms of plants and humans; green and yellow represent the respective characteristics of plants and humans. This cover has been designed using images from Freepik.com.
# Conclusions and outlook
There is no cure for Fusarium and Verticillium cotton wilt caused by FOV and V. dahliae. Currently, chemical control remains the most efficient way to combat crop diseases, and the application of emerging disease resistance breeding is the most environmentally safe control method. This review summarized the disease resistance and susceptibility genes in various types of cotton and suggested that it is possible to selectively combine or knockdown important disease-related genes to increase disease resistance in cotton [bib_ref] A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in..., Luo [/bib_ref]. Moreover, these disease-resistant genes can be used in patients undergoing reconstitution of immune function. So far, the treatment of human fusariosis mainly consists of surgical excision of the infected site, systemic antifungal drugs, and reconstitution of immune function in immunocompromised hosts [bib_ref] A Case Report of Fusariomycosis, Liu [/bib_ref] [bib_ref] Skin fusariosis: A literature review, Yang [/bib_ref] ; if we can find a way to use anti-disease proteins from cotton (such as extracellular enzymes) to assist in the treatment of patients, while avoiding side effects, it may improve the survival rates in critically ill patients and provide a new strategy for the prevention and control of fungal diseases in humans. Knowledge of Fusarium infection in humans can also be applied to cotton. Even among agricultural workers, Fusarium infection mostly occurs in the cornea, as the rest of the body is protected by clothing. A major reason for plant susceptibility to Fusarium wilt is that FOV inhabits the soil, which allows it to invade the plant through its root system. Therefore, the application of techniques such as soilless cultivation will isolate plants from the natural environment of FOV, thereby protecting the plant's vulnerable roots and greatly reducing the chances of FOV infestation.
However, the extremely complex pathogenesis of Fusarium and Verticillium cotton wilt, as well as the large breeding effort, long lead time, development of fungal resistance, and increasing national attention to pesticide residues and food safety issues, has slowed the development of disease resistance breeding and chemical control. Therefore, new ideas are required to prevent fungal diseases. Similar to the way humans and animals are vaccinated against disease, plants can also use non-pathogenic Fusarium to effectively control Fusarium disease. Some non-pathogenic Fusarium directly inhibit the growth, invasion, and colonization of pathogens by competing for nutrients in the soil, infestation sites on the surface of plant roots, and colonization sites within plant tissues [bib_ref] Mechanisms of Action and Dose-Response Relationships Governing Biological Control of Fusarium Wilt..., Larkin [/bib_ref] [bib_ref] Mechanisms involved in biological control of Fusarium wilt of cucumber with strains..., Mandeel [/bib_ref] [bib_ref] Process of tomato root colonization by a pathogenic strain of Fusarium oxysporum..., Olivain [/bib_ref]. Other non-pathogenic Fusarium can activate the plant's defense response by colonizing the plant and allowing it to acquire induced systemic resistance, thus indirectly antagonizing the pathogenic Fusarium and reducing the damage caused by it [bib_ref] Local and systemic resistance induced in watermelons by formae speciales of Fusarium..., Biles [/bib_ref]. Furthermore, there are viruses among plant pathogenic fungi that can cause fungal virulence decline. These fungal viruses can regulate the expression of the host enzymes and TFs, inhibit the synthesis of the host cytoplasmic membrane, and impair the host's transport system [bib_ref] Proteomic analysis of fungal host factors differentially expressed by Fusarium graminearum infected..., Kwon [/bib_ref] [bib_ref] Polyamines and abiotic stress tolerance in plants, Gill [/bib_ref] ; this affects the host's physiological state and potentially prevents and controls plant fungal diseases. Many fungal viruses that can inhibit Fusarium growth and mycotoxin production have been found [bib_ref] A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus, Yu [/bib_ref] [bib_ref] Molecular characterization of a dsRNA mycovirus, Fusarium graminearum virus-DK21, which is phylogenetically..., Kwon [/bib_ref] [bib_ref] Effects of the deletion and over-expression of Fusarium graminearum gene FgHal2 on..., Yu [/bib_ref] , and they are expected to be used in the prevention and control of Fusarium in the future.
Although Fusarium is a vicious pathogen, it can produce many beneficial products. L-tryptophan can induce marine Fusarium sp. L1 to produce indole alkaloids with anti-Zika virus activity [bib_ref] l-Tryptophan Induces a Marine-Derived Fusarium sp. to Produce Indole Alkaloids with Activity..., Guo [/bib_ref]. The fermentation of Fusarium sp. DCJ-A produces cyclic hexapeptide compounds that exhibit cytotoxic activity against five human tumor cells [bib_ref] Cyclic hexadepsipeptides from the fermentation of Fusarium sp. DCJ-A and their cytotoxic..., Yang [/bib_ref]. Xylanase produced by F. graminearum FGSG_03624 activates the immune response of plants and enhances their resistance to bacterial and fungal pathogens [bib_ref] The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against..., Tundo [/bib_ref]. In addition, F. graminearum Ec220 produces a xylanase with low cellulase content when grown on wheat bran [bib_ref] Fusarium graminearum as a producer of xylanases with low cellulases when grown..., Cruz-Davila [/bib_ref] , which has good commercial prospects. FOV PR-33 produces fusarinolic acid and its isomers, dehydrofusaric acid and fusaric acid, which are resistant to pathogenic bacteria and yeasts; these metabolites are potential antibacterial drugs [bib_ref] Production of antimicrobial metabolites against pathogenic bacteria and yeasts by Fusarium oxysporum..., Poleto [/bib_ref]. Bananas are highly susceptible to Fusarium infection [bib_ref] The Epidemiology of Fusarium Wilt of Banana, Pegg [/bib_ref] ; therefore, they could be considered for Fusarium cultivation for the production of compounds with industrial applications. This would provide theoretical and technological support in the fields of plant disease resistance and human medicine.
[fig] Figure 1: Functional schematic diagram of resistance genes in cotton. [/fig]
[fig] Figure 2: Regulatory network of disease resistance genes in cotton. Solid lines indicate the mechanism by which genes further affect plant disease resistance by acting on JA, SA, ET, lignin, ROS, and MAPK. Dashed lines indicate the mechanisms by which genes are regulated by JA, SA, ET, lignin, ROS, and MAPK to affect plant disease resistance. Blunt ends indicate inhibition. Arrows [/fig]
[fig] Figure 3: Similarities and differences in the resistance of plants (cotton) and humans to Fusarium. Blue represents the similarities between the defense mechanisms of plants and humans; green andFigure 3. Similarities and differences in the resistance of plants (cotton) and humans to Fusarium. [/fig]
[fig] Author: Contributions: Conceptualization, M.R. and Y.X.; writing-original draft preparation, M.M. and Y.Z. writing-review and editing, L.L. (Lulu Liu), L.L. (Lei Luo), X.H., L.Q. and F.L.; funding acquisition, F.L., M.R. and Y.X. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This work was supported by grants from the National Natural Science Foundation of China (No. U1804231 to F.L., 31972469 to M.R.) and the China Postdoctoral Science Foundation (no. 2020M672286 to Y.X.). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. [/fig]
[table] Table 1: Comparison between the pathogenic response in humans and cotton. [/table]
|
Being Born Large for Gestational Age is Associated with Increased Global Placental DNA Methylation
Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DnA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DnA methylation are lacking. the aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p < 0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p < 0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p = 0.001).Birth size is an important clinical parameter that can be used for determining the health of the newborn and is highly correlated with postpartum morbidity and complications during labour. Increases in birth weight parallel the gestational age 1 . Relating birth weight to gestational age has the advantage of evaluating the potential growth of the fetus according to the length of pregnancy 2 . Weight for gestational age is usually classified into small for gestational age (SGA), appropriate for gestational age (AGA), and large for gestational age (LGA) 3 . Abnormal fetal growth is defined as being born smaller than the 10 th percentile (SGA) or larger than the 90 th percentile (LGA) 3 . Both SGA and LGA births display an increased peri-and postnatal morbidity and mortality. Regarding maternal health, SGA and LGA births are associated with maternal disease such as hypertension, preeclampsia, and preexisting or gestational diabetes mellitus. Moreover, SGA and especially LGA births are associated with adverse obstetrical outcomes such as emergency cesarean deliveries and postpartum hemorrhages 4 . SGA newborns are at an increased risk for stillbirth, seizures, intraventricular hemorrhages, hypoxic ischemic encephalopathy, necrotizing enterocolitis, sepsis, and neonatal mortality 5 . LGA newborns display an increased risk for stillbirth, traumatic delivery, brachial plexus palsy, mechanical ventilation, and neonatal mortality 5 . Furthermore, numerous studies have demonstrated that both SGA and LGA newborns are at an increased risk for disease in later life, highlighting the importance of these anthropometric measures as surrogate parameters of fetal programming 6-9 .Adaptive processes of the fetus to adverse environmental cues during gestation can have an impact on the physiology and metabolism in later life 10,11 . An adverse intrauterine environment can be translated into being and pressure were maintain at 300 °C and 60 psig. Parameters associated with the voltage were as follows: capillary voltage 4000 V; collision energy for dC, and dG 5mdC row 7 V, 13 V and 10 V. The quantification of placental methylation was calculated based on the formula: Methylation % = [5mdC]/{[5mdC] + [dC]} over m/z signal for dC and mdC at 228.0979/112.0505 and 242.1135/126.0662, respectively.
born small for gestational age (SGA) which is an independent risk factor for metabolic diseases. Interestingly, similar associations have been demonstrated for LGA newborns, displaying an increased susceptibility for obesity, hypertension and diabetes mellitus compared to appropriate for gestational age (AGA) newborns. Overall, studies show an U-shaped relation between birth weight and type-2 diabetes, hypertension or risk of overweight in later life. However, it has been proposed that the underlying mechanisms leading to the same metabolic diseases in adult life differ between SGA and LGA newborns.
The placenta is the primary means of communication between mother and fetus during pregnancy. Placental function has been reported to be altered in pregnancies with SGA or LGA outcomes. Changes in placental function may occur due to changes in placental structure. However, the molecular pathways responsible for changes in placental structure and function in association with pregnancy outcomes have not been widely studied yet. More recent studies highlight the importance of epigenetic mechanisms, primarily DNA methylation, in placental performance during pregnancy.
Birth weight for gestational age is a complex phenotype that results from various processes and the expression of numerous genes in the genome. This might explain why association analyses between DNA methylation of certain genes and birth weight for gestational age remained elusive despite a direct connection between the gene of interest and fetal growth processes. The whole human genome contains only about 1.5% of protein encoding genes, while the remaining majority comprises of introns, repetitive elements, and other non-coding sequences. Initially regarded as "junk DNA" more recent research has revealed important functions of non-coding genomic regions and their epigenetic modification, including DNA methylation. Thus, investigating DNA methylation levels on a global scale might facilitate getting first insights into epigenetic mechanisms of complex phenotypes. However, studies investigating correlations between global DNA methylation and birth weight so far were only performed using rather small sample sizes.
Currently, various approaches to quantify global DNA methylation exist. Several studies applied genome wide array approaches. Although such assays can measure CpG island methylation of about 99% of all RefSeq genes, they only cover about 1.5% of genomic CpGs, are more specifically designed to analyze promotor methylation and neglect other genomic regions where DNA methylation might be of importance. Also newer variants of genome wide array approaches like the Illumina MethylationEPIC bead chip still exclude regions of potentially meaningful biological variation. More comprehensive methods such as whole genome bisulfite sequencing are not feasible yet for utilization with large sample sizes. Other novel sequencing based approaches that indeed are applicable for large sample sizes do so at the expense of genomic coverage and are further limited by their still quite extensive costs. Other commonly used methods measure DNA methylation in repetitive elements such as LINE-1 or Alu elements that serve as surrogate parameters for global DNA methylation. Futhermore, the absolute methyl cytosine content can be measured by using HPLC or LC-MS/MS. LC-MS/MS is reported to be the most sensitive method for absolute methyl cytosine quantification and is considered the current gold standard.
The aim of this study was to analyze placental global DNA methylation using the current gold standard method, LC-MS/MS, and to investigate whether alterations in birth weight for gestational age are associated with differences in global placental DNA methylation.
# Material and methods
clinical study. The study was conducted in 1023 mothers with singleton pregnancies delivering at the Charité obstetrics department, Berlin, Germany from 2000 to 2008. The local ethics committee approved the study and all clinical investigations were performed according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants. Data on history of diabetes in the family, the status of diabetes before or during pregnancy, smoking status before pregnancy and the ethnic background were obtained from the "Mutterpass" (German pregnancy record booklet). According to the practice guideline of the German Diabetes Assosiation (DGG) and the German Association for Gynaecology and Obstetrics (DGGG), all mothers were screened for gestational diabetes. Variables describing diabetes in the dataset were diabetes before pregnancy (preexisting diabetes mellitus according to the pregnancy record booklet) and diabetes during pregnancy (composite of diabetes before pregnancy and gestational diabetes). Gestational age was estimated from the date of the last menstrual period. Data on blood pressure during pregnancy were obtained from routine antenatal examinations. Placental samples were collected directly after delivery. One complete placental cotyledon taken from the same location was collected from all participating mothers. Placental samples were stored at −20 °C until further use for DNA isolation. Additionally, data regarding the sex of the newborn, birth weight, birth length, and head circumference were extracted into our database. DNA preparation and placental methylation quantification. DNA extraction was performed using a QIAamp DNA Mini Kit (Qiagen). RNase was added during the isolation process to eliminate the influence of RNA in the next step. Quality and quantity of the DNA solution were measured using a spectrophotometer ND-1000 (NanoDrop). Degradase plus DNAse (Zymo Research) was used in the DNA digestion process according to the manufacturer's instructions. As a control, 200 ng of the digested DNA was analyzed by agarose gel electrophoresis. DNA was prepared by adding 280 µl of 0.1% formic acid to 70 µL of digested DNA to obtain a final concentration of 2 ng/µL. Determination of placental methylation was performed by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS) as described previously. Analyses were performed with an Agilent 6530 Accurate-Mass Q-TOF instrument with Jet Stream-Interface (Palo Alto, USA). The chromatographic separation process was performed with a X-BridgeTM C18 4.6 mm × 150 mm (3.5 lm particle size) column (Waters). For gradient elution solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) were used. Flow rate of eluent was maintained constant at 0.5 ml/min. A total of 100 ng digested DNA in a volume of 50 µL was applied in every injection. The parameters used in the ESI-MS/MS step for gas temperature and gas flow rate were 250 °C and 8 L/min respectively, while the sheat nebulizer gas temperature Statistical analysis. SPSS version 20 was used for the statistical analyses. Descriptive data are given as mean plus/minus standard deviation for continuous variables or percent for dichotomous variables. SGA was defined as birth weight for gestational age below the 10th percentile, while the cutoff for LGA was above the 90th percentile, using a global reference.. For the comparison of means between groups, ANOVA was applied according to normal distribution of the data. A Bonferroni post hoc test was used to assess specific significant differences between two groups. Kruskal-Wallis test was applied for non-normally distributed data. Independence of observed associations was investigated using a multivariable ANCOVA model adjusted for suitable confounders. Parameters that were already shown to interact with global DNA methylation and/or variables that were significantly different among SGA, AGA, and LGA births were employed as confounders. For the ANCOVA model these included as diabetes during pregnancy, age of the mother, systolic blood pressure, and smoking before pregnancy. Associations between DNA methylation stratified into tertiles and the frequency for AGA, SGA or LGA was analyzed using Pearson's Chi-square test. Analysis of the independence of observed associations was performed employing a multiple logistic regression analysis. Variables known to impact on size for gestational age and/or variables that were significantly different among the three ranked global placental DNA methylation groups were included as confounders. These parameters consisted of diabetes during pregnancy, smoking before pregnancy, pre-pregnancy BMI, diastolic blood pressure, ethnicity, and sex of the child. Collinearity between confounders was controlled by using a variance inflation factor <5 50 .
Ethics approval. The study was approved by the ethical comitee of the University Hospital Charité, Berlin-Mitte, Humboldt-University Berlin, Berlin, Germany.
## Consent for publication. the final version of this manuscript was approved by all authors for submission to scientific reports
# Results
Detailed descriptive statistics of the cohort are shown in . The mean age of the mothers was 30.0 ± 5.9 years. The overall mean gestational age at birth, birth weight, and birth length index were 38.8 ± 2.0 weeks, 3355.4 ± 630.1 g, and 50.7 ± 3.2 cm, respectively. Regarding the distribution of sex, 52.3% were male and 47.7% were female newborns. Placental DNA methylation ranged from 2.01% to 4.83%, with a mean value of 3.01 ± 0.47%. Descriptive statistics of the cohort grouped according to the birth weight for gestational age are displayed in . There were 127 SGA, 796 AGA and 100 LGA newborns in this study.
LGA newborns were heavier (p < 0.001), longer (p < 0.001), and had a bigger head circumference (p < 0.001) compared to AGA and SGA newborns. Gestational age and pre-pregnancy BMI were also significantly different (p = 0.002 and p < 0.001). The distribution of sex was significantly different (p < 0.001), with SGA births showing a higher frequency of female (34.6% males; 65.4% females) and LGA births a higher frequency of male newborns (69.0% males; 31.0% females). Moreover, the distribution of the mothers who suffered from diabetes mellitus during pregnancy was significantly shifted towards LGA newborns (p < 0.001). . Descriptive statistics of all mother-child pairs and group comparison according to birth weight for gestational age; Data are given as mean ± SD or %; SGA = small for gestational age; AGA = appropriate for gestational age; LGA = large for gestational age; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure.
Scientific RepoRtS | (2020) 10:927 | https://doi.org/10.1038/s41598-020-57725-0 www.nature.com/scientificreports www.nature.com/scientificreports/ To investigate potential differences in global placental DNA methylation between the three birthweight for gestational age groups, ANOVA analyses followed by a Bonferroni post hoc test were performed. These analyses revealed a significantly higher global placental DNA methylation in LGA compared to AGA (p < 0.001) and SGA (p = 0.004) newborns. There was no significant difference in global placental DNA methylation between AGA and SGA newborns as shown in. To analyse the potential impact of various confounders on global placental DNA methylation, a multivariable ANCOVA model was calculated including diabetes during pregnancy, age of the mother, systolic blood pressure and smoking before pregnancy . This analysis demonstrated that higher levels of global placental DNA methylation are associated with LGA births independent of other confounding factors known to influence DNA methylation (p < 0.001; Partial η 2 = 0.013; 95% C.I. = 0.08-0.28; .
To further investigate the association between LGA births and increased global placental DNA methylation, tertiles of global placental DNA methylation were created and ranked into low (n = 341), moderate (n = 341), and high (n = 341) DNA methylation. Descriptive statistics of mothers ranked into tertiles according to the degree of global placental DNA methylation are shown in. Several parameters were significantly different between the three groups, including diastolic blood pressure (p = 0.017), ethnicity (p = 0.030) smoking before pregnancy (p = 0.005) and diabetes during pregnancy (p = 0.006). Moreover, Pearson's Chi-square test was performed to assess the prevalence of SGA, AGA and LGA births among the three placental DNA methylation groups. There was a significant difference (Pearson's Chi-Square: 19.051; p = 0.001) in the prevalence for SGA, AGA, and LGA births. Mothers in the group of highest global placental DNA also displayed the highest prevalence for LGA offspring (48.0% (LGA) vs. 32.4% (AGA) vs. 27.6% (SGA);. Moreover, to investigate a potential independent association between birth weight for gestational age and global placental DNA methylation, an adjusted multiple logistic regression model was calculated using birth weight for gestational age as dependent and global placental DNA methylation as independent continuous variable. The model was adjusted for important factors impacting on size for gestational age and variables that were significantly different among three groups of ranked global placental DNA methylation. These included diabetes during pregnancy, pre-pregnancy BMI, ethnicity, sex of the child, diastolic blood pressure and smoking before pregnancy. The multiple logistic regression model highlighted an independent positive association between LGA birth and global placental DNA methylation (p = 0.001; Exp(B) = 2.06; 95% C.I. = 1.35-3.16;.
# Discussion
The current study demonstrated that being born LGA is associated with a significantly higher level of global placental DNA methylation compared to AGA or SGA births. An ANCOVA model furthermore demonstrated that this association is independent of known confounders of placental DNA methylation (p < 0.001; Partial η 2 = 0.013; 95% C.I. = 0.08-0.28). To substantiate the finding of an association between LGA and higher global placental DNA methylation, global placental DNA methylation was ranked into three groups comprising of low, moderate and high DNA methylation. In these groups, the frequency of LGA births was compared. These analyses demonstrated a significantly higher frequency of LGA births in the group of mothers with the highest global placental DNA methylation levels. Furthermore, by comparing descriptive statistics of the three methylation groups, potential confounders of the association between higher global placental DNA methylation and LGA births were identified. Diastolic blood pressure, ethnicity, the prevalence of smoking before pregnancy, and the prevalence of diabetes were significantly different among the three methylation groups. Calculating a multiple logistic regression model adjusted for the identified confounders and other well-established confounders of birth weight for gestational age plus using placental methylation as a continuous variable backed results of the The average global placental methylation assessed by this study was 2.99 ± 0.47% in AGA newborns. This finding is in agreement with previous studies, demonstrating global hypomethylation in comparison to other tissues in the human body 51 . However, different to previous studies which used HPLC to quantify global placental DNA methylation, this study employed the current gold standard of global DNA methylation quantification, LC-MS/ MS, and is by far the largest study of its kind.
In a previous study we observed an association between gestational diabetes mellitus and higher levels of global placental DNA methylation in a similar cohort as the one analysed in this study. As to be expected, ranking global placental DNA methylation into 3 groups demonstrated a higher frequency of diabetes during pregnancy (composite variable encompassing mothers with overt diabetes before and during pregnancy) in the highest global placental DNA methylation tertile. As diabetes during pregnancy is strongly associated with an increased risk for LGA births and, as previously shown, is associated with increased global placental DNA methylation, any multivariable analysis had to be adjusted for maternal diabetes. However, adjusting for diabetes during pregnancy in the multivariable logistic regression model did not affect the association between increased levels of global placental DNA methylation and a higher frequency for LGA births. Furthermore, an evaluation of the study cohort excluding any mothers with diabetes (Supplemental Data) did not weaken but actually slightly strengthen the association between global placental DNA methylation and LGA births. This suggests that the association between gestational diabetes and global placental DNA methylation is an independent phenomenon and does not explain the observed association between global placental DNA methylation and LGA birth.
Several studies have been conducted to investigate the impact of global placental DNA methylation on birth weight. Most of these studies measured LINE-1 methylation as a surrogate for global DNA methylation, were placenta samples observed a higher degree of placental methylation in the low birth weight group (<2500 g) compared to the normal birth weight group (2500-4000 g). Contrary to these results, we did not observe any significant differences in global placental DNA methylation of SGA newborns. Similar to findings of the current study, Wilhelm-Benartzi et al., analyzing DNA methylation of repetitive elements in 380 placenta samples, showed that placental methylation of the Alu element AluYB8 was significantly higher in LGA offspring. Furthermore, both LINE-1 and Alu methylation levels were significantly positively associated with birth weight percentiles. Overall, the rather heterogeneous supporting and opposing findings of previous studies can only be carefully used for interpreting our current findings. Major difference of the current study is the usage of a methodology that robustly quantifies global DNA methylation, in contrast to the usage of surrogate parameters of global DNA methylation in previous studies. Furthermore, chosen sample size appears to be an important factor, given the differences in results comparing small scaleto relatively larger scale studies.
The potential biological effect of increased global placental DNA methylation in general and even more so in regards to affecting growth patterns of the offspring remains very poorly understood. The placenta does share certain phenotypic features usually found in solid neoplasms, such as cell invasion, vascular remodelling and suppression of the immune system 54 . Moreover, the placenta, similar to many neoplasms, is characterized by global DNA hypomethylation in comparison to somatic tissues. Interestingly, next to global hypomethylation, neoplastic tumors demonstrate local hypermethylation, which is often found in tumor suppressor genes. Hypermethylation of tumor suppressor genes was also demonstrated for the human placenta. Moreover, as outlined above, increases in methylation of Alu elements, was shown to be positively correlated with fetal growth. Higher methylation in these evolutionary younger transposons, was found to be relevant for placental function. Furthermore, it was demonstrated in a study in rats that the administration of the DNA methylation inhibitor 5-Azacitidine lead to significantly smaller placentas, yet again highlighting a connection between the degree of placental DNA methylation and placental growth. Placental weight, which serves as a surrogate parameter of the surface area for materno-fetal nutrient transport, is a very important determinant for fetal growth and offspring www.nature.com/scientificreports www.nature.com/scientificreports/ birth weight 60,61 . Hypothetically, above average hypermethylation of growth related regions could induce larger than average placental growth which could translate into an increased risk for LGA births. Furthermore, above average increases in DNA methylation of diverse growth related regions in the placental DNA could result in a slight net increase in global placental DNA methylation that differentiates LGA from AGA births. However, to address these issues future adequately designed studies are necessary before drawing any causal conclusions.
Findings of the current study can be interpreted by findings of studies investigating other aspects of global placental DNA methylation. Nomura et al. showed that obese women display a higher global placental DNA methylation compared to lean women. It is well established that obesity is a strong maternal risk factor for LGA offspring. In the current study we did not observe any association between pre-pregnancy BMI and global placental DNA methylation. However, different from the study of Nomura et al. in which obesity was defined as a BMI > 30 kg/m 2 and affected 30% of the participating mothers, mean pre-pregnancy BMI in the current study was 23.2 ± 4.6 kg/m 2 . It is possible that these differences in the makeup of the investigated cohorts accounted for the conflicting observation. Although increases in BMI are associated with dyslipidemia 63 , studies demonstrated BMI independent associations between the maternal blood lipid profile and LGA births. Global DNA methylation has been reported to be associated with lipid metabolism and plasma glucose concentration. It was demonstrated in a rat model that maternal dyslipidemia, induced by feeding a high fat diet, increases global placental DNA methylation. Interestingly, an intervention with dietary n-3 fatty acids reversed these alterations. Current literature suggests that the link between dietary lipids and DNA methylation might be due to their involvement in one carbon metabolism. Thus, the maternal genetic setup or consumed diet, key factors that affect blood lipid profiles, might also impact on placental lipid metabolism and DNA methylation during pregnancy. Effects of changes in the placental DNA methylation status on placental lipid metabolism or, vice versa, effects of an altered placental lipid metabolism on the placental DNA methylation status theoretically could be one factor for the positive association between DNA methylation and LGA offspring and warrant further studying.
Study strengths and limitations. One limitation of this and the majority of previous studies is that no cell type separation prior to DNA methylation analyses was performed. The placenta is composed of several different cell types ranging from fibroblast, trophoblasts to mesenchymal cells. Epigenetic patterns are cell specific and different cell types in the placenta are known to have a different DNA methylation profile. Thus, it is not possible to rule out that the obtained results were influenced by cell type heterogeneity in between samples. However, since this study used more than 1000 placenta samples, isolation and analysis of specific placental cells was not feasible. There are studies that compared the effect of different placental sampling areas and the isolation of specific placental cell types on global placental DNA methylation. Non et al. showed that sampling from different locations in the human placenta still resulted in strongly correlated degrees of LINE-1 methylation 73 . Schroeder et al. compared global DNA methylation, measured by MethylC-Seq in isolated rhesus trophoblasts to whole rhesus placenta methylation by MethylC-Seq and demonstrated a strong correlation between the two. However, studies that focus on elucidating the contribution of individual cell types to whole placental DNA methylation are still lacking. This is especially problematic for the analysis of array data, as until now accounting for mixed cell type composition, an important consideration in heterogeneous tissues such as the placenta, is still not feasible. Thus, future studies that focus on cell type specific differences in placental DNA methylation and their contribution to whole placenta DNA methylation are urgently needed.
Next to the mentioned limitations, this study has several strengths. To the best of our knowledge, it is by far the largest study of its type, investigating global placental DNA methylation in over a thousand placenta samples. More so, the current gold standard for the quantification of global DNA methylation, LC-MS/MS, was used.
# Conclusions
The current study demonstrated a significant positive association between the degree of global placental DNA methylation and being born LGA, which was independent of maternal diabetes during pregnancy, a very important risk factor for LGA births. Taken together, evidence in literature is existing that supports the observed positive association between global placental DNA methylation and being born LGA. So far the underlying mechanisms remain elusive. Hypothetically, hypermethylation of growth related DNA regions, such as tumor suppressor genes and certain transposable elements could affect placental size and function which could impact on fetal growth. Above average increases of DNA methylation in growth related DNA regions could result in pathologically increased growth. Furthermore, this might be reflected by significantly elevated levels of global placental DNA methylation. Findings of previous studies suggest a possible connection between anthropometric measurements, lipid metabolism, one-carbon metabolism, and DNA methylation. Taken together, it remains largely unknown how changes in global placental DNA methylation can affect fetal growth. Moreover, the cause of altered global placental DNA methylation levels in LGA births is still highly elusive. Thus, future studies are needed to be able to draw any more causal conclusions.
## Data availability
Data will be made available upon request to the corresponding author |
Pharmacological Actions of Myricetin in the Nervous System: A Comprehensive Review of Preclinical Studies in Animals and Cell Models
Myricetin is a natural flavonoid extracted from a variety of plants, such as medicinal herbs, vegetables, berries, and tea leaves. A growing body of evidence has reported that myricetin supplementation display therapeutic activities in a lot of nervous system disorders, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, epilepsy, and glioblastoma. Myricetin supplementation can also protect against pathological changes and behavioral impairment induced by multiple sclerosis and chronic stress. On the basis of these pharmacological actions, myricetin could be developed as a potential drug for the prevention and/or treatment of nervous system disorders. Mechanistic studies have shown that inhibition of oxidative stress, cellular apoptosis, and neuroinflammatory response are common mechanisms for the neuroprotective actions of myricetin. Other mechanisms, including the activation of the nuclear factor E2-related factor 2 (Nrf2), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), cyclic adenosine monophosphate-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF) signaling, inhibition of intracellular Ca 2+ increase, inhibition of c-Jun N-terminal kinase (JNK)-p38 activation, and suppression of mutant protein aggregation, may also mediate the neuroprotective effects of myricetin. Furthermore, myricetin treatment has been shown to promote the activation of the inhibitory neurons in the hypothalamic paraventricular nucleus, which subsequently produces anti-epilepsy effects. In this review, we make a comprehensive understanding about the pharmacological effects of myricetin in the nervous system, aiming to push the development of myricetin as a novel drug for the treatment of nervous system disorders.
# Introduction
Myricetin [fig_ref] FIGURE 1 |: The structure of myricetin [/fig_ref] is a natural flavonoid extracted from a variety of plants, such as vegetables, berries, and tea leaves. It can produce numerous pharmacological effects, including anti-inflammation [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] , anti-oxidation , anti-carcinogen [bib_ref] Molecular Mechanisms of the Action of Myricetin in Cancer, Xie [/bib_ref] , anti-diabetes [bib_ref] Antihyperglycemic Activity of Myricetin, through Inhibition of DPP-4 and Enhanced GLP-1 Levels,..., Lalitha [/bib_ref] , and cardio-protection [bib_ref] Systems Pharmacology Dissection of the Protective Effect of Myricetin against Acute Ischemia/Reperfusion-Induced..., Qiu [/bib_ref] , and display therapeutic activities in central nervous system disorders like cerebral ischemia [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] , Parkinson's disease [bib_ref] Myricetin Attenuates Neurodegeneration and Cognitive Impairment in Parkinsonism, Dhanraj [/bib_ref] , Alzheimer's disease [bib_ref] Molecular Level Insight into the Benefit of Myricetin and Dihydromyricetin Uptake in..., Liu [/bib_ref] , depression [bib_ref] Myricetin Attenuates Depressantlike Behavior in Mice Subjected to Repeated Restraint Stress, Ma [/bib_ref] , epilepsy [bib_ref] Myricetin Attenuates the Severity of Seizures and Neuroapoptosis in Pentylenetetrazole Kindled Mice..., Sun [/bib_ref] , and glioma [bib_ref] Myricetin Exhibits Antiglioma Potential by Inducing Mitochondrial-Mediated Apoptosis, Cell Cycle Arrest, Inhibition..., Li [/bib_ref]. Pharmacodynamic studies have revealed that myricetin has a series of deficits. Firstly, its oral bioavailability is only about 9.62 and 9.74%, respectively, at 2 oral doses 50 and 100 mg/kg, indicating a poor absorbing property [bib_ref] Quantitative Determination of Myricetin in Rat Plasma by Ultra Performance Liquid Chromatography..., Dang [/bib_ref]. Secondly, the stability of myricetin is easily influenced by gastrointestinal environment, which is stable in simulated gastric fluids and buffer solutions with low pH values and experience a pseudo-first-order kinetic degradation in simulated intestinal fluids and buffer solutions with high values [bib_ref] Gastrointestinal Stability of Dihydromyricetin, Myricetin, and Myricitrin: an In Vitro Investigation, Xiang [/bib_ref]. These deficits indicate that orally administration of myricetin can induce a state with low efficacy in brain delivery which may limit the application of myricetin in clinical practice. However, this hypothesis may be not definitely true, as the neuroprotective effects of myricetin produced by orally administration have been observed repeatedly in previous studies. For example, myricetin administration has been shown to reduce infarct brain volume in rat models of cerebral ischemia [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref]. Myricetin administration can also improve learning and memory impairment in animals who receive streptozotocin [bib_ref] Myricetin Protects Hippocampal CA3 Pyramidal Neurons and Improves Learning and Memory Impairments..., Ramezani [/bib_ref] , D-galactose, or scopolamine stimulation. In addition, myricetin administration can ameliorate the pathogenesis of Parkinson's disease in models in vitro and in vivo [bib_ref] Myricetin Attenuates Neurodegeneration and Cognitive Impairment in Parkinsonism, Dhanraj [/bib_ref] [bib_ref] Myricetin Reduces Cytotoxicity by Suppressing Hepcidin Expression in MES23.5 Cells, Deng [/bib_ref]. The discrepancy of myricetin between its low oral bioavailability and neuroprotective effects could be coordinated by a metabolism hypothesis: the orally administered myricetin could be converted into a metabolite that penetrates the blood-brain-barrier. Mechanistic studies have revealed that inhibition of neuroinflammation, oxidative stress, and cellular apoptosis [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] [bib_ref] The Anti-STAT1 Polyphenol Myricetin Inhibits M1 Microglia Activation and Counteracts Neuronal Death, Boriero [/bib_ref] [bib_ref] Myricetin Reduces Cytotoxicity by Suppressing Hepcidin Expression in MES23.5 Cells, Deng [/bib_ref] [bib_ref] Neuroprotective Effects of Myricetin on Epoxiconazole-Induced Toxicity in F98 Cells. Free Radic, Hamdi [/bib_ref] [bib_ref] Myricetin as a Promising Molecule for the Treatment of Post-Ischemic Brain Neurodegeneration, Pluta [/bib_ref] , activation of nuclear factor E2-related factor 2 (Nrf2) , extracellular signal-regulated kinase 1/2 (ERK1/2) [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] , protein kinase B (Akt) [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] , and cyclic adenosine monophosphate-response element binding protein (CREB), inhibition of intracellular Ca 2+ increase [bib_ref] Myricetin and Quercetin, the Flavonoid Constituents of Ginkgo Biloba Extract, Greatly Reduce..., Oyama [/bib_ref] [bib_ref] Myricetin Inhibits the Release of Glutamate in Rat Cerebrocortical Nerve Terminals, Chang [/bib_ref] , and inhibition of c-Jun N-terminal kinase (JNK) [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] or p38 [bib_ref] Myricetin Reduces Voltage Activated Potassium Channel Currents in DRG Neurons by a..., Hagenacker [/bib_ref] [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] may mediate the neuroprotective actions of myricetin. On the basis of these reports, we conclude that myricetin, dependent on its metabolites which possess neuroprotective activities, could be developed as a potential candidate for the treatment of nervous system disorders. In this review, we make a comprehensive outline for the pharmacological actions and possible mechanisms of myricetin in the nervous system by searching online literatures and databases involving in vivo animal studies and in vitro cell experiments, aiming to push the development and application of myricetin in nervous system disorder treatment.
## Pharmacological effects of myricetin in cerebral ischemia
Cerebral ischemia is a common cerebral vascular disease which occurs in conditions like stroke [bib_ref] An Updated Review of Autophagy in Ischemic Stroke: From Mechanisms to Therapies, Wang [/bib_ref] and hypoperfusion [bib_ref] Chronic Cerebral Hypoperfusion: An Undefined, Relevant Entity, Ciacciarelli [/bib_ref]. Oxido-nitrosative stress and neuroinflammation are two key pathological events that can mediate the pathogenesis of cerebral ischemia [bib_ref] Targeting Myeloperoxidase (MPO) Mediated Oxidative Stress and Inflammation for Reducing Brain Ischemia..., Chen [/bib_ref]. Inhibition of oxido-nitrosative stress and neuroinflammation may be beneficial for the treatment of cerebral ischemia. Orally myricetin administration once daily at the dose of 5 and 25 mg/kg (7 days) [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] or at the dose of 10 or 20 mg/kg [2 h before and every day after middle cerebral artery occlusion (MCAO)] has been shown to reduce neuronal apoptosis and infarct area and improve neurological deficits in a rat model of permanent MCAO via suppressing abnormally increased pro-inflammatory cytokines, malondialdehyde (MDA), and reactive oxygen species, and abnormally decreased GSH, superoxide dismutase (SOD) activity, mitochondrial membrane potential, and adenosine triphosphate (ATP) levels in ischemic brain tissues [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] [bib_ref] Myricetin Ameliorates Brain Injury and Neurological Deficits via Nrf2 Activation after Experimental..., Wu [/bib_ref] ; [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref]. These effects of myricetin may be associated with the increase in Akt activity and Nrf2 nuclear translocation and the decrease in p38 or nuclear factor-κB (NF-κB) activity [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] ; [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref]. Studies in vitro have shown that myricetin pre-incubation (10 nM, 3 h) can attenuate oxygen-glucose deprivation (OGD)-induced neuronal damage, reactive oxygen species production, and mitochondrial depolarization in SH-SY5Y cells, with a possible mechanism involving a direct binding and inhibition of myricetin to caspase-3 ; and [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref]. This indicates that reducing oxidative stress may contribute to the neuroprotective effect of myricetin in cerebral ischemia.
The Ca 2+ overloading-mediated neuronal excitotoxicity is a well-known mechanism for the pathogenesis of cerebral ischemia, during which the abnormal increase in intracellular Ca 2+ contributes largely to neuronal loss [bib_ref] Ca2+ Channel Antagonists and Neuroprotection from Cerebral Ischemia, Kobayashi [/bib_ref]. Although no direct evidence can support the downregulatory effect of myricetin on intracellular Ca 2+ in the anti- Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 797298 2 cerebral ischemia effect of myricetin, some in vitro studies may provide indirect evidence. For example, myricetin pretreatment (0.1, 0.3, 1, 3, 10 μM, 24 h) has been shown to prevent glutamateinduced nuclear fragmentation and cell death in primary cultured rat cortical neurons by suppressing the N-methyl-D-aspartic acid (NMDA) receptor-mediated Ca 2+ overloading, reactive oxygen species production, and caspase-3 activation ; and [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref]. Myricetin incubation (30 μM) can also reduce Ca 2+ influx, neuronal depolarization, and glutamate release in isolated nerve terminals induced by a potassium channel blocker 4-aminopyridine (4-AP) via blocking the N-type and P/Q-type Ca 2+ channels [bib_ref] Myricetin Inhibits the Release of Glutamate in Rat Cerebrocortical Nerve Terminals, Chang [/bib_ref] FIGURE 2 | Neuroprotective effects and possible mechanisms of myricetin in models of cerebral ischemia. Myricetin inhibits (i) OGD/R-induced inflammation, decrease in eNOS expression, phosphorylation, and activity, decrease in intracellular BH4/BH2 ratio, and decrease in intracellular GSH levels in endothelial cells via stimulation of Akt and Nrf2, (ii) glutamate-induced nuclear fragmentation and cell death in primary cultured rat cortical neurons via suppression of Ca 2+ overloading, and (iii) OGD-induced neuronal damage, reactive oxygen species production, and mitochondrial depolarization in SH-SY5Y cells via inhibition of caspase-3. Myricetin administration can also reduce neuronal apoptosis and infarct area and improve neurological deficits in a rat model of MCAO via increasing Akt activity, decreasing p38 and NF-κB activity, and increasing Nrf2 nuclear translocation. and [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref]. However, whether inhibition of Ca 2+ overloading indeed contributes to the neuroprotective actions of myricetin should be ascertained in future studies. The dysfunction of the endothelium is a known pathological event that mediates the impairment in blood-brain-barrier in cerebral ischemia. Methods that restore the function of the endothelium may help to ameliorate neuronal damages in cerebral ischemia. Myricetin pre-incubation (10, 30, or 60 μM, 24 h) can ameliorate OGD/R-induced production of pro-inflammatory cytokines, decrease in endothelial nitric oxide synthase (eNOS) expression/phosphorylation/activity, decrease in intracellular tetrahydrobiopterin (BH 4 )/BH 2 ratio, and decrease in intracellular glutathione (GSH) levels in human brain micro-vessel endothelial cell (HBMECs) [bib_ref] Myricetin Ameliorated Ischemia/reperfusion-Induced Brain Endothelial Permeability by Improvement of eNOS Uncoupling and..., Zhang [/bib_ref] ; and [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref] , which may indicate a state of eNOS uncoupling and permeability increase. These effects of myricetin may be dependent on Akt or Nrf2, as inhibition of Akt or Nrf2, both of which could be activated by myricetin, can block the improvement effect of myricetin on eNOS activity and endothelial permeability [bib_ref] Myricetin Ameliorated Ischemia/reperfusion-Induced Brain Endothelial Permeability by Improvement of eNOS Uncoupling and..., Zhang [/bib_ref] ; and [fig_ref] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in... [/fig_ref]. These results uncover a possibility that improving the functions of the endothelium may contribute to the anticerebral ischemia effect of myricetin. In view of the importance of the endothelium in brain homeostasis [bib_ref] The Endothelium, a Protagonist in the Pathophysiology of Critical Illness: Focus on..., Van Ierssel [/bib_ref] , researchers should further establish the causal relationship between the regulatory effect of myricetin on cerebral ischemia and endothelial cells in future studies.
## Pharmacological effects of myricetin in alzheimer's disease
Alzheimer's disease (AD) is a common neurodegenerative disease with progressive worsening in cognition-and memory-associated neuronal functions [bib_ref] Alzheimer Disease: Controversies in Basic Science Research, Different Theories, and Reasons for..., Rahimi [/bib_ref]. To date, no definite drugs could be available for the treatment of AD, which is partially due to the deficiency of the understanding about the pathological mechanisms of AD. Therefore, it is urgent to search novel drugs for the treatment of AD. Myricetin could be a drug for that purpose. Firstly, myricetin administration (5 or 10 mg/kg, i. p., started 1 day before stereotactic surgery, 21 days) can suppress memory impairment in a rat model of AD induced by streptozotocin [bib_ref] Myricetin Protects Hippocampal CA3 Pyramidal Neurons and Improves Learning and Memory Impairments..., Ramezani [/bib_ref] ; [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref]. Secondly, orally myricetin administration (100 mg/kg, once daily, 8 weeks) has been shown to suppress D-galactose-induced memory impairment in mice, which was indicated by the decrease in the number of platform crossings, the decrease in the time and distance spent in the target quadrant, and the increase in the time of first crossing; [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref]. Thirdly, orally myricetin administration (25 or 50 mg/kg, once daily, 6 days) can suppress scopolamine-induced decreases in platform crossings and swimming time that spent in the target quadrant in the Morris Water Maze (MWM) test in mice ; [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref].
Mechanistic studies have shown that myricetin improves learning and memory impairment in rodent models of AD likely through reversing streptozotocin-induced decrease in neuronal numbers in the hippocampus [bib_ref] Myricetin Protects Hippocampal CA3 Pyramidal Neurons and Improves Learning and Memory Impairments..., Ramezani [/bib_ref] or D-galactose-induced decrease in the phosphorylation levels of ERK1/2 and CREB; [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref] , two molecules that have neuroprotective functions. In vitro studies have reported that pre-or simultaneous administration of myricetin (0.3, 1, 3 and/or 10 μM, 48 h) can suppress Aβ 1-42induced nuclear fragmentation and caspase-3 activation in primary cultured cortical neurons, and reduce Aβ 1-40 /Aβ 1-42 levels in neuronal culture media via the promotion of α-secretase (ADAM10) expression and the inhibition of β-secretase (BACE-1) activity , suggesting that promoting neuronal survival and inhibiting Aβ accumulation may mediate the anti-AD's effects of myricetin. In addition, myricetin administration has been shown to reverse D-galactose-induced decreases in SOD activity and increases in [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref] , or scopolamine-induced increases in malondialdehyde (MDA) levels and decreases in SOD, glutathione peroxidase (GPx), and catalase activities in the hippocampus ; [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref] , suggesting that anti-oxidation may mediate the anti-AD's effects of myricetin, at least in rodent models of D-galactose or scopolamine stimulation. Iron over-accumulation has been shown to be actively involved in AD pathogenesis. Inhibition of iron accumulation may help to ameliorate AD symptoms [bib_ref] Role of Brain Iron Accumulation in Cognitive Dysfunction: Evidence from Animal Models..., Schröder [/bib_ref]. Myricetin treatment (25 or 50 mg/kg, once daily, 6 days) can reverse scopolamine-induced increases in iron contents in the hippocampus in mice via direct chelating of intracellular Fe 2+ or inhibition of transferrin receptor 1 (TrR1) expression , suggesting that inhibition of iron accumulation may contribute to the neuroprotective effects of myricetin in AD. Cholinergic hypofunction is another pathological process in AD [bib_ref] Cholinergic System and Post-translational Modifications: An Insight on the Role in Alzheimer's..., Ahmed [/bib_ref]. In mouse models of AD, myricetin administration (25 or 50 mg/kg, once daily, 6 days) can prevent scopolamine-induced increases in acetylcholinesterase (AChE) activities, thereby inducing significant increases in acetylcholine contents in the hippocampus ; [fig_ref] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models [/fig_ref] and [fig_ref] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease [/fig_ref] , suggesting that reversing cholinergic hypofunction may be a key mechanism for the anti-AD's effects of myricetin. This hypothesis could be supported by studies in vitro: myricetin incubation (0.016, 0.063, 0.25, 1, and 4 μM) can inhibit the activity of AChE in a dose-dependent manner in SH-SY5Y cells .
## Pharmacological effects of myricetin in parkinson's disease
Parkinson's disease (PD) is a neurodegenerative disease with dopaminergic neuronal degeneration in the Substantia Nigra pars compacta (SNc) and dopamine depletion in the striatum [bib_ref] Involvement of the Protein Ras Homolog Enriched in the Striatum, Rhes, in..., Serra [/bib_ref]. Besides the use of strategies that restore a normal dopamine signaling, no effective methods could be available for PD treatment in clinic. In a previous study, lateral cerebral ventricle injection of myricetin (0.5 mg/ml, 7 days) has been shown to reverse 6-hydroxydopamine (6-OHDA)-induced decreases in dopamine contents in the striatum in rats via increasing tyrosine hydroxylase expression in the substantia nigra [bib_ref] Myricetin Reduces 6-Hydroxydopamine-Induced Dopamine Neuron Degeneration in Rats, Ma [/bib_ref] ; [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref] and . In a Drosophila model of PD, myricetin administration (314 mM, 3 h before rotenone exposure, 7 days) was found to suppress rotenone-induced gait disturbance, muscular dys-coordination, and memory impairment [bib_ref] Myricetin Attenuates Neurodegeneration and Cognitive Impairment in Parkinsonism, Dhanraj [/bib_ref] ; [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref] and .
One of the mechanisms that mediate the anti-PD's effects of myricetin may be associated with anti-oxidation. Firstly, in Drosophila, myricetin treatment (314 mM, 3 h before rotenone exposure, 7 days) was found to suppress rotenone-induced increases in TBARS levels and decreases in GSH levels [bib_ref] Myricetin Attenuates Neurodegeneration and Cognitive Impairment in Parkinsonism, Dhanraj [/bib_ref] [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref]. Secondly, myricetin coincubation (50 μM) has been shown to attenuate 1-methyl-4phenylpyridinium ion (MPP + )-induced cell loss and nuclear condensation by restoring mitochondrial transmembrane potential and suppressing the production of reactive oxygen species in MES23.5 cells, a type of cells with similar properties as the neurons in the substantia nigra [bib_ref] Myricetin Attenuated MPP(+)-induced Cytotoxicity by Anti-oxidation and Inhibition of MKK4 and JNK..., Zhang [/bib_ref] ; [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref] and . Thirdly, myricetin incubation (50 μM, 30 min before rotenone treatment) in SH-SY5Y cells prevents rotenone-induced cell loss via suppressing DNA fragmentation, lipid peroxidation, and the production of hydrogen peroxide and superoxide anion (Molina-Jiménez et al., 2004; [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref] and .
Besides oxido-nitrosative stress and cellular apoptosis, iron accumulation is also known to mediate the pathogenesis of PD [bib_ref] Metals Associated Neurodegeneration in Parkinson's Disease: Insight to Physiological, Pathological Mechanisms and..., Raj [/bib_ref] ; suppression of iron accumulation may be a potential strategy for the treatment of PD. Myricetin treatment (lateral cerebral ventricle injection; 0.5 mg/ml, 7 days) can prevent 6-OHDA-induced increase of iron-staining cells in the substantia nigra of rats [bib_ref] Myricetin Reduces 6-Hydroxydopamine-Induced Dopamine Neuron Degeneration in Rats, Ma [/bib_ref] , and in MES23.5 cells, myricetin pre-treatment (1 μM, 24 h) has been reported to suppress rotenone-induced hepcidin gene transcription and decrease in ferroportin 1, an iron efflux transporter that can inhibit iron efflux [bib_ref] Myricetin Reduces Cytotoxicity by Suppressing Hepcidin Expression in MES23.5 Cells, Deng [/bib_ref] ; [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref] and , suggesting that inhibition of iron accumulation-associated pathologies in the substantia nigra may contribute to the anti-PD's effects of myricetin. In addition, myricetin incubation (10 μM, 48 h) has been shown to suppress the aggregation of the mutant α-synuclein (S87A) protein in Cos-7 cells via the activation of the ubiquitin-proteasome pathway [bib_ref] Polyphenolic Flavonoid (Myricetin) Upregulated Proteasomal Degradation Mechanisms: Eliminates Neurodegenerative Proteins Aggregation, Joshi [/bib_ref] ; [fig_ref] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models [/fig_ref] and . As the α-synuclein (S87A) mutant is tightly associated with PD [bib_ref] Mimicking Phosphorylation at Serine 87 Inhibits the Aggregation of Human α-synuclein and..., Oueslati [/bib_ref] , this finding demonstrated that inhibition of α-synuclein (S87A) aggregation may be another mechanism for the anti-PD's effects of myricetin. In fact, inhibition of protein aggregation is a featured characteristic of myricetin, as myricetin incubation (10 μM, 48 h) can suppress the aggregation of the misfolded Δ9CAT mutant protein, thermally denatured misfolded protein, mutant SOD-1 protein associated with Amyotrophic Lateral Sclerosis, and expanded (HDQ74) polyglutamine protein associated with Huntington's disease in Cos-7 cells [bib_ref] Polyphenolic Flavonoid (Myricetin) Upregulated Proteasomal Degradation Mechanisms: Eliminates Neurodegenerative Proteins Aggregation, Joshi [/bib_ref]. Catechol O-methyltransferase (COMT) is an enzyme whose inhibition can increase the bioavailability of L-dopa. Myricetin incubation (10, 20, or 50 μM) has been shown to inhibit the activity of COMT, suggesting that the previously-observed elevation of striatal dopamine levels in myricetin-treated rats is possibly mediated by the direct inhibition of myricetin on COMT activity [bib_ref] Inhibition of Catechol-O-Methyltransferase (COMT) by Myricetin, Dihydromyricetin, and Myricitrin, Zhu [/bib_ref].
## Pharmacological effects of myricetin in epilepsy
Epilepsy is a nervous system disorder characterized by the abnormal increase in neuronal excitability [bib_ref] Cortical Excitability in Epilepsy and the Impact of Antiepileptic Drugs: Transcranial Magnetic..., Hamed [/bib_ref]. How to calm-down the over-activated neurons is always a research topic. Orally myricetin administration (100 or 200 mg/kg, 26 days, 30 min prior to each pentylenetetrazol injection) can reduce seizure rates in a mouse model of epilepsy induced by pentylenetetrazole likely through up-regulating the expression of Bcl-2, Bcl-xL, brain-derived neurotrophic factor (BDNF), glutamate decarboxylase 65 (GAD65), and γ-aminobutyric acid (GABA A ), down-regulating the expression of caspase-3, Bad, and Bax, up-regulating the levels of GABA, and reducing glutamate contents and matrix metalloprotein-9 (MMP-9) activity in the hippocampus [bib_ref] Myricetin Attenuates the Severity of Seizures and Neuroapoptosis in Pentylenetetrazole Kindled Mice..., Sun [/bib_ref]. Myricetin incubation (5 μg/ml) can also promote the GABAergic activity in hypothalamic paraventricular nucleus (PVN) neurons by increasing the decay time and frequency of the inhibitory currents mediated by the GABA A receptor . These effects of myricetin may be mediated by the Ca 2+ -Ca 2+ /calmodulin dependent protein kinase II (CaMKII) signaling, as myricetin can activate the T-and L-type Ca 2+ channel and increase the levels of phospho-CaMKII . Further analysis showed that the enhancing effect of myricetin on GABAergic activity was not mediated by antagonizing the GABA A receptor benzodiazepine-binding site , suggesting that the action mode of myricetin on the GABA A receptor may be distinct from the most existing benzodiazepine-binding site agonists of GABA A receptors. In addition, the same dose of myricetin incubation can enhance a type of potassium currents in hypothalamic type-I PVN neurons in a Ca 2+ -dependent manner, and this type of currents may be the Ca 2+activated potassium current, as these currents could be activated in a relative high voltage (more positive than −40 mV) and be inactivated slowly [bib_ref] Myricetin Facilitates Potassium Currents and Inhibits Neuronal Activity of PVN Neurons, Ma [/bib_ref] , which is in accordance with the property of the typical Ca 2+ -activated potassium current [bib_ref] Electrophysiological Properties of Paraventricular Magnocellular Neurons in Rat Brain Slices: Modulation of..., Li [/bib_ref].
## Pharmacological effects of myricetin in glioma
Glioblastoma is the most common type of glioma with high proliferative activities. It can be treated by methods including chemotherapy, radiotherapy, and surgery (Cruz Da [bib_ref] A Systematic Review of Glioblastoma-Targeted Therapies in Phases II, III, IV Clinical..., Silva [/bib_ref]. The current therapies are only partially useful and the median survival time of glioblastoma patients receiving the current therapies is lesser than 18 months [bib_ref] A Retrospective Survival Analysis of Glioblastoma Patients Treated with Selective Serotonin Reuptake..., Otto-Meyer [/bib_ref]. Thus, it is urgent to search novel drugs for the treatment of glioblastoma. Myricetin may have a potential to be developed as a novel anti-tumor agent due to its remarkable anti-cancer properties. In a previous study, a sub-toxic dose of myricetin incubation (50 μM) has been shown to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of glioblastoma cells by augmenting the activation of caspases-3/-7/-8/-9 [bib_ref] Myricetin Sensitizes Malignant Glioma Cells to TRAIL-Mediated Apoptosis by Down-Regulation of the..., Siegelin [/bib_ref]. Bcl-2 and the short isoform of c-FLIP, two proteins which can inhibit TRAILtriggered cellular apoptosis, appear to mediate the sensitization effect of myricetin in glioblastoma cells, as their expression can be down-regulated by myricetin incubation (50 μM), and their overexpression can abrogate the facilitation effect of myricetin on TRAIL-induced apoptosis of glioblastoma cells [bib_ref] Myricetin Sensitizes Malignant Glioma Cells to TRAIL-Mediated Apoptosis by Down-Regulation of the..., Siegelin [/bib_ref]. In another study by [bib_ref] A Multi-Targeted Natural Flavonoid Myricetin Suppresses Lamellipodia and Focal Adhesions Formation and..., Zhao [/bib_ref] , myricetin incubation (≥20 µM) was found to inhibit glioblastoma cell proliferation, migration, and invasion by blocking the formation of lamellipodia, focal adhesions, membrane ruffles, and vasculogenic mimicry in a manner dependent on the suppression of Akt, c-Jun, Rho-associated protein kinase 2 (ROCK2), paxillin, and cortactin phosphorylation. In U251 human glioma cells, myricetin incubation (5, 15, 30, 60, 120, 240 μM) was found to alter the glioma cell shape, which is likely associated with the promotion of cellular apoptosis [bib_ref] Myricetin Exhibits Antiglioma Potential by Inducing Mitochondrial-Mediated Apoptosis, Cell Cycle Arrest, Inhibition..., Li [/bib_ref]. Medulloblastoma is a highly metastatic disease in children.
The activation of the hepatocyte growth factor (HGF) signaling is a known mechanism for the progression of medulloblastoma; its inhibition is a potential strategy for the treatment of medulloblastoma [bib_ref] Molecular Genetic Analysis of the Hepatocyte Growth Factor/MET Signaling Pathway in Pediatric..., Onvani [/bib_ref]. Myricetin incubation (2.5, 5, 10, 20 μM) can prevent HGF-induced phosphorylation of Met, a tyrosine kinase receptor for HGF, in a medulloblastoma cell line (DAOY), and HGF-induced formation of membrane ruffles of DAOY cells, thereby suppressing the migration of DAOY cells and tumor invasion.
## The other pharmacological effects of myricetin in the nervous system
Multiple sclerosis is an autoimmune disease which can induce severe oxidative stress in the nervous system; rectifying the imbalance of oxidative stress may help to ameliorate the pathogenesis of multiple sclerosis [bib_ref] Monitoring the Redox Status in Multiple Sclerosis, Tanaka [/bib_ref]. Orally myricetin administration (100 mg/kg, once daily, 5 weeks) has been shown to ameliorate hyper-locomotion, sustain the balance of mice for a longer time in a high-speed rotating cylinder, reverse spatial learning and memory damage, hinder myelin reduction, and promote myelination in cuprizone-treated mice by promoting the nuclear translocation of Nrf2 and heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQ O 1) expression, reducing MDA levels, and increasing SOD/ catalase activities . These results demonstrate that myricetin can ameliorate the neurological deficits in pathogenesis of multiple sclerosis via the Nrf-2-mediated antioxidative signaling, and myricetin could be developed as a novel drug for the treatment of multiple sclerosis. Chronic stress exposure is a common pathological factor that can induce behavioral abnormalities indicating the occurrence of various psychological disorders in the modern society. Mechanisms that mediate stress-induced behavioral abnormalities are largely unknown. The dysfunctions of the endocrine system, oxidative stress, and neurotrophic factor can be used to explain the pathogenesis of stress-induced psychological disorders [bib_ref] The Effects of Individual Psychotherapy in BDNF Levels of Patients with Mental..., Claudino [/bib_ref] [bib_ref] Revisiting the Stress Concept: Implications for Affective Disorders, Mcewen [/bib_ref] [bib_ref] The Potential of Hydrogen for Improving Mental Disorders, Satoh [/bib_ref]. Myricetin administration (50 mg/kg, i. p., 21 days) has been shown to reduce repeated restraint stress-induced increases in immobility time in mice in the forced swimming and tail suspension test likely through reducing the plasma corticosterone levels and increasing hippocampal GSP-Px activity and BDNF expression in the hippocampus [bib_ref] Myricetin Attenuates Depressantlike Behavior in Mice Subjected to Repeated Restraint Stress, Ma [/bib_ref]. Myricetin administration (40 min before each stress stimulation, 50 mg/kg, i. p., 21 days) can also inhibit repeated restraint stress-induced spatial memory in mice via reducing the levels of plasma adreno-corticotrophic hormone and increasing hippocampal BDNF expression, which was indicated by the increase in the time that spent in the target quadrant in mice exposed to chronic stress in the probe trial in the Morris water maze task [bib_ref] Protective Effects of Myricetin on Chronic Stress-Induced Cognitive Deficits, Wang [/bib_ref].
Neuroinflammation is a common pathological change that mediates the progression of nervous system disorders [bib_ref] The Role of the Inflammasome in Neurodegenerative Diseases, Piancone [/bib_ref]. Inhibition of neuroinflammation is a potential strategy for the treatment of nervous system disorders. Myricetin incubation (50 or 100 μM) has been shown to skew the hypoxia-triggered neuroinflammatory response towards an anti-inflammatory M2 phenotype in BV-2 microglia, thereby protecting the SH-SY5Y cells from death induced by conditioned media from hypoxia-stimulated microglia [bib_ref] The Anti-STAT1 Polyphenol Myricetin Inhibits M1 Microglia Activation and Counteracts Neuronal Death, Boriero [/bib_ref]. Mechanistic studies have shown that myricetin incubation can reduce the phospho-signal transducer and activator of transcription 1 (STAT1) via a direct interaction with STAT1 protein [bib_ref] The Anti-STAT1 Polyphenol Myricetin Inhibits M1 Microglia Activation and Counteracts Neuronal Death, Boriero [/bib_ref]. This finding is line with a fact that STAT1 is a pivotal factor that regulates the transition of microglia towards a pro-inflammatory M1 phenotype under hypoxia in a phosphorylation-dependent manner [bib_ref] STAT1 Drives M1 Microglia Activation and Neuroinflammation under Hypoxia, Butturini [/bib_ref]. Myricetin incubation (10 and 25 μM) can also down-regulate lipopolysaccharide (LPS)-induced increase in interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) levels in BV-2 microglia via inhibiting JNK and p38 phosphorylation [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] , which could be evidenced by a direct inhibition of LPS-induced increase in microglial numbers in the hippocampus and prefrontal cortex (50 and 100 mg/kg, i. p., once daily, 7 days) [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref].
# Conclusion
In this review, we outlined the pharmacological effects of myricetin in the nervous system. Myricetin can produce preventive and therapeutic effects in nervous system disorders through antioxidation , anti-proliferation [bib_ref] Myricetin Sensitizes Malignant Glioma Cells to TRAIL-Mediated Apoptosis by Down-Regulation of the..., Siegelin [/bib_ref] [bib_ref] A Multi-Targeted Natural Flavonoid Myricetin Suppresses Lamellipodia and Focal Adhesions Formation and..., Zhao [/bib_ref] [bib_ref] Myricetin Exhibits Antiglioma Potential by Inducing Mitochondrial-Mediated Apoptosis, Cell Cycle Arrest, Inhibition..., Li [/bib_ref] , and antineuroinflammation [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] [bib_ref] The Anti-STAT1 Polyphenol Myricetin Inhibits M1 Microglia Activation and Counteracts Neuronal Death, Boriero [/bib_ref]. The activation of the Nrf2 , ERK1/2 [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] , Akt [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] , CREB, and BDNF [bib_ref] Myricetin Attenuates the Severity of Seizures and Neuroapoptosis in Pentylenetetrazole Kindled Mice..., Sun [/bib_ref] , the inhibition of intracellular Ca 2+ increase and JNK-p38 activation [bib_ref] Myricetin and Quercetin, the Flavonoid Constituents of Ginkgo Biloba Extract, Greatly Reduce..., Oyama [/bib_ref] [bib_ref] Myricetin Reduces Voltage Activated Potassium Channel Currents in DRG Neurons by a..., Hagenacker [/bib_ref] [bib_ref] Myricetin Inhibits the Release of Glutamate in Rat Cerebrocortical Nerve Terminals, Chang [/bib_ref] [bib_ref] Myricetin against Ischemic Cerebral Injury in Rat Middle Cerebral Artery Occlusion Model, Sun [/bib_ref] [bib_ref] Inhibitory Effects of Myricetin on Lipopolysaccharide-Induced Neuroinflammation, Jang [/bib_ref] , and the suppression of mutant protein aggregation [bib_ref] Polyphenolic Flavonoid (Myricetin) Upregulated Proteasomal Degradation Mechanisms: Eliminates Neurodegenerative Proteins Aggregation, Joshi [/bib_ref] and PVN neuronal activity [bib_ref] Myricetin Facilitates Potassium Currents and Inhibits Neuronal Activity of PVN Neurons, Ma [/bib_ref] [bib_ref] Flavonoid Myricetin Modulates GABA(A) Receptor Activity through Activation of Ca(2+) Channels and..., Zhang [/bib_ref] may also mediate the neuroprotective effects of myricetin in the nervous system. However, no exact causal relationship between myricetin and its neuroprotective effects has been established. If we want to develop myricetin as a novel drug for the treatment of nervous system disorders, we should clarify the common pathway that mediates the neuroprotective effects of myricetin. We should also notice the pharmacodynamic deficits of myricetin which may restrict its application in daily-life and clinical practice, and develop novel strategies to improve the oral bioavailability of myricetin and increase the stability of myricetin in gastric fluids. These endeavors appear to be very important, as although myricetin is not being applied for the treatment of nervous system disorders, some clinical studies have revealed promising potentials for its use in clinic. For example, Emulin ™ which contains myricetin has been shown to reduce blood glucose increase in type 2 diabetic patients [bib_ref] Effect of Emulin on Blood Glucose in Type 2 Diabetics, Ahrens [/bib_ref]. In a 4-weeks randomized placebocontrolled clinical trial, the Blueberin which contains 50 mg myricetin was found to reduce fasting plasma glucose and serum C-reactive proteins levels [bib_ref] Effect of Blueberin on Fasting Glucose, C-Reactive Protein and Plasma Aminotransferases, in..., Abidov [/bib_ref]. Some other studies had reported that myricetin intake is associated with lower cancer risk in humans [bib_ref] Flavonoid Intake and Risk of Chronic Diseases, Knekt [/bib_ref] [bib_ref] Flavonoids Intake and Risk of Lung Cancer: a Meta-Analysis, Tang [/bib_ref]. Since inflammation, diabetes, and cancer are highly correlated with nervous system disorders, these previously-reported clinical studies provide indirect evidence for the development of clinical trials for myricetin in nervous system disorder treatment.
# Author contributions
JL, HX, CH, and JL wrote the paper. All authors contributed to the article and approved the submitted version.
# Acknowledgments
The Scientific Research Project of the People's Hospital of Taizhou (ZL201950).
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 797298 8
[fig] FIGURE 1 |: The structure of myricetin. [/fig]
[fig] FIGURE 3 |: Neuroprotective effects and possible mechanisms of myricetin in Alzheimer's disease-associated models. Myricetin administration can suppress (i) streptozotocin-induced memory impairment via inhibiting streptozotocin-induced decrease in neuronal numbers in the hippocampus, (ii) D-galactose-induced memory impairment via inhibiting scopolamine-induced increase in MDA levels and iron contents and decrease in SOD, GPx, and catalase activities in the hippocampus, and (iii) D-galactose-induced memory impairment via inhibiting D-galactose-induced decrease in the phospho-ERK1/2 and -CREB, decrease in SOD activity, and increase in TBARS levels in the hippocampus.Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 797298 levels of thiobarbituric acid reactive substances (TBARS)(Lei et al., 2012; [/fig]
[fig] FIGURE 4 |: Neuroprotective effects and possible mechanisms of myricetin in Parkinson's disease-associated models. Myricetin produces anti-PD's effects via suppressing 6-OHDA-induced decrease in dopamine contents and tyrosine hydroxylase expression and increase in iron-staining cells in the substantia nigra. Myricetin also has protective effects in Drosophila, MES23.5 cells, SH-SY5Y cells, and Cos-7 cells, as indicated by the concrete descriptions in this figure. Whether these effects could mediate the anti-PD effect of myricetin remain unclear.TABLE 3 | Comprehensive information about the effects of myricetin in animal models Parkinson's disease. *Suppress reactive oxygen species production Deng et al. (2021) *24 h of pretreatment *Restore mitochondrial transmembrane potential -*Reduce hepcidin gene transcription -*Increase ferroportin 1 expression Suppress the aggregation of the mutant α-synuclein (S87A) protein Cos-7 cells *10 μM Activate the ubiquitin-proteasome pathway Joshi et al. (2019) *48 h Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 797298 [/fig]
[table] TABLE 1 |: Comprehensive information about the effects of myricetin in in vivo and in vitro models cerebral ischemia. [/table]
[table] TABLE 2 |: Comprehensive information about the effects of myricetin in animal models Alzheimer's disease. [/table]
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Roles of nutrition in muscle health of community‐dwelling older adults: evidence‐based expert consensus from Asian Working Group for Sarcopenia
General muscle health declines with age, and in particular, sarcopenia-defined as progressive loss of muscle mass and strength/physical performance-is a growing issue in Asia with a rising population of community-dwelling older adults. Several guidelines have addressed early identification of sarcopenia and management, and although nutrition is central to treatment of sarcopenia, there are currently few guidelines that have examined this specifically in the Asian population. Therefore, the Asian Working Group for Sarcopenia established a special interest group (SIG) comprising seven experts across Asia and one from Australia, to develop an evidence-based expert consensus. A systematic literature search was conducted using MEDLINE on the topic of muscle health, from 2016 (inclusive) to July 2021, in Asia or with relevance to healthy, Asian community-dwelling older adults (≥60 years old). Several key topics were identified: (1) nutritional status: malnutrition and screening; (2) diet and dietary factors; (3) nutritional supplementation; (4) lifestyle interventions plus nutrition; and (5) outcomes and assessment. Clinical questions were developed around these topics, leading to 14 consensus statements. Consensus was achieved using the modified Delphi method with two rounds of voting. Moreover, the consensus addressed the impacts of COVID-19 on nutrition, muscle health, and sarcopenia in Asia. These statements encompass clinical expertise and knowledge across Asia and are aligned with findings in the current literature, to provide a practical framework for addressing muscle health in the community, with the overall aim to encourage and facilitate broader access to equitable care for this target population.
# Introduction
By 2050, a quarter of Asia's population is predicted to be ≥60 years old, while those aged ≥80 years old will account for one-fifth of all 'older adults' (≥60 years) in Asia.This trend towards an increasingly older population will inevitably lead to an increased prevalence of not just chronic diseases but also disability and loss of ability to live independently due to a multitude of underlying etiologies. Therefore, public health strategies to maintain healthy, independent living among community-dwelling older adults are of utmost importance both in terms of maintaining a fulfilled life for this age group and in managing available healthcare resources.
Age-related loss of skeletal muscle plus loss of muscle strength or/and reduced physical performance, known as 'sarcopenia', are growing public health problems in healthy community-dwelling older adults in Asia. [bib_ref] Type 2 diabetes mellitus is associated with increased risks of sarcopenia and..., Wang [/bib_ref] [bib_ref] Is osteoporosis a predictor for future sarcopenia or vice versa? Four-year observations..., Yoshimura [/bib_ref] The Asian Working Group for Sarcopenia (AWGS) set out clear statements in a 2019 expert consensus, building on previous publications in 2014 and 2016, to aid early identification of individuals at risk of sarcopenia in Asian countries. [bib_ref] Asian Working Group for Sarcopenia: 2019 consensus update on sarcopenia diagnosis and..., Chen [/bib_ref] Topics included epidemiology, screening and diagnosis, cut-off points for sarcopenia diagnosis methods and criteria, associations of sarcopenia with other diseases, as well as management of sarcopenia. However, although physical activity and a healthy diet/good nutrition are core strategies for the management and prevention of sarcopenia, the topic of nutrition was not well addressed. Therefore, this expert consensus aimed to address that gap and to set out a series of clear statements regarding clinical decision-making around nutrition in muscle health that will be specifically applicable to community-dwelling older adults living in Asia (aged ≥60 years) and contribute to the global view of care for people with sarcopenia.
## Consensus procedures
The AWGS established a special interest group (SIG) of eight experts in the Asia-Pacific region, to develop this expert consensus. The SIG convened for three meetings via videoconferencing, with active participation and attendance by all members. Prior to the first meeting, a literature search was carried out using MEDLINE to identify articles relevant to the roles of nutrition in muscle health in healthy, community-dwelling adults aged ≥60 years. Although the global cut-off for older persons is ≥65 years, we included those aged ≥60-65 as well, to account for a younger definition of 'older adults' in some Asian countries. A few studies cited do include adults below this age range, but they were only included if the mean age was ≥60 years. The search was limited to English language articles published from 2016 (inclusive) to July 2021; studies were included if they were conducted in Asia or had relevance to the Asian population (including Japan, Korea, China, Hong Kong, Taiwan, the Philippines, Vietnam, Malaysia, Thailand, Singapore, Indonesia, Laos, Myanmar, and Cambodia). Selected articles included randomized controlled trials (RCTs), meta-analyses, systematic reviews, longitudinal observational studies, cross-sectional studies, and/or retrospective studies. Case studies, and surveys, general reviews, pre-prints, and congress abstracts were excluded. The search terms were selected to avoid filtering out relevant studies; for example, middle-aged adults were initially included, and then all irrelevant studies (e.g. patients <60 years, hospitalized, with co-morbidities, biomarkers, animal studies, and outside of Asia) were de-selected. Some studies have enrolled participants younger than 60 years, but only studies with subset analysis for people aged 60 years or older were included in this consensus paper.
Key topics were established from the literature search and the first meeting was used to match these topics to key clinical questions (in PICO-Patient/Population, Intervention, Comparison, Outcome, format), with active discussion and round table agreement. The clinical questions were then matched with supporting literature (and the search expanded if specific terms were missed the first time), and based on this, a first set of draft statements were prepared. The draft statements and their supporting literature were presented at the second meeting. Active discussion took place around the strength of statement support evident in the literature, other relevant information from clinical practice, and supplementary sources (e.g. websites) that could be used to further support/inform the statements. The initial 15 draft statements and supporting text providing a rationale for each statement were further refined and sent to each SIG member for anonymous online voting, using a 5-point Likert scale:accept completely, (2) accept with some reservation, (3) accept with major reservations, (4) reject with reservations, and (5) reject completely. The percentage agreement was calculated based on a weighted scale for each vote, with >80% agreement sufficient for the statement to achieve consensus. Agreement was reached for all statements in the first round of voting, with only minor changes to the wording suggested. At the third meeting, the final wording of each statement was proposed, and a second vote took place to establish full consensus; two statements remained unchanged and did not require re-voting. All statements reached >92.5% agreement and final discussion took place with regard to information that should be included in the supporting text. The final 14 statements are detailed in [fig_ref] Table 3: Summary of studies addressing exercise in addition to nutritional supplementation in muscle... [/fig_ref] , with a literature overview providing rationale for each statement detailed below.
## Nutritional status
Malnutrition and screening With an increasingly aging Asian population, malnutrition is also a growing problem. The reported prevalence of being at risk for malnutrition in Asia among community-dwelling older adults varies from 16% to 73%, whereas that of being malnourished can be as high as 22%. [bib_ref] Frailty is a major related factor for at risk of malnutrition in..., Chang [/bib_ref] [bib_ref] A cross-sectional study of nutrient intake and health status among older adults..., Arjuna [/bib_ref] [bib_ref] Use of Mini Nutritional Assessment (MNA®) as a nutritional screening tool among..., Bullecer [/bib_ref] [bib_ref] Association between gait abnormality and malnutrition in a community-dwelling elderly population, Misu [/bib_ref] [bib_ref] Association of frailty and malnutrition with long-term functional and mortality outcomes among..., Wei [/bib_ref] [bib_ref] Malnutrition risks and their associated factors among home-living older Chinese adults in..., Wong [/bib_ref] [bib_ref] Assessment of nutritional status and risk factors for malnutrition among the elderly..., Noe [/bib_ref] [bib_ref] Malnutrition as key predictor of physical frailty among Malaysian older adults, Norazman [/bib_ref] [bib_ref] Malnutrition and sarcopenia in community-dwelling adults in Singapore: Yishun Health Study, Tan [/bib_ref] There is also a growing body of evidence that nutritional status may be a modifiable risk factor for the development of muscle health issues, including sarcopenia. Therefore, identifying individuals at risk of malnutrition, to provide early intervention, is an important public health strategy for preventing the development of sarcopenia and related complications, such as frailty. 14 Although older adults with obesity are also at risk for muscle health problems, the SIG members consider this a topic worthy of separate consideration at another forum. This consensus focuses on undernutrition as an underlying cause of sarcopenia, and therefore malnutrition as a starting point for issues pertaining to muscle health.
Several cross-sectional studies in Asia make a link between malnutrition and sarcopenia and advise early identification of older adults with associated risk factors. Relative appendicular skeletal muscle mass in community-dwelling older adults was examined in both a 'nourished cohort' (n = 400, mean age 71) and an 'at risk of malnutrition cohort' (n = 811, mean age 74) and found that the latter subgroup were four times more likely to have low muscle mass based on AWGS 2019 cut-off values. [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] [bib_ref] Factors associated with muscle mass in communitydwelling older people in Singapore: findings..., Tey [/bib_ref] In a study of older adults (≥65 years) conducted in Taiwan, participants were evaluated for nutritional status using the Mini Nutritional Assessment-Short Form (MNA-SF) and for frailty via the Study of Osteoporotic Fractures (SOF) index. [bib_ref] Frailty is a major related factor for at risk of malnutrition in..., Chang [/bib_ref] It was concluded that malnutrition risk should be evaluated in community-dwelling older adults, and frailty was identified as a major risk factor for malnutrition. [bib_ref] Frailty is a major related factor for at risk of malnutrition in..., Chang [/bib_ref] Those most at risk of malnutrition were predominantly male with lower body weight, lower body mass index (BMI), lower skeletal mass indices, and poorer muscle strength, had less energy, and more often had sarcopenia and measures of frailty, compared with well-nourished older adults. A further study examined the overlapping prevalence of malnutrition and sarcopenia and the association between parameters of malnutrition with muscle mass/strength in a community-dwelling Singaporean adult population (N = 541; 21-90 years old; average age 58.6 years). [bib_ref] Malnutrition and sarcopenia in community-dwelling adults in Singapore: Yishun Health Study, Tan [/bib_ref] Nutritional status was measured using MNA-SF and other questionnaires assessed levels of physical activity and cognition. The study concluded that being at nutritional risk/malnourished was significantly associated with 2-3 times increased risk of sarcopenia in a multivariate analysis adjusting for age. [bib_ref] Malnutrition and sarcopenia in community-dwelling adults in Singapore: Yishun Health Study, Tan [/bib_ref] Additionally, favorable Mini Nutritional Assessment (MNA) parameter scores on food intake and BMI were positively associated with greater muscle mass and handgrip strength (HGS) (P < 0.05).
A study of 301 older adults in Malaysia (mean age: 67.1 years) screened for frailty and malnutrition to find associations between the two conditions. [bib_ref] Malnutrition as key predictor of physical frailty among Malaysian older adults, Norazman [/bib_ref] The study found that the mid-upper arm circumference, calf circumference (CC), and skeletal muscle mass index were all significantly associated with malnutrition risk; lower skeletal muscle mass also independently increased the odds of being at risk for malnutrition in multivariate analysis. Similarly, a cross-sectional study in Indonesia among older adults (N = 527, mean age: 74 ± 7 years) reported a significant correlation between malnutrition risk and muscle function in terms of HGS and gait speed. [bib_ref] A cross-sectional study of nutrient intake and health status among older adults..., Arjuna [/bib_ref] All studies concluded that community screening protocols should combine screening of nutritional status and sarcopenia, as early intervention can help maintain independent and healthy living in old age.
Social and environmental factors Behavioral factors such as dietary and eating patterns play a large role in malnutrition, but social/environmental factors such as living conditions and low socio-economic status also need to be acknowledged. In studies from different Asian countries (Hong Kong, Malaysia, Myanmar, and the Philippines), older adults belonging to low-income strata uniformly comprised majority of the study population, representing up to 81.1% of participants. 7,10-12 Generally conducted in urban settings, these studies found prevalence rates of 28.1-59.4% for malnutrition risk and 1.1-21.7% for malnutrition. In contrast, these rates may even be higher among rural dwellers. In a cross-sectional study in Indonesia, rural older people were more likely than those in urban communities to be malnourished and cognitively impaired and to have associated decreases in functional capacity and independence, suggesting the roles of education and socio-economic status. [bib_ref] A cross-sectional study of nutrient intake and health status among older adults..., Arjuna [/bib_ref] The proportion of subjects at risk for malnutrition were almost twice as high in rural than urban areas while monthly income was~3.5 times lower in ruralliving than urban-living subjects. Therefore, individuals at high risk for malnutrition can potentially be identified by certain social determinants, such as those living in rural settings vs. those in urban areas, and people with low socio-economic status.
Screening tools Most studies in Asia have used the MNA or its short form (MNA-SF) to screen for malnutrition among older adults. [bib_ref] A cross-sectional study of nutrient intake and health status among older adults..., Arjuna [/bib_ref] [bib_ref] Use of Mini Nutritional Assessment (MNA®) as a nutritional screening tool among..., Bullecer [/bib_ref] [bib_ref] Association between gait abnormality and malnutrition in a community-dwelling elderly population, Misu [/bib_ref] [bib_ref] Association of frailty and malnutrition with long-term functional and mortality outcomes among..., Wei [/bib_ref] [bib_ref] Malnutrition risks and their associated factors among home-living older Chinese adults in..., Wong [/bib_ref] [bib_ref] Assessment of nutritional status and risk factors for malnutrition among the elderly..., Noe [/bib_ref] [bib_ref] Malnutrition as key predictor of physical frailty among Malaysian older adults, Norazman [/bib_ref] [bib_ref] Malnutrition and sarcopenia in community-dwelling adults in Singapore: Yishun Health Study, Tan [/bib_ref] In 2002, the European Society for Clinical Nutrition and Metabolism (European Society of Parenteral and Enteral Nutrition) recommended the MNA as a screening tool for the older adults as it is easy to put into practice and takes <10 min to complete. [bib_ref] Educational and Clinical Practice Committee, European Society of Parenteral and Enteral Nutrition..., Kondrup [/bib_ref] A subsequent systematic review deemed MNA-SF the most appropriate screening tool for community-dwelling older adults, based on the presence of extensive testing for validity and reliability as well as good results. [bib_ref] Nutritional screening in community-dwelling older adults: a systematic literature review, Phillips [/bib_ref] The review found MNA-SF, which contains only 6 questions compared with the 28-item MNA, to have high internal consistency, sensitivity (97.9-98%), and specificity (94-100%), albeit according to predominantly non-Asian studies. In Asia, the MNA-SF has demonstrated sensitivities of 81.8-100% and specificities of 74-97.3%. [bib_ref] Evaluation of Mini-Nutritional Assessment for Japanese frail elderly, Kuzuya [/bib_ref] [bib_ref] Validation of nutritional screening tools against anthropometric and functional assessments among elderly..., Suzana [/bib_ref] [bib_ref] Population-specific short-form mini nutritional assessment with body mass index or calf circumference..., Tsai [/bib_ref] It has exhibited predictive validity for sarcopenia, poor quality of life (QoL), and mortality among Asian older adults. [bib_ref] Association of frailty and malnutrition with long-term functional and mortality outcomes among..., Wei [/bib_ref] [bib_ref] Malnutrition and sarcopenia in community-dwelling adults in Singapore: Yishun Health Study, Tan [/bib_ref] [bib_ref] Elderly Nutritional Indicators for Geriatric Malnutrition Assessment (ENIGMA): development and validation of..., Ng [/bib_ref] [bib_ref] Prevalence and associated factors of sarcopenia and severe sarcopenia in older Taiwanese..., Wu [/bib_ref] The Short Nutritional Assessment Questionnaire (SNAQ©) is a three-item questionnaire assessed to have only fair validity and reliability as a screening tool for community-dwelling older adults, with a reported sensitivity of 45-88%, and specificity of 83-99% in non-Asian studies [bib_ref] Nutritional screening in community-dwelling older adults: a systematic literature review, Phillips [/bib_ref] [bib_ref] Development and validation of a hospital screening tool for malnutrition: the Short..., Kruizenga [/bib_ref] and a sensitivity of 32.9-69.2% and specificity of 61.3-73.1% in Asian studies, compared against MNA-SF, MNA, and GLIM-criteria, respectively. [bib_ref] Reliability and validity of the Japanese version of the simplified nutritional appetite..., Nakatsu [/bib_ref] [bib_ref] The Simplified Nutritional Appetite Questionnaire (SNAQ) as a screening tool for risk..., Lau [/bib_ref] [bib_ref] Accuracy of the Simplified Nutritional Appetite Questionnaire for malnutrition and sarcopenia screening..., Shimizu [/bib_ref] Therefore, with lower overall sensitivity and specificity, SNAQ is a less favourable choice for community screening. The Malnutrition Universal Screening Tool (MUST) has been evaluated in hospitalized patients, community-dwellers, and care homes, but although it is widely used in the BAPEN circle (bapen.org.uk), we did not find any studies in Asia examining its sensitivity and specificity for community-dwelling older adults and therefore cannot comment on this population.
There is limited literature to help determine who should carry out screening protocols, but from our clinical experiences, and in order to broaden access to screening, we have recommended both healthcare professionals such as physicians, dietitians, as well as 'trained personnel'. We can envision scenarios in different countries and different settings where additional personnel, such as community care providers or social workers, who may not officially be considered healthcare professionals, could effectively complete this task.
In conclusion, there is a strong link between muscle health and malnutrition, indicating that early identification of older adults at risk of malnutrition could prevent the onset of sarcopenia in many healthy, community-dwelling adults, and some subgroups at higher risk (e.g. people living in rural areas and those with low socio-economic status) may need closer observation. Please see , Consensus Statements 1-3.
## Diet and dietary patterns
## Communal eating
Specific diets and dietary patterns in older adults often lead to undernutrition and ultimately malnutrition, as discussed above. Therefore, finding ways to promote regular food intake and healthy eating in this age group is crucial to promoting and maintaining muscle health. Older adults often make simpler food choices when eating alone and are influenced by several societal factors. [bib_ref] Exploring the experience and determinants of the food choices and eating practices..., Chalermsri [/bib_ref] [bib_ref] Living with family yet eating alone is associated with frailty in community-dwelling..., Suthutvoravut [/bib_ref] [bib_ref] Food choice patterns among frail older adults: the associations between social network,..., Kim [/bib_ref] Therefore, encouraging eating in communal and social settings (within the parameters of any COVID-19 regulations), where there is a variety of food on offer, could potentially improve food choices and consumption in this age group, which will have an overall benefit on well-being and muscle health. [bib_ref] Gender differences in longevity in free-living older adults who eat-withothers: a prospective..., Huang [/bib_ref] [bib_ref] Positive effects of "textured lunches" gatherings and oral exercises combined with physical..., Kito [/bib_ref] There are a limited number of trials within Asia that have specifically addressed communal eating in relation to muscle health. However, one RCT (n = 86; average age 75.6 ± 5.6 years) in Japan examined an integrated 12 week programme of specially devised lunch gatherings containing textured foods (that are hard and cut into larger pieces, therefore require more chewing-so-called munchy lunch) in community centres, with physical exercises, on the improvement of physical function in community-dwelling older adults. [bib_ref] Positive effects of "textured lunches" gatherings and oral exercises combined with physical..., Kito [/bib_ref] A greater decrease in body fat percentage and results of the timed up-and-go test were observed in the intervention group (exercise plus munchy lunch) compared with the controls (only exercise) (P < 0.05), indirectly suggesting some benefits on overall muscle health from combining communal eating with exercise. [bib_ref] Positive effects of "textured lunches" gatherings and oral exercises combined with physical..., Kito [/bib_ref] Two other studies of note in Asia support social measures to improve overall healthy eating. [bib_ref] Eating together" is associated with food behaviors and demographic factors of older..., Ishikawa [/bib_ref] [bib_ref] Which aspects of dining style are associated with depressive mood? A study..., Takase [/bib_ref] A cross-sectional survey among 2196 Japanese older adults revealed that a higher frequency of 'eating together with someone was significantly associated with more diet satisfaction and not being frail. [bib_ref] Eating together" is associated with food behaviors and demographic factors of older..., Ishikawa [/bib_ref] In women, this higher frequency was associated with a subjective health feeling as well as higher food diversity. [bib_ref] Eating together" is associated with food behaviors and demographic factors of older..., Ishikawa [/bib_ref]
## Dairy and alternative dietary components
Although we do not have a statement specifically recommending dairy as a dietary component, we feel that it is relevant to consider the literature in the wider context of diet, particularly in the Asian population, and therefore present a brief literature overview here.
There are considerable data supporting that a diet high in dairy products is good for muscle health as they contain many of the essential components for muscle growth and development. [bib_ref] Sarcopenia: revised European consensus on definition and diagnosis, Cruz-Jentoft [/bib_ref] However, due to historic availability of dairy products in Asia, and a high prevalence of lactose intolerance in the Asian population, there is generally lower dietary consumption of dairy.Therefore, studies in Asia have focused on the benefits of the nutritional components of dairy products, for example, milk fat globule membrane (MFGM) and whey protein. However, two small-scale RCTs involving healthy older adults in Japan (total N = 86, range of mean age: 72.3-73.1 years) found limited evidence of treatment benefits from MFGM in terms of muscle mass muscle strength, and overall physical performance, although Watanabe et al. measured significant changes in motor firing rates following resistance training compared with the placebo group, and Yoshinaka et al. measured improvements in foot tapping and open-close stepping scores. [bib_ref] Effect of milk fat globule membrane supplementation on motor unit adaptation following..., Watanabe [/bib_ref] [bib_ref] Light rhythmic exercise with dietary milk fat globule membrane improves physical fitness..., Yoshinaka [/bib_ref] Therefore, both studies concluded that MFGM may enhance the benefits of light-intensity exercise.
Three RCTs have examined the effects of whey protein supplementation. [bib_ref] Effects of whey protein nutritional supplement on muscle function among community-dwelling frail..., Kang [/bib_ref] [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] Two are discussed in further detail in the later section of this article 'Protein Supplementation' and the third by Mori and Tokuda is summarized: a multidomain study in women (n = 81; average age 70.6 years) to assess whey protein supplementation along with exercise. [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] Participants were allocated to three groups: (1) exercise and protein supplementation, (2) exercise only, and (3) protein supplementation only taking part in a 24 week programme with twice-weekly intervention of exercise and/or 22.3 g of protein.
The total protein intake for all three experimental groups was adjusted to a level of ≥1.2 g/kg/day, with more during the intervention period for those assigned protein supplementation alone or in addition to exercise. An increase in skeletal muscle mass was significantly higher for those in the exercise and protein supplementation group compared with exercise only (P = 0.007) or supplementation only (P < 0.001). A similar increase was seen in the dual intervention group for HGS (P = 0.014) and gait speed (P = 0.026), compared with the protein supplementation only group. It was concluded that whey protein supplementation administered after resistance exercise could provide benefits for the prevention of sarcopenia among community-dwelling older Japanese women.
## Dietary patterns
Two Asian studies assessed dietary patterns. One cross-sectional study (n = 861; 71.0 ± 4.8 years) explored the association between dietary patterns, nutrients, and sarcopenia in community-dwelling older Chinese people across three regions. [bib_ref] Dietary pattern and dietary energy from fat associated with sarcopenia in community-dwelling..., Li [/bib_ref] Dietary intake was assessed via a questionnaire and anthropometric body measurements were taken; sarcopenia was diagnosed according to the AWGS 2014 criteria. A 'mushrooms-fruits-milk' pattern (high protein intake and low percentage of energy from fat) was found to be protective for sarcopenia, suggesting that in addition to protein, lowering the percentage energy from fat may also be considered for sarcopenia prevention and management. The second study was a prospective cohort study (mean age 72.2 and Consensus statements Malnutrition and screening 1 Initial screening for malnutrition risk should be conducted annually in older adults living in the community. Older adults who present with low body mass index (BMI), unintentional weight loss, and low muscle mass or exhibit poor muscle strength at any time should be assessed for malnutrition. 2 Community screening should be carried out by healthcare professionals or trained personnel and use standard tools. Older adults identified as being malnourished or at risk of malnutrition should be referred to a relevant healthcare professional for further assessment. Those at risk of malnutrition should be re-screened every 3 months. 3 Older adults in certain settings such as those living in rural areas, in social isolation, and with low socio-economic status may be at higher risk for malnutrition and should be screened more often than once a year. Diet and dietary patterns 4. Opportunities for communal and social eating among older adults, using a variety of food types, may improve healthy eating, overall well-being, and muscle health, with observance of public health measures for COVID-19. 5. When possible, nutritional counselling around good dietary practice for those at risk of malnutrition or sarcopenia should be provided by healthcare professionals prior to dietary enrichment or supplementation. Nutritional supplementation 6. To maintain sufficient protein intake, we recommend a daily protein intake of ≥1.0 g/kg BW for healthy older adults and ≥1.2 g/kg BW for those with sarcopenia and/or frailty. This target protein intake should be achieved primarily by diet, and where that is not possible, then protein supplementation can be considered. 7. For older adults who are candidates for supplementation, high-quality protein, amino acids such as leucine and L-carnitine, or oral nutritional supplement (ONS) containing beta-hydroxy-beta-methylbutyrate (HMB) may be considered and should be taken according to the specific prescribing information. 8. Determination of serum 25-OH vitamin D levels can be considered in patients at risk of malnutrition or sarcopenia. Oral vitamin D supplementation (800-1000 IU/day) may be beneficial for older adults with vitamin D insufficiency. Higher doses may be required for those who are deficient in 25-OH vitamin D. Lifestyle interventions 9. Nutritional supplementation in older adults, combined with an exercise regimen, is recommended for additional benefit in the management and prevention of sarcopenia. 10. When feasible, the exercise component of the combined intervention should be varied to include resistance training, moderate-intensity aerobic exercise, and balance training. Each exercise session should be structured (e.g. warm-up-resistance/balance-aerobic-cool-down) and have a frequency of two to three times per week. 11. Where possible, the combined intervention should be tailored, preferably guided by a trained personnel and in a group exercise setting, either delivered in-person or remotely via suitable videoconferencing platforms. Outcomes and assessments 12. Screening or assessment using appropriate measurements such as weight, body mass index (BMI), calf circumference (CC), hand grip strength (HGS), or the 5-time chair stand test (CST) can be performed at follow-up appointments to monitor outcomes and response to initiated interventions, referring to a physician when required. 13. Measurement of health-related quality of life (QoL) and impairments in instrumental activities of daily living (IADL) are additional measures that may be used to assess outcomes in response to initiated interventions. Impacts of COVID-19 14. Ongoing public health measures for COVID-19 (lockdown/circuit-breakers/social distancing) are an increased risk factor for malnutrition and sarcopenia; therefore, more attention should be paid to improved nutrition and exercise during the COVID-19 pandemic.
Please note that all statements refer to healthy community-dwelling older adults (age ≥ 60 years).
## Awgs consensus for nutrition in muscle health
76.2 years for non-sarcopenic and sarcopenic at baseline), which examined adherence to dietary patterns relating to the Diet Quality Index-International (DQI-I) and the Mediterranean Diet Score. [bib_ref] A prospective cohort study to examine the association between dietary patterns and..., Chan [/bib_ref] A cross-sectional analysis (n = 3957) was used to assess the associations between dietary patterns and prevalent sarcopenia, and a longitudinal analysis (n = 2948) for the 4 year incidence of sarcopenia with adjustment for sociodemographic and lifestyle factors. Higher DQI-I, a higher 'vegetables-fruits' and a higher 'snacks-drinks-milk product' dietary pattern score were all associated with lower odds of having sarcopenia in Chinese older men. However, the same effect was not observed in women, and there was no association of dietary patterns with 4 year incidence of sarcopenia with either sex. Therefore, the currently available literature does not support a specific recommendation for a particular dietary pattern in relation to muscle health. This is also difficult given varying diets between countries, but the findings do relate to the general recommendation for balanced meals comprising 1/4 protein, 1/4 wholegrains, and 1/ 2 fruit and vegetables (adapted from My Healthy plate, www. healthub.sg).
## A pro-inflammatory diet
A diet with pro-inflammatory potential has been associated with a higher risk of sarcopenia, [bib_ref] Dietary inflammatory potential and risk of sarcopenia: data from national health and..., Geng [/bib_ref] [bib_ref] Association between inflammatory potential of the diet and sarcopenia/its components in community-dwelling..., Son [/bib_ref] [bib_ref] How dietary patterns are related to inflammaging and mortality in community-dwelling older..., Chan [/bib_ref] and therefore, it follows that a diet with anti-inflammatory potential, that is, diets including specific antioxidant-rich food, would be preventative for this condition. In a cross-sectional study (n = 2451), the consumption of dietary chilli and sweet pepper (antioxidant rich) were evaluated using a self-administered food frequency questionnaire. [bib_ref] Consumption of chilies and sweet peppers is associated with lower risk of..., Wang [/bib_ref] Higher consumption of chilli and sweet pepper was found to significantly correlate with a lower risk of sarcopenia, suggesting that capsaicins and capsiates (the active anti-inflammatory compounds in these foods) may be beneficial to prevent onset of sarcopenia. Additionally, some benefit of a diet rich in betaine was observed in a 4 year community-based study (n = 1996; aged 50-75 years), which found that higher levels of serum betaine correlated with greater levels of lean percentage body mass (LM%) and a decreased risk of having lower LM%. [bib_ref] Serum betaine is inversely associated with low lean mass mainly in men..., Huang [/bib_ref] A diet rich in red meat or processed meat has been linked with having a high pro-inflammatory index; therefore, the source of protein for muscle build-up needs to be considered carefully. [bib_ref] Dietary pattern analysis and biomarkers of low-grade inflammation: a systematic literature review, Barbaresko [/bib_ref] In a study by Peng et al., a 25% daily calorie intake of protein resulted in an improved 6 min walking distance, but at the expense of higher C-reactive protein. [bib_ref] Protein-enriched diet improved muscle endurance and marginally reduced intramuscular adiposity: results from..., Peng [/bib_ref] Animal vs. plant proteins were compared in terms of supporting build-up of lean muscle mass in a systematic review and meta-analysis. [bib_ref] Animal protein versus plant protein in supporting lean mass and muscle strength:..., Lim [/bib_ref] From the 18 RCTs analysed, it was concluded that the protein source did not significantly affect changes in absolute lean mass or muscle strength, but animal protein did favor the per cent lean mass, and was especially prominent in younger adults (<50 years). There was no link between protein source and benefits from resistance exercise training. Therefore, the protein source, dose, and frequency are potentially important, in particular so it does not inadvertently drive inflammation. This is further discussed in the 'Protein Supplementation' section but also adds strength to the need for nutritional counselling ahead of prescribed dietary enrichment and supplementation.
## Nutritional supplementation
## Proteins, amino acids, and beta-hydroxy-betamethylbutyrate
Protein energy malnutrition increases the risk of sarcopenia among older adults. [bib_ref] Protein intake and muscle health in old age: from biological plausibility to..., Landi [/bib_ref] Besides insufficient protein intake from anorexia of aging, older adults may have an aging-related decreased anabolic response to dietary proteins (i.e. anabolic resistance), reducing their ability to maintain muscle mass. [bib_ref] Protein intake and muscle health in old age: from biological plausibility to..., Landi [/bib_ref] [bib_ref] Association between anorexia of ageing and sarcopenia among Japanese older adults, Tsutsumimoto [/bib_ref] Accordingly, because older people may need more dietary protein than recommended levels for younger adults (0.8 g/kg BW/day), the following guidance on protein consumption has been advocated in previous literature: 1.0-1.2 g/kg BW/day for healthy older adults, 1.2-1.5 g/kg BW/day for those with acute or chronic disease, and 2.0 g/kg BW/ day for those with frailty, severe illness, or malnutrition. [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref] One study comparing the effects of regular dietary protein (15% of total calorie per meal) vs. high dietary protein (25% of total calorie per meal) in community-dwelling older adults found that the estimated glomerular filtration rate was significantly decreased in the high protein group (from 103.5 ± 12.6 pre-test to 99.2 ± 15.5 ml/min/1.73m 2 post-test, P = 0.03). The high protein group also demonstrated an increase in high-sensitivity C-reactive protein. [bib_ref] Protein-enriched diet improved muscle endurance and marginally reduced intramuscular adiposity: results from..., Peng [/bib_ref] No differences were observed between the high protein group and the regular protein group in terms of muscle mass or strength, although 6 min walking distance was significantly increased in the high protein group. In another trial of older adults with sarcopenia, a target protein consumption of 1.2-1.5 g/kg BW/day led to increased appendicular muscle mass index, regardless of treatment group (supplementary group received vitamin D-enriched and leucine-enriched whey protein supplement in addition to meeting dietary target). [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] A cross-sectional analysis of 3213 community-dwelling middle-aged and older adults (mean age: 60.7 years) in China found that participants with a total protein intake of >0.96 g/kg BW/day were less likely to have low muscle mass by skeletal muscle index compared with those ingesting ≤0.96 g/kg WB/day; the lowest risk of having low muscle mass was in subjects with ≥1.68 g/kg BW/day of protein intake (odds ratio 0.3, 95% confidence interval 0.2-0.4). [bib_ref] Amount rather than animal vs plant protein intake is associated with skeletal..., Li [/bib_ref] Collectively, this points to an unquantified role of increasing dietary protein intake in place, or in addition, to supplementation, noting potential adverse events (AEs) at higher levels of intake. [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] [bib_ref] Protein-enriched diet improved muscle endurance and marginally reduced intramuscular adiposity: results from..., Peng [/bib_ref] Older adults presenting with renal impairment need to be advised separately on safe protein levels, but as this consensus specifically targets healthy older adults, we have not addressed this here.
Essential amino acids, and principally leucine, represent the major stimuli for protein synthesis [bib_ref] Signaling pathways involved in translational control of protein synthesis in skeletal muscle..., Anthony [/bib_ref] ; hence, the amino acid profile of dietary protein sources may be an important consideration during supplementation. The muscle-building potential of protein supplementation is also affected by digestion and absorption kinetics, with some debate pertaining to possible differences between 'fast' proteins (e.g. whey) over 'slow' proteins (e.g. casein) in older populations, although overall the main aim is to meet the necessary protein requirements. [bib_ref] The rate of protein digestion affects protein gain differently during aging in..., Dangin [/bib_ref] [bib_ref] Whey protein stimulates postprandial muscle protein accretion more effectively than do casein..., Pennings [/bib_ref] To evaluate the effects of protein supplementation on muscle health, multiple small-scale RCTs 39,41,59-61 and one large-scale RCT [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] have been conducted in five Asian countries (China, Korea, Malaysia, Singapore, and Taiwan) with a total of 1320 subjects. See [fig_ref] Table 2: Summary of studies addressing supplementation in muscle health [/fig_ref] for a summary of the relevant studies.
In general, the trials involved older adults aged ≥50 years (range of mean age: 58.4-74.8 years). Nutritional status ranged from non-malnourished 59 to medium-high nutritional risk as defined by the MUST, [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] although most participants had low malnutrition risk at baseline by MNA. Three studies [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] [bib_ref] A high whey protein, vitamin D and E supplement preserves muscle mass,..., Bo [/bib_ref] [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] were performed solely among sarcopenic subjects, whereas three studies [bib_ref] Effects of whey protein nutritional supplement on muscle function among community-dwelling frail..., Kang [/bib_ref] [bib_ref] Efficacy of L-carnitine supplementation on frailty status and its biomarkers, nutritional status,..., Badrasawi [/bib_ref] [bib_ref] Oral nutritional supplement with β-hydroxy-β-methylbutyrate (HMB) improves nutrition, physical performance and ameliorates..., Peng [/bib_ref] were exclusive to pre-frail/frail subjects. Only one study included communitydwelling independently ambulating older adults with a high functional ability and little or no co-morbidities. [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] Protein supplementation in three trials was primarily by whey protein 1,39,60 though in one study leucine was an additional ingredient with a duration of 10-26 weeks [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] and in another the protein supplement contained whey and leucine but was predominantly casein (50%). [bib_ref] Leucine-enriched protein supplementation increases lean body mass in healthy Korean adults aged..., Kang [/bib_ref] While the methods of preparation differed, the supplements provided approximately 40-60 g of protein per day, divided into 2-3 doses before meals or 30 min after exercise. Additionally, we found three trials in which the intervention consisted mainly of amino acids: one trial on the lysine-derivative L-carnitine (500 mg three times a day) [bib_ref] Efficacy of L-carnitine supplementation on frailty status and its biomarkers, nutritional status,..., Badrasawi [/bib_ref] and two trials 15,63 using an oral nutritional supplement (ONS) containing the leucine derivative beta-hydroxy-beta-methylbutyrate (HMB) 1.5-3 g/day divided into two doses.
There was insufficient evidence of benefits from casein supplementation in terms of muscle health, 61 but in the rest of the RCTs, protein supplementation was associated with significantly greater improvements in muscle strength, muscle mass, and physical performance, compared with controls or no intervention. Furthermore, studies with primarily whey-based supplementation improved QoL, [bib_ref] A high whey protein, vitamin D and E supplement preserves muscle mass,..., Bo [/bib_ref] and supplementation with L-carnitine improved frailty scores. [bib_ref] Efficacy of L-carnitine supplementation on frailty status and its biomarkers, nutritional status,..., Badrasawi [/bib_ref] In a double-blind, randomized, placebo-controlled trial consisting of 811 older adults at risk of malnutrition in Singapore (the SHIELD study), participants were asked to consume either an ONS containing HMB or placebo twice a day for 180 days, along with dietary counselling as the standard of care. There was a significant improvement in malnutrition risk and nutritional outcomes such as weight, BMI, mid-upper arm circumference, and vitamin D levels in the intervention group compared with the placebo group. The intervention group was also found to have stringer leg strength and greater HGS in females. [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] Another study among community-dwelling older Chinese adults with sarcopenia showed that an exercise programme with or without an HMB containing ONS significantly improved leg extension and results from the five-stand chair test, and those that received ONS had additional benefits of increased total lean muscle mass and lower limb muscle mass. [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] The secondary analysis of this study showed that T-cell-specific inflammatory gene expression was changed significantly after 12 weeks of intervention in the group with ONS, which was associated with improved leg extensions in older adults with sarcopenia. [bib_ref] Peripheral blood T cell gene expression responses to exercise and HMB in..., Ma [/bib_ref] No serious AEs were reported in any of the RCTs, with only constipation cited as an AE in two studies. [bib_ref] A high whey protein, vitamin D and E supplement preserves muscle mass,..., Bo [/bib_ref] [bib_ref] Leucine-enriched protein supplementation increases lean body mass in healthy Korean adults aged..., Kang [/bib_ref] Benefits of protein supplementation in terms of muscle strength occurred in both males and females [bib_ref] Effects of whey protein nutritional supplement on muscle function among community-dwelling frail..., Kang [/bib_ref] and were observed among older adults with sarcopenia or low muscle mass, 15,41,60 pre-frail/frail patients, 60-62 patients at mediumhigh nutritional risk, [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] as well as patients with no malnutrition. [bib_ref] Efficacy of L-carnitine supplementation on frailty status and its biomarkers, nutritional status,..., Badrasawi [/bib_ref] Notably, the presence of co-interventions may have confounded some of the results.
In three studies, 39,61,62 all subjects were instructed to perform regular exercise, while in four RCTs, all participants were given nutritional counselling, such as advice on meal compositions or prescription of specific levels of protein intake. [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] [bib_ref] Effects of whey protein nutritional supplement on muscle function among community-dwelling frail..., Kang [/bib_ref] [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] [bib_ref] Oral nutritional supplement with β-hydroxy-β-methylbutyrate (HMB) improves nutrition, physical performance and ameliorates..., Peng [/bib_ref] The issue of confounding was addressed in a double-blind RCT, which provided dietary counselling in both arms of the clinical study as standard care and included the use of a placebo in the control arm and ONS in the intervention arm. [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] In conclusion, sufficient dietary protein intake is crucial for improving muscle health, but high-quality protein supplements and ONS containing HMB are beneficial where this is not possible. Please see , Consensus Statements 6 and 7.
In a distinct study, some benefit to muscle health was found with a unique chicken-based ONS. [bib_ref] The benefits of a novel chicken-based oral nutritional supplement for older adults:..., Assantachai [/bib_ref] This double-blind RCT (n = 38, mean age: 81.5 years) compared older adults receiving either the chicken-based ONS or a similarly flavored placebo over a period of 6 months and divided participants into higher or lower level of physical activity. The higher-level group had a significantly improved usual gait speed compared with the lower-level physical activity group (indicating that physical activity improved usual gait speed but was not related to the ONS). Significant changes were observed in two bone markers at baseline and 6 months in the intervention group, indicating improved bone resorption. Therefore, beneficial effects on age-related bone resorption were observed for this ONS, but it did not directly benefit sarcopenia-related measures. This is therefore insufficient evidence to inform a consensus statement, but it will be interesting to see further data from this supplement as it emerges.
## Vitamins
Vitamin D triggers genomic and non-genomic pathways in muscle cells that preserve muscle function through various mechanisms, including maintenance of calcium homeostasis and proliferation of muscle fibres. [bib_ref] Fall prevention and vitamin D in the elderly: an overview of the..., Annweiler [/bib_ref] [bib_ref] Mechanisms of vitamin D on skeletal muscle function: oxidative stress, energy metabolism..., Dzik [/bib_ref] Historically, the clinical link between vitamin D and muscle health comes from cases of severe vitamin D deficiency that caused myopathies and severe muscle weakness or pain. [bib_ref] Fall prevention and vitamin D in the elderly: an overview of the..., Annweiler [/bib_ref] [bib_ref] Improving the vitamin D status of vitamin D deficient adults is associated..., Sinha [/bib_ref] Increasing evidence indicates muscle-bone crosstalk and an integrative role for vitamin D in this relationship. [bib_ref] Vitamin D, muscle and bone: integrating effects in development, aging and injury, Girgis [/bib_ref] Observational studies indicate that lower levels of vitamin D result in reduced muscle mass and strength. [bib_ref] Low vitamin D and high parathyroid hormone levels as determinants of loss..., Visser [/bib_ref] A recent meta-analysis of predominantly non-Asian trials found insufficient evidence for benefit from vitamin D supplementation in terms of muscle mass or function, [bib_ref] Vitamin D and muscle health: a systematic review and meta-analysis of randomized..., Bislev [/bib_ref] although it has been highlighted that there were a number of limitations of the included studies, such as vitamin-D replete populations, small sample sizes, and inconsistent intervention methods in terms of dose and metabolites. [bib_ref] Vitamin D deficiency 2.0: an update on the current status worldwide, Amrein [/bib_ref] In Asia, a limited number of recent clinical trials directly assessed the effects of vitamin D supplementation on muscle health parameters. In one RCT involving Japanese older adults (N = 130, mean age: 70.5 years) who received either oral vitamin D3 alone (1000 IU/day), exercise alone, or a combination of the two interventions for 24 weeks, the mean baseline serum vitamin D levels varied by treatment group (range: 27.2-29.0 ng/mL). [bib_ref] The impact of exercise and vitamin D supplementation on physical function in..., Aoki [/bib_ref] Compared with exercise alone and exercise plus vitamin D, there was insufficient evidence of benefit from vitamin D alone in terms of muscle mass, muscle strength, and physical performance, despite a significant increase in serum vitamin D levels from baseline. In several other RCTs, vitamin D was supplemented orally at a dose of 260-1600 IU/day in combination with various types of supplements (e.g. whey, leucine, and HMB), with or without exercise, for a duration of 12 weeks. [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] [bib_ref] Leucine-enriched protein supplementation increases lean body mass in healthy Korean adults aged..., Kang [/bib_ref] [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Exercise and nutritional supplementation on communitydwelling elderly Japanese women with sarcopenic obesity:..., Kim [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] Although most of these trials demonstrated benefits from the intervention overall, they were not specifically designed to examine benefit from vitamin D alone, and so it is difficult to attribute independent benefit of vitamin D based on these data.
Multiple cross-sectional studies in Asia (total N = 11 769) may provide indirect evidence of a role for vitamin D supplementation in muscle health. [bib_ref] White blood cell counts, insulin resistance, vitamin D levels and sarcopenia in..., Kim [/bib_ref] [bib_ref] Serum 25-hydroxyvitamin D and elderly skeletal muscle mass and function in urban..., Meng [/bib_ref] [bib_ref] The most effective factors to offset sarcopenia and obesity in the older..., Oh [/bib_ref] [bib_ref] Vitamin D is related to handgrip strength in adult men aged 50..., Wang [/bib_ref] Two studies conducted in China [bib_ref] Serum 25-hydroxyvitamin D and elderly skeletal muscle mass and function in urban..., Meng [/bib_ref] [bib_ref] Vitamin D is related to handgrip strength in adult men aged 50..., Wang [/bib_ref] and one in Japan 80 among middleaged and older adults (mean age: 75.4 years) found a significant, positive correlation between serum vitamin D levels and HGS. The odds of having low HGS increased four-fold among community-dwelling older adults with vitamin D insufficiency (serum level < 20 ng/mL) compared with vitamin D-sufficient subjects. [bib_ref] Low serum 25-hydroxyvitamin D is associated with low grip strength in an..., Kitsu [/bib_ref] Two studies have shown that serum levels of vitamin D were also significantly associated with sarcopenia among Korean older adults (range of mean age: 67.4-74.5 years). [bib_ref] White blood cell counts, insulin resistance, vitamin D levels and sarcopenia in..., Kim [/bib_ref] [bib_ref] The most effective factors to offset sarcopenia and obesity in the older..., Oh [/bib_ref] Specifically, subjects with serum vitamin D levels ≤ 18.67 ng/mL were twice as likely to have sarcopenia than those with levels ≥ 23.73 ng/mL, 78 although stratified analyses by sex have shown conflicting results across different studies. [bib_ref] White blood cell counts, insulin resistance, vitamin D levels and sarcopenia in..., Kim [/bib_ref] [bib_ref] Serum 25-hydroxyvitamin D and elderly skeletal muscle mass and function in urban..., Meng [/bib_ref] [bib_ref] Vitamin D is related to handgrip strength in adult men aged 50..., Wang [/bib_ref] [bib_ref] Low serum 25-hydroxyvitamin D is associated with low grip strength in an..., Kitsu [/bib_ref] Despite their observational nature, these studies lend evidence to the correlation between vitamin D status and muscle health and are also indicative of the value of screening vitamin D levels in community-dwelling older adults. In these studies, the commonly applied criteria to classify vitamin D status were <20 ng/mL (i.e. μg/L) for deficiency, 20-29.9 ng/mL for insufficiency, and ≥30 ng/mL for sufficiency. [bib_ref] Serum 25-hydroxyvitamin D and elderly skeletal muscle mass and function in urban..., Meng [/bib_ref] [bib_ref] Low serum 25-hydroxyvitamin D is associated with low grip strength in an..., Kitsu [/bib_ref] The cut-offs reflect recommendations of the Endocrine Society in 2011, in which the evidence mostly originates from non-Asian populations. [bib_ref] Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical..., Holick [/bib_ref] Interestingly, data from Asia suggest that different ethnic cultures, linked, for example, with clothing choices, may influence vitamin D levels, [bib_ref] Prevalence and determinants of suboptimal vitamin D levels in a multiethnic Asian..., Man [/bib_ref] indicating a possible need for population-specific reference values. However, in Japan, after analysis of country-level data, an expert panel proposed definitions of vitamin D deficiency/insufficiency, which closely matched the aforementioned values.To assess vitamin D status, the majority of studies in Asia measured serum levels of 25-hydroxyvitamin D or 25-OH vitamin D via radioimmunoassay, [bib_ref] White blood cell counts, insulin resistance, vitamin D levels and sarcopenia in..., Kim [/bib_ref] [bib_ref] Serum 25-hydroxyvitamin D and elderly skeletal muscle mass and function in urban..., Meng [/bib_ref] [bib_ref] The most effective factors to offset sarcopenia and obesity in the older..., Oh [/bib_ref] [bib_ref] Low serum 25-hydroxyvitamin D is associated with low grip strength in an..., Kitsu [/bib_ref] which may not be routinely available in community-based testing facilities. A recent systematic review did not find any studies directly evaluating the benefits of screening for vitamin D deficiency.Hence, routine testing of vitamin D levels in community-dwelling older adults may be undertaken upon careful consideration of feasibility and applicability, and in resource-limited settings, a more targeted approach may be reasonable, for example, screening for suspected osteoporosis.
The role of vitamin E in sarcopenia has drawn attentions due to its antioxidant and anti-inflammatory properties. [bib_ref] Potential roles of vitamin E in age-related changes in skeletal muscle health, Chung [/bib_ref] However, we did not find any RCTs in Asia that directly evaluated vitamin E supplementation in terms of muscle health outcomes. Two cross-sectional studies examined serum levels and dietary vitamin E but provided inconclusive evidence for vitamin E supplementation. [bib_ref] Low serum vitamin E level associated with low hand grip strength in..., Kim [/bib_ref] [bib_ref] Dietary intake of vitamin E and fats associated with sarcopenia in community-dwelling..., Otsuka [/bib_ref] Therefore, evidence to support a role of vitamin supplementation in Asian studies was only found for vitamin D. Please see , Consensus Statement 8.
## Lipids
Although proteins are the building blocks for muscle, lipids such as omega-3 or n-3 polyunsaturated fatty acids are suggested to affect muscle health indirectly by preventing low-grade inflammation. [bib_ref] The role of omega-3 in the prevention and treatment of sarcopenia, Dupont [/bib_ref] Significantly lower levels of serum n-3 fatty acids were linked with sarcopenia and higher dietary intake of fats (66.7-143.8 g/day vs. 6.8-41.4 g/day) correlated with lower odds of sarcopenia. [bib_ref] Dietary intake of vitamin E and fats associated with sarcopenia in community-dwelling..., Otsuka [/bib_ref] Overall, we found scarce evidence to support a statement around dietary/supplementary lipid intake in muscle health, but the findings support a balanced and healthy diet to maintain muscle health and so are relevant in the broader context.
## Lifestyle interventions in addition to nutrition
Aside from aging, other pathologic factors that affect muscle quantity and quality include inactivity (e.g. sedentary behaviour and physical inactivity) and malnutrition (e.g. undernutrition). In a retrospective cohort study among 552 older adults (mean age 74.6 years) in Japan, age, obesity, and malnutrition were independent risk factors for sarcopenia. [bib_ref] Prevalence and risk factors of sarcopenia in community-dwelling older adults visiting regional..., Kurose [/bib_ref] This study also revealed that the number of daily conversations, which was an index of physical, cognitive, and social function, independently predicted sarcopenia. [bib_ref] Prevalence and risk factors of sarcopenia in community-dwelling older adults visiting regional..., Kurose [/bib_ref] Collectively, these findings suggest a role in muscle health for multi-domain interventions that address physical, cognitive, and social aspects on top of nutritional factors.
Several RCTs (total number of subjects = 1200), conducted in three countries in Asia (Hong Kong, Japan, and Taiwan), have evaluated the efficacy of combined exercise and nutritional supplementation in terms of improvement in muscle health. [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] The impact of exercise and vitamin D supplementation on physical function in..., Aoki [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] [bib_ref] The effects of exercise and milk-fat globule membrane (MFGM) on walking parameters..., Kim [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] Although some of the aforementioned studies described in the 'Protein' section also included exercise, we have focused on studies here that had a separate exercise group, so that the specific benefit of exercise could be assessed. Please see [fig_ref] Table 3: Summary of studies addressing exercise in addition to nutritional supplementation in muscle... [/fig_ref] for a summary of studies addressing exercise in addition to supplementation, in muscle health.
One RCT was conducted exclusively among sarcopenic patients, 62 one among non-sarcopenic patients, [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] one among pre-frail/frail patients, [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] and one among robust patients. [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] Most studies involved older participants (range of mean age: 60.9-84.2 years) and a predominantly female study population, although two trials recruited solely female participants. [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] The effects of exercise and milk-fat globule membrane (MFGM) on walking parameters..., Kim [/bib_ref] With regard to baseline BMI, the majority of the trials enrolled non-obese subjects (range of mean BMI: 18.8-25.5 kg/m 2 ).
Despite the variety in exercise regimens applied, the combined intervention in the RCTs generally included resistance training, using body weight in-chair exercises or resistance bands. Apart from two studies, [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] resistance exercises were mixed with other types, including aerobic and balance training. A few studies employed specific approaches such as progressive (i.e. a structured, gradual increase in the weights used), [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] The effects of exercise and milk-fat globule membrane (MFGM) on walking parameters..., Kim [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] predominantly home-based 40,73,90 group (i.e. multiple people exercising together), 62,92 and personalized programmes. [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] The sessions ranged from 5 to 60 min, with frequencies ranging from 2 to 7 times per week, for a duration of 3-6 months, and were initially supervised, and in virtually all RCTs, the exercise sessions were structured.
In terms of the nutritional component, protein or derivatives were the major component of the additional supplement [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] although the component, that is, whey, branched-chain amino acids, HMB, or a combination, and the amount differed per trial. In one of these studies, [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] the experimental arm received a co-intervention in the form of nutritional management, whereby protein intake was maintained at 1.2 g/kg BW/day; individuals with a baseline protein consumption of <1.2 or >1.3 g/kg BW/day were excluded. The mean baseline protein intake among participants in other trials was 1.5-1.8 g/kg BW/day, depending on the study arm, [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] 60.8-64.9 g of protein/day, which increased by 7.2-8.6 g/day in the intervention arms after 6 months of supplementation with skimmed milk and mixed nuts. [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] One trial 91 provided the intervention groups with MFGM as the nutritional supplement while another trial gave vitamin D. [bib_ref] The impact of exercise and vitamin D supplementation on physical function in..., Aoki [/bib_ref] All studies reported at least one muscle health parameter as an outcome of interest (i.e. muscle mass, muscle strength, or physical performance).
In the majority of RCTs, combined exercise and nutrition resulted in greater improvements in muscle mass, muscle strength, and physical performance, compared with no intervention. [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] [bib_ref] The effects of exercise and milk-fat globule membrane (MFGM) on walking parameters..., Kim [/bib_ref] Benefits from combined exercise and nutrition were observed as early as 1 month from the start of intervention [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] and lasted up to 3 months after the intervention. [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] The combined intervention also had benefits in terms of walking parameters, 91 flexibility, 90 levels of physical activity, and activities of daily living (ADL). [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] Two RCTs did not show sufficient evidence of benefits from combined exercise and nutrition in terms of at least one muscle health parameter. [bib_ref] The impact of exercise and vitamin D supplementation on physical function in..., Aoki [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] However, Aoki et al. was the only study that did not supplement with a macronutrient, whereas Woo et al. enrolled a younger study population (mean age: 61 years) compared with the rest of the studies, which may have contributed to this result. Data from two studies, [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] as well as two subgroup analyses, [bib_ref] Peripheral blood T cell gene expression responses to exercise and HMB in..., Ma [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] revealed that, regardless of sarcopenia status, combined exercise and nutrition had benefits on muscle health.
Both RCTs that applied only resistance training demonstrated benefits from combined exercise and nutrition, [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] while the benefit was not consistent among the studies that added aerobic, balance, flexibility, or gait training. In terms of approach, evidence of benefit was consistent among studies that employed an individualized programme. [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] There were no studies that specifically addressed who should guide such exercise programmes, but in order to widen access, we feel this can be carried out by a range of 'trained personnel', such as exercise instructors, physiotherapists, and community care providers. Therefore, there is a reasonable level of evidence, among Asian studies, to support additional benefits from including an exercise component along with other dietary/nutritional interventions, in selected subjects. This is also supported by the WHO guidelines on physical activity and sedentary behavior, and the ICFSR guidelines. [bib_ref] International Clinical Practice Guidelines for Sarcopenia (ICFSR): screening, diagnosis and management, Dent [/bib_ref] Please see , Consensus Statements 9, 10, and 11. It is important to note that any additional exercise as part of a multi-domain intervention should be in addition to maintaining basic levels of daily physical activity such as walking to the shops, to maintain general overall health.
## Outcomes and assessments
Randomized controlled trials in Asia have evaluated the effects of nutritional supplementation through a variety of outcome measures such as body composition, muscle mass, muscle function, instrumental activities of daily living (IADLs), and QoL. Among community-dwelling older adults, practicality is an important consideration in choosing tests to monitor the effects of a given intervention. For instance, BMI and weight are simple measurements in clinical practice that may translate to improvements in physical performance and ADLs. In contrast, some validated tests that directly assess muscle health may require complex equipments. For example, appendicular skeletal muscle mass was measured via bioelectrical impedance analysis or dual energy X-ray absorptiometry in the majority of RCTs but may be applicable only in healthcare facilities or clinical research settings. [bib_ref] Impact of specialized oral nutritional supplement on clinical, nutritional, and functional outcomes:..., Chew [/bib_ref] [bib_ref] Effect of milk fat globule membrane supplementation on motor unit adaptation following..., Watanabe [/bib_ref] [bib_ref] Effect of whey protein supplementation after resistance exercise on the muscle mass..., Mori [/bib_ref] [bib_ref] Effects of adequate dietary protein with whey protein, leucine, and vitamin D..., Lin [/bib_ref] [bib_ref] A high whey protein, vitamin D and E supplement preserves muscle mass,..., Bo [/bib_ref] [bib_ref] The impact of exercise and vitamin D supplementation on physical function in..., Aoki [/bib_ref] [bib_ref] Exercise and nutritional supplementation on communitydwelling elderly Japanese women with sarcopenic obesity:..., Kim [/bib_ref] [bib_ref] Synergistic effect of bodyweight resistance exercise and protein supplementation on skeletal muscle..., Yamada [/bib_ref] One relatively easy test to perform is rhe determination of CC, which is the maximum value measured with a non-elastic tape on both calves or a dominant or non-dominant calf as specified and displays moderate-to-high sensitivity (73-92%) and specificity (50-88%) in detecting sarcopenia or low skeletal muscle mass. [bib_ref] Low serum vitamin E level associated with low hand grip strength in..., Kim [/bib_ref] [bib_ref] Calf circumference as a screening instrument for appendicular muscle mass measurement, Hwang [/bib_ref] [bib_ref] Calf circumference as a surrogate marker of muscle mass for diagnosing sarcopenia..., Kawakami [/bib_ref] [bib_ref] Large calf circumference indicates nonsarcopenia despite body mass, Kusaka [/bib_ref] In 2019, the AWGS recommended CC as a screening tool for sarcopenia in the community, using a cut-off of <34 cm for males and <33 cm for females. [bib_ref] Asian Working Group for Sarcopenia: 2019 consensus update on sarcopenia diagnosis and..., Chen [/bib_ref] A cross-sectional study among 139 Japanese older adults (mean age: 76.7 ± 6.6 years) has shown that these cut-offs achieved a sensitivity of 83.3% and specificity of 62.8% in predicting sarcopenia. [bib_ref] Changes in the screening efficacy of lower calf circumference, SARC-F score, and..., Ito [/bib_ref] While the AWGS has similarly advocated the use of HGS (with cut-offs of 28 and 18 kg for males and females, respectively), to identify possible sarcopenia in the community, 4 its measurement entails access to a dynamometer, which may not be routinely available. In addition, for those with a hand abnormality or limitation, knee extension or quadriceps strength can be used as an alternative strength measurement with high levels of sensitivity and specificity for sarcopenia diagnosis. [bib_ref] Asian Working Group for Sarcopenia: 2019 consensus update on sarcopenia diagnosis and..., Chen [/bib_ref] [bib_ref] Diagnostic accuracy of quadriceps strength-based criteria compared to handgrip-based criteria for diagnosing..., Assantachai [/bib_ref] The AWGS has also endorsed several physical performance tests to diagnose sarcopenia, including the 6 m walk for establishing gait speed and the Short Physical Performance Battery for judging impaired mobility. [bib_ref] Asian Working Group for Sarcopenia: 2019 consensus update on sarcopenia diagnosis and..., Chen [/bib_ref] However, there may be limited space in community healthcare clinics to allow the distance involved in these two tests. Among the physical performance tests, the 5-time chair stand test (CST) has been recognized by AWGS as a surrogate for gait speed in the community, with a cut-off of ≥12 s for low physical performance. [bib_ref] Asian Working Group for Sarcopenia: 2019 consensus update on sarcopenia diagnosis and..., Chen [/bib_ref] Apart from a chair, the CST does not necessitate any advanced apparatus or wide space to accomplish and has been shown to have a sensitivity of 75% and specificity of 94% for predicting low gait speed. [bib_ref] Usefulness of chair stand time as a surrogate of gait speed in..., Nishimura [/bib_ref] In a more recent cross-sectional study involving 678 Japanese older adults (mean age: 74.7 ± 7.2 years), the 30 s CST had a sensitivity of 75.0-76.4% and specificity of 71.7-76.8% for diagnosing sarcopenia, using 17 and 15 stands as cut-offs for males and females, respectively. [bib_ref] The 30-s chair stand test can be a useful tool for screening..., Sawada [/bib_ref] Based on these findings, a cross-sectional study concluded that sit-to-stand tests better represent the physical performance compared with muscle strength, the 30 s CST may be a suitable alternative to the 5-time CST. [bib_ref] Performance on sit-to-stand tests in relation to measures of functional fitness and..., Yee [/bib_ref] To measure impairments in IADLs, most RCTs in Asia have utilized a five-item questionnaire assessing the ability of community-dwelling older adults to (1) walk two to three blocks outdoors on level ground, (2) climb 10 steps without resting, (3) prepare own meals, (4) do heavy housework like scrubbing floors or washing windows, and (5) shop for groceries or clothes. [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] In studies that performed a health-related QoL assessment, it was quantified most frequently by the health survey short forms SF-12 and SF-36, both of which were divided into physical and mental component scores. [bib_ref] Protein-enriched diet improved muscle endurance and marginally reduced intramuscular adiposity: results from..., Peng [/bib_ref] [bib_ref] A high whey protein, vitamin D and E supplement preserves muscle mass,..., Bo [/bib_ref] [bib_ref] Effects of exercise and nutrition supplementation in community-dwelling older Chinese people with..., Zhu [/bib_ref] [bib_ref] Individualized homebased exercise and nutrition interventions improve frailty in older adults: a..., Hsieh [/bib_ref] [bib_ref] Randomized controlled trial of exercise and nutrition supplementation on physical and cognitive..., Woo [/bib_ref] Recently, a Chinese version of a sarcopenia-specific QoL questionnaire (SarQOL®) has also been validated, exhibiting high correlation with SF-36. [bib_ref] Psychometric properties of the Chinese version of the sarcopenia and quality of..., Le [/bib_ref] Although there are several blood-based measurements to examine nutritional outcomes in muscle health, these may be difficult to perform in community healthcare settings and thus we have excluded them in this consensus. Please see , Consensus Statements 12 and 13 for statements relating to outcome measures.
## Impacts of covid-19
Sarcopenia has been an increasing problem during the COVID-19 pandemic, as a result of sedentary behavior imposed during circuit breakers and lockdowns globally. [bib_ref] Letter to the editor: COVID-19 quarantine in older people: the need to..., Bonadias Gadelha [/bib_ref] [bib_ref] Sarcopenia during COVID-19 lockdown restrictions: long-term health effects of short-term muscle loss, Kirwan [/bib_ref] In Asia, a retrospective cohort study among 121 adult pa-tients in Korea with COVID-19 (median age 62 years) showed that low skeletal muscle index at baseline independently predicted longer hospital stay while also increasing the risk of mortality. [bib_ref] Prognostic implication of baseline sarcopenia for length of hospital stay and survival..., Kim [/bib_ref] This finding highlights the importance of maintaining muscle health among older adults during the COVID-19 pandemic. Acknowledging the direct health effects of COVID-19 on older people, as well as the indirect consequences of corresponding public health measures, the AWGS released recommendations for Asian older adults, including adequate nutrition and exercise to enhance physical resilience in the context of the pandemic. [bib_ref] COVID-19 and older people in Asia: Asian Working Group for Sarcopenia calls..., Lim [/bib_ref] Although essential, public health strategies, such as social distancing and community quarantine, can impact not only the psychosocial well-being of older adults but also their physical activity and dietary habits. [bib_ref] COVID-19 and older people in Asia: Asian Working Group for Sarcopenia calls..., Lim [/bib_ref] In a systematic review of four studies from China, the prevalence rates of malnutrition risk among older adults with COVID-19 ranged from 41.1% (n = 58) to 100% (n = 4) while the prevalence of malnutrition was 52.7% (n = 96). [bib_ref] Nutritional risk screening tools for older adults with COVID-19: a systematic review, Silva [/bib_ref] Altogether, the emerging body of evidence from the COVID-19 pandemic emphasizes the relevance of nutritional management for muscle health among community-dwelling older adults. Please see , Consensus Statement 14.
# Conclusions
Our research identified key topics regarding muscle health, with robust data from cross-regional studies in Asia. Although there was considerable heterogeneity among studies, and a mix of cross-sectional studies and RCTs, we were able to expand on this with evidence from previous guidelines and our own clinical experience and expertise, to develop a set of 14 statements that guide practice from screening strategies and assessment methods, to impacts of dietary nutrition, supplements, exercise, and COVID-19. Of note, it is clear that muscle health and good overall nutrition are intimately linked, and at screening, there is a need to assess these factors in combination. Our aim is for these consensus statements to provide a practical framework, with the hope of broadening healthcare options for community-dwelling older adults, leading to improved overall muscle health in this target population. reviewing topics and statements at all stages of the process, and voting. In addition, all members provided views and perspectives from their clinical experience to support the statements. All authors contributed to writing the manuscript and reviewed the draft several times. The authors certify that they comply with the ethical guidelines for authorship and publishing of the Journal of Cachexia, Sarcopenia and Muscle.
# Funding
Medical writing support, in accordance with Good Publication Practice 3 ethical guidelines, was provided by Fiona Clare Chaplin and Tristan Marvin Uy of MIMS Pte Ltd and was funded by the Abbott Nutrition, and Taiwan Association for Integrated Care.
## Conflicts of interest
G.D. is a member of the Scientific Advisory Board at Abbott Australia and TSI Pharmaceuticals and holds a research grant from TSI Pharmaceuticals but has no other conflicts of interest to declare beyond this. HL.C.D. provides medical and lay lectures for Abbott but has no other conflicts of interest beyond this. M.A. received research funding from Astellas Pharma, Bayer HealthCare, Boehringer Ingelheim, Chugai Pharmaceutical, Daiichi Sankyo, Eli Lilly Japan, Eisai, Kracie Pharma, Mitsubishi-Tanabe Pharma, MSD, Novartis Japan, Ono Pharmaceutical, Sanofi, Takeda Pharmaceutical, Teijin Pharma, and Tsumura and lecture fees from Daiichi Sankyo, Mitsubishi-Tanabe Pharma, MSD, Sumitomo Dainippon Pharma, and Takeda Pharmaceutical. He has no other conflicts of interest to declare. S.T.H.C. has previously received grant co-funding, travel grant, and honoraria from Abbott Nutrition. L.-K.C., J.W., H.A., and P.A. have no conflicts of interest to declare.
[table] Table 2: Summary of studies addressing supplementation in muscle health [/table]
[table] Table 3: Summary of studies addressing exercise in addition to nutritional supplementation in muscle health [/table]
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A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation
## Supplementary materials and methods
Identification of proteins in L. japonicus roots.
# Plant material
L. japonicus wild-type Gifu B-129 (EMS 467.2; BC1; M5) seeds were germinated and grown in the greenhouse as previously described . Inoculation of a subset of wild type plants were performed 7 days after germination using the Mesorhizobium loti MAFF303099 strain. Wild type roots tissues were harvested 17 days after germination.
## Protein isolation and in-solution tryptic digestion.
For proteomics study, protein extraction from roots were performed as previously described . Briefly, the tissue was harvested into pre-chilled mortar (-20°C) and homogenized in ice-cold extraction buffer (0.1 M TrisHCl (pH 8.0); 30% sucrose; 10 mM DTT; 1/100 phosphatase inhibitor cocktail 1 and 2 from Sigma) and the protein concentration was measured using the Bradford method . Proteins were extracted using phenol/SDS and precipitated with 100 mM ammonium acetate in methanol followed by washes in 100 mM ammonium acetate in methanol and 80% acetone. Four biological replicates were performed
The protein pellet was dried and dissolved in a buffer (7 M urea, 2 M thiourea and 200 mM TEAB) followed by determining the protein concentration using Qubit® Quantitation Kit (Invitrogen), following the manufacturer's instructions (between 60 and 100 µg protein per biological replica). Disulfide bridges were reduced using 10 mM DTT for 1 hour at 25 o C. Following, free thiol alkylation was performed using 40 mM iodacetamide for 40 min at room temperature in the dark. Samples were diluted to 1 M urea and digested overnight with trypsin (Promega) (1:20, w/w) at 25 o C. After ! 2 digestion, a final concentration of 2% formic acid was added and the tryptic peptides were clean-up by home-made chromatographic Poros 50 R2 (PerSeptive Biosystems) and
Poros Oligo R3 (Applied Biosystems) reverse phase microcolumns as previously described and dried. Peptides were resuspended in 200 mM TEAB and duplicates of peptide concentration were measured for all 20 samples using Qubit® Quantitation Kit (Invitrogen) and the four samples with highest concentration for each root tissue (between 33 and 65 µg peptide per biological replica) were used for iTRAQ labeling.
## Itraq labeling
Labeling of the peptides with the 4-plex iTRAQ reagents (AB SCIEX) was performed according to the manufacturer's recommendations. For each sample, the tryptic peptides were dissolved in 30 µl of 200 mM TEAB, pH 8.5 and to each iTRAQ 4-plex reagent vial 70 µl of ethanol was added and mixed with a biological replica and incubated at room temperature for 1 hour. Then, the samples were acidified with 1% formic acid. The iTRAQ labeling and label peptides were separated using a TSK Amide-80 HILIC column in a microHPLC system as previously described . For each run, 10 µg of peptides were loaded and fractionated in either one fraction (total) or 10 sub-fractions (fractionated) and dried. For each of the four iTRAQ labeling set, four and one technical replicas for the total and fractionated samples were performed, respectively.
## Reversed phase nano-lc and mass spectrometry analysis
For the MS analysis, labeled peptide mixtures were dissolved in 0.1% formic acid and either 1 µg (total) or one sub-fraction (fractionated) was loaded onto a C18-reversed phase pulled needle capillary column (18 cm length, 100 µm inner diameter, packed in-house with ReproSil-Pur C18-AQ 3 µm resin). The samples were analyzed by an EASY-nano LC system (Proxeon Biosystems) coupled online to an ESI-LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Peptides were eluted using a gradient from 100% phase A (0.1% formic acid) to 35% phase B (0.1% formic acid, 95% acetonitrile) for 120 (total) or 100 min (fractionated), 35% to 100% phase B for 5 min and 100% B for
# Analysis of proteomics
Raw data were inspected in Xcalibur v.2.1 (Thermo Scientific). Database searches were performed using Proteome Discoverer 1.3 with Mascot v.2.3 algorithm against an inhouse Lotus database version 2.5 (39,734 sequences; 11,779,282 residues). The searches were performed with the following parameters: MS accuracy 10 ppm, MS/MS accuracy 0.6 Da for CID and 0.1 Da for HCD, trypsin digestion with two missed cleavage allowed, fixed carbamidomethyl modification of cysteine and variable modification of oxidized methionine. iTRAQ 4plex (monoisotopic mass = 144.102) for N-terminus and Lys were set as variable modification. Number of proteins and protein groups and, numbers of peptides were estimated using Proteome Discoverer, false discovery rates around 1% and peptide rank 1was applied as a cut-off limit. iTRAQ quantification was performed using Proteome Discoverer based in reporter ion integration within 50 ppm window.
[formula] 0 4 0 ! 42 [/formula]
Gifu1, Gifu2, and Gifu3 correspond to non-treated wild-type L. japonicus Gifu root samples in biological replicates 1, 2 and 3, respectively, with the total number of spectra with Mascot ion scores above 20 indicated. Highly abundant root proteins are highlighted in yellow.
## ! 43
Supplemental
[table] Table 3: Protein identified from L. japonicus roots. chr1.CM0591.520.r2.d Lj1g0116620.1,Lj1g0116650.1,Lj1g0116650.2,Lj1g0128900.1, Lj1g0129290.1,Lj1g2626180.1,Lj1g2626190.1,Lj1g2626200.1, Lj1g2626210.1,Lj1g3716560.1,Lj1g3716640.1,Lj1g3716810.1 Proteins were extracted from wild-type L. japonicus roots in four biological replicates (BR), labeled, and identified using MS. (+) indicates a positive ID according to the parameters described in the supplementary materials and methods. LjT48C16.160.r2.d Lj0g0045929.1,Lj0g0045959.1,Lj0g0075949.2,Lj0g0075949.3, Lj0g0075959.1,Lj0g0075959.2,Lj0g0075959.3,Lj0g0075959.4, Lj0g0150369.2,Lj0g0293869.1,Lj0g0334169.1,Lj0g0361519.1 LjT48C16.170.r2.d Lj0g0045929.1,Lj0g0045959.1,Lj0g0075949.2,Lj0g0075949.3, Lj0g0075959.1,Lj0g0075959.2,Lj0g0075959.3,Lj0g0075959.4, Lj0g0361519.1 [/table]
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Successful MRI-Guided Focused Ultrasound Uterine Fibroid Treatment Despite an Ostomy and Significant Abdominal Wall Scarring
We present a case of successful magnetic resonance imaging-guided focused ultrasound surgery (MRgFUS) of a uterine fibroid in a patient with extensive anterior abdominal wall surgical scars from two longitudinal laparotomies, a total colectomy and ileostomy. This case demonstrates that MRgFUS can be safely used in patients with an ostomy and significant abdominal wall scarring, but careful pretreatment planning and positioning during treatment is needed.
## Case report
MRI-guided focused ultrasound (MRgFUS) is a noninvasive method of thermal ablation, which, through MRI guidance, allows for 3D treatment planning and feedback of temperature deposition in the area to be treated. MRgFUS was FDA approved for fibroid treatments in 2004 [bib_ref] Sustained relief of leiomyoma symptoms by using focused ultrasound surgery, Stewart [/bib_ref]. Obstruction in the near-field of the focused ultrasound beam, such as from an ostomy bag, or indeed extensive abdominal wall scar tissue (which has different acoustic properties), could lead to increased absorption of acoustic energy and skin burns [bib_ref] Full thickness abdominal burn following magnetic resonance guided focused ultrasound therapy, Leon-Villapalos [/bib_ref]. We discuss MRgFUS of a symptomatic uterine fibroid in a patient with longitudinal abdominal scarring and ileostomy and discuss how we circumnavigated these potential obstructions.
A nulliparous 48-year-old premenopausal woman with a symptomatic uterine fibroid presented for MRgFUS. She had menometrorrhagia, bulk symptoms, urinary frequency, and fatigue. Her fibroid symptom severity score and healthrelated quality of life questionnaire (transformed UFS-SSS QOL) [bib_ref] Focused ultrasound treatment of uterine fibroid tumors: safety and feasibility of a..., Stewart [/bib_ref] [bib_ref] The UFS-QOL, a new diseasespecific symptom and health-related quality of life questionnaire..., Spies [/bib_ref] [bib_ref] Uterine leiomyomas: MR imaging-guided focused ultrasound surgery-results of different treatment protocols, Fennessy [/bib_ref] [bib_ref] The responsiveness of the uterine fibroid symptom and healthrelated quality of life..., Harding [/bib_ref] was 93.8 (on a scale of 0-100), indicating very symptomatic disease. She had declined hysterectomy, having already had two laparotomies in the past for total colectomy and ileoanal pouch formation for inflammatory bowel disease, and subsequent lysis of adhesions and ileostomy. She was fearful of possible complications related to adhesions and pelvic floor compromise should she undergo additional surgery. In addition, as a self-employed woman, she was most interested in a minimally invasive intervention requiring the least amount of recovery time.
On physical examination, there was a right lower quadrant ileostomy and a well-healed vertical scar extending from above the umbilicus to the mons pubis. A firm, nontender pelvis mass in the left lower quadrant was appreciated up to the level of the umbilicus. Diagnostic MRI demonstrated the uterus measuring 9.7 × 10.0 × 9.5 cm with a single anterior intramural fibroid measuring 8.8 × 7.6 × 6.4 cm. The fibroid was homogenous and isointense to muscle on the T1-weighted images and of low signal intensity on the T2-weighted images and demonstrated mostly homogeneous enhancement post intravenous gadolinium injection.
After review of the screening MR images (Figures 1(a) and 1(b)) and consultation with the hospital ostomy service, a plan was made to attach the ostomy bag off-center, cut back the wafer/base plate, and rotate the bag off to the side such that it lay away from the center of the abdomen. The : Pretreatment screening axial T2 weighted screening MR images (a, b) and axial T2 weighted images on the day of treatment (c, d). On screening images, the ostomy is evident in the right lower quadrant (grey arrows), and longitudinal abdominal scarring is seen in the subcutaneous tissue in the midline (black arrows) which overlies the uterine fibroid (F). The ostomy bag can also be seen to overlie the anterior abdominal wall (white thick arrows). On the day of treatment, the ostomy bag was placed off to the side and was no longer visible on the images. The skin of the anterior abdominal wall was pulled off to the right side such that the ostomy and scar are now out of the treatment window and the fibroid (F) is accessible for treatment. patient was asked to fast from midnight the night before and to maintain a liquid diet in the 24 hours prior to treatment to decrease ostomy output.
On the day of the procedure, the patient was positioned prone and slightly off-center on the MRI table, such that the left lower anterior wall was positioned over the gel pad and the ostomy off to the right side and out of the treatment path. The skin of the anterior abdominal wall was pulled towards the ostomy, such that the midline longitudinal scars were also out of the treatment field, increasing the size of the acoustic or treatment window and subsequently allowing treatment of a greater fibroid volume .
During treatment, as always, careful attention was paid to the thermal maps to ensure no heat builds up outside of the fibroid. Following delivery of multiple high power sonications to the treatment area, intravenous gadolinium was administrated and showed a 4.1 × 5 × 4.7 cm area of nonenhancement, consistent with necrosis, within the fibroid. No abnormal areas of enhancement within the subcutaneous tissue or the regions of the scar were identified [fig_ref] Figure 2: T1 spoiled gradient recalled [/fig_ref].
Three months post treatment, the patient reported marked symptom improvement with a decrease in bulk symptoms and increase in energy. Her transformed UFS-SSS QOL decreased from 93.8 to 50. No skin changes, abnormality, or any changes in ostomy function were reported.
# Discussion
To our knowledge, this case report is the first to describe MRgFUS in a patient with an ostomy and a history of multiple abdominal surgeries. This case posed a number of potential challenges: the presence of an ostomy, longitudinal abdominal scarring and multiple abdominal surgeries, which pose a threat of underlying adhesions. MRgFUS treatment of patients with horizontal scars [bib_ref] A novel method to aid in the visualization and treatment of uterine..., Zaher [/bib_ref] , usually secondary to cesarean sections or myomectomies [bib_ref] A novel method to aid in the visualization and treatment of uterine..., Zaher [/bib_ref] , can be managed by angling the ultrasound beam superior or inferior to the horizontal Pfannenstiel scar. However, longitudinal scars are more problematic as they are usually midline and the beam has to penetrate from either side of a usually extensive scar. This case demonstrates how thorough individualized, patient-specific planning is paramount to a successful treatment. The presence of an overlying ostomy bag and stoma, coupled with longitudinal scars, are not a contraindication to successful MRgFUS fibroid treatment, which is a treatment option to be considered by patients with such medical problems.
[fig] Figure 2: T1 spoiled gradient recalled (SPGR) echo sequences in the axial (a, c) and sagittal (b, d) planes, post administration of intravenous gadolinium gadopentate. Imaging acquired pretreatment (a, b) demonstrates homogenous enhancement of the fibroid (F), and post treatment (c, d) there is a large area of nonenhancement within the fibroid (outlined in white), consistent with a treatment effect. There is no evidence for enhancement in the scar tissues of the anterior abdominal wall. (P): Pubic bone; (S): spine. [/fig]
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“This is hard to cope with”: the lived experience and coping strategies adopted amongst Australian women with pelvic girdle pain in pregnancy
Background: Women with pregnancy-related pelvic girdle pain (PPGP) report diminished ability to perform physical activities and experience higher rates of mood disorders, such as anxiety and depression, than pregnant women without PPGP. Despite these physical and psychological impacts, little is known about the lived experiences of PPGP amongst Australian women and the ways in which they cope. Situated within biographical disruption and social support theories, this study sought to gain a conceptual understanding of the experience and impact of PPGP on daily life, and how women cope with this condition during pregnancy.Methods:A qualitative research design, situated within a phenomenological framework, using individual, semistructured interviews consisting of open-ended questions was used with a flexible and responsive approach. Purposive sampling of pregnant women attending a single hospital included 20 participants between 14 and 38 weeks gestation, classified with PPGP as per recommended guidelines, with a mean (SD) age of 31.37 (4.16) years. Thematic analysis was performed where interview data was transcribed, coded, grouped into meaningful categories and then constructed into broad themes.Results: Three themes were identified: 1. a transformed biography; 2. coping strategies; and 3. what women want. The pain experienced created a dramatic change in women's lives, making the pregnancy difficult to endure. Women utilised social support, such as family, to help them cope with pain, and a self-care approach to maintain a positive mindset and reduce stress. Although a few women received support from healthcare professionals, many reported a lack information on PPGP and limited societal recognition of the condition. Women wanted early education, personalised information and prompt referral to help them cope with PPGP.Conclusions: Findings from this study highlighted the complexity of living with PPGP as women attempted to deal with the unexpected impact on daily life by seeking support from partners and families, while also struggling with societal expectations. Although women with PPGP used a number of coping strategies, they sought greater support from healthcare professionals to effectively manage PPGP. These findings have important implications for the provision of health care to women living with PPGP.
# Background
Pregnancy tends to be perceived as a joyous time in women's lives. Society portrays a picture of bliss and happiness during pregnancy, often highlighting the positive moments such as feeling the baby's first movements "kicking" and the pregnancy "glow". However, there are unpleasant physical symptoms associated with pregnancy, including nausea, fatigue, heartburn and musculoskeletal pain which do not attract the same amount of media attention [bib_ref] Incidence of pregnancy-related discomforts and management approaches to relieve them among pregnant..., Nazik [/bib_ref]. Of these physical symptoms, pregnancy-related pelvic girdle pain (PPGP) is a common pain reported universally during pregnancy [bib_ref] The severity and impact of pelvic girdle pain and low-Back pain in..., Gutke [/bib_ref]. Pain is located within the pelvic area between the posterior iliac crest and gluteal folds, with or without leg pain [bib_ref] European guidelines for the diagnosis and treatment of pelvic girdle pain, Vleeming [/bib_ref]. Although prevalence rates globally have been reported to range from 7 [bib_ref] Pregnancy-related pelvic girdle pain in the Netherlands, Van De Pol [/bib_ref] to 84% [bib_ref] Etiology and prognosis of pregnancy-related pelvic girdle pain: design of a longitudinal..., Bastiaanssen [/bib_ref] , a prevalence rate of 44% was determined recently in an Australian population [bib_ref] Prevalence and factors associated with pelvic girdle pain during pregnancy in Australian..., Ceprnja [/bib_ref].
Women with PPGP report a significant reduction in their ability to perform daily activities, such as standing, walking and turning in bed, and face major challenges in their role as a mother, undertaking household chores and work-related tasks [bib_ref] The severity and impact of pelvic girdle pain and low-Back pain in..., Gutke [/bib_ref] [bib_ref] European guidelines for the diagnosis and treatment of pelvic girdle pain, Vleeming [/bib_ref] [bib_ref] Women's experiences of pregnancy related pelvic girdle pain: a systematic review, Mackenzie [/bib_ref] [bib_ref] Pregnancy-related lumbopelvic pain: listening to Australian women, Pierce [/bib_ref] [bib_ref] Struggling with daily life and enduring pain": a qualitative study of the..., Persson [/bib_ref] [bib_ref] Health-related quality of life and physical ability among pregnant women with and..., Olsen [/bib_ref] [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] [bib_ref] The pelvic ring of pain: pregnant women's experiences of severe pelvic girdle..., Elden [/bib_ref]. However, PPGP does not only cause physical impacts. Women with PPGP experience higher rates of mood disorders, such as anxiety and depression, suffer sleep disturbances, and have a lower quality of life than their pregnant counterparts who do not experience pain [bib_ref] Pregnancy-related pelvic girdle pain in the Netherlands, Van De Pol [/bib_ref] [bib_ref] Women's experiences of pregnancy related pelvic girdle pain: a systematic review, Mackenzie [/bib_ref] [bib_ref] Health-related quality of life and physical ability among pregnant women with and..., Olsen [/bib_ref] [bib_ref] A qualitative exploration of the views and experiences of women with pregnancy..., Clarkson [/bib_ref].
Despite the physical and psychological impact on daily life, little is known about the lived experiences of PPGP and the ways women cope with it. Coping has been defined as any cognitive or behavioural attempt, successful or unsuccessful, to manage conditions that are perceived as difficult or induce stress. In order to understand the lived experience and coping strategies adopted by women, a qualitative approach is essential to allow for a deeper exploration and understanding from the perspective of women who live with PPGP. A recently published systematic review of eight studies describing the experiences of women found that pain had a major impact on women's lives and families [bib_ref] Women's experiences of pregnancy related pelvic girdle pain: a systematic review, Mackenzie [/bib_ref]. However, of the eight studies, only three included women classified with PPGP using recommended guidelines [bib_ref] Struggling with daily life and enduring pain": a qualitative study of the..., Persson [/bib_ref] [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] [bib_ref] The pelvic ring of pain: pregnant women's experiences of severe pelvic girdle..., Elden [/bib_ref]. These three studies, all conducted in Sweden, reported that women struggled with daily life, avoided movement at home and work, and redistributed household tasks to partners, parents and other relatives [bib_ref] Women's experiences of pregnancy related pelvic girdle pain: a systematic review, Mackenzie [/bib_ref] [bib_ref] Struggling with daily life and enduring pain": a qualitative study of the..., Persson [/bib_ref] [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] [bib_ref] The pelvic ring of pain: pregnant women's experiences of severe pelvic girdle..., Elden [/bib_ref]. Although these studies reveal valuable information about the Swedish experience of coping with PPGP, there has been limited investigation into PPGP using qualitative methodology in Australian women, and the strategies Australian women use to deal with pain remain unknown.
Previous studies have identified that women value support of health care professionals and the interventions that they provide, as well as information about the condition [bib_ref] Women's experiences of pregnancy related pelvic girdle pain: a systematic review, Mackenzie [/bib_ref] [bib_ref] A qualitative exploration of the views and experiences of women with pregnancy..., Clarkson [/bib_ref]. However, it has been suggested that current health care services do not meet the needs of women struggling to cope with PPGP [bib_ref] The severity and impact of pelvic girdle pain and low-Back pain in..., Gutke [/bib_ref] [bib_ref] Pregnancy-related lumbopelvic pain: listening to Australian women, Pierce [/bib_ref] [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] [bib_ref] The pelvic ring of pain: pregnant women's experiences of severe pelvic girdle..., Elden [/bib_ref] [bib_ref] The role of physiotherapy in managing pregnancy related pelvic girdle pain, Ceprnja [/bib_ref]. In an Australian study, only 25% of women with lumbopelvic pain received any type of treatment for their condition, despite telling their health care provider about their pain [bib_ref] Pregnancy-related lumbopelvic pain: listening to Australian women, Pierce [/bib_ref]. The reasons for low treatment rates are unknown, but a lack of knowledge about PPGP has been suggested to result in poor management and outcomes [bib_ref] Women's experiences of pregnancy related pelvic girdle pain: a systematic review, Mackenzie [/bib_ref] [bib_ref] The role of physiotherapy in managing pregnancy related pelvic girdle pain, Ceprnja [/bib_ref]. In order to deliver healthcare that truly meets women's needs, efforts must be made to explore what existing and /or additional supports women feel may assist them to cope with PPGP. This has not been extensively examined previously worldwide.
Thus, this paper aimed to explore the lived experience of Australian women with PPGP including the impacts on daily life, the coping strategies they adopted, and what additional supports they may require to help them better cope with PPGP. Knowledge gained from this qualitative enquiry will inform healthcare professionals of the care needs of the women they seek to support during pregnancy.
## Theoretical frameworks
This study is situated within biographical disruption theory [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref] and social support theory. These theoretical lenses allow for examination and interpretation of the responses from women which are complex, real-world problems.
Biographical disruption theory, coined by British sociologist Michael [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref] , provides insight into how people respond and adapt to illness, suggesting that it is a "major kind of disruptive illness" [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref]. The theory highlights that biographical disruption does not result from a disorder, but from the ways that disorder impinges on one's physical ability to engage with daily life [bib_ref] Embodiment and the foundation of biographical disruption, Engman [/bib_ref]. Suffering from pain during pregnancy can bring disorder to a woman's life and impact on her ability to perform activities of daily living. [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref] also suggested that attention needs to be provided to the actions that individuals actively seek to counter the impact on their lives [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref]. Therefore, the strategies cultivated by women to help them cope with PPGP were explored. Keywords: Pregnancy, Women, Pelvic girdle pain, Qualitative, Interview, Experience, Coping Social support has been theorised as needing to be mobilized in response to the changes in life and social relationships [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref]. In social support theory, the effects of stressful life events on health are reduced through the supportive actions of others or the belief that support is available. Supportive actions may enhance coping and are categorised into four behaviours including emotional, instrumental, informational and appraisal. Thus, the social support theory provided real-world knowledge about what women need to be able to cope with PPGP.
# Methods
## Design and setting
A qualitative research approach provided a rich description of the lived experience of women living with PPGP, allowing for women to describe their experiences of how PPGP impacted on daily life and the strategies used for coping with PPGP [bib_ref] Qualitative description: the poor cousin of health research, Neergaard [/bib_ref]. Qualitative research was deemed vital as little is known about the lived experience of women with PPGP in Australia. This study was situated within a phenomenological framework in order to understand what the first-hand experience means for women with PPGP. In line with the phenomenological framework, the semi-structured individual interviewing method was used to collect the data in this study. The method allowed the participants to articulate their lived experiences in detail.
This study was conducted at Westmead Hospital in Sydney, Australia, from November 2019 to February 2021. Westmead Hospital is a large teaching and tertiary referral government funded hospital in an urban centre with over 5200 births recorded annually. The hospital has a catchment area that includes women from a diverse range of socio-economic and ethno-cultural backgrounds, educational levels and working status [bib_ref] Prevalence and factors associated with pelvic girdle pain during pregnancy in Australian..., Ceprnja [/bib_ref].
## Participants
Women attending Westmead Hospital for antenatal care were provided with written and verbal information about the aims and methods of the study by the first author (DC) and assured of their confidentiality and privacy being maintained. Potential volunteer participants were also informed that they could withdraw from the study at any time without their ante-natal or health care being affected.
Women were included if they were over 18 years of age, between 14 and 38 weeks gestation, classified with PPGP and had a sufficient command of the English language to be able to be able to provide written and informed consent and complete the interview. All participants were classified as having PPGP according to recommended guidelines, which included a physical examination [bib_ref] European guidelines for the diagnosis and treatment of pelvic girdle pain, Vleeming [/bib_ref]. Participants were excluded if they self-reported any medical or obstetric complication(s) that may have affected pregnancy including pre-eclampsia, eclampsia, serious intellectual or psychiatric impairment, systemic disease(s), or recent spinal fracture, trauma or surgery.
Stratified purposive sampling was used to ensure that the sample was representative of women with PPGP who attended Westmead Hospital such that participants were intentionally selected according to the needs of the study [bib_ref] Purposeful sampling for qualitative data collection and analysis in mixed method implementation..., Palinkas [/bib_ref] [bib_ref] Sampling in qualitative research. Purposeful and theoretical sampling; merging or clear boundaries?, Coyne [/bib_ref]. Attempts were made to include women with a culturally and linguistically diverse background who were able to speak English. This ensured that a range of voices were heard and provided opportunities for women to be able to share their individual experiences who may otherwise not be included in this type of research. In addition, a broad sample of women was sought, including those who had a partner and those who did not, women of differing paid employment status, and there were no restrictions placed on parity.
## Sample size
Using the qualitative approach, the sample size required was determined when saturation of themes was achieved such that the collection of new data did not add any further information on the aims of the study. Studies situated within the phenomenological methodology have suggested that this requires between 5 and 25 participants. In this study, it was planned that at least 25 women would be interviewed to ensure richness of the data with a broad and diverse sample. However, no new information emerged after the tenth participant interview. After discussion amongst the research team, the decision was made to continue to collect data in order to further probe and explore themes to be certain that that no further information became available with more interviews and to allow for deeper understating of issues raised by women. Previous research has reported that "code saturation" may indicate when researchers have "heard it all", but "meaning saturation" is needed to "understand it all". Thus, our final sample included 20 women with PPGP in order to develop a richly textured understanding of the lived experience of women with PPGP. Ethical approval was granted by the Westmead Hospital (2019/ETH02528) and Western Sydney University (H12532) human research ethics committees.
## Procedure
The first author (DC) contacted each participant by telephone to offer a face-to-face interview or the option to complete a written diary to explore their lived experiences. The written diary method offered was originally planned to allow the participants to document their experiences in a diary at a time convenient for them, and described in the published protocol [bib_ref] How do Australian women cope with pelvic girdle pain during pregnancy? A..., Ceprnja [/bib_ref]. However, all participants chose to attend a face-to-face interview. The interview was scheduled at a mutually convenient date and time at Westmead Hospital with each participant receiving reimbursement for the cost of transport or parking to the value of AUD$20.00.
On attendance, each participant completed a written questionnaire to determine anthropomorphic characteristics (age, height, body mass) and information about their current pregnancy, including weeks of gestation, parity, pregnancy type (singleton, multiples), and current pain level using a visual analogue scale [bib_ref] A critical review of visual analogue scales in the measurement of clinical..., Wewers [/bib_ref]. Further information that was collected included country of birth, self-identified ethnicity, marital status, current physical activity level, and work status.
## Interviews
An interview guide consisting of open-ended questions was used with a flexible and responsive approach to ensure that the same range of topics were discussed with each participant [fig_ref] Table 1: Interview Guide Questions How do you see your pelvic girdle pain? Tell... [/fig_ref]. Key questions were followed by further prompts as relevant to the individual interviewee. Although, follow up questions differed slightly between participants, using an interview guide ensured the key topics were covered in all interviews. The interview guide was developed by the researchers and the questions aimed to determine the lived experience of having PPGP. Women were questioned about how PPGP impacted their daily life, what changes to the performance of daily activities they had made, and how they felt they coped, including what strategies they used to help them cope with PPGP. Women were also asked about what other support(s) or information should be provided to help them be able to better cope with PPGP.
The interviews were digitally recorded and all participants were assigned a coded number and pseudonym. The interviews ranged between 45 to 60 min in duration. The interviewer made written notes during and after each interview which included observations, thoughts and ideas about the participants' responses. These notes were then discussed amongst the research team to determine whether changes to the interview guide were needed to further explore the responses and themes offered by women. For example, based on a response by a participant early in the study, a question was added to the interview guide asking women about their views on judgement by others whilst experiencing pain during pregnancy. Similarly, women were questioned as to whether they felt they needed to plan ahead as a coping strategy when trying to deal with PPGP.
The interviews were transcribed verbatim by the first author (DC) within a week of completion and the transcript was then provided to participants. Member checking ensured that participants were able to review their responses and make edits if they felt more information was needed or to reword text they were not comfortable being included.
# Data analysis
Thematic analysis was conducted whereby each recorded interview was listened to several times to make sense of the data and the interview as a whole [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref]. A realist approach was adopted to explore women's experiences, meaning data were accepted at face value, participant's responses were taken to be a true reflection of their experiences, and quotes were interpreted literally [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref]. Open coding was conducted by naming sections of the participants' narratives in the text. There was regular discussion between all authors to ensure thorough and consistent coding patterns of the data. To enhance trustworthiness, codes were then grouped to form meaningful categories as agreed upon by all authors. Further, analysis of three transcripts was conducted by two authors (DC and AG) independently to see if similar categories emerged. The next step was to construct broader themes from the categories. Two authors met initially and discussed themes (DC and PL) with further discussion and comparison amongst all authors, moving back and forth between text and categories to enrich data credibility. As a consequence, some of the original categories were combined, while others were separated until the final framework of broad themes was established. An audit trail was also adopted to ensure the rigour of the study, providing detailed clarification of the reasons for analytical and theoretical choices. In accordance with accepted qualitative methods, data collection, transcription and analysis was carried out concurrently to be able to determine when saturation was reached [bib_ref] Analysing qualitative data, Pope [/bib_ref]. The consolidated criteria for reporting qualitative research (COREQ) checklist was followed [bib_ref] Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews..., Tong [/bib_ref]. A published protocol details the rationale and proposed methodological approach of the design for this study [bib_ref] How do Australian women cope with pelvic girdle pain during pregnancy? A..., Ceprnja [/bib_ref]. Changes to the methods were needed due to the COVID-19 pandemic. As a consequence, the study deviated from the published protocol by only utilising individual interviews as physical distancing measures, in line with government guidelines, restricted the use of focus groups.
# Results
Twenty women with a mean (standard deviation (SD)) age of 31(4.3) years between 22 and 39 weeks' gestation participated in this study. All women were married or in a de-facto relationship. Women self-identified their ethnicity as being Australian (40%), from the subcontinent (25%), Asian (15%), Middle Eastern (10%) or European (10%). All women reported singleton pregnancies, and more than half the women (55%) had not had a previous pregnancy over 24 weeks gestation. All women reported completing high school with 65% having tertiary qualifications. Of the 20 women, 15 reported being in paid employment. Low levels of physical activity were reported amongst the sample, with 40% of women seldom participating in sport or exercise (less than once per week) and a further 30% reporting participation only once per week. The mean (SD) pain reported was 68 (23) mm on a visual analogue scale.
Three broad themes were identified and incorporated a number of subthemes following thematic analysis of the interviews [fig_ref] Table 2: Broad themes and subthemes [/fig_ref]. Verbatim quotes from the interviews are used to support these themes using pseudonyms to protect the identity of participants.
## A transformed biography
The pain experienced by these participants created a dramatic change in their lives. Women's narratives suggested that they underwent an unexpected and transformed biography that had a significant impact on their day to day activities, with an additional sense of being judged by society.
## An unexpected reality
Women found the pain due to PPGP to be a surprise and were unprepared for the impact it had on their lives, being different to what they anticipated and resulting in a view that they had not been realistic in their expectations of pregnancy. The pain made the pregnancy harder, and women felt the pain robbed them of enjoyment during this time of their lives, highlighted by the following: Women expressed a range of feelings regarding PPGP including annoyance and frustration, with many believing it was unfair and questioning "why me?". Women described feeling unhappy, sad and depressed in response Women were perturbed about the changes to their mood, describing themselves as irritable and bad-tempered. Unexpected sentiments of guilt were also raised by some women, with respect to how they were feeling towards their unborn child and with some feeling they had let their existing family down. Often, women questioned their roles as a mother and wife, and were dismayed to feel that they were not able to do a good job in either role.
[formula] " [/formula]
## "i am feeling guilty about how i am feeling about this baby. thinking … maybe better to not have baby. as a mother, there's always guilt. for my son now we are not doing any activities, which is not fair for him. imagine as a mum how you are feeling, your child is just watching tv and you are just lying down. i wanted to help but i can't do it" (leah)
## Impact on the day to day
All women expressed that pain limited their physical activity with a decreased tolerance for walking, standing, bending and lifting. The pain also interfered with their ability to perform activities of daily living such as cooking, cleaning, shopping and driving. Movement was slower and it took a longer time to complete tasks: "Makes things harder and I have to allow more time to do things. It's harder to do the housework, that needs a bit of effort" (Rose).
Some women discussed the impact of pain on their social life. They did not do as much because of the pain due to physical demands, such as standing, when socialising and psychologically not feeling in the mood to socialise. Women could not maintain their normal exercise levels, and this change in physical activity was difficult to accept.
## "i'm used to being really active and fit, and now with the pain it limits what i can do so i'm not feeling like myself, you know not as fit and active as i am usually like. getting used to this change is hard for me to take" (peta)
## Perceived judgement
Women did not want to complain for fear of being viewed as a "whinger", with many feeling that they would be judged by others if they revealed that they were struggling during their pregnancy or showed any weakness, such as having pain. Hence, they did not want to reveal the truth of their experience, instead wanting to show the world they were having a "good" pregnancy. Despite selfidentifying as being strong and resilient, women often expressed that they were at the mercy of society's judgement because of their gender.
"I'm a strong person, not really bothered by social media and stuff normally, but I feel a pressure in pregnancy to show a certain side to the world … and it can be challenging to deal with. There is judgement of women everywhere and this is just one form of it I guess" (Sofia)
Women also went to great lengths to avoid appearing ungrateful for being pregnant. Women expressed that telling others about their pain may be judged as taking pregnancy for granted. Some spoke of how fortunate they were to be pregnant in the context of other women experiencing fertility struggles, yet found the experience of pain to be no less difficult to deal with.
## "i don't want to appear ungrateful, because like i am lucky to have two and now be having three on the way, but … i guess it was just hard and unexpected" (kitty)
## Coping strategies
Despite the disruption to their biography, women attempted to find ways to cope with the pain and several coping mechanisms were described.
## Social support
Social support was essential to the women. They expressed a reliance on others for assistance to perform tasks and chores that they would have normally performed themselves. Their partners were their biggest support, helping with household activities such as cooking, cleaning and shopping. Women valued the moral support and felt fortunate for the tangible assistance provided by partners and family: "My husband and family help out all the time, I can't really ask for more" (Carly).
Some women were reluctant to ask for help to avoid being a burden to others. Additionally, women were less likely to receive help if their partner was busy at work, whilst others felt they did not receive sufficient support if their family was not near. A couple of women reported a lack of social support with their needs being dismissed by family, friends and colleagues.
"No empathy from others, people just expect you to deal with it. I think it is more about people just dismissing the pain and not paying any attention to it. I think for me it is just ignored really" (Sara)
## Self-care
Women attempted to stay positive with an optimistic outlook where possible. They tried to not push themselves too hard, and advocated for a self-care approach to coping with the focus on their emotional, physical and spiritual wellbeing. They spoke of the importance of being kind to themselves by resting when needed and not feeling guilty about taking time out for themselves. In the face of a lack of options offered to them, women felt the need to practice self-preservation.
"I think you need to take care of yourself and try and keep the pain to a level that's not too bad. There's not a lot else you can do, but you can look after yourself " (Rose)
Focusing inwards, women used affirmative self-talk to stay positive through their pregnancy and tried not to let trivial or insignificant things bother them.
## "i think it is important to stay calm and have a positive outlook so that the little things don't get to you" (leanne)
By organising themselves and planning ahead, women felt more in control of being able to cope with tasks that needed to be performed. Although this reduced spontaneity, women placed value on making choices that suited them. The demands of the household were difficult to meet, so women prioritised what needed to be done and set themselves lower standards in order to reduce some of the stress on themselves: "I am trying not to put pressure on myself for things to be perfect" (Jennifer).
Women looked forward to a time when they no longer had pain, highlighting that a hopeful mindset helped them maintain perspective and endure the pain.
"I think I cope because I know there is an end point in sight, like it's not like I'm going to be pregnant forever so I know there is an end point coming. I think this helps me cope. I just focus on getting through this pregnancy, you know like ... just get through to the other side and then it will be better" (Kitty)
## Care from health care professionals
Women sought care from midwives, doctors and physiotherapists to help manage their pain. Specifically, women reported care from physiotherapists including massage, prescription of exercise and stretches, provision of pelvic belts, and education. Women were very appreciative of the practical support afforded by physiotherapy. Most often, the midwife made the referral to physiotherapy and women were grateful for their efforts towards organising care. However, some women described having to take the initiative themselves by actively seeking referrals to physiotherapy.
"I had to ask three times to get a referral to physiotherapy. They didn't refer me straight away. I asked twice, thrice and then they said we will send you to physio" (Emma).
Many suggested they would give other women suffering from PPGP advice to seek help early as they had found it easier to deal with the pain.
"I would say go and get help early. Ask to see the physio and don't leave until they give you the referral. See the physio and do the stretches. The stretches are really the best thing. And you don't know what you should do without the physio" (Eloise)
Women largely attempted to avoid taking medication, as they were unsure about the safety of medication during pregnancy or the effects it may have on their baby. For many, medication was a last resort if there were no other options.
## What women want
Although women with PPGP reported using a number of coping strategies, they sought more help from health care services. Having their pain validated was important to women, together with a desire for personalised information which was meaningful to them.
## Acknowledgement by others
Support and acknowledgement of the pain was considered integral to women's experiences when coping with pain. Women felt cared for if they had a good relationship with midwives, doctors and physiotherapists. They felt reassured when examined by someone they considered an expert and greatly appreciated the recognition, support and care provided.
"Yes, the physio saw me and gave me information, had a look at my back and pelvis. I am not saying you come out of the pain like magic or something, but at least you feel supported" (Cassie)
In contrast, women who did not receive an assessment were not satisfied with their health care and did not feel comforted: "No one checked where the pain was or looked at it. How can I feel reassured that it is nothing serious when no one even looked at it?" (Sofia).
Some women felt they were brushed off by health care workers, saying their "concerns were dismissed", the pain was "normalised" and they were left to "fend for themselves". Some went even further stating that they felt the focus was entirely on the baby in pregnancy, with a lack of attention towards the mother.
"I think some recognition of the pain and some empathy would have been nice. Like as soon as you mention you have pain to the midwife or doctor, it's just shut down. Like the conversation ends. No one follows up, they don't ask you anything more about it. I think there is no focus on the mother in pregnancy, it's all around the baby" (Sara)
## More information
A small number of women received information to meet their healthcare needs. Those who received information found it increased their knowledge about PPGP, assisting them to manage the pain. However, the majority of women reported a dearth of information regarding PPGP, with some suggesting they had not received any information at all so they were "in the dark" about PPGP. Many commented that receiving information earlier during their pregnancy about PPGP in the form of written material and brochures may have been helpful. However, some indicated a preference for education and advice to be tailored to their individual needs, including the opportunity to ask questions.
"I gather that everyone is busy in the hospital, but it doesn't take much to ask about the pain and then give information to the person so they can know about it and ask questions. I guess that would help make us feel empowered" (Sara)
Other women reported seeking information on the internet due to the limited material from health care services or providers. A few women found information from the internet helpful, yet most suggested that it was hard to find credible information from Australian sources. Women spoke of having to "trawl through a lot of information", and being unsure of what was good information, preferring hospital sources of information for their trustworthiness and credibility.
"You are not sure who you can trust on the internet and what information is true. I much prefer to ask at the hospital … you know … ask people who I can trust" (Georgia)
# Discussion
The findings of this study revealed that PPGP transformed the biography of the women as it was an unexpected reality of pregnancy. The experience of pain made the pregnancy difficult, both psychologically and physically. Women reported a number of strategies that helped them cope including having social support and accessing health care services. These findings are supported by previous studies conducted in Sweden and England [bib_ref] Struggling with daily life and enduring pain": a qualitative study of the..., Persson [/bib_ref] [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] [bib_ref] The pelvic ring of pain: pregnant women's experiences of severe pelvic girdle..., Elden [/bib_ref] [bib_ref] A qualitative exploration of the views and experiences of women with pregnancy..., Clarkson [/bib_ref] , with the further finding that women expressed self-care as a strategy to coping with pain during pregnancy.
As theorised by the biographical disruption theory [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref] , the impacts of pain were both unexpected and unwanted, and caused a change in the physical ability of women to engage with daily life. The pain also had negative effects on women's body perception, self-identity and psychological wellbeing, which all combined to further disrupt their biography [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref]. As Bury posited, in response to the changes in life and social relationships, resources must be mobilized [bib_ref] Chronic illness as biographical disruption, Bury [/bib_ref]. In this study, resources in the form of support from others, including partners, family members and health care professionals, assisted women to cope with the interruption caused by PPGP. In particular, help from partners and families with household chores was paramount.
Women with PPGP placed a high value on credible information and empathetic care from healthcare services. This is consistent with previous studies of people in pain who have a desire for personalised information [bib_ref] People with low back pain want clear, consistent and personalised information on..., Lim [/bib_ref] and in studies of pregnant women with gestational diabetes who expressed a need for clear information from trustworthy sources [bib_ref] Women with gestational diabetes mellitus want clear and practical messages from credible..., Harrison [/bib_ref]. Women also appreciated treatment by physiotherapists, preferring an active approach to management rather than a pharmacological one. Thus, the provision of information early in pregnancy and timely referrals to relevant healthcare professionals for personalised management may lessen the impact of biographical disruption to women's lives.
Women's ability to cope with PPGP was hampered by a perception of judgement from others. Women commented that they felt like failures if they were not seen as having a "good" pregnancy. They were also keen to not "complain" or "whinge" for fear of being labelled as weak or demanding. In the face of this perception of judgement, many women were reluctant to reveal the truth of their experiences, hiding their pain from others, suffering in silence and not seeking support to cope with pain. While not directly captured in this study, the perception of this external judgement has been identified previously to impact on women's moods [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref]. Judgement of women is not a new phenomenon, but our work in pregnancy identifies the need to address community attitudes enabling women to voice their concerns without fear of repercussions and seek required support.
Expanding on the concept of self-care as a coping strategy in Australian women, self-care has been defined as positive actions and the adoption of behaviours to minimise the impact of illness [bib_ref] Self-care research: where are we now? Where are we going?, Riegel [/bib_ref]. To combat the negative emotional effects associated with pain, such as low mood and anxiety, in our study, women adopted self-care approaches such as a positive mindset, and prioritised their needs to reduce pressure on themselves and minimise stress. Women may assume this approach because they perceive there to be limited options to help them cope, or to exert their own independence and take responsibility towards self-management of pain, perhaps motivated by a maternal desire to be able to nurture their growing baby.
Australian women discussed their experience of pain as creating some strain on their family, although it was not to the degree described by Swedish women in [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] who spoke of "breaking points" in relationships [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref]. The reasons for the differences in this finding may be due to ethno-cultural differences in the sample populations or the practice of self-care emphasized by the women in the current study. Possibly, Australian women may be reducing the stress placed on family relationships by utilising self-care approaches to maximise coping. Further investigation of the outcomes afforded by self-care is clearly warranted [bib_ref] Self-care research: where are we now? Where are we going?, Riegel [/bib_ref]. Nonetheless, health services may need to explore opportunities to promote self-care behaviours to women with PPGP, which may include provision of education, skills training and e-health options, to improve patient outcomes.
# Strength and limitations
The methodology for sampling ensured a large cohort of women from various backgrounds who provided rich, authentic experiences. The catchment of the hospital in Western Sydney includes women from a wide range of ethno-cultural backgrounds with over 50% of residents born overseas [bib_ref] Prevalence and factors associated with pelvic girdle pain during pregnancy in Australian..., Ceprnja [/bib_ref]. This diversity is common to many urban cities in Australia and globally, thus providing a voice to women who may otherwise not be heard to tell their stories of pain during pregnancy. The thorough process of probing the emerging narratives provided opportunities for more women to share their lived experiences regarding themes, leading to increased confidence with saturation of information in this study. Women expressed gratitude for having the opportunity to share their experiences, provide opinions and articulate concerns, which was empowering and helped validate their pain, common to previous qualitative studies investigating PPGP [bib_ref] Struggling with daily life and enduring pain": a qualitative study of the..., Persson [/bib_ref] [bib_ref] Life's pregnant pause of pain: pregnant women's experiences of pelvic girdle pain..., Elden [/bib_ref] [bib_ref] The pelvic ring of pain: pregnant women's experiences of severe pelvic girdle..., Elden [/bib_ref] [bib_ref] A qualitative exploration of the views and experiences of women with pregnancy..., Clarkson [/bib_ref].
There are a few limitations to the cohort of participants. For example, all pregnancies were singletons. The majority (90%) of participants were in the third trimester, thus the impacts of PPGP may be different earlier in pregnancy. However, we know PPGP prevalence increases with advancing gestation [bib_ref] Prevalence and factors associated with pelvic girdle pain during pregnancy in Australian..., Ceprnja [/bib_ref] , hence the reported themes will be common to most women experiencing pain. All the participants were married or in de-facto relationships, hence the findings may have been different if single women were included as they may report different social support(s) or a lack thereof. The use of focus groups may have added to the richness of the information, but were not able to be conducted due to the social distancing restrictions during the COVID-19 global pandemic. Therefore, it is not known if the information offered in a group setting would be different to the responses provided in a one-to-one interview.
## Implications for health care
The findings point to the complexity of living with PPGP as women struggle to complete household tasks, wrangle with feelings of guilt and frustration, whilst trying to meet societal expectations placed on them. The plight of women, as told in this study, highlights the need to improve attitudes and approaches in caring for pregnant women, both in the community and in the health care sector.
Public health approaches, such as education campaigns, may be required to promote the important role partners and families have in supporting pregnant women, and aid change in community attitudes towards women, enabling them to seek assistance without fear of judgement. Government policies may also need to be examined, such as flexible working arrangements and childcare rebates, to ensure adequate social support is provided in pregnancy. It is essential that health care services provide timely education, endorse self-care approaches, and adopt patientfocused models of care, including expert assessment and referral pathways, to ensure all women have access to the right care, by the right person, at the right time.
# Conclusion
The unexpected reality of pain and associated limitations in physical capability, compounded by feelings of judgement from others, creates a need for timely intervention and resources for women with PPGP. Women expressed that this was not how they had envisaged their pregnancies would be and experienced a disruption that dramatically transformed their daily lives and social interactions. Women rely on social supports to cope with the emotional and functional impacts of PPGP on daily life, and advocate for a self-care approach to maintain a positive mindset and reduce stress. The findings suggest women seek more from health care professionals, including early education, personalised information, and prompt referrals for treatment, to assist in the management of PPGP. There is a clear need for the provision of empathetic community support and responsive healthcare services to support women with pain to better cope during pregnancy and improve their experiences at this critical time of their lives.
[table] Table 1: Interview Guide Questions How do you see your pelvic girdle pain? Tell me about your pelvic girdle pain. What is it like? Can you explain to me what having pelvic girdle pain in pregnancy means to you? How do you feel about having pelvic girdle pain in your pregnancy? Tell me about how it makes you feel. What feelings does the pain bring out? How does having pelvic girdle pain in pregnancy affect your daily life? Does the pain make certain activities harder? How does having pelvic girdle pain in pregnancy affect you at home? With family? At work? In social situations? What changes to the performance of daily activities have you made as a result of having pelvic girdle pain in pregnancy? How do you do things differently? Do you change positions more often? Why do you have to do this? How does this help? Do you rest more? How does this help? Tell me about what activities you have stopped doing or do less of because of having the pain? In your view, do you feel you can cope with having pelvic girdle pain in pregnancy? Do you feel you are able to cope? How do you see this? What does coping mean to you? What do you do to help you cope better? Do you ask others for help at home or at work? Do you outsource tasks, such as cleaning, online shopping? Have you changed tasks at work? Do you limit your activity? Do you receive any treatment? Use a pelvic belt? What supports have you received to help you cope with pelvic girdle pain? From where? Any supports from midwives, physiotherapists, doctors or other health care professionals? Tell me about the supports. What about any information from ante-natal education programs, brochures, fact sheets, online resources? Other people, such as relatives, friends, and work colleagues? What other things do you think would help you cope with pelvic girdle pain better? What about more information? Earlier information? Tell me about what would you like from health care professionals? From family? Friends? Work colleagues? What about referrals for treatment? Medication? Braces? Complimentary/alternate treatments, such as acupuncture? [/table]
[table] Table 2: Broad themes and subthemes [/table]
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Transcription Factor AhR, Cytokines IL-6 and IL-22 in Subjects with and without Peri-Implantitis: A Case Control-Study
J.A.S.) † These authors contributed equally to this work.Abstract: Peri-implantitis is a plaque-associated condition characterized by mucosal inflammation and subsequent progressive loss of supporting bone; it is caused by bacterial biofilm, but the host response triggered by bacterial stimulation promotes the release of cells and mediators that culminate in tissue destruction. The Aryl-hydrocarbon Receptor (AhR) is associated with IL-22 production by Th22 and Th17 CD4+ Th cells. The presence of IL-6 may promote the Th22 phenotype. The present case-control study evaluated the gene expression of AhR, IL-22, and IL-6 in the peri-implant tissues of healthy and peri-implantitis patients. Tissue biopsies were collected from thirty-five volunteers (15 healthy and 20 with peri-implantitis). A real-time PCR reaction was utilized to assess the AhR, IL-22, and IL-6 gene expression levels relative to the reference gene (GAPDH). The results were analyzed using the Mann-Whitney test with a significance level of 5%. Higher levels of gene expression of AhR and IL-6 were detected in peri-implantitis tissues. The IL-22 gene expression levels did not differ between groups. In conclusion, higher gene expression levels for AhR and IL-6 were detected in the soft tissues of peri-implantitis patients. IL-22 did not vary between conditions, which may indicate the loss of the immunomodulatory role of IL-22 in periimplantitis.
# Introduction
Dental implants have been used as a method for the rehabilitation of missing teeth, in which the long-term results reach notorious levels of success [bib_ref] The long-term efficacy of currently used dental implants: A review and proposed..., Albrektsson [/bib_ref]. Despite well-established protocols, some failures can occur and lead to implant loss [bib_ref] Clinical trials of endosseous implants: Issues in analysis and interpretation, Listgarten [/bib_ref] [bib_ref] Clinical and microbiological determinants of ailing dental implants, Tabanella [/bib_ref] [bib_ref] Factors related to peri-implantitis-A retrospective study, Renvert [/bib_ref]. Dental implants, like teeth, can be colonized by bacteria [bib_ref] Comparative biology of chronic and aggressive periodontitis vs. peri-implantitis, Heitz-Mayfield [/bib_ref]. The establishment of a submucosal dysbiotic process [bib_ref] The severity of human peri-implantitis lesions correlates with the level of submucosal..., Kroger [/bib_ref] causes an inflammatory response in the body that leads to alterations of the tissues that protect and support the implants [bib_ref] Are peri-implantitis lesions different from periodontitis lesions?, Berglundh [/bib_ref] , triggering clinical signs of illness. Two inflammatory diseases (mucositis and peri-implantitis) mediated by bacterial dysbiosis have been described around dental implants [bib_ref] Peri-implant health, peri-implant mucositis, and peri-implantitis: Case definitions and diagnostic considerations, Renvert [/bib_ref].
The knowledge of the factors that mediate the pathogenesis of peri-implantitis has recently evolved to the point of suggesting a division of the peri-implant conditions into subtypes according to the identified triggering factors [bib_ref] Distinguishing predictive profiles for patient-based risk assessment and diagnostics of plaque induced,..., Canullo [/bib_ref] [bib_ref] Classification Systems for Peri-implantitis: A Narrative Review with a Proposal of a..., Canullo [/bib_ref]. Thus, confirming that there is an infectious origin in peri-implantitis, the pathogenesis of the disease in the context of the microbiome/host interrelation are beginning to be elucidated, as well as the role of microorganisms in the functioning of epithelial barriers immunity around implants.
For periodontitis, the dysregulation of the immune response has been described as an important part of tissue damage progression [bib_ref] Current understanding of periodontal disease pathogenesis and targets for host-modulation therapy, Hajishengallis [/bib_ref]. That regulation of immune response is given by cytokines with pro-and anti-inflammatory characteristics [bib_ref] Expression of IL-6, IL-10, IL-17 and IL-33 in the peri-implant crevicular fluid..., Severino [/bib_ref]. An imbalance in the production of cytokines can result in a destructive and progressive inflammatory response, thus determining the severity of the disease [bib_ref] Immunohistological study of lesions induced by Porphyromonas gingivalis in a murine model, Gemmell [/bib_ref]. This may also play a fundamental role in the establishment and progression of peri-implant disease.
The aryl hydrocarbon receptor (AhR) is a latent, evolutionarily highly conserved cytoplasmic transcription factor whose expression is variable (and poorly characterized in many tissues) but which is highly expressed in epithelial barriers, especially in intestinal epithelial cells (IECs) and in cells of the gut-associated immune system (organ barriers) [bib_ref] The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology, Esser [/bib_ref]. It is activated by low molecular weight molecules (barrier permeable) of different chemical nature, both xenobiotic and endogenous, which produces cell/ligand-specific transcriptomic changes, as well as changes in cell functions [bib_ref] How Ah Receptor Ligand Specificity Became Important in Understanding Its Physiological Function, Murray [/bib_ref].
It has been documented that AhR could be involved in disease tolerance and may function as a sensor of bacterial danger [bib_ref] Aryl hydrocarbon receptor control of a disease tolerance defence pathway, Bessede [/bib_ref]. There may be crosstalk (complex formation) with other proteins, including nuclear factor-kB (NFkB). AhR deficiency has been related to a decrease in IL-22 levels and therefore susceptibility to infections.
IL-22, which is a member of the Il-10 family, is recognized for its dual character, that is, protector and mediator of the pathogenesis of multiple infectious/inflammatory diseases [bib_ref] Interleukin-22 and connective tissue diseases: Emerging role in pathogenesis and therapy, Xuan [/bib_ref] [bib_ref] Microbiota-Dependent Effects of IL-22, Sabihi [/bib_ref]. As a protector agent it is part of the defense mechanisms against pathogens, as it acts on epithelial cells and induces their production of antimicrobial peptides [bib_ref] The Aryl Hydrocarbon Receptor (AHR) as a Potential Target for the Control..., Pernomian [/bib_ref] , contributes to wound healing, and stimulates tissue regeneration [bib_ref] The emerging roles of AhR in physiology and immunity, Hao [/bib_ref] [bib_ref] IL-17 and IL-22 in immunity: Driving protection and pathology, Eyerich [/bib_ref] ; on the other hand, as a mediator of pathogenic process it has been associated with various inflammatory diseases due to their association with osteoclastic differentiation and resorption activity (In vitro) [bib_ref] Increased levels of the T-helper 22-associated cytokine (interleukin-22) and transcription factor (aryl..., Diaz-Zuniga [/bib_ref].
Some non-lymphoid cells (fibroblasts, mast cells, macrophages, and neutrophils) can also produce IL-22 in different diseases [bib_ref] In vivo dioxin favors interleukin-22 production by human CD4+ T cells in..., Brembilla [/bib_ref] [bib_ref] TLR2-activated human langerhans cells promote Th17 polarization via IL-1beta, TGF-beta and IL-23, Aliahmadi [/bib_ref] , and innate lymphoid cells (ILCs), which reside at barrier surfaces, are also a main source of this cytokine [bib_ref] Interleukin-22 and connective tissue diseases: Emerging role in pathogenesis and therapy, Xuan [/bib_ref].
It is also reported that AhR in the presence of immunological stimulation by proinflammatory cytokines or activation of Toll receptors regulates the expression of cytokine/ chemokine genes, especially IL-6 [bib_ref] Inflammatory signaling and aryl hydrocarbon receptor mediate synergistic induction of interleukin 6..., Hollingshead [/bib_ref] , contributing to the function of the epithelial barrier.
The aim of the present case control study was to investigate the levels of gene expression of Ahr, IL-22, and IL-6 in the peri-implant soft tissues of peri-implantitis and healthy patients.
# Materials and methods
## Study population
This case-control study included partially or totally edentulous individuals who presented at least one implant-supported restoration in function for more than 2 years, as previously described [bib_ref] Treg and TH17 link to immune response in individuals with peri-implantitis: A..., Giro [/bib_ref]. This earlier study evaluated the levels of gene expression of the levels of RORγT and FOXP3 gene expression around healthy and diseased implants. Briefly, for inclusion in the study, the patients had to meet the following inclusion criteria: absence of lesions in the oral cavity, good oral hygiene, and indication of anti-infective surgical treatment for peri-implantitis (peri-implantitis group). Exclusion criteria were pregnancy or lactation; systemic diseases that could interfere with peri-implant tissues (osteoporosis, immune disorders, hepatitis, diabetes); use of systemic antibiotics 3 months prior to the sample collection; chronic use of medications that could interfere with immuneinflammatory response (e.g., corticosteroids, non-steroidal anti-inflammatories, immunosuppressive drugs, bisphosphonates) 3 months prior to the sample collection; chronic use of antimicrobial rinses (e.g., chlorhexidine, essential oils, cetylpyridinium chloride, triclosan). The experimental protocol was approved by the Research Ethics Committee of the University of Guarulhos (CAAE #0007.0.132.000-10) and the patients signed a free and informed consent form.
## Clinical parameters
Measurements of bleeding on probing (BoP), suppuration, probing depth (PD) in mm, and clinical attachment level (CAL) in mm were determined at six sites per implant for all the subjects included in the study. The PD and CAL measurements were recorded to the nearest mm using a North Carolina periodontal probe (PCPNU-15, Hu-Friedy, Chicago, IL, USA).
## Peri-implant tissue collection
The peri-implant tissue collection sites met the following criteria:
## -
Healthy group: dental implants scheduled to surgical procedures for non-diseaserelated reason procedures such as dental implant placement next to other implants, soft tissue grafting to modify peri-implant tissue phenotype. - Peri-implantitis group: In order to obtain a biopsy of an area representative of the periimplant inflammatory process, the mucosal tissue was removed around the implant with advanced peri-implantitis (PD ≥ 5 mm, bleeding on probing and/or suppuration, mobility and impairment of 2/3 of bone support). The tissue around a single implant was obtained from each individual with peri-implantitis.
# Gene expression analysis
## Rna extraction
Immediately after the biopsies were performed, the peri-implant mucosal tissue samples were packed in an RNAlater ® solution (Ambion Inc., Austin, TX, USA) to prevent RNA degradation. Samples were incubated at 4 - C for 24 h and then stored at −20 - C until extraction. First, the RNA later solution was aspirated, and the tissue was packaged in liquid nitrogen for shredding. The triturated sample was then placed in TRIZOL reagent (Gibco BRL, Life Technologies, Rockville, MD, USA), homogenized for 30 s, and incubated for 5 min at room temperature. After this period, chloroform (Sigma, St. Louis, MO, USA) was added, and the samples were vortexed and centrifuged at 11,500 rpm for 15 min at 4 - C. The aqueous portion was transferred to another tube to which isopropanol was added, stirred, incubated for 20 min at −20 - C, and centrifuged as described above. RNA samples were subsequently resuspended in approximately 50 µL of diethylpyrocarbonate (DEPC) treated water and stored at −70 - C. Finally, the RNA concentration was determined by means of a spectrophotometer. Next, 1 µg of total RNA was evaluated for quality by 1% agarose gel electrophoresis.
## Dnase treatment
Total RNA samples were treated for disposal of any DNA residue with DNAse (DNAfree TM, Ambion Inc., Austin, TX, USA) as recommended by the manufacturer. Buffer solution and DNAse turbo were added to the tubes with the extracted RNA, based on the previously evaluated RNA concentration. After shaking and centrifugation, the samples remained incubated at 37 - C for 30 min. Finally, the inactivator was added and the solution was stirred and centrifuged. Total RNA was again quantified by means of aspectrophotometer.
## Reverse transcription
A total of 1 µg of the total DNA free RNA sample was used for cDNA synthesis. Reactions were performed to a final volume of 30 µL using the First-Strand cDNA Synthesis Kit (Roche Diagnostic Co., Indianapolis, IN, USA) following the manufacturer's recommendations. Initially, the samples were incubated for 10 min at 25 - C and then for 60 min at 42 - C. After the second incubation step, the samples were incubated for 5 min at 95 - C and then for 5 min at 4 - C for cooling. The reagents used and their respective concentrations were buffer solution (1×), MgCl2 (5 mM), deoxynucleotides (1 mM), randomized primers (3.2 µg), RNAse inhibitor (50 U), and AMV reverse transcriptase (20 U).
## Real-time pcr (rt-pcr) gene expression analysis 2.5.1. primer design
The GAPDH (glycerin-aldehyd-3-phosphat-dehydrogenase, reference gene) primers for AhR, IL-22, and IL-6 were designed with the help of a program developed specifically for the preparation of primers for the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). All primers were checked for specificity by melting curve analysis, always using positive and negative controls. [fig_ref] Table 1: Target genes, primers sequences, amplification profile, and amplicon size during Real Time... [/fig_ref] shows the primer sequence, reaction profile, and amplicon size.
## Reaction optimization
The efficiency for each gene was optimized before the start of the reactions. Concentrations ranging from 2.5 to 5 M for each pair of primers were used to determine under which conditions the reaction presented the best efficiency, as suggested by the equipment manufacturer, and 5 µM was chosen.
## Rt-pcr reactions
RT-PCR reactions were performed with the LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany) using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany). The reaction profile was determined following the protocol suggested by the equipment manufacturer. For each analysis, water was used as a negative control, and the reaction product was quantified using the manufacturer's software (LightCycler Relative Quantification Software-Roche Diagnostics GmbH). GAPDH gene expression levels were used as reference (housekeeping) for normalization of values.
# Statistical analysis
Statistical analysis was performed using the Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA). Initially, the data were analyzed for normality using the Kolmogorov-Smirnov test and when the absence of normal values was detected, nonparametric statistical methods were used. Differences in gender frequency were assessed using Fisher's exact test. Mean Student's t-test evaluated age. All demographic data were presented as mean and standard deviation, except gender. The Mann-Whitney test performed comparisons of the levels of gene expression of the AhR transcription factor and the cytokines IL-6 and IL-22. The results were expressed as mean and standard deviation. The level of significance was set at 5% (p < 0.05).
# Results
A total of 35 patients participated in this study, 16 females and 19 males. Initially, 35 samples were obtained and divided between the two experimental groups (healthy, n = 15, and peri-implantitis, n = 20). The periodontal parameters were collected for both groups and presented in the . Diseased implants presented more clinical inflammation when compared with non-diseased implants (p < 0.05). . Mean ± SD of the periodontal parameters and clinical characteristics of evaluated implant supported restoration from control (healthy, n = 15 subjects) and test (diseased, n = 20 subjects) groups. Mann-Whitney U test (* p < 0.05); ns: no significant.
## Health
Peri-Implantitis During the RNA extraction processes, three samples (belonging to the peri-implantitis group) that did not have the necessary quality were excluded from proceeding with the analysis. In the real-time PCR step (RT-PCR), all samples showed expression of the reference gene (GAPDH). In total, 15 samples for the healthy group and 17 samples for the peri-implantitis group were included.
The results for the gene expression levels of the transcription factor AhR and the cytokines IL-6 and IL-22 regarding the reference gene GAPDH are shown in [fig_ref] Figure 1: Gene expression levels of the transcription factor Aryl-hydrocarbon Receptor [/fig_ref]. The highest levels of gene expression of the AhR-1 transcription factor and the IL-6 cytokine were found in the peri-implantitis group compared to the healthy group (p = 0.024 and p = 0.001, respectively). The analysis of IL-22 expression levels for the healthy and periimplantitis groups did not reveal significant differences between groups (p = 0.46).
# Discussion
Barrier organs such as mucosa from the oral-gut axis and skin, which are in continuous contact with external agents, including possible infectious agents, must be able to differentiate between physiological and pathological agents and subsequently activate immune responses according to the type of stimuli [bib_ref] Oral Versus Gastrointestinal Mucosal Immune Niches in Homeostasis and Allostasis, Suarez [/bib_ref]. Mucosal tissues surrounding implants are no exception to this rule, and innate and adaptive host responses within the oral mucosa have been associated with the progression of peri-implant disease.
To evaluate possible alterations in the function of the epithelial barrier around implants that may be related to the occurrence of peri-implantitis, the gene expression of factors associated with the differentiation of Th cells in the Th22 subpopulation (AhR, IL-22, and IL-6) were evaluated in peri-implant soft tissues. Increased mRNA expression levels for Ahr and IL-6 were detected in diseased peri-implant tissues compared to healthy tissues. In addition, similar levels of IL-22 gene expression were observed in healthy and peri-implantitis patients, which shows a pattern contrary to what has been described for periodontal disease studying the Th22 T cells subpopulation [bib_ref] Increased levels of the T-helper 22-associated cytokine (interleukin-22) and transcription factor (aryl..., Diaz-Zuniga [/bib_ref] [bib_ref] IL-22-expressing CD4(+) AhR(+) T lymphocytes are associated with RANKL-mediated alveolar bone resorption..., Cortez [/bib_ref].
The activators of AhR are, among others, natural substances found in yeasts and bacteria, stress factors and substances such as hydrogen and oxygen metabolites, metals, oxidized low-density lipoproteins, ozone, indoles, and even arachidonic acid metabolites such as lipoxin A4 [bib_ref] The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology, Esser [/bib_ref]. This recognition of the microbiota and host-generated tryptophan metabolites has been proposed to explain the role of the AhR in innate immune signaling within barrier tissues in response to the presence of microorganisms.
Since peri-implantitis is an inflammatory disease initiated by bacteria, the changes in AhR expression and its activation may be directly related to the dysbiosis condition. A reciprocal interaction between the microbiota and the AhR has also been described where the microorganisms generate AhR activators, and consequently, the AhR-mediated host response regulates the microbiota through quorum-sensing activity [bib_ref] How Ah Receptor Ligand Specificity Became Important in Understanding Its Physiological Function, Murray [/bib_ref] ; this constitutes in itself a mechanism through which the epithelia try to control the changes in the microbiome in search of preventing the disease or its progression.
The responses activated by AhR may depend on the tissue environment, such as that generated by effector immune responses [bib_ref] The Aryl-Hydrocarbon Receptor Protein Interaction Network (AHR-PIN) as Identified by Tandem Affinity..., Tappenden [/bib_ref]. Epithelial barriers include multiple immune cells, many of which express differential levels of AhR: low levels in naïve T cells, helper T cells Th1 and Th2, and regulatory T cells, but high levels in Th17 cells and in both the interleukin (IL)-17/IL-22-producing and IL-17/IL-22-non-producing subsets of peripheral gd T cells. AhR is even recognized as a marker of the Th22 subpopulation of CD4+ T cells [bib_ref] Identification of a human helper T cell population that has abundant production..., Teranishi [/bib_ref]. It is believed that Th17 cells that express ROR-γT are responsible for mediating the synthesis of AhR. This transcription factor, in turn, is necessary for the activation of cytokine production by some cell populations, such as the secretion of IL-22 by Th17 cells [bib_ref] Aryl hydrocarbon receptor (AHR) functions: Balancing opposing processes including inflammatory reactions, Bock [/bib_ref].
It is known that the mucosa acts as a protective physical barrier against the entry of microorganisms. In this context, it has been described that IL-22 is a cytokine produced by cells of the innate immune response, such as monocytes, dendritic cells, and natural killers. This cytokine has protective actions against the entry of bacteria and fungi, and it increases the proliferation of epithelial cells and the tissue repair [bib_ref] Identification of a human helper T cell population that has abundant production..., Teranishi [/bib_ref] [bib_ref] IL-22 increases the innate immunity of tissues, Wolk [/bib_ref] [bib_ref] Border patrol: Regulation of immunity, inflammation and tissue homeostasis at barrier surfaces..., Sonnenberg [/bib_ref] [bib_ref] IL-22 defines a novel immune pathway of antifungal resistance, De Luca [/bib_ref] [bib_ref] Th22 cells represent a distinct human T cell subset involved in epidermal..., Eyerich [/bib_ref]. IL-22 has distinct characteristics from other cytokines, as it is the only cytokine secreted by immune cells that acts primarily on non-immune epithelial cells with a unidirectional signaling flow [bib_ref] Biological and pathological activities of interleukin-22, Perusina Lanfranca [/bib_ref].
Despite the protective functions described for IL-22, studies in a murine model of periodontitis progression establishing the presence of CD4+ AhR+ subpopulations that produce IL-22 in periodontal tissues reported a higher detection of these cells in periodontal lesions when compared with uninfected controls, and their association with alveolar bone loss [bib_ref] IL-22-expressing CD4(+) AhR(+) T lymphocytes are associated with RANKL-mediated alveolar bone resorption..., Cortez [/bib_ref].
Thus, it would be expected that in the presence of an infectious process, the activation of AhR would increase the production of IL-22 in search of modulating the inflammatory process and preventing further damage. In the present study, however, it was found that despite the increase in the gene expression of AhR, there was no significant increase in IL-22 gene expression pattern. Studies carried out to explain the host/microbiome relationship in other inflammatory conditions have found a reduction in the expression of IL-22 related to dysbiotic processes.
In a murine model of alcoholic liver disease, ethanol-associated dysbiosis reduced AhR activation levels, and thus intestinal Il-22 production was also decreased [bib_ref] Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to..., Hendrikx [/bib_ref]. This could lead us to think of alterations in the regulation of inflammation mediated by Il-22 induced by dysbiotic changes that occur in peri-implantitis, which could be related to differences observed in the progression of periodontal and peri-implant diseases, where a faster progression pattern has been described for the infection around implants when compared to the infection around teeth.
The analysis of IL-6 gene expression in peri-implant tissues with peri-implantitis revealed the presence of increased levels of this cytokine compared to healthy tissues. In agreement with the data obtained in the present study, [bib_ref] Expression of IL-6, IL-10, IL-17 and IL-33 in the peri-implant crevicular fluid..., Severino [/bib_ref] evaluated the levels of cytokines IL-6, IL-10, IL-17, and IL-33 in the peri-implant crevicular fluid by ELISA. Increased levels of IL-6 were detected compared to healthy peri-implant tissue [bib_ref] Expression of IL-6, IL-10, IL-17 and IL-33 in the peri-implant crevicular fluid..., Severino [/bib_ref]. In 2016, Duarte et al. conducted a systematic review of the cytokines involved in the pathogenesis of peri-implantitis. The analysis of 18 articles indicated moderate evidence of increased levels of pro-inflammatory cytokines, such as IL-6 [bib_ref] Could cytokine levels in the peri-implant crevicular fluid be used to distinguish..., Duarte [/bib_ref]. More recently, Diaz-Zuniga et al.
(2017) [bib_ref] Differential human Th22-lymphocyte response triggered by Aggregatibacter actinomycetemcomitans serotypes, Diaz-Zuniga [/bib_ref] conducted an in vitro study with monocyte-derived dendritic cells and CD4+ T cells from donors stimulated with A. actinomycetemcomitans. Increased levels of IL-6 produced by dendritic cells, and IL-22 and AhR by CD4+ T cells, were detected [bib_ref] Differential human Th22-lymphocyte response triggered by Aggregatibacter actinomycetemcomitans serotypes, Diaz-Zuniga [/bib_ref]. These results are partially in line with those found in the present study.
Finally, the cross-sectional study design does not allow a clear impact of the markers on the progression of peri-implant diseases. This limitation could impact on the gene expression markers and further prospective studies could clarify the role of transcription factor AhR in soft tissue sealing.
# Conclusions
In conclusion, higher gene expression levels of the transcription factor AhR and the cytokine IL-6 were detected in the soft tissues of peri-implantitis patients, which can be interpreted as a reflection of the dysbiotic condition present around implants with periimplantitis that exacerbated the inflammatory process. In peri-implant tissues, the low expression of IL-22 genes could imply an alteration in the modulation of the immune response at the level of the peri-implant mucosa.
This communication highlights the need for more studies on the role and modulation of IL-22 in peri-implant tissue loss.
## Institutional review board statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Guarulhos University (protocol code #0007.0.132.000-10).
# Informed consent statement:
Informed consent was obtained from all subjects involved in the study.
# Data availability statement:
The data of this study can be released by email by the author responsible for correspondence upon request.
[fig] of 10, Figure 1: Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 6 Cont. [/fig]
[fig] Figure 1: Gene expression levels of the transcription factor Aryl-hydrocarbon Receptor (AhR) (A), interleukin (IL)-6 (B), and IL-22 (C) according to the expression of the reference gene (GAPDH: glycerin-aldehyde-3-phosphate-dehydrogenase). * Statistically significant difference assessed using Mann-Whitney test (p < 0.05). [/fig]
[fig] Author: Contributions: Conceptualization, G.G., M.F.B. and J.A.S.; methodology, M.F.B., L.R.L.M. and K.G.-L.; formal analysis, N.C.d.S. and L.J.S.; investigation, J.A.S. and G.G.; resources, J.A.S.; writing-original draft preparation, G.G., J.A.S., L.J.S. and N.C.d.S.; writing-review and editing, G.G, J.A.S., M.F.B., L.J.S., N.C.d.S., L.R.L.M. and K.G.-L.; project administration, J.A.S.; funding acquisition, J.A.S. All authors have read and agreed to the published version of the manuscript. Funding: J.A.S. was funded by CNPq # 301527/2006-7, # 504392/2010-7 and 311368/2019-0; FAPESP # 2008/07154-5. [/fig]
[table] Table 1: Target genes, primers sequences, amplification profile, and amplicon size during Real Time PCR reaction AhR, Aryl-hydrocarbon Receptor; GADPH, glycerin-aldehyd-3-phosphatdehydrogenase; IL, interleukin. [/table]
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The Robot-Assisted Extravesical Anti-reflux Surgery: How We Overcame the Learning Curve
Management of vesicoureteral reflux (VUR) has evolved over the past several decades, with a trend toward a decrease in surgical management. In spite of this, ureteral reimplantation remains a commonly performed procedure by pediatric urologists in selected cases. Although the basic tenets of the ureteral reimplant procedure remain the same, the extra-vs. intravesical approach, and the traditional open vs. minimally invasive approach remain the primary options to correct reflux. Considering the advantages conferred by the robotic surgery platform, many leading centers have preferentially adopted robot-assisted laparoscopic extravesical anti-reflux surgery, or in common surgical parlance, the robot-assisted laparoscopic ureteral reimplantation (RALUR), over pure laparoscopic or open approaches. Predicated on our experience of performing over 170 cases of RALUR, we have made technical modifications which we posit reduce the morbidity of the procedure while offering acceptable outcomes. This review highlights the evolution and establishment of RALUR as a standardization of care in the surgical management of VUR at our institution. In particular, we emphasize the technical nuances and specific challenges encountered through the learning curve in hopes of facilitating this process for others.
# Introduction
Over the past two decades, both the evaluation and therapeutic interventions for vesicoureteral reflux (VUR) have undergone extensive evolutions, with a clear trend toward non-operative management and a focus on voiding dysfunction as the major risk factor for urinary tract infection (UTI) [bib_ref] The decline of the open ureteral reimplant in the United States: National..., Kurtz [/bib_ref].
In spite of significant investigations aimed at identifying risk factors for the development of recurrent urinary tract infections and predicting the potential for VUR resolution and/or the development of renal scarring, our ability to do so remains limited. Still, when conservative management fails, and febrile UTIs or significant renal scarring occurs, children do require surgical management.
The surgical principles for the correction of VUR remain consistent, several years after its initial description [bib_ref] Ureterovesical anastomosis: the description and evaluation of a technique, Paquin [/bib_ref]. In order to prevent VUR the length of the anti-refluxing tunnel is extended, traditionally in a 5:1 ratio of tunnel length to width of the ureter. While the open ureteral reimplant has been the traditional gold standard repair for VUR, the minimally invasive approaches, such as sub-ureteral injections or laparoscopic and robotic assisted laparoscopic ureteral reimplant (RALUR), have been established as viable alternatives [bib_ref] The decline of the open ureteral reimplant in the United States: National..., Kurtz [/bib_ref].
Although initial enthusiasm for RALUR has waned for some due to concerns about a steep learning curve which can potentially increase the risk of patient morbidity or persistent VUR [bib_ref] Editorial comment regarding: Prospective multicenter study on robot-assisted laparoscopic extravesical ureteral reimplantation..., Gargollo [/bib_ref] , the well-established benefits of the robotic approach encourage its use [bib_ref] Prospective multicenter study on robot-assisted laparoscopic extravesical ureteral reimplantation (RALUR-EV): Outcomes and..., Boysen [/bib_ref]. Benefits of the robotic vs. the open approach include an improved field of vision via magnification intraperitoneal visualization of the ureters and bladder, improved cosmesis for the patient, and a more rapid recovery in the immediate postoperative period due to an extravesical approach. In our experience several technical modifications mitigate the aforementioned risk of RALUR while maximizing patient outcomes.
In this review, we seek to explore the evolution of extravesical RALUR-as it is the most widely adapted robotic approach-and describe technical modifications that render this procedure reproducible across surgeons and lessen the learning curve trajectory.
## Evolution of ralur
Since its first description by , the technical aspects of the RALUR have undergone several modifications. Along with the initial descriptions of robotic assisted pediatric urologic procedures, Peters highlighted the advantages of the robotic system, the need for evolution and evaluation [bib_ref] Robotically assisted surgery in pediatric urology, Peters [/bib_ref]. The surgical principles illustrated for both the robotic-assisted intravesical (RAIVUR) and extravesical techniques were adapted to the new platform while adhering to the principles of contemporary open surgical procedures. The intravesical techniques were favored in bilateral cases due the concerns of urinary retention with bilateral extravesical reimplantation [bib_ref] Robotically assisted surgery in pediatric urology, Peters [/bib_ref].
Since then, while the RAIVUR confers advantages of decreased hematuria and bladder spasms compared to the open approach, it failed to gain widespread adaptation, largely due to technical challenges such as insufflation leaks through the larger trocar hiatus and limited working space of a small bladder [bib_ref] Intravesical robotically assisted bilateral ureteral reimplantation, Peters [/bib_ref] [bib_ref] Robotic assisted laparoscopic ureteral reimplantation in children: case matched comparative study with..., Marchini [/bib_ref]. Only three groups have reported their experience with RAIVUR, with modest success rates (83, 92, and 100% reflux resolution in 6, 19, and 3 patients, respectively) and highly variable complication rates (17, 52, and 0%) [bib_ref] Intravesical robotically assisted bilateral ureteral reimplantation, Peters [/bib_ref] [bib_ref] Robotic assisted laparoscopic ureteral reimplantation in children: case matched comparative study with..., Marchini [/bib_ref] [bib_ref] Early experience in roboticassisted laparoscopic bilateral intravesical ureteral reimplantation for vesicoureteral reflux..., Chan [/bib_ref].
Meanwhile, the Lich-Gregoir technique has become popular for the robotic approach due to its ready adaptability to the technology. This trans-abdominal approach provides excellent visualization of the retrovesical space, particularly when compared to the open approach. The magnification of the robotic camera facilitates meticulous detrusor dissection, which combined with judicious use of energy devices limits the potential for collateral damage to the nerve bundles of the bladder [bib_ref] Nerve sparing robotic extravesical ureteral reimplantation, Casale [/bib_ref].
As with other minimally invasive approaches, RALUR confers the significant benefits of minimally invasive surgery including a shorter convalescence, reduced hospital stay, and improved cosmesis. The realization of the potential improved experience for patients has led to more widespread acceptance for utilization of RALUR. This had led to more centers having a higher number of RALUR over open ureteral reimplants, including ours, and this is reflected in publication trends as well .
A negative postoperative voiding cystourethrogram (VCUG) remains the gold standard to declare surgical success after a ureteral reimplantation. But, traditionally high success rates of the open approach obviated a post-operative VCUG in practice. Similarly, with accumulating experience with the RALUR, and pilot studies have demonstrated that our technique delivers reliable VCUG proven success [bib_ref] Robot-assisted laparoscopic ureteral reimplantation: a single surgeon comparison to open surgery, Schomburg [/bib_ref]. Our institution now defines surgical success as a lack of postoperative febrile UTI and a negative VCUG, if obtained in the postoperative period [bib_ref] Is robot-assisted laparoscopic bilateral extravesical ureteral reimplantation associated with greater morbidity than..., Srinivasan [/bib_ref]. Potential short term complications reported after RALUR include minor self-limiting adverse events such as bladder spasms, hematuria, and GI disturbances. Other reported complications include urinary extravasation, UTIs, incisional hernia, ureteral obstruction resulting in anuria, and the rare complication of ureteral strictures.
Early reports for RALUR are typical for most new techniques: they are comprised of single institution experiences with small patient numbers. To compound this, there is a lack of uniformity in data reporting, including the age at the time of repair, the indication for ureteral reimplantation (VUR vs. obstruction), and the degree of reflux at the time of surgery. Bladder and bowel dysfunction has proven to be a significant risk factor for surgical failure yet remains under reported in the literature [bib_ref] Bladder dysfunction and vesicoureteral reflux, Sillén [/bib_ref]. Overview of the literature review [fig_ref] TABLE 1 |: A literature review of RALUR-EV [/fig_ref] suggests, in the first decade of utilization of RALUR, there were inconsistencies in reported success (66.7-100%) and complication (0-100%) rates . It is notable that higher Clavien grade complications occurred in reports following smaller patient cohorts. However, recent growing evidence in literature has been relatively consistent in proving the safety and efficacy of RALUR in prospective multi-institutional collaborative efforts [bib_ref] Prospective multicenter study on robot-assisted laparoscopic extravesical ureteral reimplantation (RALUR-EV): Outcomes and..., Boysen [/bib_ref].
## The learning curve
The learning curve refers to variations in the productivity of a new surgical procedure or surgeon over a specific time period that leads to achieving a consistent level of expertise to meet contemporary standards. Defining a learning curve is a challenging concept. Although some authors claim that proxies such as operative time, complication rates, and functional outcomes are inadequate measures to assess a true learning curve, most reports have defaulted to these as practical measures of surgical success [bib_ref] Evaluating the learning curve for robotassisted laparoscopic radical cystectomy, Pruthi [/bib_ref] [bib_ref] Learning curve using robotic surgery, Kaul [/bib_ref].
As outlined above, the heterogeneity of the published literature on RALUR on key variables such as patient demographics, grades of reflux, comorbidities, in addition to variable study catchment periods makes assessment of a true learning curve difficult.
In order to trace the influence of a learning curve to surgical parameters and outcomes for RALUR at our institution, we have compared the failure rates (need for secondary anti-reflux surgery), radiographic reflux resolution rate, operative time, and/or complication rates over defined time frames (yearly trends) for a series of consecutive cases [fig_ref] TABLE 2 |: Summary of institutional RALUR data [/fig_ref]. We feel as though a single institution center with a relatively high surgical volume as well as 5 different surgeons provides a unique opportunity to carry out this analysis. A review of data from a prospectively maintained database from 2012 till October 2018, includes six surgeons performing RALUR on 170 patients, of which 60 were bilateral. One hundred twenty-seven were female patients. In our series, the average age at surgery was 5.9 years with a general trend toward higher age at surgery each year, contrary to the literature on national data [bib_ref] The decline of the open ureteral reimplant in the United States: National..., Kurtz [/bib_ref]. A Majority of RALUR were done for dilating VUR (Grade 3 or higher) with breakthrough UTIs or renal scarring (100 cases), 36 cases had a duplex anomaly, 23 obstructive megaureter and 10 bladder diverticula. Additionally, 18 cases had a prior history of failed sub-ureteric injection. Among VUR cases, 40% had high grade VUR (IV and V). The operative time varied depending on the number of ureters, need for cystoscopy, retrograde pyelography, placement of a suprapubic tube, and other concomitant procedures depending on the associated pathology (nephrectomy, heminephrectomy, etc.). For unilateral procedures without any concomitant procedure the mean operative time was 161 min (49 cases), and it was 208 min for bilateral cases (48 cases). Operative time includes time of first incision or procedure start, to procedure end time as recorded by the nursing staff. The operative time did not vary significantly between the first and last quarters of consecutive case series . Blood loss was minimal in most of the cases from the beginning.
Over the follow up period of 1 to 75 months (mean 23, median 20 months), six cases had transient urinary retention and four cases needed surgery for port site hernia (we now meticulously close fascia even for 5 mm port sites under direct vision). Four cases of ureteral obstruction were noted based on increased dilation of calyces on renal ultrasound with symptoms of flank pain with nausea; of which three cases resolved with cystoscopy and ureteral stenting for 6 weeks and, in another case, required open ureteral reimplantation. We previously reported our surgical outcomes on an initial cohort and found that postoperative febrile UTIs occurred in 15.8% of unilateral cases compared with 20% of bilateral cases (p = 0.61) [bib_ref] Is robot-assisted laparoscopic bilateral extravesical ureteral reimplantation associated with greater morbidity than..., Srinivasan [/bib_ref]. Surgical failure, denoted by postoperative febrile UTI and a positive VCUG was noted in five (8.7%) of the unilateral cases vs. three (8.6%) of the bilateral RALUR cases (p = 0.98). The updated demographic and clinical information is summarized in [fig_ref] TABLE 2 |: Summary of institutional RALUR data [/fig_ref].
Although the intraoperative surgical time has remained consistent over the years, we note that the number and complexity of complications are decreasing over subsequent years, concomitant to a reduced need for secondary interventions. Indeed, these results encouraged increasing use of RALUR at our institution with the increase in expertise and confidence level. Backed by comparable outcomes there has been an increase in the proportion of RALUR cases as compared to open ureteral reimplants . We expect, also, that being an academic teaching institute with many surgeons and trainees, inherent variations in the proficiency levels with atypical learning curve patterns would affect continued improvement in specific parameters.
## Technical modifications to achieve a successful ralur
Based on our experiences with RALUR, in addition to standard steps [fig_ref] TABLE 3 |: Standard steps of RALUR [/fig_ref] , we have adopted several key technical modifications that we believe have improved our institutional outcomes [fig_ref] TABLE 4 |: Challenges and cautions/modifications [/fig_ref]. Proper case selection is vital and must consider patient age, toilet training status, and the presence or absence of dysfunctional elimination. If there is a concern for secondary reflux due to a neurogenic bladder, this must be worked up prior to intervention. If significant bowel and/or bladder dysfunction persists in spite of adequate therapy, a suprapubic tube should be considered in order to ensure proper, low-pressure post-operative voiding prior to removing within a week. In order to avoid collateral damage to the detrusor muscle and the nerve plexus, meticulous dissection and judicious use of electrocautery is vital [fig_ref] FIGURE 3 |: Operative steps of RALUR [/fig_ref]. We recommend that estimating tunnel length is inaccurate-and overestimated-when the bladder is even slightly distended, hence we now delineate the tunnel length-ensuring a measurement of tunnel length five times the diameter of the distal ureter-while the bladder is completely drained with a foley catheter [fig_ref] FIGURE 3 |: Operative steps of RALUR [/fig_ref]. We regularly utilize a hitch stitch not only to aid detrusor dissection but also to mark the direction of proposed tunnel [fig_ref] FIGURE 3 |: Operative steps of RALUR [/fig_ref]. We use only the tip of the hook to cauterize the identified bleeding spots and spread the muscles bluntly, rather than cutting those layers [fig_ref] FIGURE 4 |: Operative steps of RALUR [/fig_ref].
Once the tunnel length of the detrusor is delineated, we begin dissecting the tunnel midway between the hitch stitch and ureterovesical junction until the bladder mucosal layer is defined [fig_ref] FIGURE 4 |: Operative steps of RALUR [/fig_ref]. Utilizing this window, we find that further progression proximally and distally proceeds more easily without the risk of inadvertent cystotomies [fig_ref] FIGURE 4 |: Operative steps of RALUR [/fig_ref]. We avoid medial and inferior dissection close to the VUJ, which poses damage to the nerve plexus and may increase the risk of detrusor injury and urinary retention. The ureters must be adequately mobilized in a cephalad direction in order to remove any proximal tension that may tease the ureter out of the tunnel.
Once the detrusorrhaphy begins, we prefer interrupted suturing with long term absorbable sutures (5-0 polydiaxone) in a "bottom up" approach, starting from uretero-vesical junction and then moving distally toward the hitch stitch (dome), as it allows for a tailored formation of the tunnel depending on the available ureter length and amount of tension. This technique can be confusing for the beginner and requires careful passing of suture underneath the ureter and back toward the initial bite Refluxing stumps in cases of ectopic ureteral insertion Adequate exposure, resection of residual, and closure of the stump.
Multiple post site scars Multiple port site scars HidES (Hidden incision for endoscopic surgery) groin ports, hide umbilical camera port within umbilical crease; only two working 5 mm ports; no assist port.
Injury to vas and vessels Poor field of vision Preservation of uterine vessels; Under-vision dissection distal to the vas deferens. Starting the distal ureteral dissection with good hemostasis to maintain optimum visibility [fig_ref] FIGURE 3 |: Operative steps of RALUR [/fig_ref].
Frontiers in Pediatrics | www.frontiersin.org side [fig_ref] FIGURE 4 |: Operative steps of RALUR [/fig_ref] to allow placement of the ureter deep in the tunnel with close approximation of the detrusor edges. It is important to include ureteral adventitia in the initial and ending detrusor closure stitch. In cases of dismembered reimplants, solitary kidney, tapered or complex reimplants we prefer to leave a double J stent, but we do not stent typical RALUR cases. We also do not routinely place a drain. We leave a urinary catheter overnight and discharge the patient once they are able to void, leaving residuals that are <25% of the expected bladder capacity. [fig_ref] TABLE 4 |: Challenges and cautions/modifications [/fig_ref] summarizes the common pitfalls and technical modification adopted to address those concerns.
## Challenges
In their recent review, Baek et al. analyzes the reasons for slower adoption of RALUR in comparison to the widespread and quickly adopted robotic prostatectomy among adult counterparts. The steeper learning curve and concerns about the efficacy compared to open reimplants were oft-cited reasons, and the authors suggested that the procedure be deferred to a later point in the robotic experience [bib_ref] Lessons learned over a decade of pediatric robotic ureteral reimplantation, Baek [/bib_ref]. However, from our learning curve experience, we deduce that careful adherence to outlined steps allows the RALUR to be safely and reproducibly performed. Standardization of salient steps with adequate training and judicious use of electrocautery will ensure the avoidance of common pitfalls.
Although there are debates regarding the efficacy of the RALUR in comparison to open ureteral reimplantation, we must be cautious while comparing the historical reports to the contemporary outcomes as the population characteristics (age group, voiding dysfunction, etc.) have been changing. Eventually, the trends suggest RALUR inexorably will be adopted more widely, and reports and ongoing multi-institutional consortiums will continue to provide further evidence regarding its safety and affirming its efficacy.
# Conclusions
RALUR utilization has increased since its inception, but concerns over the procedure's technical difficulty, safety, risk of urinary retention and outcomes has limited its widespread use. Herein, we demonstrate that the learning curve of RALUR can be shortened with specific modifications predicated on experience, and that with technical adaptations, clinically significant improvements in surgical outcomes may be expected. In appropriately selected patients and with adequate preparation, we posit that the RALUR is a safe and effective technique that confers the well-described advantages of minimally invasive surgery to the treatment of VUR.
## Data availability
All datasets generated for this study are included in the manuscript and/or the supplementary files.
# Author contributions
ARS conceived the idea, laid the platform for the review with substantial additions to the manuscript, along with proofreading. RS prepared the initial manuscript and collected the raw data. KS prepared the literature review and part of data abstraction. CL and AKS reviewed the manuscript and made significant corrections.
[fig] FIGURE 1 |FIGURE 2 |: Trends in publications and number of RALURs performed at our institute. Source: PubMed search report of terms "Open ureteral reimplantation" and "Robot-assisted ureteral reimplantation" as on 23 July 2018. Institutional-OR time trends over 2012 to 2018. [/fig]
[fig] FIGURE 3 |: Operative steps of RALUR. (A) Small window created in the broad ligament to access the ureter directly. (B) Ureteral mobilization: gentle handling by grasping only the ureteral adventitia. (C) Marking the detrusor tunnel in a collapsed bladder in line with the ureter. (D) Hitch stitch at the distal end of tunnel marking. [/fig]
[fig] FIGURE 4 |: Operative steps of RALUR. (A) Making a detrusor window till bluish bladder mucosa is delineated. (B) Detrusor muscle separated with direct pinpoint electrocautery tip combined with blunt spreading of the muscle fibers. (C) Detrusor tunnel opened proximally to ureterovesical junction. (D) Passing suture underneath the ureter to advance the ureter into the detrusor trough (left to right). [/fig]
[table] TABLE 1 |: A literature review of RALUR-EV. # Clinical or radiological success rates. *Some reports did not report complications as per Clavien-Dindo grading directly; The reports were graded based on the description. Some authors included UTIs in the complications. There were no Grade-4 complications related to surgery reported in any of the studies. In many series, same patients have had complications of different grades, hence they cannot be summated. [/table]
[table] TABLE 2 |: Summary of institutional RALUR data: patient characteristics and operative outcomes. [/table]
[table] TABLE 3 |: Standard steps of RALUR. [/table]
[table] TABLE 4 |: Challenges and cautions/modifications. [/table]
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Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification
PCR inhibition is frequent in medical microbiology routine practice and may lead to falsenegative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.
# Introduction
Molecular biology and particularly real-time PCR (qPCR) has revolutionized the biological diagnosis of infectious diseases. Nonetheless, PCR may fail because it is based on an enzymatic reaction susceptible to various mechanisms of inhibition. Inhibition of PCR reaction is frequent in clinical microbiology and exposes to the risk of false negative results, hence PCR inhibition screening is recommended. PCR inhibition appears as a hardly predictable event and data about its actual frequency in routine practice of clinical biology laboratories are scarce. Differential susceptibility of each type of qPCR to different inhibitors and heterogeneity of sample matrixes make its detection non trivial. Many methods to detect PCR inhibition have been reported and some guidelines have been published. The use of PCR controls with a defined quantity of DNA molecules to check for the presence of a Cp switch due to the presence of inhibitors in the sample extract is relevant; however it is risky since technicians may have to manipulate and distribute target DNA, thus increasing the risk of inter-well contamination. 'Internal' amplification controls, based on alien DNA added at a low concentration in the specimen before DNA extraction, and of which the presence must be checked in a duplex PCR together with the target, is also highly relevant. Most of commercial inhibition controls are based on this principle. The rationale of using a human gene as an extraction or inhibition control is less acceptable because the human DNA target is present in high quantity in the sample as compared to the target DNA. Importantly, there is presently no consensus for PCR inhibition detection in routine practices. For example, in the single field of molecular diagnosis of toxoplasmosis in France, seven different methods are used to detect PCR inhibition in 30 laboratories. Most of them use either a pathogen pathogen-specific PCR or a human gene amplification method. The former is an internal control made of either genomic T. gondii DNA or a plasmid containing the targeted pathogen DNA sequence. It has one major drawback which is the increased risk of false positive results due to increased amounts of amplicons and airborne contamination of reaction wells. Amplification controls may be prepared from genomic DNA (as in our Toxoplasma-PCR assay) or from the target DNA sequence cloned in a plasmid (as in our Pneumocystis-PCR assay). One development of the plasmid strategy is to clone a chimeric sequence to be amplified by the same primers than the PCR target but detected by specific probe(s). In the second type of method, albumin, beta-globin or human RNase P genes are targeted. This method is found attractive since (i) they can be implemented in all qPCR assays involving human samples; and (ii) they constitute a complete process control for DNA extraction and amplification. Yet, human gene qPCR cycle of positivity (Cp) depends also on the initial human DNA content or cellularity in the clinical sample, which is highly variable. Indeed, DNA content or cellularity depends on the size/volume of the sample, the matrix, i.e. the nature of the sample, such as blood or cerebrospinal fluid, and on the pathophysiological state of the patients. The control of DNA extraction is another critical step of molecular diagnosis, but was not explored in our study. In addition, it is reported in the literature that PCR methods differing by their primers and/or amplified sequence have variable susceptibility to inhibitors. These studies tend to invalidate the assumption that absence of inhibition in a qPCR targeting any human gene has a good predictive value for assessing inhibition in a qPCR targeting a pathogen. Consequently, it is critical to assess the performances of human gene-based PCR inhibition screening methods in clinical samples in clinical microbiology routine practice. In this study, (i) we analyse the frequency of PCR inhibition in a large range of clinical samples and (ii) we show that human gene-based qPCR methods are not efficient to affirm the presence/absence of PCR inhibitors.
# Methods
We retrospectively analysed Toxoplasma and Pneumocystis qPCR tests for all the clinical samples analysed in 2016 in the Department of Parasitology-Mycology at the academic hospital of Montpellier (Montpellier, France).
## Dna extraction
The DNA extraction method was dependent on the PCR assay and the sample matrix. For Pneumocystis-PCR, all types of respiratory samples were analysed, including bronchoalveolar lavage fluid (BALF), sputum, bronchoaspiration, nasal aspiration and pleural fluid; DNA was extracted using QIAamp DNA minikit (Qiagen) according to the manufacturer specifications. For Toxoplasma-PCR, the DNA extraction method depended on the matrix. For low cellular samples such as cerebrospinal fluids (CSF), aqueous humor, amniotic fluids and crystal clear BALF, DNA was extracted using the Tween-Nonidet-NaOH method. When Pneumocystis and Toxoplasma qPCRs were performed on the same sample, QIAamp DNA minikit (Qiagen) was used. Other samples were processed using protein precipitation solution (A795A; Promega, France). As recommended, DNA extracts were then stored at-20˚C prior to PCR for best preservation.
## Toxoplasma and pneumocystis qpcr assays
The Toxoplasma qPCR targeted the 'rep529' sequence (AF146527) and was performed as described by Reischl et al.. The Pneumocystis qPCR targeted the major surface glycoprotein gene (AF372980) and was performed as described by Fillaux et al.. We used LC4801 Probe Master 2X (Roche1) for both qPCR and Uracil-N-Glycosylase (Roche1) for Toxoplasma qPCR. Each PCR well contained 15 μL of mix and 5 μL of DNA extract for Toxoplasma qPCR and 18 μL of mix and 2 μL of DNA extract for Pneumocystis qPCR. Amplification and detection occurred in LightCycler1 480 (Roche) and raw fluorescence data were analysed using the LightCycler1 480 software release 1.5.0 (Roche). Cycles of positivity were determined by the second derivative method.
## Pcr controls and definition of inhibition
Each PCR plate contained one well for negative control (sterile water) and one for positive control (calibrated positive sample). Cp values of positive plate controls were plotted into Levey-Jennings charts and interpreted according to the Westgard rules. DNA extraction control was performed in routine practice by qPCR targeting the human albumin gene. Amplification controls were made of reaction wells containing pathogen DNA on top of the patient DNA extract. For the Toxoplasma qPCR assay, this pathogen-specific amplification positive control was prepared from a 10 5 T. gondii tachyzoites/mL freeze-dried standard as described in Varlet-Marie et al.; 2 μL of T. gondii DNA extract were added to obtain a final concentration of 1.5 T. gondii genome/tube for which the expected Cp value is 35.2 ±1.5. The control was performed in duplicate. A sample was considered inhibited if both amplification control wells showed a Cp>38. If only one well showed a Cp>38, we considered this as irrelevant and due to stochastic events. For Pneumocystis-qPCR, the amplification control was made of the pathogen target DNA sequence, which had been cloned into pGEM-T easy1. Plasmid DNA maxipreparation (Qiagen Maxiprep1) was then diluted in order to reach a Cp of 30.1 ±1.2, and 1 μL of this dilution was added to the reaction tube. A Pneumocystis-qPCR reaction was considered inhibited if the Cp difference between amplification control and the positive plate control was �3. In this study, albumin-qPCR was considered as inhibited if the difference between the Cp of albumin-qPCR (Cp alb ) obtained for a sample and the mean Cp alb for the matrix considered was �3.
## Data management
PCR results and bio-clinical data were exported from LightCycler1 480 release 1.5.0 software (Roche1) and DxLab1 software (MedaSys1) respectively, to spreadsheets (S1 and S2 Figs).
## Determination of the pcr efficiencies
PCR efficiencies of the techniques were determined using a standard curve generated by performing a logarithmic serial dilution of DNA extracts. The standard curves demonstrating a linear relationship between the logarithm of the copy number and the Cp value, allows to determine the PCR efficiency using: Efficiency = -1+10exp(-1/slope).
## Statistics
Data were analyzed using R 3.4.0 and RStudio 1.0.143 softwares. We calculated the Youden's index considering the pathogen-specific amplification control as the reference method to detect PCR inhibition. The Youden's index combines sensitivity and specificity into a single measure (Sensitivity + Specificity-1) and its value ranges between -1 and 1. A value of 1 indicates that there are neither false positive nor false negative results. The best Cp alb cut-off values of albumin-qPCR to detect inhibition were defined using the Youden's index. Sensitivities and specificities of these Cp alb cut-offs were next compared by binomial tests to the 95% theoretical value in order to assess their inferiority. Correlation of Cp alb to leucocyte counts was assessed using Spearman's rank correlation. Holm correction was performed for all the tests. Samples without Cp alb value due to a flat amplification curve were excluded from the graph and the correlation. A p value <0.05 was considered as significant.
## Ethical approval and informed consent
This work was carried out in accordance with the relevant guidelines and regulations; it does not include potentially identifying patient/participant information. The study corresponds to a non-interventional retrospective study and according to the French Health Public Law (CSP Art L1121-1.1), such studies are exempt from informed consent requirement and do not require approval by an ethics committee.
# Results and discussion
## General description
The Dept. of Parasitology-Mycology is a regional and national reference centre which coordinates the "Molecular biology pole" of the National Reference Center for Toxoplasmosis (http:// cnrtoxoplasmose.chu-reims.fr). The laboratory is accredited according to ISO 15189:2012 for the Toxoplasma and Pneumocystis PCR assays. The Toxoplasma-qPCR assay is being used in routine since July 2009, >17000 clinical samples have been routinely tested, and PCR efficiency is regularly controlled and found at �97.5%. For the Pneumocystis-qPCR assay, the corresponding features are March 2013, >2500, and 90 ±9%. PCR efficiency of the albumin qPCR was 98.5%. These laboratory-developed qPCR assays may therefore be considered as robust and finely optimized.
During the year 2016, 3152 samples were referred to the laboratory as part of the routine activity: 2225 samples were analysed by Toxoplasma-qPCR, 964 by Pneumocystis-qPCR, and 37 samples were analysed by both methods (S1 . Two samples analysed by Toxoplasma-qPCR were excluded from this study, one due to cancellation of the analysis and another due to identity problem (S1 and S2 Figs). Most Toxoplasma-qPCR analysed samples were blood samples (1874 out of 2225), followed by 95 CSF and 114 postnatal samples. No Pneumocystis-qPCR samples had to be excluded from the analysis; the 964 Pneumocystis-qPCR samples comprised 787 BALF and 141 sputa samples. The percentage of positive samples for the Toxoplasma-qPCR was 1.7%, ranging from 0.3% for the blood to 12% for the amniotic fluid samples. For Pneumocystis-qPCR, the percentage of positive sample was 7.1%.
## Inhibition rates in clinical samples as assessed from the pathogen-specific amplification control
Of the 3152 analysed samples, 62 (2.0%) showed evidence of PCR inhibition as assessed from the pathogen-specific amplification control: 45 samples for Toxoplasma-qPCR and 17 for Pneumocystis-qPCR. However, this percentage varied from 1.8-15% depending on the matrix. In 61/62 inhibited samples, tenfold dilution of the DNA extract released inhibition, confirming that the unexpected Cp values were due to PCR inhibitors. The remaining sample was a cord blood tested for the presence of Toxoplasma for which PCR remained inhibited despite the dilution, showing flat amplification curves for both the target-and the albumin-specific inhibition controls before and after tenfold dilution. No additional dilution was applied to this sample due to the risk of reduced sensitivity. Interestingly, blood samples, which are reported elsewhereto be prone to PCR inhibition did not exhibit the highest inhibition rate in our study. Samples like sputum or placenta were also prone to PCR inhibition, probably because it is difficult to optimize a DNA extraction protocol for heterogeneous matrixes with highly different structures and properties from one sample to another. Using a similar pathogen-specific control method to detect PCR inhibitors in BALF samples, Döskaya et al. found a 23.8% inhibition rate for Pneumocystis PCRbut a large study of Buckwalter et al. reported inhibition rates under 1% on 386,706 samples. These discrepant results illustrate that PCR inhibition measurement is a problem of variable importance in routine practices, probably depending on the ability of extraction methods to remove inhibitors, on the susceptibility of PCR methods to inhibitors and on the performance of PCR inhibition detection.
## Performances of pcr inhibition detection using albumin-qpcr
409/3152 samples (12.9%) exhibited a Cp alb above the threshold, i.e. above the mean Cp for the matrix concerned + 3. Thus, (i) the albumin-qPCR identified many more samples as containing inhibitors than the pathogen-specific controls, and this was so in both qPCR assays, i.e. 285 vs. 45 for the Toxoplasma assay, and 127 vs. 17 for the Pneumocystis assay. In addition, (ii) it is noteworthy that only 13 and 3 samples were detected as inhibited using both inhibitordetection methods, for the Toxoplasma and Pneumocystis assays, respectively. We therefore conclude that (i) the human gene-based qPCR detected inhibitors much more often than the pathogen-specific qPCR and (ii) it most often did not detect them correctly. Considering this poor correlation between pathogen-specific controls and albumin qPCR to detect PCR inhibitors, we wished to determine Cp alb thresholds more adapted to our assays. To do so, we calculated the sensitivity, specificity of the albumin qPCR to detect PCR-inhibiting samples using the pathogen-specific control as reference and the Youden's index (Sensitivity + Specificity-1) and we varied the Cp alb threshold (see Methods and. We could not determine a Cp alb threshold allowing to obtain 95% sensitivity and 95% specificity, whether this strategy was used for all matrixes together or for each type of matrix (S3 and S4 Figs). We therefore concluded that no Cp alb cut-off values can be determined to efficiently detect pathogen-specific inhibitions for both pathogen-specific PCR assays. To check the influence of the cellularity of clinical samples on Cp alb values, we compared white blood cell counts to albumin qPCR results for blood samples. We analysed 1690/1874 blood samples; the remaining 184 samples were excluded from the analysis due to the absence of whole blood cell count on the same day as Toxoplasma-PCR. Cp alb values were correlated to leucocyte counts (p<0.0001; Spearman's rank correlation), but for a given white blood cell count a large range of Cp alb was observed. The lack of sensitivity and specificity of albumin PCR to detect target specific inhibitions may be explained by a differential susceptibility of each qPCR assay to inhibitors and by the variable quantity of human DNA in clinical samples. These results prevent using human gene-based PCR, e.g. albumin, betaglobin or human RNase P genes, as PCR inhibition detection method. We based our demonstration on two models, i.e. Toxoplasma and Pneumocystis but these results should be expanded to the detection of other pathogens in human samples. Another widely used method to search for inhibitors is the use of commercial "universal" controls. These commercial controls are made of exogenous DNA and are added either in the sample before extraction or with the DNA extract in the PCR mix. Differential susceptibility to inhibitors and efficiency discrepancies between PCR assays should also prove problematic in this approach. Indeed, the size and GC rate of the amplicons of the foreign DNA used will have an impact on the detection of the inhibition. So, implementation of one of these controls should be avoided until their performances have been assessed in routine practice.
In conclusion, pathogen-specific amplification controls appear to be a method of choice for screening the presence of inhibitors in a PCR assay for infectious diseases as compared to the use of a human gene-based qPCR.
Supporting information S1 Spreadsheet of raw data. |
Clinical characterization of data-driven diabetes subgroups in Mexicans using a reproducible machine learning approach
Laboratory samples were taken, and body composition was assessed using a SECA mBCA-515 medical body composition analyzer calibrated for the Hispanic population. Genotyping was also carried out to assess carrier status for the rs1334232 risk variant in SLC16A11 in all subjects. In a subset of subjects, the presence of diabetic kidney disease (DKD) was assessed using the albumin-to-creatinine ratio (ACR), diabetic neuropathy (DN) was assessed using the modified Michigan questionnaire with physical examination (n=1,123) and diabetic retinopathy was evaluated using a standardized ophthalmological examination (DR, n=353). To assess non-alcoholic fatty liver disease (NAFLD) the fatty-liver index (FLI) was used (14). In the overall UIEM-SIGMA cohort, SNNN-Model 2 was used for diabetes subgroup classification and the SNNN-Model 4 for subjects treated with insulin.A subsample of study participants from the SIGMA-UIEM cohort (n=67) underwent additional phenotyping (15, Supplementary Material). Subjects had been diagnosed with T2D for<5 years, had HbA1c<8%, and were treated only with metformin. Insulin sensitivity was assessed using the M-values from euglycemic hyperinsulinaemic clamps (EHC), adjusted for body weight or insulin levels (M-I). To evaluate the acute insulin response to glucose (AIRg), a frequently-sampled intravenous glucose tolerance test (FSIVGTT) was performed. Subcutaneous and visceral adipose tissue areas (SFA, VFA) were quantified using magnetic resonance imaging (MRI); intra-pancreatic and intrahepatic triglyceride (IHTG) content was determined using MRI spectroscopy.Metabolic Syndrome cohortWe performed a prospective observational cohort study including Mexican adults living in large urban settings of central Mexico including Mexico City, Cuernavaca, Leon, Toluca and Aguascalientes to evaluate incidence of T2D, arterial hypertension and cardiovascular diseas. Study sample comprised apparentlyhealthy adults ≥20 years, who resided for >6 months in the evaluated city, and without plans to move to other city in the short term, whose grandparents and parents were born in Mexico. We excluded individuals with previously diagnosed diabetes, cardiovascular disease, cerebral vascular disease, pregnancy, alcoholism (≥10 servings of alcohol per week), acute stress event or any condition that could potentially endanger her life in the three following years. Participants (n=7, were identified and evaluated at their workplaces (offices of the federal government or private companies) (n=3246), homes (n=189) or during a visit of a relative to a medical unit (n=2709).All assessments were performed after a 9-12hr fasting period. The evaluation consisted in a clinical examination using standardized questionnaires, anthropometric measurements and a blood draw. Demographic information and a medical history, including personal and family history of the most common chronic diseases, were obtained. The evaluation included a 24-hour diet recall, 7-day food frequency questionnaire, the three-factor eating questionnaire (16), the short version of the International physical activity questionnaire (IPAQ) (17) and for adults ≥50 years, an assessment of their functionality and depression. Participants were informed about their results and were advised to visit a primary care physician to seek for treatment if required. They were contacted after a three-year period (±6 months) and invited to repeat the evaluation using the same tools and methods. Multiple approaches were applied to cases that were not reachable at the place in which they were originally invited to participate, including phone calls, e-mail messages, telegrams, invitations through friends or relatives, and visits to the workplace. The response rate was 80.7% (n=6,166). Clinical data and blood samples were collected from 10,052 participants at baseline from 2007-2011. We excluded 2,416 individuals who had either undiagnosed T2D (n=429) or declined permission to be included in the follow-up (n=1987). Consequently, our study sample considered for the primary end-point was 7,636 participants. The follow-up visit was performed 29.5±9.7 months later (2010-2013); 6,166 patients were reached for the second evaluation. Twenty-two deaths were recorded among participants. The study was approved by the Ethics Committee of the Instituto Nacional de Ciencias Médicas y Nutrición and all participants signed an informed consent form.
All serum samples were kept frozen until processed in a central laboratory certified by the External Comparative Evaluation of Laboratories Program of the College of American Pathologists. Clinical chemistry parameters and the lipid profile were measured using commercially available reagents (Synchron CX5 delta, Beckman Coulter). Immunonephelometric methods were applied for the measurement of apolipoprotein B (IMMAGE, Beckman Coulter) and C reactive protein . Insulin concentrations were measured using an ELISA method (AxSYM, Abbott).
Incident diabetes (ID) was defined if a previously healthy subject (fasting plasma glucose (FPG) <126mg/dL) at baseline had a medical diagnosis of T2D or started treatment with a glucose-lowering drug after follow-up and/or had a fasting glycemia ≥126mg/dL in the second visit. Incident impaired fasting glucose (IFG) was defined by FPG in the range 100-125mg/dL in the final visit for individuals that had the same variable <100 mg/dL at baseline. Hypercholesterolemia was defined by the presence of a total cholesterol concentration >200mg/dL or being under statin therapy. Metabolic syndrome and its components were defined according to IDF and ATP-III recommendations.
## Sigma-uiem subcohort
The UIEM-SIGMA subcohort is an open-population cohort aimed at to characterize carriers and non-carriers of SLC16A11 variants associated with increased risk for type 2 diabetes (T2D) in Mexicans. The UIEM-SIGMA cohort includes individual with and without . We included subjects with T2D and complete information for clustering (n=1521). In a subset of patients with T2D from this cohort, we assessed diabetic kidney disease (DKD) using albumin-to-creatinine ratio (ACR) and diabetic neuropathy (DN) using the modified Michigan questionnaire with physical examination (n=1,123) and diabetic retinopathy using a standardized ophthalmological examination . Ophtalmologists evaluated vision acuity, ruled-out diabetic retinopathy and macular edema using a no-mydriatic camera for retinal review. Pupillary pharmacological dilation was performed when photographs had poor quality For the deep-phenotyping subcohort we included Mexican-mestizo subjects with parents and grandparents born in Mexico, aged 20-79 years, with BMI 18-34.9 kg/m 2 . Individuals with T2D, HbA1c concentration <8% and without insulin treatment were eligible for the study. No subject smoked tobacco had cardiovascular disease, diabetes complications, or an acute infection. Subjects with >3% weight loss in the last three months, taking medications or with conditions that could interfere with insulin secretion and action, high-performance athletes, with alcohol consumption more than 2 units per day in men or 1 unit in women were also excluded. Subjects provided written informed consent before participating in this study, which was approved by the Comité de Ética en Investigación of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ).
Intervention studies were conducted and completed within the course of a one-month period. First, to identify carriers of the risk haplotype of the SCL16A11 variant samples were genotyped using a Quant Studio 12K Flex Real-Time PCR platform from Thermo Fisher Scientific.
Body composition analysis: Body fat mass (FM) and fat-free mass (FFM) were determined using dualenergy X-ray absorptiometry (DXA, GE Healthcare) and were standardize using height squared in meters. Subcutaneous and visceral adipose tissue areas (SAA, VAA) were quantified using magnetic resonance imaging, and the subcutaneous/intra-abdominal fat ratio was calculated. Intra-pancreatic and intra-hepatic triglycerides content was determined using MRI spectroscopy (Philips Achieva 3 Teslas). In the overall UIEM-SIGMA cohort, we performed body composition analyses using a SECA mBCA-515 medical body composition analyzer calibrated for Hispanic population.
Insulin sensitivity: Participants were instructed to fast for at least 12 hours before the study and subjects with T2D were instructed to suspend oral treatment three days before the procedure. The study was not performed if FPG >250 mg/dL. A catheter was inserted into a forearm vein to infuse dextrose and insulin and a second catheter into a forearm vein in the contralateral hand was inserted in a retrograde fashion to obtain arterialized blood samples using a hot box. Insulin was infused at a rate of 50 mU/m 2 body surface area (BSA)/min BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open Diab Res Care doi: 10.1136/bmjdrc-2020-001550 :e001550.
(initiated with a priming dose of 200 mU/m 2 /min for 5 min and then 100 mU/m 2 /min for 5 min). Euglycemia (~100 mg/dL) was maintained by a variable infusion of 20% dextrose. During the clamp procedure, blood samples were drawn every 10 minutes during the final 30 minutes to determine glucose and insulin concentrations. Insulin sensitivity was determined as the glucose infusion rate (M value) during the final 30 minutes adjusted for weight and for steady state insulin levels (M-I index).
Insulin secretory response: Participants were instructed to fast for 12 hours before the procedure. Two intravenous catheters were placed in antecubital veins (one in each arm). Blood samples were withdrawn at −10, and 25 minutes for measurement of serum glucose and insulin. Glucose was administered intravenously at a dose of 0.3 g/kg for 60 seconds beginning at time 0. The MINMOD Millenium computer package6, 7 was used to estimate the acute insulin response to glucose (AIRg). The AIRg represents the acute insulin response and is defined as the area under the serum insulin curve between 0 and 10 minutes.
Laboratory methods: Plasma glucose concentration was measured by an automated glucose analyzer (Yellow Springs Instruments Co.) Serum insulin concentration was measured by using a chemiluminescent immunoassay (Beckman Coulter Access 2) and HbA1c levels with HPLC (Variant II Turbo, BIORAD). Lipid concentrations (cholesterol, triglycerides, and HDL cholesterol), apolipoprotein AI, apolipoprotein B, uric acid, creatinine, hepatic enzymes and C reactive protein (CRP) were measured using colorimetric assays (Unicel DxC 600 Synchron Clinical System Beckman Coulter). Plasma adiponectin, leptin, and fibroblast growth factor (FGF)-21 concentrations were determined by performing ELISA assays (Merck Millipore).
## Population stratification:
A principal components analysis was performed on 32 ancestry informative markers genotypes, previously validated against whole genomic data using EIGENSTRAT software 9. The top two principal components were used as covariates in the linear regression model to correct for ancestry.
## Ensanut medio camino
ENSANUT Medio Camino 2016 is a population-based survey with probabilistic, multi-stage, stratified and cluster sampling, with regional representativeness (north, center, Mexico City and south) and by urban and rural strata. It comprised a sample of 9,474 homes, and the population interviewed was 29,795 individuals, of whom 8,824 were adults 20 years and older. The survey period was conducted from May to October 2016. The ENSANUT 2016 questionnaire is the same as the one applied in 2006 and 2012. In detail, the diagnostic questions for previous medical diagnosis of diabetes, hypertension and high cholesterol are similar. Weight and height measurements were taken using the same protocols as on previous occasions. A blood sample was taken in a stratified random representative subsample of this population (n=4,180).
For this evaluation, we included subjects with self-report of previous medical diagnosis of T2D or taking oral T2D medications or having either FPG ≥126mg/dL or HbA1c ≥6.5% regardless of time since T2D diagnosis. Plasma glucose concentration was measured by an automated glucose analyzer (Yellow Springs Instruments Co.) Serum insulin concentration was measured by using a chemiluminescent immunoassay (Beckman Coulter Access 2) and HbA1c levels with HPLC (Variant II Turbo, BIORAD). Lipid concentrations (cholesterol, triglycerides, and HDL cholesterol) and uric acid were measured using colorimetric assays (Unicel DxC 600 Synchron Clinical System Beckman Coulter).
## Caipadi cohort
The CAIPaDi program consists of two phases. The first phase comprises an initial and 3 visits one month apart each one taking place in a single 7 hours shift. The interventions include medical care, diabetes education, nutrition, physical activity, psychological evaluation, psychiatric assessment, eye exam, foot and dental care. These are delivered by one nurse, two endocrinologists, a diabetes educator (DE), a nutritionist, an ophthalmologist, a psychologist, a psychiatrist, a physical activity instructor and a dentist. Each intervention follows a procedure manual and has: 1) a specific goal, 2) a self-management strategy and 3) prespecified indicators. Each session is 30 to 60 minutes long; some of them are group meetings in which a predesigned dynamic is executed. Blood test, EKG, weight and height are obtained at arrival; blood test BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open Diab Res Care doi: 10.1136/bmjdrc-2020-001550 :e001550. results are available in 2 hours and attached to every medical record so specialists can adapt and adjust the treatment according to their results.
The second phase consists of annual evaluations where all interventions from the initial phase are reinforced. A continuous at-distance support system was implemented to maintain communication with patients via email, phone calls, text messages, and through the hospital's webpage. (http://innsz.mx/opencms/contenido/departamentos/CAIPaDi). The patients for this study were enrolled from November 1st, 2013 to September 30th, 2019. Inclusion criteria were: type 2 diabetes patients, ≤ 5 years of diagnosis, without disabling complications (blindness, renal failure, stroke, limb amputations, ischemic heart disease) and non-smokers; when smokers, patients attended a Smoking Cessation Clinic as part of the treatment for 6 months before entering the program given the negative impact of smoking in diabetes. If selected, patients received a phone call and an e-mail with the information of their first visit appointment. The Institutional Ethics and Research Committees from the National Institute of Medical Sciences and Nutrition Salvador Zubirán (INCMNSZ for its name in Spanish) approved this study and it was registered in ClinicalTrials.gov (NCT02836808). All patients signed an informed consent form. Each visit was held at the CAIPaDi Center.
Procedures: Participants could participate in groups of 10 people in individual sessions depending on the intervention, with a close relative being encouraged to participate with them. Every one of these interventions followed a procedure manual and included a checklist of the main actions to be implemented and variables to be measured. The aim of visit 1 was to obtain a complete assessment of the patient and provide basic information to start the required changes. On visit 2, patients underwent a problem-oriented evaluation, where the recommendations were selected based on patient´s profile. Visit 3 focused on the identification of potential barriers that may impede metabolic control achievement and visit 4 aimed to reinforce the knowledge already acquired and evaluate the initial results of interventions. During visits 5 and 6, the barriers and their proposed solutions were reviewed. In summary, a collaborative, iterative process was applied in each intervention. To evaluate the competencies acquired in every visit, a structured exam was applied to each patient asking them to undertake activities related to self-care. All interventions were applied in every Laboratory procedures: Fasting concentrations of glucose, creatinine, lipids and HbA1c (Bio-Rad Variant II Turbo HbA1c Kit 2, with HPLC method) were assessed in each visit. Albuminuria/creatinuria ratio (ACR) (SYNCHRON CX system with colorimetric method) was used for screening diabetic nephropathy at baseline and annual visits. The laboratory is certified by ISO 90001:2015 and the College of American Pathologist. Body composition was assessed by bioimpedance (body composition analyzer JAWON medical ioi353).
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) |
Severe immune thrombocytopenia after COVID-19 vaccination: Report of four cases and review of the literature
## Letter to the editor
## Severe immune thrombocytopenia after covid-19 vaccination: report of four cases and review of the literature
To the editor, From December 2020, four COVID-19 vaccines have been worldwide administered under emergency use; in last months' cases of vaccinerelated ITP have been reported [bib_ref] A case of immune thrombocytopenia after BNT162b2 mRNA COVID-19 vaccination, King [/bib_ref] [bib_ref] Thrombocytopenia following Pfizer and moderna SARS-CoV-2 vaccination, Lee [/bib_ref] [bib_ref] Severe, refractory immune thrombocytopenia occurring after SARS-CoV-2 vaccine, Helms [/bib_ref] [bib_ref] COVID-19 vaccination associated severe immune thrombocytopenia, Shah [/bib_ref] raising concern about this possible complication and the management of patients with preexisting autoimmune cytopenias.
Herein we report four cases of ITP following COVID-19 vaccination and a brief literature review.
## #1:
A 27-year-old man presented with hematomas and epistaxis, 10 days after receiving the first dose of BNT162b2 mRNA vaccine (Comirnaty). Blood tests showed a low platelet count (1 × 10 9 /L), with normal WBC e Hb. Peripheral blood film did not show platelet clumping, schistocytes or blasts; no significant findings were observed in biochemical and coagulation tests. He was diagnosed with ITP and therapy with prednisone 1 mg/kg/d was started. Because of no response after 48 h and persistent hemorrhagic diathesis the patient received IVIG 1 g/kg/day for 2 consecutive days with rapid response. However, when we started steroid tapering, platelets soon decreased; therefore, we shifted to dexamethasone 40 mg/d for 4 days every 2 weeks: he underwent 3 cycles obtaining complete response. #2: A 63-year-old man was admitted to hospital because of epistaxis and hematomas developed 14 days after receiving the first dose of ChAdOx1-S (Vaxzevria) vaccination. Medical history was positive for diabetes mellitus and arterial hypertension. Blood tests revealed severe thrombocytopenia (2 × 10 9 /L) with only mild leukocytosis and a normal Hb. Coagulation times, kidney and liver function tests were normal. Autoimmune diseases, viral infections or heamatologic malignancies were excluded. In the hypothesis of ITP, steroid therapy with methilprednisolone 1 mg/kg/d was started with an immediate improvement of platelet count. After 3 weeks, steroid tapering was started and continued maintaining complete response. The second dose of vaccination mRNA-1273 Moderna vaccine (Spikewax) was performed after 9 weeks from the first one without any worsening of the platelet count. #3: A 39-year-old woman with a history of ITP completely resolved with dexamethasone and IVIG in 2012 and 2019, six days after receiving the second dose of BNT162b2 mRNA vaccine (Comirnaty), presented to emergency room with ecchymosis; her platelet count was 1 × 10 9 /L: she received methylprednisolone 1 mg/kg/d and IVIG 0,4 g/kg/d for 5 consecutive days obtaining a partial response (platelets 41 × 10 9 /L). One week later she developed petechiae on her right arm and platelets were 3 × 10 9 /L; IVIG 1 g/kg/d were again given for 2 consecutive days and TPO-mimetic eltrombopag 50 mg/ d started with initial response; dosage needed to be increased to 75 mg/d the following week because of a new drop of platelets. Afterwards platelets collapsed again to 5 × 10 9 /L: she therefore started romiplostim obtaining partial response. She is ongoing evaluation for splenectomy. #4: A 24-year-old male with a history ITP successfully treated with IVIG, in 2018 was diagnosed with wAHA: a bone marrow and a lymph node biopsy excluded a lymphoproliferative syndrome. High doses steroids, IVIG and rituximab led to full recovery. In 2021, 21 days after the first dose of Modena mRNA vaccine he presented with a platelet count of 2 × 10 9 /L. The rest of the blood count and LDH were normal. He had petechiae in the oropharynx and bilateral upper extremities. A tb CT was negative for lymphoproliferative disorders. The patient received IVIG 0,4 g/kg/day for 5 days and methilprednisolone 1 mg/kg/day. Despite an initial response, 17 days after he presented with bruises and a platelet count of 5 × 10 9 /L. He received platelet transfusion and IVIG 1 g/kg/day for 2 days. He continued steroid therapy with a fast improvement in platelet count. A bone marrow biopsy yielded no evidence of lymphoproliferative disorder. Five weeks platelet count is normal and the steroid tapering is still ongoing.
Paulsen et al. reported 4 patients with severe ITP after having received of ChAdOx1 nCoV-19 adenoviral vector vaccine: three of four patients had a past medical history of autoimmune disorders or preexisting mild thrombocytopenia but a stable platelet count; time from vaccination was 2-11 days and all patients obtained complete response to treatment. Also Pfizer-Biontech mRNA vaccine have been related to ITP: a platelet drop was described 3-18 days after administration of first or second dose and generally successfully treated with steroids with or without IVIG. Some had a previous diagnosis of ITP with stable platelet count at the moment of vaccine administration [bib_ref] A case of immune thrombocytopenia after BNT162b2 mRNA COVID-19 vaccination, King [/bib_ref] [bib_ref] Thrombocytopenia following Pfizer and moderna SARS-CoV-2 vaccination, Lee [/bib_ref]. Moderna vaccine-related ITP cases have been described, in a case severe and refractory prompting therapy with dexamethasone, IVIG, platelet transfusions, rituximab and eltrombopag [bib_ref] Thrombocytopenia following Pfizer and moderna SARS-CoV-2 vaccination, Lee [/bib_ref] [bib_ref] Severe, refractory immune thrombocytopenia occurring after SARS-CoV-2 vaccine, Helms [/bib_ref]. Reports of ITP following Johnson and Johnson COVID-19 vaccine are present in literature too [bib_ref] COVID-19 vaccination associated severe immune thrombocytopenia, Shah [/bib_ref]. An overall incidence of thrombocytopenia, including ITP, after vaccination with Pfizer-BioNTech COVID-19 Vaccine or Moderna COVID-19 Vaccine is described in the case-series reported to the Vaccine Adverse Event Reporting System published on June 2021. The reporting rate of thrombocytopenia was 0.80 per million doses for both vaccines, less than the annual incidence rate of 3.3 ITP cases per 100,000 adults [bib_ref] Thrombocytopenia including immune thrombocytopenia after receipt of mRNA COVID-19 vaccines reported to..., Welsh [/bib_ref].
Our report describes four more cases of severe ITP following COVID-19 vaccination with both mRNA and adenoviral vector vaccine. Two patients had a new onset of severe ITP while two had a history of ITP but stable platelet values. Of note, while the first two were well managed Contents lists available at ScienceDirect Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd with oral corticosteroids, in the latter two cases thrombocytopenia was more difficult to manage: they experienced relapses and one of them failed treatment with steroids, IVIG and eltrombopag, obtaining only partial remission with romiplostim. This, together with available literature data, underlies that hematologic monitoring in patients with ITP is advisable before and after vaccination [bib_ref] SARS-CoV-2 vaccination in patients with autoimmune cytopenias: the experience of a reference..., Fattizzo [/bib_ref]. To date, it is not defined which vaccine should be administered to patients who developed ITP after the first dose, as all of them (mRNA and adenoviral vector) have been linked to possible ITP: from our experience, it appears reasonable to complete vaccination carefully monitoring CBC in order to early recognize a platelet drop.
As vaccination against COVID-19 infection is still ongoing it is mandatory to collect more data and, waiting for more information and guidelines, it appears safer to pay attention to possible hemorrhagic events and carefully monitor patients with previous history of ITP as their management could be demanding and difficult as showed by our experience. Anyway, there are no doubts that, considering mortality and morbidity risk after COVID-19 infection, benefits of vaccination outweigh the risk to develop ITP as a vaccine-related adverse event.
https://doi.org/10.1016/j.bcmd.2021.102615 Received 30 September 2021; Accepted 1 October 2021 Blood Cells, Molecules and Diseases 92 (2021) 102615 |
miR-155 is involved in Alzheimer’s disease by regulating T lymphocyte function
Alzheimer's disease (AD) is considered the most common cause of sporadic dementia. In AD, adaptive and innate immune responses play a crucial role in clearance of amyloid beta and maintenance of cognitive functions. In addition to other changes in the immune system, AD alters the T-cell responses that affect activation of glial cells, neuronal cells, macrophages, and secretion of pro-inflammatory cytokines. These changes in the immune system influence AD pathogenesis. Micro-RNA (miRNA)-155 is a multifunctional miRNA with a distinct expression profile. It is involved in diverse physiological and pathological mechanisms, such as immunity and inflammation. Recent studies indicate that miR-155 regulates T-cell functions during inflammation. In this article, we summarize recent studies describing the therapeutic potential of miR-155 via regulation of T cells in AD. Further, we propose that regulation of miR-155 might be a new protective approach against AD pathogenesis.
# Introduction
Alzheimer's disease (AD) is a major cause of dementia in humans, and about 27 million people suffer from this disorder [bib_ref] An estimate of the worldwide prevalence and direct costs of dementia in..., Wimo [/bib_ref] [bib_ref] A review: inflammatory process in Alzheimer's disease, role of cytokines, Rubio-Perez [/bib_ref]. Neuroinflammation, a pathological hallmark of AD, occurs in susceptible regions in the AD brain [bib_ref] Interleukin-1 in the genesis and progression of and risk for development of..., Griffin [/bib_ref] [bib_ref] Cytokines in neuroinflammation and Alzheimer's disease, Cacquevel [/bib_ref] [bib_ref] Association between the interleukin-1beta polymorphisms and Alzheimer's disease: a systematic review and..., Bona [/bib_ref] [bib_ref] A review: inflammatory process in Alzheimer's disease, role of cytokines, Rubio-Perez [/bib_ref] and plays an important role in AD progression [bib_ref] Peripheral inflammatory mechanisms modulate microglial activation in response to mild impairment of..., Ke [/bib_ref]. During AD, neuroinflammation increases the concentrations of pro-inflammatory cytokines [bib_ref] Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices, Bauer [/bib_ref] [bib_ref] Detection of interleukin-6 and alpha 2-macroglobulin immunoreactivity in cortex and hippocampus of..., Strauss [/bib_ref] [bib_ref] Patients with Alzheimer's disease display a pro-inflammatory phenotype, Remarque [/bib_ref] and percentage of activated immune cells [bib_ref] Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro..., Lombardi [/bib_ref] [bib_ref] Lymphocyte subset patterns and cytokine production in Alzheimer's disease patients, Speciale [/bib_ref] [bib_ref] Increased activity of Th-17 and Th-9 lymphocytes and a skewing of the..., Saresella [/bib_ref]. Furthermore, it regulates accumulation of inflammatory molecules and activated glial cells in the surroundings of amyloid plaques in the brain of patients with AD and animal models [bib_ref] Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices, Bauer [/bib_ref] [bib_ref] Systemic inflammation, infection, ApoE alleles and Alzheimer disease: a position paper, Fillit [/bib_ref] [bib_ref] In-vivo measurement of activated microglia in dementia, Cagnin [/bib_ref]. However, the functions of both the inflammatory and immune components need to be further investigated in AD pathogenesis [bib_ref] Autoimmunity and age-associated cognitive decline, Lal [/bib_ref] [bib_ref] Innate immunity, local inflammation and degenerative disease, Mcgeer [/bib_ref] [bib_ref] Four easy pieces: interconnections between tissue injury, intermediary metabolism, autoimmunity and chronic..., Steinman [/bib_ref]. Adaptive immune cells such as T and B lymphocytes play important roles in inflammatory responses in the AD brain. Several studies report that differentiation of cluster of differentiation (CD) 3+ T-cells in AD hippocampal parenchyma is increased compared with controls [bib_ref] Occurrence of T cells in the brain of Alzheimer's disease and other..., Togo [/bib_ref] and that T-cells are activated and infiltrate into the AD brain. In addition, subsets of T-cells in blood circulation as well as in the brain parenchyma are altered in AD [bib_ref] T-cells in Alzheimer's disease, Town [/bib_ref]. During infiltration, T cells produce interferon gamma (IFN-α) that leads to the deposition of amyloid beta peptides (Aβ) and subsequently, cognitive dysfunction [bib_ref] IFN-gamma production by amyloid beta-specific Th1 cells promotes microglial activation and increases..., Browne [/bib_ref]. MicroRNAs (miRNAs) are single-stranded, ∼22 nucleotide long non-coding RNAs that regulate gene expression [bib_ref] Genomics of microRNA, Kim [/bib_ref]. Several miRNAs are expressed in the brain and are involved in inflammation and microglia activation [bib_ref] miR-155 gene: a typical multifunctional microRNA, Faraoni [/bib_ref] [bib_ref] MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein..., Junker [/bib_ref] [bib_ref] Post-transcriptional regulation of miR-27 in murine cytomegalovirus infection, Buck [/bib_ref] , cell cycle regulation [bib_ref] The let-7 microRNA represses cell proliferation pathways in human cells, Johnson [/bib_ref] [bib_ref] MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and..., Schultz [/bib_ref] and in apoptosis [bib_ref] Upregulation of miR-23a-27a-24-2 cluster induces caspase-dependent andindependent apoptosis in human embryonic kidney..., Chhabra [/bib_ref]. Recent studies report that miRNAs are also associated with the T cell functions, such as T cell activation and development [bib_ref] Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB..., Gatto [/bib_ref] [bib_ref] MiR-146a and NF-kappaB1 regulate mast cell survival and T lymphocyte differentiation, Rusca [/bib_ref] [bib_ref] miR-146a controls the resolution of T cell responses in mice, Yang [/bib_ref]. Among a number of miRNAs, miR-155 reportedly regulates inflammatory and immune responses via modulation of suppressor of cytokine signaling 1 (SOCS1;, activator protein 1, and signal transducers and activators of transcription 5 (STAT5; [bib_ref] STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma, Kopp [/bib_ref]. It is observed to be associated with multiple processes, such as regulation of IFN-γ signaling and thus, CD8+ T-cell activation [bib_ref] The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling, Gracias [/bib_ref] , T cell development [bib_ref] Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory..., Kohlhaas [/bib_ref] [bib_ref] Inositol phosphatase SHIP1 is a primary target of miR-155, O'connell [/bib_ref] , cell-cell interactions [bib_ref] MicroRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts, Martin [/bib_ref] , and macrophage activation [bib_ref] MicroRNA-155 is induced during the macrophage inflammatory response, O'connell [/bib_ref]. Recent research demonstrates that the expression of several miRNAs change in AD [bib_ref] RNA in brain disease: no longer just ''the messenger in the middle, Nelson [/bib_ref] [bib_ref] MicroRNAs (miRNAs) in neurodegenerative diseases, Nelson [/bib_ref] [bib_ref] Computational challenges in miRNA target predictions: to be or not to be..., Barbato [/bib_ref] [bib_ref] MicroRNA-219 modulates NMDA receptormediated neurobehavioral dysfunction, Kocerha [/bib_ref] ; including in the brain tissue and cerebrospinal fluid [bib_ref] Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative..., Cogswell [/bib_ref]. Accordingly, miR-155 expression has been observed to be altered in brain tissue from patients with AD [bib_ref] Tumour necrosis factor-alpha (TNF-alpha) and miRNA expression in frontal and temporal neocortex..., Culpan [/bib_ref]. It enhances neuroinflammation in AD progression in a triple transgenic mouse model of AD [bib_ref] Early miR-155 upregulation contributes to neuroinflammation in Alzheimer's disease triple transgenic mouse..., Guedes [/bib_ref]. In this review, we present a new perspective regarding the regulatory role of miR-155 in T-cell functions and thus, AD progression.
## T-cell response in ad
In AD, interaction between the central nervous system and immune system is facilitated by lymphocytes in the blood and by immune mediators [bib_ref] Systemic and acquired immune responses in Alzheimer's disease, Britschgi [/bib_ref]. During an inflammatory response, immune cells in the blood migrate and infiltrate the brain. However, the level of T-cells in the brain is significantly lower in AD than in other neurodegenerative diseases, such a multiple sclerosis or Parkinson's disease [bib_ref] The role of helper T cell subsets in autoimmune diseases, Lafaille [/bib_ref] [bib_ref] Role of Th1 and Th2 cells in autoimmune demyelinating disease. Braz, Nagelkerken [/bib_ref]. In normal, unaffected patients, there are few T-cells in the brain; however, due to disruption of the blood brain barrier (BBB), this number increases in the AD brain, specifically in the hippocampus and temporal cortex [bib_ref] Alzheimer's disease, autoimmunity and inflammation. The good, the bad and the ugly, Sardi [/bib_ref]. T-cells are derived from lymphoid stem cells in the bone marrow and mature in the thymus [bib_ref] Positive and negative selection of T cells, Starr [/bib_ref]. Based on the expression of surface molecules such as CD3, CD4, and CD8, the development of T-cells in the thymus has been divided into three stages: initial, intermediate, and final [bib_ref] Positive and negative selection of T cells, Starr [/bib_ref]. Mature T-cells are classified into naïve, effector, and memory T-cells. Each subset expresses specific surface molecules, such as the C-C chemokine receptor type 7 (CCR7), CD45RA, CD70, and CD27 [bib_ref] Four functionally distinct populations of human effector-memory CD8+ T lymphocytes, Romero [/bib_ref] [bib_ref] Differentiation associated regulation of microRNA expression in vivo in human CD8+ T..., Salaun [/bib_ref]. Based on cytokine profiles, T helper (Th) cells are divided into Th1, Th2, Th9, and Th17 cells depending on the activity of other immune cells and based upon their ability to produce various cytokines [bib_ref] Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from..., Harrington [/bib_ref] [bib_ref] MicroRNA-mediated regulation of T helper cell differentiation and plasticity, Baumjohann [/bib_ref]. A study of immune parameters in AD reports a decrease in the percentage of naïve T-cells, an increase in the number of memory T-cells and CD4+ Tcells, and a reduction of regulatory T-cells (Tregs) compared with the control group [bib_ref] Dramatic shifts in circulating CD4 but not CD8 T cell subsets in..., Larbi [/bib_ref]. Furthermore, a clinical study of AD reports a significant reduction of naïve CD4+ T-cells in these patients and an increase in number of late-differentiated memory T-cells [bib_ref] Immune profiling of Alzheimer patients, Pellicano [/bib_ref]. Xue and colleagues report a significant reduction of CD3+ T-cells, but marginal changes in CD4+ and CD8+ T-cell subsets in AD [bib_ref] Alterations in lymphocyte subset patterns and co-stimulatory molecules in patients with Alzheimer..., Xue [/bib_ref]. Richartz-Salzburger and colleagues confirmed the decrease of CD3+ and CD8+ T-cell number, but showed a minor increase in CD4+ cells in AD [bib_ref] Altered lymphocyte distribution in Alzheimer's disease, Richartz-Salzburger [/bib_ref]. Several studies report that CD45RO+ T-cell expression increases in the brains of patients with AD [bib_ref] Occurrence of T cells in the brain of Alzheimer's disease and other..., Togo [/bib_ref] [bib_ref] Association between APOE epsilon4 allele and increased expression of CD95 on T..., Lombardi [/bib_ref]. Further, Lombardi and colleagues [bib_ref] Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro..., Lombardi [/bib_ref] showed an increase in the CD4+ Th and CD25+ Treg subsets in patients with AD. Other studies report that CD45RO+ T-cell expression increases in the amyloid-beta peptide (Aβ), a marker of AD, has been reported to stimulate the macrophage inflammatory protein (MIP)-1α expression in peripheral T-cells and its receptor C-C chemokine receptor type 5 (CCR5) expression in brain endothelial cells. These alterations in signaling help T-cells cross the BBB. In addition, accumulation of Aβ in AD stimulates microglia, which secrete granulocyte macrophage-colony stimulating factor (GM-CSF) to regulate antigen presentation [bib_ref] Local and systemic GM-CSF increase in Alzheimer's disease and vascular dementia, Tarkowski [/bib_ref]. Furthermore, circulating Aβ-reactive T-cells are observed in patients with AD [bib_ref] Microgliamediated nitric oxide cytotoxicity of T cells following amyloid beta-peptide presentation to..., Monsonego [/bib_ref]. Interestingly, animal studies using APP/PS1 mice demonstrate that Aβ-reactive Th1 cells stimulate microglial activation [bib_ref] IFN-gamma production by amyloid beta-specific Th1 cells promotes microglial activation and increases..., Browne [/bib_ref] and decrease Aβ pathology [bib_ref] Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like..., Butovsky [/bib_ref] [bib_ref] Abeta-specific T-cells reverse cognitive decline and synaptic loss in Alzheimer's mice, Ethell [/bib_ref] [bib_ref] Abeta-induced meningoencephalitis is IFN-gamma-dependent and is associated with T cell-dependent clearance of..., Monsonego [/bib_ref] [bib_ref] T cells specifically targeted to amyloid plaques enhance plaque clearance in a..., Fisher [/bib_ref]. By secreting Th2-type cytokines (downregulate proinflammatory responses), Aβ-reactive Tcells reduce development of AD symptoms [bib_ref] Nasal administration of amyloid-beta peptide decreases cerebral amyloid burden in a mouse..., Weiner [/bib_ref] [bib_ref] Local and systemic GM-CSF increase in Alzheimer's disease and vascular dementia, Tarkowski [/bib_ref]. Additionally, astrocytes secrete transforming growth factor-beta (TGF-β) that promotes Th2 responses and thus, alleviates Aβ pathology in an AD animal model [bib_ref] TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice, Wyss-Coray [/bib_ref]. Interestingly, a co-culture (T-cell and microglia) study demonstrates that Th1 cells upregulate expression of major histocompatibility complex (MHC) class II and CD40, markers of antigen-presenting cells in microglia [bib_ref] Functional maturation of adult mouse resting microglia into an APC is promoted..., Aloisi [/bib_ref]. Aβ-reactive Th1 cells increase the secretion of inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) and promote the expression of MHCII and CD86 in microglia [bib_ref] Activation of mixed glia by Abeta-specific Th1 and Th17 cells and its..., Mcquillan [/bib_ref]. Th1 and Th17 cells increase microglial production of inflammatory cytokines and expression of MHCII, CD80, and CD86 [bib_ref] Infiltration of Th1 and Th17 cells and activation of microglia in the..., Murphy [/bib_ref]. In addition, hyperpermeability of the BBB in AD increases the infiltration of circulating immune cells, such as T-cells [bib_ref] Occurrence of T cells in the brain of Alzheimer's disease and other..., Togo [/bib_ref] [bib_ref] Increased T-cell reactivity and elevated levels of CD8+ memory Tcells in Alzheimer's..., Schindowski [/bib_ref]. In patients with AD, T-cell migration into the brain is followed by enhanced expression of MHC I and II in activated microglia [bib_ref] Altered blood-brain-barrier function in Alzheimer's disease?, Mattila [/bib_ref] [bib_ref] Embryonic stem cells and retinal repair, Vugler [/bib_ref]. In AD, T-cells also participate in various activities, such as expression of neurotrophic factors [bib_ref] Neurogenesis and neuroprotection induced by peripheral immunomodulatory treatment of experimental autoimmune encephalomyelitis, Aharoni [/bib_ref] [bib_ref] Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like..., Butovsky [/bib_ref] [bib_ref] The neuroprotective effect of inflammation: implications for the therapy of multiple sclerosis, Hohlfeld [/bib_ref] and neurogenesis [bib_ref] Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like..., Butovsky [/bib_ref] [bib_ref] IFN-gamma enhances neurogenesis in wild-type mice and in a mouse model of..., Baron [/bib_ref] [bib_ref] Interferon-gamma differentially affects Alzheimer's disease pathologies and induces neurogenesis in triple transgenic-AD..., Mastrangelo [/bib_ref] [bib_ref] CD4-positive T lymphocytes provide a neuroimmunological link in the control of adult..., Wolf [/bib_ref]. Taken together, we propose that T-cells are one of the key regulators of pathological processes in AD. Thus, control of these cells may provide an effective treatment strategy for alleviating the pathogenesis of AD.
MicroRNA miRNAs are short, approximately 22 nucleotide-long, noncoding RNAs [bib_ref] MicroRNAs: genomics, biogenesis, mechanism and function, Bartel [/bib_ref] that regulate gene expression by stimulating either mRNA degradation or their translational repression by binding to the 3 -untranslated region of target mRNAs [bib_ref] Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation, Bagga [/bib_ref] [bib_ref] Posttranscriptional gene silencing by siRNAs and miRNAs, Filipowicz [/bib_ref] [bib_ref] The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation, Chen [/bib_ref]. Similar to pre-mRNAs, a pri-miRNA sequence contains a CAP structure and ploy-A tail. Pri-miRNAs are transcribed by both RNA polymerase I and II [bib_ref] Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways, Lee [/bib_ref]. miRNAs play important roles in diverse mechanisms including cell proliferation, development, and differentiation [bib_ref] MicroRNA biogenesis and cancer, Gregory [/bib_ref]. They are not restricted to the cytoplasm, and are also functional in the nucleus [bib_ref] Trafficking of mature miRNA-122 into the nucleus of live liver cells, Foldes-Papp [/bib_ref] [bib_ref] Analysis of microRNA knockouts in mice, Park [/bib_ref]. In humans, over 2500 miRNAs have been identified [bib_ref] MicroRNA and cancer-a brief overview, Acunzo [/bib_ref] , and most are located at chromosomal regions exhibiting amplification, deletion, or translocation in various diseases, including cancer [bib_ref] MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias, Calin [/bib_ref] [bib_ref] MicroRNA expression profiles classify human cancers, Lu [/bib_ref] [bib_ref] A microRNA expression signature of human solid tumors defines cancer gene targets, Volinia [/bib_ref] , leukemia [bib_ref] MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias, Calin [/bib_ref] [bib_ref] MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like..., Lawrie [/bib_ref] [bib_ref] MicroRNA gene expression in malignant lymphoproliferative disorders, Xu [/bib_ref] , diabetes [bib_ref] miR-194 promotes burn-induced hyperglycemia via attenuating IGF-IR expression, Yu [/bib_ref] , cardiovascular disease [bib_ref] The emerging role of microRNAs in cardiovascular disease, Maegdefessel [/bib_ref] , and AD [bib_ref] Epigenetic drug discovery for Alzheimer's disease, Cacabelos [/bib_ref] [bib_ref] Circulating miRNAs as potential biomarkers in Alzheimer's disease, Galimberti [/bib_ref]. Interestingly, miRNAs are also involved in the regulation of T-cell development, maturation, differentiation, and function [bib_ref] Dynamic regulation of miRNA expression in ordered stages of cellular development, Neilson [/bib_ref] [bib_ref] Costimulationdependent expression of microRNA-214 increases the ability of T cells to proliferate..., Jindra [/bib_ref]. T-cells play an important role in the adaptive immune response. miR-155 is involved in multiple processes [bib_ref] Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB..., Gatto [/bib_ref] [bib_ref] Inositol phosphatase SHIP1 is a primary target of miR-155, O'connell [/bib_ref] , including inflammation [bib_ref] Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible..., Tili [/bib_ref] , immunity [bib_ref] Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory..., Kohlhaas [/bib_ref] [bib_ref] MiR-155 is overexpressed in patients with atopic dermatitis and modulates T-cell proliferative..., Sonkoly [/bib_ref] [bib_ref] The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling, Gracias [/bib_ref] [bib_ref] STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma, Kopp [/bib_ref] and regulatory mechanisms in numerous diseases. The present review therefore emphasizes the role of miR-155 in T-cell alterations during AD pathology.
## Mir-155 and t-cell responses in ad
Several studies report that the expression of miR-155, mediated by Toll-like receptors, increases in monocytic cell lines during lipopolysaccharide (LPS)-induced inflammation [bib_ref] NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of..., Taganov [/bib_ref] [bib_ref] MicroRNA-155 is induced during the macrophage inflammatory response, O'connell [/bib_ref]. miR was shown to regulate acute inflammation after pathogen recognition by Toll-like receptors on monocytes or macrophages; thus, it was involved in innate immunity [bib_ref] NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of..., Taganov [/bib_ref] [bib_ref] MicroRNA-155 is induced during the macrophage inflammatory response, O'connell [/bib_ref]. In addition, inflammatory cytokines such as IFN-α, γ, and TNF-α also strongly stimulate miR-155 expression. These findings indicate that miR-155 is a component of the innate immune response that depends on functions of numerous inflammatory mediators [bib_ref] MicroRNA-155 is induced during the macrophage inflammatory response, O'connell [/bib_ref]. Interestingly, miR-155-null mice exhibit reduced IL-2 and IFN-γ production, indicating that it is necessary for T-cell responses [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref]. In recent in vivo studies, elevated levels of miR-155 were observed following T-cell stimulation through the T-cell receptor (TCR; [bib_ref] Regulation of the germinal center response by microRNA-155, Thai [/bib_ref] [bib_ref] The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling, Gracias [/bib_ref]. miR-155 is also required for development and generation of T cells after TCR activation in vivo [bib_ref] CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of..., Georgantas [/bib_ref] , and also for T-cell response, such as dendritic cell-T-cell interactions [bib_ref] Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible..., Tili [/bib_ref] [bib_ref] MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development, O'connell [/bib_ref]. miR-155-deficient mice exhibit impaired antigen-presentation by dendritic cells as wells as defective dendritic cell-T-cell interactions [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref]. Consequently, miR-155-null mice lack adequately activated T-cells [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref].
Further, miR-155 regulates BBB permeability in central nervous system neuroinflammatory disorders by regulating cellcell interaction molecules in mouse brains [bib_ref] MicroRNA-155 negatively affects blood-brain barrier function during neuroinflammation, Lopez-Ramirez [/bib_ref]. As discussed above, miR-155 is associated with T-cell functions by regulating the TCR and inflammatory cytokine production. These evidence suggest that miR-155 is involved in T-cell immune functions and thus, in the inflammation during AD. Therefore, we summarize the multiple roles of miR-155 in functions of different T-cell types.
## Th1, th2 and th17 cells
Recent in vitro studies report that the expression of miR-155 is up-regulated in activated T-cells [bib_ref] Identification and characterization of human BIC, a gene on chromosome 21 that..., Tam [/bib_ref] [bib_ref] A role for Dicer in immune regulation, Cobb [/bib_ref]. Thai et al. observed that miR-155-deficient mice have reduced germinal center function, T-cell dependent immune responses, and cytokine production [bib_ref] Regulation of the germinal center response by microRNA-155, Thai [/bib_ref]. In addition, the immune responses in miR-155-deficient mice are diverted toward a Th2 pattern, with a significant increase of IL-10, which mediates immunosuppressive effects against cell-mediated responses [bib_ref] Regulation of the germinal center response by microRNA-155, Thai [/bib_ref]. In addition, T-cells from miR-155-null mice show an increased tendency to differentiate into Th2 type cells; they enhanced Th2-type cytokine production when cultured in vitro [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref]. On the other hand, elevated levels of miR-155 in activated CD4+ T-cells induce Th1 cell differentiation by targeting the IFN-γ receptor alpha chain [bib_ref] Micro-RNA-155 inhibits IFN-gamma signaling in CD4+ T cells, Banerjee [/bib_ref] , and miR-155 deficient CD4+ T-cells are more likely to polarize toward Th2 cells [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref] [bib_ref] Micro-RNA-155 inhibits IFN-gamma signaling in CD4+ T cells, Banerjee [/bib_ref]. miR-155 specifically targets c-Maf, affecting activation of Th2 specific cytokine IL-4 [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref]. A reduced number of IFN-γ-producing cells lacking miR-155 results in T-cell dysfunction and antigen-presentation defects [bib_ref] Inositol phosphatase SHIP1 is a primary target of miR-155, O'connell [/bib_ref]. Phosphatidylinositol 3, 4, 5-trisphosphate 5-phosphatase 1 (SHIP1) has also been suggested as a functional target of miR-155 in CD4+ T cells e.g., macrophages [bib_ref] Inositol phosphatase SHIP1 is a primary target of miR-155, O'connell [/bib_ref] and dendritic cells [bib_ref] MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development, O'connell [/bib_ref]. The levels of SHIP1 are reduced in miR-155 −/− mice. SHIP1 suppresses Th1 responses [bib_ref] T cell-specific deletion of the inositol phosphatase SHIP reveals its role in..., Tarasenko [/bib_ref] and T-cells by modulating IFN-γ production [bib_ref] Epistasis between microRNAs 155 and 146a during T cell-mediated antitumor immunity, Huffaker [/bib_ref]. In human CD4+ Tcells, miR-155 targets the IFN-γ receptor alpha subunit and regulates proliferation of the Th1 and Th2 subsets is involved in the T cell response. Th1 cells up-regulate expression of major histocompatibility complex (MHC) class II and CD86 in antigen presenting cells such as macrophages. Aβ-reactive Th1 cells increase the secretion of inflammatory cytokines such as IFN-γ and TNF-α. miR-155 is associated with multiple process including the interaction between dendritic cells and T cells, and the regulation of Th17 and CD4+ T cell differentiation. It is also involved in regulating proliferation of Th1, Th2, and CD8+ T cells, and survival of Treg cells. 2010). Th17 cells are a newly defined subset of CD4+ T-cells that modulate autoimmunity by producing pro-inflammatory cytokines, including IL-17, IL-21, and IL-22 [bib_ref] IL-23 drives a pathogenic T cell population that induces autoimmune inflammation, Langrish [/bib_ref] [bib_ref] IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells, Korn [/bib_ref] [bib_ref] Interleukin-17 and type 17 helper T cells, Miossec [/bib_ref]. miR-155-deficient mice are characterized by reduced numbers of Th17 cells, and thus, suggest that miR-155 is required for Th17 differentiation [bib_ref] MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development, O'connell [/bib_ref] [fig_ref] FIGURE 2 |: miR-155 is associated with specific transcription genes regulating activation of T cells [/fig_ref]. Taken together, miR-155 appears to regulate the differentiation, proliferation, and activation of Th1, Th2, and Th17 cells in the inflammatory state.
## Treg cells
Treg cells play an important role in regulating the immune response and preventing autoimmunity [bib_ref] Regulatory T-cell physiology and application to treat autoimmunity, Tang [/bib_ref]. Both mouse and human Treg cells express a set of miRNAs [bib_ref] A role for Dicer in immune regulation, Cobb [/bib_ref] [bib_ref] Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3..., Rouas [/bib_ref] [bib_ref] Comprehensive analysis of miRNA expression in Tcell subsets of rheumatoid arthritis patients..., Smigielska-Czepiel [/bib_ref]. miR-155 has been reported to regulate the development of Treg cells by inducing forkhead box P3 (Foxp3), which regulates Treg cell survival in vivo [bib_ref] Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory..., Kohlhaas [/bib_ref] [bib_ref] Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1..., Lu [/bib_ref]. In line with this finding, miR-155 knock-out mice are observed to have reduced Treg cell numbers. Consequently, they had reduced STAT5 phosphorylation and IL-2 receptor signaling due to insufficient SOCS1 suppression [bib_ref] Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1..., Lu [/bib_ref]. Other studies postulate that miR-155 deficiency results in reduced numbers of Treg cells due to decreased proliferation and increased apoptosis [bib_ref] Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses, Lu [/bib_ref] [bib_ref] The miR-17 approximately 92a cluster of microRNAs is required for the fitness..., Skinner [/bib_ref] [fig_ref] FIGURE 2 |: miR-155 is associated with specific transcription genes regulating activation of T cells [/fig_ref]. Nevertheless, miR-155 appears to modulate the activation and proliferation of Treg cells during inflammation. The evidence suggests that miR-155 also regulates the Treg cell-mediated inflammation during AD.
## Cd8+ t-cells
Differentiation of naïve CD8+ T-cells into effector or memory cytotoxic T-cells (CTLs) depends upon activation following interaction with antigen-presenting cells [bib_ref] Dicer controls CD8+ T-cell activation, migration and survival, Zhang [/bib_ref]. A deficiency of miR-155 decreases CD8+ T-cell responses, whereas miR-155 overexpression increases CD8+ T-cell responses during inflammation [bib_ref] The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling, Gracias [/bib_ref] [bib_ref] Micro-RNA 155 is required for optimal CD8+ T cell responses to acute..., Lind [/bib_ref]. Antigen-specific CD8+ T cells lacking miR-155 show increased phosphorylation of STAT1 in response to Type I interferon signaling [bib_ref] The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling, Gracias [/bib_ref]. Inhibition of STAT1 and interferon regulatory factor 7 (IRF7) partially ameliorates the immune dysfunction of miR-155 deficient CD8+ T-cells in vivo [bib_ref] The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling, Gracias [/bib_ref]. report that miR-155 deficient CD8+ T-cells exhibit improved immune systems following SOCS1 overexpression. Taken together, miR-155 appears to affect the activation of CD8+ T-cells, which are involved in the expression of STAT1, IRF7, and SOCS1 during inflammation.
# Conclusions
Inflammatory and immune responses play a crucial role in AD pathogenesis. Thus, an appropriate regulation of diverse T-cell types may alleviate AD related severe pathologies. miR-155 controls characteristics such as survival, differentiation, proliferation, and activation of Th1, Th2, Th17, Treg, and CD8+ T-cells during inflammation. Admittedly, miR-155 is not easy to identify the absolute beneficial function or the absolute negative function on inflammation caused in AD through T cell regulation, suggesting that it is associated with the various T cell type responses and the complicated T cell signaling. However, this review suggests promising approaches for AD treatment, involving control of miR-155. Although findings from clinical studies are still in the preliminary stages, further studies involving modulation of miR-155 levels could enable development of effective treatments for AD.
# Author contributions
JS obtained the information and wrote the preliminary draft. JEL reviewed and revised the manuscript. JS and JEL revised details of the manuscript and provided overall supervision.
[fig] FIGURE 2 |: miR-155 is associated with specific transcription genes regulating activation of T cells. miR-155 regulates the development of Treg cells by inducing FOXP3, which plays an important role in Treg cell survival in vivo, and regulates the phosphorylation of STAT5 and SOCS1. SHIP1 increases the survival of T cells by modulating IFN-γ production. [/fig]
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Study protocol: a randomised controlled trial of the effects of a multi-modal exercise program on cognition and physical functioning in older women
Background: Intervention studies testing the efficacy of cardiorespiratory exercise have shown some promise in terms of improving cognitive function in later life. Recent developments suggest that a multi-modal exercise intervention that includes motor as well as physical training and requires sustained attention and concentration, may better elicit the actual potency of exercise to enhance cognitive performance. This study will test the effect of a multi-modal exercise program, for older women, on cognitive and physical functioning. Methods/design: This randomised controlled trial involves community dwelling women, without cognitive impairment, aged 65-75 years. Participants are randomised to exercise intervention or non-exercise control groups, for 16 weeks. The intervention consists of twice weekly, 60 minute, exercise classes incorporating aerobic, strength, balance, flexibility, co-ordination and agility training. Primary outcomes are measures of cognitive function and secondary outcomes include physical functioning and a neurocognitive biomarker (brain derived neurotrophic factor). Measures are taken at baseline and 16 weeks later and qualitative data related to the experience and acceptability of the program are collected from a sub-sample of the intervention group.Discussion: If this randomised controlled trial demonstrates that multimodal exercise (that includes motor fitness training) can improve cognitive performance in later life, the benefits will be two-fold. First, an inexpensive, effective strategy will have been developed that could ameliorate the increased prevalence of age-related cognitive impairment predicted to accompany population ageing. Second, more robust evidence will have been provided about the mechanisms that link exercise to cognitive improvement allowing future research to be better focused and potentially more productive.
# Background
Ageing is associated with decline in cognitive functions that are critical to independence, social engagement, and quality of life. Unchecked, this normal and gradual deterioration can advance to clinical cognitive impairment which, in turn, carries a higher risk for progression to dementia [bib_ref] Occurrence of cognitive impairment and dementia in the community: a 9-year-long prospective..., Caracciolo [/bib_ref] [bib_ref] Executive decline and dysfunction precedes declines in memory: the Women's Health and..., Carlson [/bib_ref]. As the proportion of people aged over 65 years continues to expand, it is estimated that, by 2050, dementia could affect some 106.2 million people globally [bib_ref] Forecasting the global burden of Alzheimer's disease, Brookmeyer [/bib_ref]. Age-related cognitive decline affects far more people than dementia [bib_ref] Incidence of dementia and cognitive impairment, not dementia in the United States, Plassman [/bib_ref] ; so, there is much to be gained at the economic, social and individual levels if an effective, inexpensive approach to age-related cognitive impairment, can be found.
Exercise training has been identified as a promising countermeasure, to age-related cognitive decline [bib_ref] Protocol for Fit Bodies, Fine Minds: a randomized controlled trial on the..., O'dwyer [/bib_ref]. The search for definitive evidence has seen a trend away from non-experimental and quasi-experimental research toward randomised controlled trials (RCTs). Systematic reviews of this literature suggest that formal exercise training resulting in increased cardiorespiratory fitness can improve cognitive performance in older people [bib_ref] Vanhees L: Physical activity and enhanced fitness to improve cognitive function in..., Angevaren [/bib_ref] [bib_ref] Fitness effects on the cognitive function of older adults: a meta-analytic study, Colcombe [/bib_ref] [bib_ref] A meta-regression to examine the relationship between aerobic fitness and cognitive performance, Etnier [/bib_ref]. However, there is still much to be clarified. While cardiovascular exercise demonstrates a moderate effect on cognition, according to a Cochrane review, there is insufficient evidence that cognitive improvements can be attributed to improved cardiovascular fitness [bib_ref] Vanhees L: Physical activity and enhanced fitness to improve cognitive function in..., Angevaren [/bib_ref]. The suggestion is that other factors may be involved and more recently a number of areas have been identified as important targets for future research. These include the motor aspect of exercise [bib_ref] Motor training induces experience-specific patterns of plasticity across motor cortex and spinal..., Adkins [/bib_ref] [bib_ref] Healthy mind in healthy body? A review of sensorimotor-cognitive interdependencies in old..., Schaefer [/bib_ref] ; and combined modality exercise interventions to address the limitations of repeatedly evaluating a single component of exercise training in relation to cognition [bib_ref] Physical and motor fitness are both related to cognition in old age, Voelcker-Rehage [/bib_ref]. Accordingly this study will adopt a novel approach by combining key emerging themes from the extant literature [bib_ref] Multi-modal exercise programs for older adults, Baker [/bib_ref]. Specifically, this study is a RCT of a multi-modal exercise intervention, based on a broad definition of fitness which includes motor (balance, co-ordination, agility, proprioception, flexibility and reaction time) [bib_ref] Physical and motor fitness are both related to cognition in old age, Voelcker-Rehage [/bib_ref] and physical (cardiovascular and resistance) components.
The mechanisms that underpin exercise-induced cognitive gains are also receiving greater attention. This focus has largely been propelled by evidence that indicates that the brain retains a life-long capacity to change and adapt in response to environmental and experiential stimuli, including exercise training [bib_ref] Neuronal and cognitive plasticity: a neurocognitive framework for ameliorating cognitive aging, Greenwood [/bib_ref]. This capacity, referred to as neuroplasticity, has been demonstrated in exercise trained ageing animals [bib_ref] Activity-dependent regulation of neuronal plasticity and self repair, Kempermann [/bib_ref] [bib_ref] Running enhances neurogenesis, learning, and long-term potentiation in mice, Van Praag [/bib_ref] [bib_ref] Exercise enhances learning and hippocampal neurogenesis in aged mice, Van Praag [/bib_ref] ; and the prospect of similar possibilities in older humans has attracted significant interest [bib_ref] Motor training induces experience-specific patterns of plasticity across motor cortex and spinal..., Adkins [/bib_ref] [bib_ref] Plasticity of brain networks in a randomized intervention trial of exercise training..., Voss [/bib_ref]. As yet, the mechanisms of human brain plasticity (changes in brain structure and function) and cognitive plasticity (changes in cognitive performance), are poorly understood. There are, however, theoretical indications that these changes may be more likely to occur as a result of interventions that involve novelty and complexity that necessitates mental effort [bib_ref] Physical and motor fitness are both related to cognition in old age, Voelcker-Rehage [/bib_ref] [bib_ref] Activity-dependent regulation of neuronal plasticity and self repair, Kempermann [/bib_ref] ; otherwise known as cognitive load [bib_ref] Load theory of selective attention and cognitive control, Lavie [/bib_ref]. Significantly sustained mental effort is a feature of motor skills acquisition. In addition, there is some evidence, mostly from animal studies, that exercise-induced brain changes may be mediated by neurotrophins (proteins that play a role in neurone development), such as, brain-derived neurotrophic factor (BDNF) [bib_ref] Plasticity of brain networks in a randomized intervention trial of exercise training..., Voss [/bib_ref]. BDNF may play a key role in brain plasticity by regulating the growth, maintenance and survival of neurons in the adult brain. Mixed results from the few human studies indicate that poorly understood gender effects may obscure the interpretation of BDNF levels as a biomarker of cognitive function [bib_ref] Oude Voshaar RC: Serum brain-derived neurotrophic factor: determinants and relationship with depressive..., Bus [/bib_ref]. Recently, Coelho et al. [bib_ref] Physical therapy intervention (PTI) increases plasma brain-derived neurotrophic factor (BDNF) levels in..., Coelho [/bib_ref] found higher levels of plasma BDNF in response to an exercise intervention in older women (before 351 ± 68 pg/ml and after 593 ± 79 pg/ml; p < 0.001). Accordingly this study will focus on older women.
A much less emphasised but fundamental subject in the extant literature is the question of the relevance of the tasks involved in traditional neurocognitive testing. It is unclear how well performance in various neurocognitive tests translates into the functional abilities and mental processes required for competency in the instrumental activities of daily living for older adults. Furthermore, the extent to which exercise-induced cognitive change correlates with (and mitigates) the processes that characterise age-related cognitive decline, is far from established. The notion of ecological validity or the extent to which test outcomes predict real-life behaviour [bib_ref] Ecological validity of measures of executive functioning, Odhuba [/bib_ref] , is not reflected in the present literature, but is increasingly being considered in RCTs generally, by the inclusion of a qualitative evaluation aimed at clarifying the everyday relevance and acceptability of study interventions and their intended or unintended outcomes.
A combination or multi-modal exercise intervention that includes motor as well as physical training and requires sustained attention and concentration, may better elicit the actual potency of exercise to enhance cognitive performance, in later life. Conducted as a randomised controlled trial, the study being undertaken will possess the design parameters with the greatest capacity to test the efficacy of such an intervention. Supplementary qualitative data provides greater insight into the clinical, functional and ecological (every-day) relevance of the intervention. In gathering early data into the effects of exercise on cognitive functioning in women (negating the gender effect) and on BDNF levels (as a biomarker of brain plasticity); more robust evidence will have been provided about the mechanisms that link exercise to cognitive improvement. If these questions can be answered future research may be more precisely focused and potentially more productive.
## Aims of the study
The study focuses on women aged 65-75 years, and aims to:
1. test the effect of a complex, multi-modal exercise program on cognitive functions, physical functioning and a biomarker (BDNF) of brain plasticity; 2. explore the associations between changes in cognitive functions and changes in BDNF, over time and; 3. describe the experience of individuals participating in a multi-modal exercise program.
A multi-modal exercise program has been developed by the first author (SV) in consultation with physiotherapists, exercise physiologists and psychologists. The effects of this exercise program are being evaluated in a randomized controlled trial. The theoretical basis of the program incorporates current understandings derived from the literature on ageing, cognitive neuroscience, psychology and exercise science.
# Methods /design
## Study design
The study is a 16-week randomised controlled trial of a multi-modal exercise program for women aged 65-75 years. The study is designed to assess the effect of an exercise intervention combining physical fitness training (aerobic and strength) and motor fitness training (balance, coordination, agility, reaction time and flexibility). Participants are randomly allocated to the intervention group or a no exercise wait list control group (See . Measurement of primary and secondary outcomes will take place two weeks prior to commencing the exercise training study (baseline) and within one week of completing the 16 weeks exercise or control period. The protocol has been approved by the Griffith University Human Research Ethics Committee (GU Ref No: PES/05/12/HREC).
## Setting
The study is being conducted in community halls on the Gold Coast which is located in South Eastern Queensland, Australia.
## Study population
The program is designed for non-cognitively impaired, community dwelling older adults. Volunteers are recruited through media coverage, local organisations such as 'Probus' and 'Senior Citizens' and Government agencies such as the Gold Coast City Council. To be considered for the study, individuals must be:
1. female and aged between 65 and 75 years; 2. doing less than 60 minutes of formal exercise training each week; and 3. able to attend classes twice each week on weekday mornings.
## Eligibility criteria
Exclusion criteria include: a score below 31 on the Telephone Interview of Cognitive Status (TICS) [bib_ref] Detection of dementia in the elderly using telephone screening of cognitive status, Welsh [/bib_ref] ; inability to obtain written clearance from a General Practitioner; a diagnosis of dementia or Parkinson's disease; the inability to walk 20 meters unaided and head injury within the previous 12 months.
## Informed consent
Informed written consent is obtained before baseline testing takes place.
## Sample size
One hundred participants will be recruited for the study. Sample size is determined using a power calculation to detect between group differences in the primary outcome measures. Previous randomised controlled trials of exercise interventions have shown effect sizes between 0.24 and 1.17, in similar populations, for cognitive performance outcome measures, as a result of aerobic interventions [bib_ref] Vanhees L: Physical activity and enhanced fitness to improve cognitive function in..., Angevaren [/bib_ref]. To obtain 80% statistical power with an α level of 0.05, using a t-test for two independent groups and an effect size of 0.6, the sample size required in each of the two groups is 36 participants (G*Power 3.1) [bib_ref] Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses, Faul [/bib_ref] [bib_ref] A flexible statistical power analysis program for the social, behavioral, and biomedical..., Faul [/bib_ref]. Samples of 50 for both the intervention and control groups are recruited, to allow for attrition.
## Randomisation
An automated randomisation service will allocate participants to one of two groups (intervention group or 'wait-list' controls). The randomisation service provided by The Clinical Trials Coordinating Centre (CTCC) The design of the study.
(Griffith University, Queensland, Australia) is accessed either by telephone or using web-based technology.
## Blinding
Exercise instructors are aware of the allocation of participants; however, all data collectors are blinded to group allocation. Data analysis is conducted by the first author (SV) on a de-identified database.
## Intervention
## Exercise training intervention
Participants in this group receive a multi-modal exercise class, twice weekly, for a period of 16-weeks. Classes are conducted for 60 minutes and the overall program is designed to include progressions and variations (See [fig_ref] Table 1: Overview of three phases of exercise intervention program [/fig_ref]. Each session includes aerobic (cardiovascular), strength (resistance) and motor fitness (balance, co-ordination, flexibility and agility) training in accordance with the ACSM Guidelines for Exercise Testing and Prescription(See [fig_ref] Table 2: Specifics of exercise intervention [/fig_ref] for details). The cardiovascular component is set to music and choreographed movements are cued in random rather than serial order. Strength training incorporates progressive weight training and weight bearing exercises involving the major muscle groups. Motor training involves both static and dynamic balance, coordination and agility requiring manoeuvring around and stepping over objects. Reaction time training includes ball activities and flexibility training involves dynamic and static stretches for all major muscle groups (See [fig_ref] Table 2: Specifics of exercise intervention [/fig_ref]. Sessions are conducted by accredited fitness instructors, with a maximum of 20 participants and an instructor-toparticipant ratio of 1:10.
## Control group
The control group is on a waiting list to attend the 16-week exercise program which commences at the end of the study; and after the post intervention measures have been completed by both groups. The classes are offered free of charge. Control group participants are contacted by telephone every 4 weeks to foster an ongoing sense of engagement in and relevance to the study.
## Intervention fidelity
Two instructors are present in each class. Only these two instructors will administer the intervention. At random intervals an independent assessor will observe all classes and monitor for content consistency using a check-list based on the explicit components of the exercise intervention protocol. In addition, instructors will maintain a log of activities in each class and these logs will also be reviewed by the independent assessor to check for intervention fidelity.
## Adherence to intervention protocol
Trained fitness instructors document attendance at each class. Intervention group are asked to plan to attend at least 85% of classes and are followed up by telephone if they are absent for two consecutive classes. In order to maximise participant contact and follow-up participants in both groups are asked to provide at least two sets of contact details; direct and via a friend or family member.
## Assessment protocol screening
Information sessions about the study are conducted at a number of local organisations that are attended by older adults. Individuals who express interest in the study at the information sessions are asked for their contact details and initial checks of age group and the ability to walk 20 meters unaided are completed. Individuals with preliminary eligibility are issued with General Medical Practitioner (GP) clearance forms which are subsequently used should the screening telephone interview reveal the requirement for GP clearance.
The formal screening process is conducted by telephone and takes approximately 15 minutes. It includes the TICS and the Pre-Activity Readiness Questionnaire (PAR-Q). Individuals who are otherwise eligible and who report a serious medical condition are asked to take clearance forms to their GPs to obtain written permission to participate. Once individuals have been assessed as being eligible to participate they are invited to attend a data collection and randomisation session where demographic and health information is collected along with baseline outcome measures.
## Baseline demographics, current activity levels and health information
Details of age, education, marital status, occupation and language used at home are collected by interview. Participants are asked to give details of any medical conditions that may affect physical activity (using the PAR-Q).
They are asked to complete the 21 item Depression, Anxiety and Stress Scale (DASS-21) which has good internal consistency as well as convergent and discriminant validity, especially for the depression scale [bib_ref] Psychometric properties of the Depression Anxiety and Stress Scale-21 in older primary..., Gloster [/bib_ref]. In addition, all participants will be fitted with a pedometer for five days to record activity levels at baseline.
## Outcome measurement
The primary outcome measure is neurocognitive test performance related to the processes of working memory, inhibition, shifting, verbal fluency and simple and complex reaction time i.e. executive function. These functions are highly associated with age-related cognitive decline, mobility and the ability to perform activities of daily living [bib_ref] Structural brain changes in aging: courses, causes and cognitive consequences, Fjell [/bib_ref] [bib_ref] Declining executive control in normal aging predicts change in functional status: the..., Royall [/bib_ref] [bib_ref] Olde Rikkert MGM: Executive functions are associated with gait and balance in..., Van Iersel [/bib_ref]. A recent randomised controlled trial of a resistance training program used similar primary outcome measures and demonstrated an effect for a twice a week exercise program [bib_ref] Resistance training and executive functions: a 12-month randomized controlled trial, Liu-Ambrose [/bib_ref]. The secondary outcome measures are blood serum levels of BDNF, physical and functional capacity including the 6 minute walk test, the 'timed up and go' test and the one-leg stance test of balance; and anthropometric values related to girth circumference at the waist and hip, resting heart rate and blood pressure. Physical functioning and cognitive assessment are conducted in the community (in halls) by exercise physiologists and psychologists, respectively. Anthropometric measurements are completed by a registered nurse. Cognitive testing requires less than 60 minutes and precedes physical assessment which lasts for 30-40 minutes. Participants are transported to an accredited blood collection facility. Blood samples are then conveyed to a laboratory for assay of plasma Brain Derived Neurotrophic Factor (BDNF).
## Primary outcome measures neurocognitive tests
California older adult stroop test (inhibition) Stroop tests assess the ability to suppress a habitual response in favour of an unusual response. They generally demonstrate good test-retest reliability (0.90, 0.83 and 0.91 for the three parts of the test)and minimal susceptibility to practice effects. The COAST is a Stroop test developed specifically for use with older populations [bib_ref] Yoash-Gantz R: California Older Adult Stroop Test (COAST): development of a stroop..., Pachana [/bib_ref]. Participants are required to first name colours (Colour), then read colour name words (Word); and then name ink colours when the names of colours are printed in a different (non-corresponding/incongruent) colour ink. During the incongruent condition, the two conflicting sources of colour information cause a competing effect (Interference) which is most typically observed as a prolonged reaction time compared to the neutral or congruent conditions [bib_ref] A comparison of label-based review and ALE meta-analysis in the Stroop task, Laird [/bib_ref]. Controlled oral word association test (COWAT) (verbal fluency) Different forms of the COWAT test measure verbal fluency and also draw on executive function and memory. Participants are given one minute to generate as many words as possible that begin with a specific letter. This task is repeated three times with three different letters (e.g. F, A, S). These tests generally demonstrate inter-scorer reliability of 0.70, retest reliability of 0.88 and concurrent validity.
Letter-number sequencing test (LNS) (working memory) The Letter-Number Sequencing testmeasures working memory as well as sequencing, attention and concentration abilities. The participant is read a combination of numbers and letters and is asked to recall the numbers first in ascending order and then the letters in alphabetical order. Each item consists of two trials, and each trial is a different combination of numbers and letters. There are seven items ranging from 2-letter/number sequences (e.g., B-7) to 8-letter/number sequences (e.g., S-2-L-8-B-1-G-7). The maximum score possible is 24 points. The validity and reliability of this test is well established in older adults with test-retest reliability in the range of 0.70 to 0.80 and a factor loading of 0.62 with the Working Memory Index.
Psychomotor speed (simple reaction time and complex reaction time) Reaction Time, the time between presentation of a stimulus and initiation of a response, is said to reflect psychomotor and processing speed; and is assessed with 1-choice and 4-choice stimulus conditions. Using computer based testing the first stimulus condition requires identification of a single object (e.g., yellow circle). The second stimulus condition requires the discrimination of a single object, amongst 4 object choices. Simple reaction time (SRT) and 4-choice reaction time (CRT) are calculated as the time (milliseconds) from perception (release of press-pad) to touch of appropriate stimulus on the screen. Split half reliability of up to 0.90 has been reported for an 18 task trialTrail making test (TMT) (part A & B) (executive function and shifting) The TMT is considered to be a test of visual search, attention, flexibility of thinking, motor function, and executive function. Part B of the TMT requires the individual to mentally shift between two well-rehearsed sequences (numbers and letters) as quickly and as accurately as possible. Shifting ability represents the capacity to adapt to changes in the environment by switching from one mental set to another.
In Part A, the task is to connect the numbers in ascending order (i.e.1-2-3-4. . .). Part B involves alternately connecting numbers and letters in ascending order (for the numbers) and sequential order for the alphabet letters (i. . The score derived for each trail is the number of seconds required to complete the task. Reliability tests for the TMT range from 0.60 to 0.90and the test is particularly useful for detecting early stages of dementia.
## Secondary outcome measures
Brain derived Neurotrophic factor (BDNF) Brain derived neurotrophic factor (BDNF) is a protein and the most abundant neurotrophin in the brain, with an important role in brain neurogenesis, synaptic plasticity, learning and memory [bib_ref] The positive impact of physical activity on cognition during adulthood: a review..., Ratey [/bib_ref] [bib_ref] Neuroplasticity -exerciseinduced response of peripheral brain-derived neurotrophic factor: a systematic review of..., Knaepen [/bib_ref]. The brain has been found to be the main source of BDNF concentrations in venous blood (circulating BDNF) [bib_ref] Evidence for a release of brain-derived neurotrophic factor from the brain during..., Rasmussen [/bib_ref]. Participants have their usual breakfast (1-2 hours before venipuncture) and are asked to refrain from exercise for 36 hours prior to blood draw. Eight milliliters of venous blood is drawn into EDTA tubes in the morning, to control for diurnal range variation in plasma BDNF levels [bib_ref] Measurements of brain-derived neurotrophic factor: methodological aspects and demographical data, Trajkovska [/bib_ref]. The samples are processed according to the manufacturer's specifications. Plasma is obtained by centrifugation of blood tubes for 15 min at 2000 × g and 24°C and is aliquoted and stored at −80°C until measured. Plasma samples are assessed for BDNF concentrations using a commonly used [bib_ref] Measurements of brain-derived neurotrophic factor: methodological aspects and demographical data, Trajkovska [/bib_ref] commercially available two-site sandwich enzyme-linked immunoabsorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).
## Physical functioning: six minute walk test (6mwt)
The Six-minute walk test is a functional validated measure of aerobic fitness (exercise capacity); and is based on the number of meters walked in six minutes. The 6MWT is performed indoors, along a 30 meter walking course that is flat and straight with a starting line and turnaround point clearly marked. The total distance walked is tallied using pre-marked intervals as a guide. The 6MWT is reported to have strong test-retest reliability when used before and after cardiac rehabilitation programs [bib_ref] Repeated six-minute walk tests for outcome measurement and exercise prescription in outpatient..., Bellet [/bib_ref]. A recent study has confirmed that while there is a learning effect that can increase the distance walked; when used as an outcome measure, one measurement is sufficient to show change in performance over time [bib_ref] Repeated six-minute walk tests for outcome measurement and exercise prescription in outpatient..., Bellet [/bib_ref].
Physical functioning: Timed Up-and-Go This is a clinical performance based measure of mobility, lower extremities function and fall risk. It is normally distributed, related to executive function and suitable for the assessment of healthy older adults [bib_ref] Properties of the 'Timed Upand Go' test: More than meets the eye, Herman [/bib_ref]. The TUG test (TUGT) will be conducted using a chair with arms and a seat height of 46 cm placed upon a flat, surface with cones marking the 3 m turning point [bib_ref] Predicting the probability for falls in community-dwelling older adults using the Timed..., Shumway-Cook [/bib_ref]. Subjects are instructed: "On the word 'go' , get up and walk as quickly and as safely as possible to cross the line marked on the path, turn around, walk back and sit down" [bib_ref] Predicting the probability for falls in community-dwelling older adults using the Timed..., Shumway-Cook [/bib_ref]. The activity will be timed from the subject's back leaving the back of the chair to the return of the subject to this same position. Using a standardised protocol, each subject will be required to perform one untimed, practice TUGT followed by two timed TUGTs (i.e. TUGT1 and TUGT2). At least four minutes of seated rest occurs between each TUGT.
Physical functioning: balance test (the one-leg stance test) The one-leg stance test [bib_ref] An ataxia test not requiring rails, Fregly [/bib_ref] requires participants to stand unassisted on one leg with hands on hips. Participants are asked to stand on one preferred leg, flex the opposite knee allowing the foot to clear the floor; then balance on the one leg for as long as possible. Timing begins when the leg is lifted and is timed in seconds until the person returns the non-weight bearing foot to the ground. The test has good reliability in older adults (ICC =0.92) [bib_ref] The relevance of clinical balance assessment tools to differentiate balance deficits, Mancini [/bib_ref].
Anthropometric measures Weight is measured using a digital flat weighing scale with a non-slip glass platform. Height (using a stadiometer), waist circumference and hip circumference are measured in centimeters. The waist is measured at the smallest circumference around the torso between the end of the xiphisternum and the top of the iliac crests. The hips are measured at the greatest circumference between the iliac crests and the upper femur. Resting blood pressure is measured using a sphygmomanometer (Welch Allyn 767 Series mobile aneroid sphygmomanometer) and stethoscope. Resting heart rate is calculated by palpation of the radial pulse for one minute. The anthropometric measurements are conducted by a registered nurse.
## Analyses
The data analysis for the randomised controlled trial will be undertaken on an intention-to-treat basis. To characterize the sample in this study, means or proportions and standard deviations (SDs) will be calculated by randomisation group for multiple baseline demographic, cognitive functioning and anthropometric variables. Unadjusted means and SDs for each cognitive performance measure obtained at the baseline and follow-up visits will be calculated. Missing outcome data will be imputed using multiple imputation [bib_ref] Intention-to-treat in randomized controlled trials: recommendations for a total trial strategy, Polit [/bib_ref]. Analysis of covariance will be undertaken for 16 week follow-up outcome measure scores, controlling for baseline cognition (TICS), and using baseline outcome values as covariates. Ninety-five per cent confidence intervals and effect sizes (standardised mean differences) will be calculated for each outcome measure. Differences in follow-up secondary outcome measure scores will be assessed for normality of distribution and either parametric or nonparametric statistical analyses applied as appropriate (e.g. ANCOVA, logistic regression etc.). Hierarchical multiple regression analyses will be used to investigate associations between plasma BDNF levels and a range of independent variables including demographic, anthropometric, cognitive functioning and physical functioning variables.
## Qualitative component
A qualitative component is embedded in the study to elicit information about the participants' experience of the intervention and perception of changes to physical and cognitive function. This component of the study will employ a triangulation approach to the incorporation of qualitative and quantitative data at the stage of results interpretation [bib_ref] Three techniques for integrating data in mixed methods studies, O'cathain [/bib_ref].
Face to face, audiotaped, individual interviews are undertaken with a sub-set of the study population allocated to the intervention group. Interviews are conducted with 12 to 15 participants who give written informed consent. The final number of interviews will be determined by data saturation which will be revealed during concurrent analysis. The interviews commence with questions such as, "Please describe a typical exercise class", "What aspects of the class did you find most/ least enjoyable?" "What aspects of the class did you find least/most beneficial?" "Why?" "What changes if any, have you noticed about yourself that you think may relate to class attendance?" Verbatim transcription of data will be undertaken and thematic analytic techniques will be used to analyse the data.
# Discussion
A future study, involving multi-modal exercise training and conducted in the form of a randomised controlled trial would be expected to extend previous work in a number of significant ways. Firstly, an adequately powered randomised controlled trial would fulfil a methodological recommendation that has consistently been made in systematic reviews of the extant literature. Secondly, to our knowledge, there are currently no studies where this particular design has been used to test whether the combination of cardiovascular, strength and motor fitness training can elicit cognitive effects in later life. Thirdly, a theory-driven intervention design allows a targeted approach that increases the likelihood of eliciting improved cognitive performance in the very regions of the brain that have most pertinence to age-related decline. Adding motor exercise training to an exercise intervention deliberately introduces the ingredient of movement based novelty and complexity [bib_ref] Physical and motor fitness are both related to cognition in old age, Voelcker-Rehage [/bib_ref] [bib_ref] Activity-dependent regulation of neuronal plasticity and self repair, Kempermann [/bib_ref]. Motor training requires sustained concentration and attention and is therefore inherently cognitively demanding [bib_ref] Practice of contemporary dance improves cognitive flexibility in aging, Coubard [/bib_ref].
Increased cognitive load engages higher order cognitive processes that largely activate the prefrontal cortex and it is this area of the brain that is said to most evidence the early declines associated with the ageing process [bib_ref] The positive impact of physical activity on cognition during adulthood: a review..., Ratey [/bib_ref]. Fourthly, while a properly designed randomised controlled trial is the gold standard for assessing the efficacy of an intervention in terms of statistical significance, the real-life relevance of outcomes may be better reflected in terms of the clinical significance of the study results. To this end, this study will triangulate psychometric data with qualitative data, to better reflect the impact of the intervention on the everyday experience of older women. Finally, there is still a gap in the literature regarding the mechanisms that might underpin the cognitive effects of exercise. Animal models have generated great interest in the role of BDNF and human studies demonstrate promise, however, more evidence is needed. Together the particular combination of design characteristics proposed for this study is expected to make a unique contribution to the current body of knowledge by furthering our understanding of the relationships between multi-modal exercise, age, cognitive function and BDNF in older women.
[table] Table 1: Overview of three phases of exercise intervention program [/table]
[table] Table 2: Specifics of exercise intervention [/table]
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Neuro-Immune Regulation in Inflammation and Airway Remodeling of Allergic Asthma
Allergic asthma is a common chronic inflammation of the airways and causes airway remodeling eventually. For a long time, investigators have been focusing on the immunological mechanism of asthma. However, in recent years, the role of neuroregulation in the occurrence of asthma has gradually attracted investigators' attention. In this review, we firstly describe neuro-immune regulation in inflammation of allergic asthma from two aspects: innate immunity and adaptive immunity. Secondly, we introduce neuro-immune regulation in airway remodeling of asthma. Finally, we prospect the role of pulmonary neuroendocrine cells in the development of asthma. In general, the amount of researches is limited. Further researches on the neural regulation during the occurrence of asthma will help us clarify the mechanism of asthma more comprehensively and find more effective ways to prevent and control asthma.
# Introduction
Allergic asthma is a chronic inflammation of the airways caused by repeated exposure to allergens such as dust, mites and pollens [bib_ref] The Immunology of Asthma, Lambrecht [/bib_ref]. It is a disease with high heterogeneity [bib_ref] Asthma Phenotypes: The Evolution From Clinical to Molecular Approaches, Wenzel [/bib_ref]. The hallmark of asthma is airway hyperresponsiveness (AHR) with symptoms of cough, shortness of breath, wheeze and chest tightness. Asthma is a serious global problem with the prevalence rate of 5.8% in children under in adults, and fatality rate of 1.3/100,000 (https://www.cdc.gov/nchs/fastats/ asthma.htm [Accessed . Asthma can be divided into Type 2-High and Type 2-Low subtypes according to whether T helper 2 (Th2) cells are dominant [bib_ref] The Basic Immunology of Asthma, Hammad [/bib_ref]. Type 2-High asthma, also known as allergic asthma, is characterized by large numbers of Th2 cells and group 2 innate Abbreviations: AHR, airway hyperresponsiveness; Th2, T helper 2; ILC2s, group 2 innate lymphoid cells; TSLP, thymus stromal lymphopoietin; ACh, acetylcholine; ASMs, airway smooth muscle cells; DCs, dendritic cells; CGRP, calcitonin generelated peptide; PNECs, pulmonary neuroendocrine cells; NT4, neurotrophin 4; OVA, ovalbumin; 5-HT, 5hydroxytryptamine; TGF-b, transforming growth factor b; b 2 -AR, b2-adrenergic receptor; NGF, nerve growth factor; EETs, eosinophil extracellular traps; NMU, neuromedin U; VIP, vasoactive intestinal peptide. lymphoid cells (ILC2s). By secreting type 2 cytokines, these two types of cells can initiate a series of signaling cascades [bib_ref] Revisiting Type 2-High and Type 2-Low Airway Inflammation in Asthma: Current Knowledge..., Robinson [/bib_ref] [bib_ref] Immunologic Mechanisms in Asthma, Boonpiyathad [/bib_ref]. It starts with the trigger of allergen which is captured by antigenpresenting cells such as dendritic cells. Then Th2 cells in lymph nodes are induced and activatedwith an antigen specific manner. In addition, injured epithelial cells release cytokines such as IL-25, IL-33, and thymus stromal lymphopoietin (TSLP), which can directly recruit and activate Th2 cells and ILC2s in an antigen-nonspecific manner [bib_ref] Bronchial Allergen Challenge of Patients With Atopic Asthma Triggers an Alarmin (IL-33,..., Wang [/bib_ref] [bib_ref] Intelectin Contributes to Allergen-Induced IL-25, IL-33, and TSLP Expression and Type 2..., Yi [/bib_ref]. These two types of cells produce a large number of type 2 cytokines and activate effector cells such as B cells, basophils and eosinophils, thus initiate the pulmonary inflammatory response [bib_ref] Human Eosinophils Constitutively Express a Unique Serine Protease, PRSS33, Toyama [/bib_ref] [bib_ref] Involvement and Possible Role of Eosinophils in Asthma Exacerbation, Nakagome [/bib_ref] , causing pathological changes such as AHR, excess mucus secretion, and airway remodeling [bib_ref] Contribution of Airway Eosinophils in Airway Wall Remodeling in Asthma: Role of..., Kuo [/bib_ref].
Although immune response plays a vital role in the occurrence and development of allergic asthma, neuro regulation, especially neuro-immune regulation in asthma has drawn more and more attention in recent years. Just as skin and intestine, the lung is an important organ that connects the body with the environment. Airway epithelial cells are exposed to all kinds of external irritants from air sources during breath. The lung is the first organ to sense and recognize dangers by alarming the body immediately and responding to these foreign invaders. During this process, nervous and immune system cooperate closely. They share many similarities, such as the same universal distribution almost all over the body, common transmitting mediators such as neurotransmitters and cytokines. In addition to exercising their basic innate defense function, the innate nervous/immune system of the body can also continuously promote adaptive regulation with the changes of external environment [bib_ref] Neuromodulation by the Immune System: A Focus on Cytokines, Salvador [/bib_ref]. Nervous and immune system interact with each other. Sensory nerve fibers express cytokine receptors, which can sense cytokines and send messages to the brain via the autonomic nervous system [bib_ref] Neuromodulation by the Immune System: A Focus on Cytokines, Salvador [/bib_ref] [bib_ref] Pain and Immunity: Implications for Host Defence, Baral [/bib_ref] [bib_ref] The Meningeal Lymphatic System: A New Player in Neurophysiology, Mesquita [/bib_ref]. Neurotransmitters can also regulate immune responses through neural receptors on the surface of immune cells [bib_ref] The Neuropeptide NMU Amplifies ILC2-Driven Allergic Lung Inflammation, Wallrapp [/bib_ref] [bib_ref] Calcitonin Gene-Related Peptide Negatively Regulates Alarmin-Driven Type 2 Innate Lymphoid Cell Responses, Wallrapp [/bib_ref]. This review focuses on neuro-immune regulation in inflammation and airway remodeling of allergic asthma.
## Neuro-immune regulation in inflammation of allergic asthma
The lung is a highly innerved organ. Nerves in the lung can be divided into sensory or afferent nervous system and motor or efferent nervous system according to the signal direction travelling within the nerve [bib_ref] Overview of Innate Lung Immunity and Inflammation, Riches [/bib_ref] [bib_ref] KCNQ/M-Channels Regulate Mouse Vagal Bronchopulmonary C-Fiber Excitability and Cough Sensitivity, Sun [/bib_ref]. Sensory nerves from the airways relay stretch stimuli, mechanoreceptors, and chemical stimuli, chemoreceptors, along afferent sensory fibers or vagus nerves to the central nervous system [bib_ref] Overview of Innate Lung Immunity and Inflammation, Riches [/bib_ref]. Sympathetic, parasympathetic, non-adrenergic and non-cholinergic parasympathetic nerves constitute the motor pathways of the lung. The parasympathetic nerve releases acetylcholine (ACh) and activates muscarinic M3 receptors on airway smooth muscle cells (ASMs), resulting in the effects of bronchial contraction [bib_ref] Control of Neurotransmission by NaV1.7 in Human, Guinea Pig, and Mouse Airway..., Kocmalova [/bib_ref] [bib_ref] Airway and Lung Remodelling in Chronic Pulmonary Obstructive Disease: A Role for..., Roth [/bib_ref]. The pulmonary sympathetic nerve innervates the blood vessels and submucosal glands of the bronchus and antagonizes parasympathetic action by noradrenaline [bib_ref] Reflex Regulation of Airway Sympathetic Nerves in Guinea-Pigs, Oh [/bib_ref]. Non-adrenergic non-cholinergic parasympathetic nerves may mediate bronchiectasis through vasoactive intestinal peptides and nitric oxide [bib_ref] Neurotransmitters in Parasympathetic Ganglionic Neurons and Nerves in Mouse Lower Airway Smooth..., Balentova [/bib_ref].
## Neuro-immune regulation in innate immune cells
Like a guard, dendritic cells (DCs) are the first to sound the alarm when allergens invade. Allergen capture by antigen-presenting cells such as DC cells is the beginning of allergic asthma. Nervous system regulates DCs by neuropeptides. Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by pulmonary neuroendocrine cells (PNECs) mainly in lung. It inhibits maturation of DCs by CGRP receptors expressed in DCs themselves, reducing the activation and proliferation of antigenspecific T cells [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. In mouse model of ovalbumin (OVA) induced asthma, CGRP-pretreated DCs alleviate inflammation of allergic asthma with reduced eosinophil numbers in bronchoalveolar lavage fluid [bib_ref] The Neuropeptide Calcitonin Gene-Related Peptide Affects Allergic Airway Inflammation by Modulating Dendritic..., Rochlitzer [/bib_ref]. Interestingly, the nerve cells can also be a whistleblower to allergens thus giving instructions to DCs. A study using skin allergen exposure model found that allergens can directly activate transient receptor potential vanilloid 1 positive sensory neurons in skin, causing itch and pain behaviors. Activated neurons release neuropeptides such as substance P, which can induce the migration of CD301b + DCs into the draining lymph nodes, where the differentiation of Th2 cells is initiated [bib_ref] Substance P Release by Sensory Neurons Triggers Dendritic Cell Migration and Initiates..., Perner [/bib_ref]. Whether the nervous system is also the first responder of allergens during asthma, thereby triggering the accumulation of dendritic cells and subsequent immune responses needs further exploration. If so, inhibition of sensitization by the allergen to sensory nerve fibers could potentially reduce the activation of pulmonary inflammation during asthma. Besides, antigens can also lead to the variation of innervation, regulating inflammatory infiltration in tissues in an age-related manner. In neonatal mice, exposure to antigens could elevate neurotrophin 4 (NT4) levels of ASMs thus increase ASMs innervation through NT4/TrkB signaling. As a result, persistent AHR appears in adulthood (28) [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. Therefore, in addition to the familiar antigen-presentation process, the interaction between the nervous system and antigens might be more important during the initial phase of inflammation.
In asthma, mast cells accumulate within or close to epithelium and smooth muscle under the stimulation of allergen [bib_ref] The Role of Mast Cells in the Structural Alterations of the Airways..., Carter [/bib_ref]. Mast cells could mediate acute inflammatory response of asthma by secreting a large number of pro-inflammatory and pro-airway constrictor mediators [bib_ref] The Role of Mast Cells in the Structural Alterations of the Airways..., Carter [/bib_ref] [bib_ref] Mast Cells and Their Progenitors in Allergic Asthma, Mendez-Enriquez [/bib_ref] [bib_ref] Mast Cell-Mediated Orchestration of the Immune Responses in Human Allergic Asthma: Current..., Ali Komi [/bib_ref] [bib_ref] Thirdhand Smoke Component can Exacerbate a Mouse Asthma Model Through Mast Cells, Yu [/bib_ref]. Mast cell is now recognized to be involved in nerve regulation during the development of asthma-with muscarinic M3 receptors on its surface [bib_ref] Mast Cell-Derived Serotonin Enhances Methacholine-Induced Airway Hyperresponsiveness in House Dust Mite-Induced Experimental..., Mendez-Enriquez [/bib_ref]. Bronchial stimulation test by methacholine provocations is a classic method used for clinical diagnosis of asthma. Mast cells may play the key role in this response. Under the stimulation of methacholine, mast cells release 5-hydroxytryptamine (5-HT), interact with 5-HT2 receptors in parasympathetic nerves, thus release ACh [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. ACh, an endogenous neurotransmitter, enhances bronchoconstriction and AHR in house dust mite induced experimental asthma [bib_ref] Mast Cell-Derived Serotonin Enhances Methacholine-Induced Airway Hyperresponsiveness in House Dust Mite-Induced Experimental..., Mendez-Enriquez [/bib_ref]. Therefore, mast cells and parasympathetic neurons cooperate to form a vicious circle that amplifies the bronchial constriction of choline. Asthma gets worse as the result. Why are mast cells involved in parasympathetic regulation of ASMs? Why the parasympathetic nerve manipulates mast cells to enhance its control on ASMs? Is it the result of the body's rapid response to foreign substances? Studies showed that the interference of mast cells in ASMs occur with the development of ASMs. Following allergen exposure during early-life, mast cells in lung can produce amount of NT4, which is essential for the innervation of ASMs during development [bib_ref] Mast Cell-Derived Neurotrophin 4 Mediates Allergen-Induced Airway Hyperinnervation in Early Life, Patel [/bib_ref] , causing longterm airway dysfunction. Mast cells can also produce transforming growth factor b (TGF-b) to induce b2-adrenergic receptor (b 2 -AR) phosphorylation in ASMs, thereby reducing the airway dilation effect of b 2 -AR agonists (35) [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. Therefore, during the occurrence of allergic asthma, mast cells are activated by immunity and affect ASMs under nerve regulation, resulting in persistent airway contraction and AHR. Obviously, mast cells are closely related to the nervous system. Mast cells seem to control the distribution and function of nerves. Besides inflammatory factors, are mast cells also regulated by neurotransmitters? After all, signals travel much faster in nerves. More research is expected.
Eosinophil is accepted as an important kind of effector cells in the development of allergic asthma. Extensive eosinophil infiltration around the airway is considered a hallmark of allergic asthma [bib_ref] Multiple Biological Aspects of Eosinophils in Host Defense, Eosinophil-Associated Diseases, Immunoregulation, and..., Kanda [/bib_ref] [bib_ref] The Eotaxin Chemokines and CCR3 Are Fundamental Regulators of Allergen-Induced Pulmonary Eosinophilia, Pope [/bib_ref] [bib_ref] Current Concepts of Severe Asthma, Ray [/bib_ref]. However, growing evidence suggests that eosinophils are inextricably linked to the nervous system during the development of asthma. Tropomyosin receptor kinase A is a high affinity receptor for nerve growth factor (NGF). In a tropomyosin receptor kinase A-knock-in mouse model, eosinophils are found to migrate to airway during inflammation via eotaxin-1 [bib_ref] Regulation of Eosinophil Recruitment and Allergic Airway Inflammation by Tropomyosin Receptor Kinase..., Dileepan [/bib_ref]. Moreover, eosinophils are closely related to nerve fibers in asthma [bib_ref] Eosinophil and Airway Nerve Interactions in Asthma, Drake [/bib_ref] [bib_ref] Eosinophil and Airway Nerve Interactions, Kingham [/bib_ref]. Under the action of VLA-4 and CD11b, eosinophils adhere to VCAM-1 and ICAM-1 on parasympathetic fibers (41) [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. It was found that in guinea pigs [bib_ref] Antibody to VLA-4, But Not to L-Selectin, Fryer [/bib_ref] and monkeys (43) during allergy, eosinophil binding to VCAM-1 or ICAM-1 is important in airway hyperreactivity. Eosinophils are then activated and release major basic protein (MBP), which is an antagonist of M2 muscarinic receptor in human, thus enhancing parasympathetic mediated bronchoconstriction (40) [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. Besides, airway and peripheral blood eosinophils are associated with increased airway innervation in asthma patients. Eosinophils increase airway sensory innervation in mice and humans, which is remarkable in moderate persistent asthma, thus leading to AHR [bib_ref] Eosinophils Increase Airway Sensory Nerve Density in Mice and in Human Asthma, Drake [/bib_ref]. Strikingly, a novel study suggests that eosinophils play a much more interesting role in neuro-immune regulation than we previously have known. Eosinophil extracellular traps (EETs) are web-like DNA traps generated from eosinophils [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. In asthma tissues from human and mice, PNECs are surrounded by EETs and activated via the CCDC25-ILK-PKCa-CRTC1 pathway [bib_ref] Eosinophil Extracellular Traps Drive Asthma Progression Through Neuro-Immune Signals, Lu [/bib_ref]. It appears that PNECs are one type of the target cells of eosinophils, but previous studies have shown that eosinophils are influenced by Eosinophils are then activated and release MBP, which is an antagonist of M2 muscarinic receptor, thus enhancing parasympathetic mediated bronchoconstriction. Besides, eosinophils increase airway innervation. Under the stimulation of methacholine, mast cells release 5-HT, interacting with 5-HT 2 receptors in parasympathetic nerves, thus release ACh (neuronal ACh), enhancing bronchoconstriction. Mast cells also produce NT4 following allergen exposure during early-life, increasing ASMs innervation through NT4/TrkB signaling, causing long-term airway dysfunction. Mast cells also produce TGF-b to induce b2-AR phosphorylation in ASMs, thereby causing b2-AR agonists resistance. Neuropeptides generate form neurons and neuroendocrine cells such as NMU and VIP activate ILC2s. CGRP generated mainly from PNECs inhibits maturation of DCs, it may have bidirectional effects on ILC2s. Besides neuronal ACh, non-neuronal ACh released from epithelia cells and macrophages mainly acted in small airways. ACh activate muscarinic 3 acetylcholine receptor on ASMs and fibroblast cells, causing airway contraction and airway remodeling. NGF released from neurons, epithelial cells, ASMs and other immune cells acts on fibroblasts leading to fibrosis. NGF can also activate Th2 cells and promote the differentiation of B cells into plasma cells. EETS, Eosinophil extracellular traps; PNEC, pulmonary neuroendocrine cell; MBP, major basic protein; 5-HT, 5-hydroxytryptamine; ACh, acetylcholine; NT4, neurotrophin 4; ASMs, airway smooth muscle cells; NGF, nerve growth factor. cells such as PNECs [bib_ref] Pulmonary Neuroendocrine Cells Amplify Allergic Asthma Responses, Sui [/bib_ref]. Such interactive network regulation is not unusual for sophisticated organisms like mammals, and suggests that we need to take a dialectical view of disease.
ILC2s are important players in both neural and immune regulation. They play a vital role in asthma by releasing type 2 cytokines. Besides respond to alarmins IL-25, IL-33 and TSLP which are released by epithelium, ILC2s could also be regulated by other stimuli. ILC2s are bidirectional regulated by the nervous system [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. In vitro, Nmur1 is highly expressed in ILC2s purified from mice, and neuropeptides neuromedin U (NMU) activate ILC2s by interacting with NMUR [bib_ref] The Neuropeptide NMU Amplifies ILC2-Driven Allergic Lung Inflammation, Wallrapp [/bib_ref] [bib_ref] The Neuropeptide Neuromedin U Stimulates Innate Lymphoid Cells and Type 2 Inflammation, Klose [/bib_ref] [bib_ref] Neuronal Regulation of Type 2 Innate Lymphoid Cells via Neuromedin U, Cardoso [/bib_ref]. In house dust induced airway inflammation model, NUM amplifies allergic inflammation with the combination of IL-25 [bib_ref] The Neuropeptide NMU Amplifies ILC2-Driven Allergic Lung Inflammation, Wallrapp [/bib_ref]. Vasoactive intestinal peptide (VIP) is another neuropeptide that belongs to non-adrenergic, non-cholinergic (NANC) system. VIP is one of the most potent endogenous bronchodilators and is proposed to have anti-inflammatory effects [bib_ref] Inhalable Powder Formulation of a Stabilized Vasoactive Intestinal Peptide (VIP) Derivative: Anti-Inflammatory..., Misaka [/bib_ref]. However, recent study find it expressed at high levels in nodose ganglion neurons in the lung and has a positive stimulating effect on ILC2. Under the stimulation of IL-5 and OVA, the generation of VIP increase in nodose ganglion neurons both in vitro and in vivo. VIP may promote ILC2s activation partly by VIP-VPAC2 axis thus amplify the airway inflammatory [bib_ref] Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation, Talbot [/bib_ref]. ILC2s are found resided in proximity to PNECs in naïve mice, and when cultured with CGRP and IL-33, ILC2s are activated with increased IL-5 production (46). However, another study analyzed single-cell RNA-seq atlas of lung ILCs and found that ILC2s express both CGRP and its encoding genes, and endogenous CGRP derived from neurons and neuroendocrine cells could negatively regulate IL-33 or IL-25 driven pulmonary ILC2s response, inhibit the production of ILC2-derived type 2 cytokines, thus reduce tissue damage under certain conditions [bib_ref] Calcitonin Gene-Related Peptide Negatively Regulates Alarmin-Driven Type 2 Innate Lymphoid Cell Responses, Wallrapp [/bib_ref]. In a study of Nippostrongylus brasiliensis infection mouse model also showed that a subset of ILC2s in lung express CGRP receptor components, and CGRP limits ILC2s response and worm clearance [bib_ref] Neuropeptide CGRP Limits Group 2 Innate Lymphoid Cell Responses and Constrains Type..., Nagashima [/bib_ref]. This suggests the complexity of CGRP function and ILC2s microenvironment in different disease models. Many neuro-derived factors can inhibit type 2 immune responses mediated by ILC2s. b 2 -AR is an important member of the adrenergic nervous system. b 2 -AR gene is detected in murine and human ILC2s. In N. brasiliensis and Alternaria alternata induced murine lung inflammation models, ILC-deficient mice exhibit increased levels of ILC2s in the lung after infection, which could be inhibited by b 2 -AR agonist treatment. b 2 -AR agonist is an important class of medicine in the treatment of asthma. It can reduce bronchial constriction by targeting b 2 -AR on ASMs. Whether b 2 -AR signaling also alleviate asthma symptoms by dampening ILC2 responses needs further study. a7-nicotinic acetylcholine receptor (a7nAChR) is thought to have anti-inflammatory effect in inflammatory diseases [bib_ref] Nicotinic Acetylcholine Receptor Alpha7 Subunit is an Essential Regulator of Inflammation, Wang [/bib_ref]. a7nAChR is found expressed on murine ILC2s and a7nAChR agonist inhibited ILC2s function with decreased IL-5 and IL-13 production in vitro. By giving intranasal recombinant mouse IL-33 to Rag2deficient mice (devoid of T and B cells), ILC2s are induced and a7nAChR agonist treatment could ameliorate ILC2s-mediated AHR by decreasing the expression of GATA-3, a key transcription factor of ILC2s [bib_ref] Nicotinic Acetylcholine Receptor Agonist Attenuates ILC2-Dependent Airway Hyperreactivity, Galle-Treger [/bib_ref]. In conclusion, ILC2s express a variety of neuropeptide receptors. As the innate immune cells in the lung, ILC2s become the pivot of lung neuroimmune regulation.
We know that there are other types of innate immune cells, like macrophages, basophils and neutrophils. A special group of macrophages have been found near the large bronchi and airway nerves, named nerve-and airway-associated macrophages [bib_ref] Identification of a Nerve-Associated, Lung-Resident Interstitial Macrophage Subset With Distinct Localization and..., Ural [/bib_ref]. This kind of macrophages can activate neurons to produce colony stimulating factor 1 (CSF1) by producing bone morphogenic protein 2, and CSF1 is the necessary signal to maintain its survival. So, nerve fibers provide nutrients for the survival of macrophages. But researches on their relationship with nervous system is still insufficient.
## Neuro-immune regulation in adaptive immunocytes
As we mentioned above, large numbers of Th2 cells is a hallmark of Type 2-high asthma. Th2 cells, induced and activated in lymph nodes by DCs in an antigen specific manner, produce large amounts of type 2 cytokines thus activate further immune cascades. Stimulated by Th2 cells, B cells mature into plasma cells and secrete IgE in response to cytokines IL-4 and IL-13 [bib_ref] Allergen-Specific IgG(+) Memory B Cells are Temporally Linked to IgE Memory Responses, Hoof [/bib_ref]. IgE interacts with cells that have receptors FcϵRI and CD23, such as mast cells, basophils, DCs and ASMs, causing acute allergic response and AHR [bib_ref] The High Affinity IgE Receptor (FcepsilonRI) Expression and Function in Airway Smooth..., Redhu [/bib_ref].
Differentiation of T cells into Th2 cells is a landmark in the development of allergic asthma, and the subsequent production of large amounts of type 2 cytokines cause persistent inflammatory changes. These are also important molecular indicators for determining asthma severity and evaluating the effectiveness of interventions in various studies. T cells are certainly an important battleground for the nervous system to participate in immune regulation. As a transient receptor potential ankyrin channel, transient receptor potential ankyrin 1 (TRPA1) is widely expressed in sensory neurons. It can be triggered by wide variety of stimuli and release amounts of inflammatory neuropeptides and neurotransmitters, facilitating the communication between the nervous system and the immune system [bib_ref] Mammalian Transient Receptor Potential TRPA1 Channels: From Structure to Disease, Talavera [/bib_ref]. Furthermore, adaptive immune cells may directly participate in the battlefield of neuroimmune response through TRPA1. The expression of TRPA1 is increased in lung tissues and CD4 + T cells in an OVA-induced mouse model of asthma, accompanied with the development and exacerbation of asthma [bib_ref] The Neuro-Immune Interaction in Airway Inflammation Through TRPA1 Expression in CD4+ T..., Li [/bib_ref]. It provides a complement to non-neural sources of TRPA1. As we have discussed before, the neurotransmitter VIP regulates the immune system by acting on its receptor VPAC2. With the help of VPAC2 expressed on T cells, VIP promotes the differentiation of CD4 + T cells into Th2 cells and increase the production of IL-5 and IL-13 correspondingly (50). NGF has been suggested to play an important role in neuroimmune regulation in airway inflammation by overwhelming studies [bib_ref] NGF and Its Receptors in the Regulation of Inflammatory Response, Minnone [/bib_ref] [bib_ref] Brain-Derived Neurotrophic Factor in the Airways, Prakash [/bib_ref] [bib_ref] Small Interfering RNA Targeting Nerve Growth Factor Alleviates Allergic Airway Hyperresponsiveness, Chen [/bib_ref]. The aggravating role of NGF in lung inflammation can be explained partly by regulating T and B cells. NGF exacerbates inflammation and airway remodeling by enhancing Th2 in OVA sensitized asthma in rat model [bib_ref] Nerve Growth Factor Exacerbates Allergic Lung Inflammation and Airway Remodeling in a..., Yang [/bib_ref]. Suppression of NGF could reduce Th2 immune response, decrease inflammatory infiltration and airway response in murine asthma model [bib_ref] Blockage of Nerve Growth Factor Modulates T Cell Responses and Inhibits Allergic..., Shi [/bib_ref]. The above effects in lung inflammation resulting from inhibition of NGF is also observed in mice infected with respiratory syncytial virus (RSV) [bib_ref] Neutralization of Nerve Growth Factor (NGF) Inhibits the Th2 Response and Protects..., Wu [/bib_ref]. And RSV infection in infants is considered a risk factor for the development of asthma [bib_ref] Role of Viral Infections in the Development and Exacerbation of Asthma in..., Jartti [/bib_ref] [bib_ref] Integrated Omics Endotyping of Infants With Respiratory Syncytial Virus Bronchiolitis and Risk..., Raita [/bib_ref]. It seems that NGF is a broad and powerful molecule involved in neuroimmune inflammation. It is worth noting that the regulation of adaptive immunity by the nervous system may partly explain the higher incidence of allergic asthma in children. It is found that during postnatal development in mice and humans, sympathetic nerves in the lung in mice and humans undergo a transition from dopaminergic type to adrenergic type. In this process, dopamine is bound to a specific dopamine receptor, DRD4, then IL-2-STAT5 signaling is upregulated and histone trimethylation is induced at Th2 gene loci, as a result, differentiation of CD4 + T cell to Th2 cell is promoted [bib_ref] Age-Related Dopaminergic Innervation Augments T Helper 2-Type Allergic Inflammation in the Postnatal..., Wang [/bib_ref]. Thus, the dopamine-DRD4 pathway augments Th2 inflammation in the lung of young mice in allergen exposure models, which is not so evident in the lungs of adult mice dominated by the adrenergic type. NGF can also affect the function of plasma cells in the development of asthma. In a mouse model of OVA induced allergic asthma, plasma cells from airways and spleen express different patterns of neurotrophins receptors, and TrkA is only expressed on pulmonary plasma cells. In vitro, NGF promotes survival of pulmonary plasma cells by increasing transcription factors in plasma cells (X-box binding protein 1 and NF-kB subunit RelA) responsible for production of immunoglobulins. Consistently, anti-NGF treatment reduce the number of pulmonary plasma cells and serum IgE [bib_ref] Nerve Growth Factor and Neurotrophin-3 Mediate Survival of Pulmonary Plasma Cells During..., Abram [/bib_ref]. B cell activation and subsequent production of large amounts of antigen-specific IgE are known to be characteristic of allergic asthma. However, studies on neural regulation on B cells or plasma cells in asthma are handful and years in advance. Interestingly and excitingly, FcϵR1, a highaffinity IgE receptor, is found expressed on vagal nociceptor neurons in lung in an OVA sensitized mouse model. It means that this kind of neurons can sense invading allergens directly, inducing allergic inflammation [bib_ref] FcepsilonR1-Expressing Nociceptors Trigger Allergic Airway Inflammation, Crosson [/bib_ref]. This finding is an important addition to the understanding of neuro-immune regulation in the development of asthma, and more research is expected.
## Neuro-immune regulation in airway remodeling of allergic asthma
Airway remodeling, the ultimate pathological change in the development of many pulmonary diseases, is one of the most characteristic pathological features of persistent asthma and main reason for hospital care [bib_ref] Mucus Plugs in Patients With Asthma Linked to Eosinophilia and Airflow Obstruction, Dunican [/bib_ref]. ASMs and fibroblasts play important roles in airway remodeling, and they are closely related to the nervous system.
ASMs express receptors for IgE and respond to IgE directly, causing airway obstruction in severe asthma. ASMs is the main kind of effector cell of AHR and airway remodeling, especially in small airways [bib_ref] The Role of the Small Airways in the Pathophysiology of Asthma and..., Bonini [/bib_ref]. ASMs becomes innervated by parasympathetic fibers since embryogenesis, and its development partly rely on ACh secreted from parasympathetic nerves [bib_ref] Targeting Acetylcholine Receptor M3 Prevents the Progression of Airway Hyperreactivity in a..., Patel [/bib_ref]. In asthma, the airway tone is increased mainly because of ASMs contraction, induced by ACh [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. ACh is derived from two ways--neuronal (the classic) and non-neuronal way. The former is released from nerves like vagus and parasympathetic nerves, the latter comes from epithelial cells and macrophages, which is the main source in small airways [bib_ref] Long-Acting Muscarinic Antagonists and Small Airways in Asthma: Which Link?, Cazzola [/bib_ref]. ACh activated muscarinic 3 acetylcholine receptor on ASMs and fibroblast cells, causing airway contraction and airway remodeling [bib_ref] Long-Acting Muscarinic Antagonists and Small Airways in Asthma: Which Link?, Cazzola [/bib_ref] [bib_ref] Pharmacology and Therapeutics of Bronchodilators Revisited, Matera [/bib_ref]. It is unclear whether the ACh from these two sources is different. Besides, just as we discussed, eosinophils increase parasympathetic ACh release by inhibiting M2 receptor function. An increase in airway eosinophils specifically enhances bronchial constriction in mice, independent of changes in muscarinic M3 receptors or 5-HT receptors in ASMs [bib_ref] Lung Eosinophils Increase Vagus Nerve-Mediated Airway Reflex Bronchoconstriction in Mice, Nie [/bib_ref]. Apparently, the body considers neuromodulation to be an efficient and economical way. Even without the involvement of nerve fibers, various cells of the body communicate with each other through neurotransmitters. During evolution for over tens of millions of years, human beings and other mammals have developed such elaborate structures. Making full use of the intrinsic transmitters and receptors not only enables us to respond quickly to the environment, but also is more cost-effective --far more efficient and economic than evolving one or more extra regulatory systems.
Excessive collagen deposition and subepithelial fibrosis is another striking feature of airway remodeling [bib_ref] Immunologic and Non-Immunologic Mechanisms Leading to Airway Remodeling in Asthma, Fang [/bib_ref] [bib_ref] Airway Remodeling in Asthma: What Really Matters, Fehrenbach [/bib_ref] [bib_ref] Pathobiology of Severe Asthma, Trejo Bittar [/bib_ref]. Almost all these mechanisms are associated with the activation of fibroblasts and its trans-differentiation into myofibroblasts [bib_ref] Enhanced Asthma-Related Fibroblast to Myofibroblast Transition is the Result of Profibrotic TGF-Beta/Smad2/3..., Wnuk [/bib_ref] [bib_ref] TGF-Beta1 Evokes Human Airway Smooth Muscle Cell Shortening and Hyperresponsiveness via Smad3, Ojiaku [/bib_ref]. TGF-b1 is identified as one of the important factors in this process [bib_ref] TGF-Beta Signaling in Lung Health and Disease, Saito [/bib_ref]. However, more and more researches reveal that neural regulation also plays a vital role [fig_ref] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma [/fig_ref]. Previous studies have shown that NGF was highly increased during allergic asthma, especially in chronic phase [bib_ref] Nerve Growth Factor Induces Type III Collagen Production in Chronic Allergic Airway..., Kilic [/bib_ref]. And NGF induced the production of type III collagen in fibroblasts by activating p38 MAPK in a TGF-b1 independent way, thus promoting fibrosis [bib_ref] Nerve Growth Factor Induces Type III Collagen Production in Chronic Allergic Airway..., Kilic [/bib_ref]. Interestingly, activated airway epithelium is a major source of NGF in allergic airway inflammation [bib_ref] Airway Epithelial Cells Produce Neurotrophins and Promote the Survival of Eosinophils During..., Hahn [/bib_ref]. And NGF can also be produced by ASMs and infiltrated immune cells including mast cells, T cells, eosinophils [bib_ref] Human Eosinophils Produce Neurotrophins and Secrete Nerve Growth Factor on Immunologic Stimuli, Kobayashi [/bib_ref] [bib_ref] The Neurotrophins Nerve Growth Factor, Brain-Derived Neurotrophic Factor, Neurotrophin-3, and Neurotrophin-4 Are..., Nassenstein [/bib_ref] [bib_ref] Upregulation of Nerve Growth Factor Expression by Human Airway Smooth Muscle Cells..., Freund [/bib_ref] [bib_ref] Differential Regulation of Neurotrophin Expression in Human Bronchial Smooth Muscle Cells, Kemi [/bib_ref]. It seems that when a man is going down-hill, everyone will give him a push.
Besides, macrophage may promote the development of pulmonary fibrosis (91) by affecting nerves in the lung. In a bleomycin inhaled mouse model, macrophage-derived neuronal guidance proteins such as netrin-1 is involved in pulmonary adrenergic nerve remodeling and promotes pulmonary fibrosis (92).
## Regulation of pnecs in allergic asthma
PNECs are a rare and evolutionarily conserved type of lung epithelial cells, accounting for about 0.5% of airway epithelial cells [bib_ref] Number and Proliferation of Neuroendocrine Cells in Normal Human Airway Epithelium, Boers [/bib_ref] , 0.01% of total lung cells [bib_ref] A Molecular Cell Atlas of the Human Lung From Single-Cell RNA Sequencing, Travaglini [/bib_ref]. They were firstly described in 1954 [bib_ref] Argyrophilia of Bright Cell System in Bronchial Tree in Man, Feyrter [/bib_ref]. They scatter among lung epithelial cells, or exist in clusters, forming neuroepithelium bodies (NEBs) consisting of 5-20 PNEC cells [bib_ref] Formation of a Neurosensory Organ by Epithelial Cell Slithering, Kuo [/bib_ref] [bib_ref] Functional Characterization of Pulmonary Neuroendocrine Cells in Lung Development, Injury, and Tumorigenesis, Song [/bib_ref] [bib_ref] A Confocal Microscopic Study of Solitary Pulmonary Neuroendocrine Cells in Human Airway..., Weichselbaum [/bib_ref]. PNECs are located along the airway epithelium in the trachea and lung in humans and rodents, while NEBs are mainly distributed at the airway branch points [bib_ref] Formation of a Neurosensory Organ by Epithelial Cell Slithering, Kuo [/bib_ref] , where inhalation particles gathered [bib_ref] Early Airway Structural Changes in Cystic Fibrosis Pigs as a Determinant of..., Awadalla [/bib_ref]. PNECs originate from the endoderm or basal cells [bib_ref] Airway Stem Cells Sense Hypoxia and Differentiate Into Protective Solitary Neuroendocrine Cells, Shivaraju [/bib_ref] [bib_ref] Airway Basal Stem Cells Generate Distinct Subpopulations of PNECs, Mou [/bib_ref]. PNECs are the first to differentiate in the lung [bib_ref] Formation of a Neurosensory Organ by Epithelial Cell Slithering, Kuo [/bib_ref]. The differentiated PNECs gradually move towards the bronchial bifurcation to form NEBs. Nerve fibers then enter the NEBs to innervate them, and innate lung immune cells such as ILC2 settle around the NEBs [bib_ref] Formation of a Neurosensory Organ by Epithelial Cell Slithering, Kuo [/bib_ref]. Although the population of PNECs is very limited, their function can be significant. PNECs have both neural and endocrine properties. They are the only innervated cells in the lung epithelium and the cytoplasm of PNECs is rich in core vesicles containing many kinds of substances like amines, amine metabolizing enzymes, purines, neuroendocrine markers, functional proteins and so on [bib_ref] Recent Advances and Contraversies on the Role of Pulmonary Neuroepithelial Bodies as..., Cutz [/bib_ref]. In vitro, PNECs release their vesicle contents under the stimulation of oxygen, mechanical stretching and chemical spines, thereby inducing corresponding pathophysiological changes [bib_ref] Pulmonary Neuroendocrine Cells: Physiology, Tissue Homeostasis and Disease, Noguchi [/bib_ref]. Innervation of PNECs appear in rabbit embryos on the 16th day [bib_ref] Innervation of Pulmonary Neuroendocrine Cells and Neuroepithelial Bodies in Developing Rabbit Lung, Pan [/bib_ref]. The specific form of PNECs and nerve interaction varies in different species [bib_ref] Formation of a Neurosensory Organ by Epithelial Cell Slithering, Kuo [/bib_ref] , which is still not well understood. Present study shows that the nerve innervating PNECs may be sensory nerve, and its cell body is located in the vagal ganglion or dorsal root ganglia [bib_ref] Vagal Sensory Neuron Subtypes That Differentially Control Breathing, Chang [/bib_ref]. PNECs are considered as the most important "sense" cells in the airways. They can sense oxygen changes in the airways [bib_ref] Directed Migration of Pulmonary Neuroendocrine Cells Toward Airway Branches Organizes the Stereotypic..., Noguchi [/bib_ref]. The number of PNECs increases in hypoxic environment, and plays a protective role in the surrounding airway epithelial cells [bib_ref] Airway Stem Cells Sense Hypoxia and Differentiate Into Protective Solitary Neuroendocrine Cells, Shivaraju [/bib_ref]. Some members of the olfactory receptor family are found expressed in human PNECs, thus PNECs can sense a variety of chemical stimuli [bib_ref] Chemosensory Functions for Pulmonary Neuroendocrine Cells, Gu [/bib_ref]. Besides, they can also sense mechanical stimuli [bib_ref] Mechanical Stretch-Induced Serotonin Release From Pulmonary Neuroendocrine Cells: Implications for Lung Development, Pan [/bib_ref] [bib_ref] Consider the Lung as a Sensory Organ: A Tip From Pulmonary Neuroendocrine..., Garg [/bib_ref]. When the airway epithelium is damaged, PNECs act as progenitor cells to repair the damaged epithelium [bib_ref] Functional Characterization of Pulmonary Neuroendocrine Cells in Lung Development, Injury, and Tumorigenesis, Song [/bib_ref] [bib_ref] Neuroepithelial Bodies of Pulmonary Airways Serve as a Reservoir of Progenitor Cells..., Reynolds [/bib_ref] [bib_ref] Neuroepithelial Body Microenvironment is a Niche for a Distinct Subset of Clara-Like..., Guha [/bib_ref]. PNECs are not the key cells to maintain lung development [bib_ref] Pulmonary Neuroendocrine Cells Amplify Allergic Asthma Responses, Sui [/bib_ref] , but are necessary in many lung diseases including asthma [bib_ref] Pulmonary Neuroendocrine Cells Amplify Allergic Asthma Responses, Sui [/bib_ref] [bib_ref] Hyperplasia of Pulmonary Neuroendocrine Cells in a Case of Childhood Pulmonary Emphysema, Alshehri [/bib_ref] [bib_ref] Clinical Characteristics of Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia: A Retrospective Analysis, Almquist [/bib_ref] [bib_ref] Fragmentation of Small-Cell Lung Cancer Regulatory States in Heterotypic Microenvironments, Schaff [/bib_ref].
PNECs may serve as the center of neuro-immune regulation in the asthmatic lung [bib_ref] Early Life Allergen-Induced Mucus Overproduction Requires Augmented Neural Stimulation of Pulmonary Neuroendocrine..., Barrios [/bib_ref]. PNECs activate ILC2s by secreting neuropeptides thus amplify the allergen stimulation of immune cells in asthma [bib_ref] Pulmonary Neuroendocrine Cells Amplify Allergic Asthma Responses, Sui [/bib_ref]. PNECs are the main source of gammaaminobutyric acid (GABA) in the lung. GABA secreted by PNECs can promote the transformation of club cells around PNECs into goblet cells. In addition, GABA leads to excess mucus secretion in airway goblet cells by acting on GABA type a and GABA type b receptors [bib_ref] Pulmonary Neuroendocrine Cells Secrete Gamma-Aminobutyric Acid to Induce Goblet Cell Hyperplasia in..., Barrios [/bib_ref] and worse symptoms in asthma. Adjacent to ILC2s, PNECs can directly stimulate ILC2s to produce IL5, IL-13 and other cytokines by secreting CGRP, and then trigger Th2 response (46) .
# Conclusion
The occurrence of asthma involves multi-factors such as heredity, environment and so on. Neuro-immune regulation is ubiquitous in the development of asthma and airway remodeling. The link between the nervous system and the immune system is intricate and interlocking. This review provides a new view for the study of the pathogenesis of allergic asthma and the search for effective treatment from the perspective of nerve regulation on immunity. The nervous system is closely related to the various cells of the FIGURE 2 | Effects of PNEC cells on surrounding cells in allergic asthma. In murine asthma model, PNECs secret CGRP to active ILC2s and further promote the differentiation of Th2 cells. Type 2 cytokines such as IL-5 and IL-13 secreted by ILC2s act on eosinophils and goblet cells. Eosinophils in turn activate PNECs by releasing EETs. Besides, PNECs secret GABA to promote transformation of club cells near PNECs into goblet cells. GABA leads to excess mucus secretion in airway goblet cells by acting on GABA type a and GABA type b receptors, thus worse symptoms in asthma. EETS, Eosinophil extracellular traps; PNECs, pulmonary neuroendocrine cells; GABA, gamma-aminobutyric acid; ASMs, airway smooth muscle cells.
immune system, but it is not clear whether neuro-immune regulation is the cause or the result for the development of asthma. With the discovery of PNECs, the function of neuroregulation in the physiological and pathological aspects in asthma has been paid more attention gradually. However, related investigations are still very handful. There are still many questions to be answered. For example, most of the signaling pathways and key molecules that mediate neuro-immune regulation are not yet clear, and the function of some cells and molecules is controversial.
With the novel perspective of neuro-immune regulation, it is our hope to screen out the high-risk population of asthma in early childhood, help find a simpler and more specific monitoring method of asthma treatment, as well as develop new asthma treatments. While for now, more research is expected and this is definitely a field worth further exploration.
# Author contributions
[fig] FIGURE 1 |: Neuro-immune interactions in inflammation and airway remodeling of allergic asthma. Eosinophils migrate to airway during inflammation via eotaxin-1. They release EETs surrounding and activating PNECs. Under the action of VLA-4 and CD11b, eosinophils adhere to VCAM-1 and ICAM-1 on parasympathetic fibers. [/fig]
[fig] FUNDING: We are very grateful for the financial support from the National Natural Science Foundation of China (grant No. 81970029), Shaanxi Province Natural Science Foundation (Project No. 2021JQ-024) and fundamental research funds for the central universities (xjh012020026). [/fig]
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Data fusion detects consistent relations between non-lesional white matter myelin, executive function, and clinical characteristics in multiple sclerosis
We examined the influence of dysfunctional, non-lesional white matter on cognitive performance in multiple sclerosis (MS). Forty-six MS subjects were assessed using MRI-based myelin water imaging (MWI), and average myelin water fraction (MWF) values across 20 white matter regions of interest (ROIs) were determined. A datafusion method, multiset canonical correlation analysis (MCCA), was used to investigate the multivariate, deterministic joint relations between MWF, executive function, and demographic and clinical characteristics. MCCA revealed one significant component (p = 0.009) which consisted of three linked profiles, with a pairwise correlation between the MWF and cognitive profiles of r = 0.37, a correlation between MWF and demographics profiles of r = 0.31, and between cognitive and demographics profiles r = 0.64. White matter ROIs representing long-range intra-hemispheric tracts and ROIs connecting the two hemispheres were positively related through their individual profiles to overall cognitive performance, education and female gender, while age, EDSS, and disease duration were related negatively. Surprisingly, lesions within the ROIs had a negligible effect on overall relations between imaging, cognitive, and demographic variables. These findings indicate that there is a strong association between a pattern of MWF values and cognitive performance in MS, which is modulated by age, education, and disease severity. Moreover, this consistent relation involves multiple white matter regions and is separate from the influence of lesions.
# Introduction
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system with a wide spectrum of motor and non-motor impairments such as fatigue, vision impairments, balance issues, and cognitive impairments that greatly affect quality of life. A hallmark of MS is the deterioration of the white matter (WM) microstructural integrity, due to inflammation, edema, axonal loss, and demyelination. Lesions, or focal plaques, typically seen as hyperintensities on proton density or T2 weighted magnetic resonance images, are often characterised by a high degree of demyelination. In addition to focal loss of myelin in lesions, a general decrease in overall myelin can be observed in non-lesional tissue, both in diffusely abnormal white matter (DAWM) and normal-appearing white matter (NAWM) (MacKay and [bib_ref] Magnetic resonance of myelin water: an in vivo marker for myelin, Mackay [/bib_ref].
MS disease manifestations are highly variable in both symptom presentation and radiological MRI markers . Lesions appearing similar to each other on conventional MRI may not correspond to consistent patterns of clinical symptoms. Besides lesion location, the extent of lesioned tissue also provides limited information about behavioural consequences. This mismatch between radiological markers and disease manifestation has been termed the 'clinical-radiological paradox' and has hampered the ability to robustly infer disease status and course based on MRI lesion characteristics alone .
Despite much research, there is still limited understanding of the relations between cognitive impairments and WM lesions in MS, likely because higher cognitive functioning requires a distributed network of https://doi.org/10.1016/j.nicl.2019.101926 Received 23 November 2018; Received in revised form 29 May 2019; Accepted 30 June 2019 multiple brain regions acting in concert [bib_ref] Towards a network theory of cognition, Mcintosh [/bib_ref] [bib_ref] Interpretation of neuroimaging data based on network concepts, Mcintosh [/bib_ref] , as opposed to independent activity in discrete loci. A crucial aspect of effective communication between distinct brain regions is the myelination of axons in the WM, as speed and coherence of signal transmission is a critical factor in complex motor and cognitive function. In previous in vivo research inferring relations between MRI white matter integrity measures and cognition, most relied on measures only partially associated with myelin content such as the diffusion tensor imaging (DTI) measures of fractional anisotropy (FA) and radial diffusivity [bib_ref] Cognitive processing speed in older adults: relationship with white matter integrity, Kerchner [/bib_ref] [bib_ref] Language, aging, and cognition: frontal aslant tract and superior longitudinal fasciculus contribute..., Rizio [/bib_ref] [bib_ref] White matter microstructure and cognitive function, Roberts [/bib_ref]. Other studies have also utilized the magnetic transfer ratio (MTR) as a measure of WM integrity in disease [bib_ref] Reduced magnetisation transfer ratio in cognitively impaired patients at the very early..., Faiss [/bib_ref] and healthy aging [bib_ref] Magnetization transfer ratio relates to cognitive impairment in normal elderly, Seiler [/bib_ref] While there is some degree of correspondence between these measures and myelination of underlying tissue, they do not directly quantify myelin content nor are they specific to it [bib_ref] Is diffusion anisotropy an accurate monitor of myelination? Correlation of multicomponent T2..., Mädler [/bib_ref] [bib_ref] Is the magnetization transfer ratio a marker for myelin in multiple sclerosis?, Vavasour [/bib_ref]. External factors such as direct axonal damage, or underlying architecture of WM fibre bundles can affect DTI measures [bib_ref] Evidence of demyelination in mild cognitive impairment and dementia using a direct..., Bouhrara [/bib_ref]. Similarly, a change in myelination may not be quantitatively reflected in these measures, rendering them non-specific to myelin. In contrast, the myelin water fraction (MWF) gained from in vivo T2 relaxation studies has a very good correspondence to stained myelin content examined histopathologically, the gold standard of assessing myelination [bib_ref] Myelin water imaging in multiple sclerosis: quantitative correlations with histopathology, Laule [/bib_ref] [bib_ref] Myelin water imaging of multiple sclerosis at, Laule [/bib_ref]. While MWF has been extensively utilized in MS research, a direct link to cognitive performance is still lacking.
Previous studies exploring the association between WM microstructure and cognitive performance have also largely attempted to relate performance on a particular cognitive test to a measure in a specific WM region; however, it is far more likely that several brain regions jointly engage in a given task. Moreover, since it is difficult to design cognitive tests that selectively probe one particular aspect of cognition in isolation, changes in myelin markers will probably have widespread downstream effects across multiple cognitive tests and domains. Thus, methods that accommodate multivariate clinical and imaging data, to assess the joint relations between two or more sets of variables may be more advantageous.
In this study we have tried to address the aforementioned limitations by 1) using a myelin specific measure of WM integrity 2) using a data-driven multivariate approach suitable for fusion of cognitive performance, demographic and clinical data in a cohort of MS subjects. We use the multivariate, data-driven method of multiset canonical correlation analysis (MCCA) to examine the associations between overall myelin content and cognitive profiles, and clinical variables such as age, gender, years of education, and disease severity. We hypothesized that a significant association would exist between overall cognitive performance across multiple tests, overall MWF values, and demographic variables.
# Materials & methods
This study received ethical approval from the University of British Columbia Clinical Research Ethics Board, and all subjects provided written, informed consent. We enrolled a total of 46 subjects (35F/ 11 M) diagnosed with relapsing-remitting multiple sclerosis (RRMS) based on the McDonald 2005 criteria [bib_ref] Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria, Polman [/bib_ref] , with an average ( ± standard deviation) age of 42.9 ± 10.9 years [fig_ref] Table 1: Demographics, clinical, and cognitive measures [/fig_ref]. All imaging data were acquired on a Philips (Netherlands) Achieva 3 T MRI scanner with an 8 channel head coil. We acquired a full brain 3DT1weighted scan for structural references with an inversion recovery MPRAGE sequence TI = 808 ms, TR = 1800 ms and an isotropic voxel size of 1mm 3 . T 2 relaxation data were collected using a modified GRASE sequence with 32 echoes with 10 ms echo spacing and TR = 1000 ms. Twenty slices were acquired at 5 mm slice thickness and reconstructed to 40 slices at 2.5 mm. The in-plane voxel size was 1x1mm. A dual echo PDw/T 2 w scan with TE1 = 8.4 ms, TE2 = 80 ms, TR = 2800 ms and voxel size of 0.97 × 0.97x5mm 3 was used for lesion identification.
The multi-echo GRASE sequence was analyzed using in-house MATLAB code which uses an NNLS fitting methods to approximate the multi-exponential decay curve with a number of basis functions, resulting in one whole cerebrum MWF map per subject. The algorithm includes correction for stimulated echoes as well as a regulariser to make the fit more robust against noise in the time domain [bib_ref] Applications of stimulated echo correction to multicomponent T2 analysis, Prasloski [/bib_ref]. The calculation of MWF was performed as described in [bib_ref] Applications of stimulated echo correction to multicomponent T2 analysis, Prasloski [/bib_ref].
WM lesions were delineated semi-automatically utilizing the PDw/ T 2 w images. A radiologist with extensive experience in MS lesion identification digitally marked all lesions with seed points using a custom-built software interface. T 2 lesions were then segmented using a previously validated method [bib_ref] Optimizing the use of radiologist seed points for improved multiple sclerosis lesion..., Mcausland [/bib_ref] that automatically computes the extent of each marked lesion using a customized Parzen window classifier to estimate the intensity distribution of the lesions.
We used 20 WM ROIs, part of the FSL (FMRIB, Oxford) package, which cover the majority of the WM and delineate major WM tracts . A full list of ROI names can be found in supplementary table T1. In order to extract the average MWF per ROI, the ROIs were nonlinearly registered to each subjects' native space using the registration parameters obtained from registering the MNI template to the first echo of the multi-echo T2 data, and the FNIRT program of FSL. All registrations were visually checked for accuracy and if [fig_ref] Table 1: Demographics, clinical, and cognitive measures [/fig_ref].
necessary, registrations were re-performed with adjusted parameters to ensure an appropriate alignment between images. Once the ROIs were registered, a white matter mask (obtained from the 3DT1 image with FASTand registered to the multi-echo data with FLIRT, was applied to ensure only WM voxels were being considered for analysis. The MWF averages of all WM ROIs comprised the imaging set X nxk with n = 46 subjects and k = 20 WM ROIs (features). All subjects were assessed with a cognitive battery evaluating performance in processing speed, working memory, executive function and attention domain. We administered the subtests of the Wechsler Adult Intelligence Scale-IV (WAIS IV)that included digit span, arithmetic, letter number sequencing, symbol search, and coding. Composite index scores from the WAIS-IV were obtained for use in the analysis including Working Memory Index (WMI), which utilized scores from digit span, arithmetic, and letter number sequencing subtests. The WAIS-IV Processing Speed Index (PSI) was based on the symbol search and coding subtests. In addition to WAIS IV, we further assessed Verbal Letter Fluency Test (FAS), and Trail-Making Test, (TMT A and B) [bib_ref] Effect of physical layout in performance of the trail making test, Arnett [/bib_ref] to evaluate executive function and attention. Detailed explanations of the evaluated abilities of each test can be found in our previous study [bib_ref] Cognitive performance in subjects with multiple sclerosis is robustly influenced by gender..., Lin [/bib_ref]. In the end, WMI, PSI, FAS, and the TMT A/B raw scores formed the cognitive set Y nxl with n = 46 subjects and l = 5 cognitive features.
The subjects age, gender, years of education, Kurtzke Expanded Disability Status Scale (EDSS), and disease duration (DD) in years were collated to form the demographic set Z nxo with n = 46 subjects and o = 5 demographic and disease severity features.
# Statistical analysis
# Multivariate correlation analysis
One method to relate two sets of multivariate data is canonical correlation analysis (CCA) [bib_ref] Relations between two sets of Variates, Hotelling [/bib_ref] , with the goal of finding linear combinations of the original variables that are maximally correlated. In other words, CCA finds linear transformations (canonical vectors) for each set, such that the correlation between the projections of the original data (canonical variates) onto these canonical vectors are maximised. In the case of more than two groups of datasets, an extension of CCA, multiset CCA (MCCA) [bib_ref] Canonical analysis of several sets of variables, Kettenring [/bib_ref] can be employed. MCCA identifies a correlation structure among canonical variates of multiple datasets by a series of linear transformations so that they are maximally correlated. The projections are called canonical variates (P i ), with i = X, Y, Z for each respective set, and can be viewed as condensed representations, or profiles of each set, while sharing commonalities across sets. Further profiles/canonical variates can be extracted with new sets of canonical vectors such that they have maximum correlation among them but are uncorrelated to the prior canonical variates. Canonical loadings are often used to assess the contribution of the original variables to the canonical variates in a set, and are defined as the correlation between each variable and the canonical variate.
# Methodological considerations
All three sets, X (MWF), Y (cognitive scores), and Z (demographics) served as the input to the MCCA model. To avoid overfitting, we utilized principal component analysis (PCA) to reduce the dimensions of each data set to a common dimensionality of five components prior to the MCCA step. The significance of MCCA components was assessed with a nonparametric permutation test in which the order of subjects was permuted and MCCA was performed again. This procedure was done 1000 times to generate a null distribution of pairwise correlation values and the original correlations were assessed against this distribution to define significance. To estimate the robustness of the loadings, we performed a leave-one-out cross validation.
## Effect of lesions
In order to investigate the effects of lesion tissue in this methodology, we computed two measures of 'lesion contribution' per ROI. The first measure, 'lesion percentage' is the ratio of lesional voxels to total number of voxels for a given ROI. The second measure, 'subject lesions' is the count of subjects that had at least one lesion in a particular ROI. We performed multiple linear regression with the two lesion contribution measures as predictors and MCCA loadings on ROIs as outcome variables. As a second test, MCCA was performed twice. Once as described above, and repeated, but specifically excluding voxels that were contained within the lesions, by subtracting the lesion mask from the ROI mask prior to calculating the average MWF per ROI. We then compared the results from the MCCA when lesion tissue was included and when it was excluded.
## Post-hoc tests
In order to determine the biological significance of the significant canonical variate, we performed different post-hoc analyses. First, we employed k-means clustering on the weighted values from the X, Y, Z (i.e. MWF, cognition and demographic) data sets constituting the significant canonical variate. As per design, these combinations were ones that resulted in the largest correlation between data sets. In the clustering we were looking for a differentiation between mildly and moderately affected subjects, thus we limited the number of clusters to two. We used the non-parametric Kruskal-Wallis test to compare the two groups with regards to their disease state.
For further validation of the clusters, and to demonstrate that the canonical variate had biological meaning, we used an independent measure, the average whole brain cortical thickness based on the high resolution 3DT1 sequence and obtained from Freesurfer [bib_ref] FreeSurfer, Fischl [/bib_ref] , to test for differences between the two clusters.
# Results
## Multiset canonical correlation analysis
We found one significant MCCA component relating imaging, cognitive, and demographic variables (permutation test p = 0.009). The pairwise correlations between the profiles were r xy = 0.37, r xz = 0.31, and r yz = 0.64, with r ij and i,j being the imaging, cognitive, or demographic profile [fig_ref] Figure 2: Fig [/fig_ref]. [fig_ref] Figure 3: Loadings of individual features per set when lesions were included in the... [/fig_ref] shows the canonical loadings, which are Pearson's correlations between the profiles and the original features in their respective set and reflect the shared variance between the original features and its profile. Error bars are 95% confidence intervals, determined with a leave-one-out cross validation.
All features across all three sets contributed significantly to their respective profiles, as suggested by the confidence intervals not spanning zero. At the same time, there was considerable variability among the loadings, indicating a distinct hierarchy of contributions between features towards each profile. Three WM ROIs display a negative loading, while the remaining 17 ROIs showed a positive loading on the WM profile. In the cognitive profile, WMI, PSI, and FAS showed positive loadings whereas TMT A and B exhibited a negative loading. This opposite contribution of cognitive tests to the cognitive profile was expected since higher scores of the WMI, PSI, and FAS indicate better performance, while the opposite is true for the TMT A/B tests as higher scores signify worse performance. The variables of the demographic set demonstrated a mix of positive and negative loadings, with gender and education showing positive contributions to this profile. In contrast, age, EDSS, and disease duration loaded negatively onto this profile. [fig_ref] Figure 4: Scatter plots of canonical variates with color coding of different variables from... [/fig_ref] illustrates the opposing contributions to their respective profiles from some of the variables showing some of the highest loadings from the cognitive and demographic sets. The figure displays the profiles with subjects color-coded according to their EDSS score [fig_ref] Figure 4: Scatter plots of canonical variates with color coding of different variables from... [/fig_ref] , their PSI score [fig_ref] Figure 4: Scatter plots of canonical variates with color coding of different variables from... [/fig_ref] , and their TMT A score [fig_ref] Figure 4: Scatter plots of canonical variates with color coding of different variables from... [/fig_ref]. Correlations of the canonical variates or profiles. Top: A 3D representation of the correlation between canonical variates. Bottom: The pairwise correlations between canonical variates P are: 0.38 between set X (MWF) and Y (cognitive), 0.31 between set X and set Z (demographics and disease severity), and 0.64 between set Y and set Z. The overall significance of the component was p = 0.009, assessed with a permutation test. [fig_ref] Table 2: Comparison of clinical and cognitive variables between cluster [/fig_ref]. shows the WM ROIs with a substantial lesion contribution (in this case measured by how many subjects presented a lesion in an ROI), that also had a significant loading on the WM profile.
# Post hoc results
After a k-means clustering looking for two clusters, cluster one was comprised of 36 subjects and cluster two included 10 subjects [fig_ref] Figure 6: Using k-means on canonical variates of significant component looking for two clusters [/fig_ref]. The clustering separated subjects into a mildly affected (average EDSS = 2.09, average DD = 9.32 years) and a moderately affected (average EDSS = 3.80, average DD = 19.10 years) group with p = 0.0065 for EDSS and p = 0.0093 for DD [fig_ref] Table 2: Comparison of clinical and cognitive variables between cluster [/fig_ref]. As expected, cluster 2, with greater EDSS and higher DD, exhibited greater cortical thinning (2.61 mm vs 2.51 mm p = 0.0143, for cluster 1 and cluster 2, respectively). Color coded ROIs that showed lesions in at least 50% of subjects (red) and 33% of subjects (orange) with some of the largest loadings onto the WM profile. The bilateral thalamic radiation in blue showed lesions in at least 50% of subjects but did not show large loadings. Even though there was a substantial lesion contribution in those ROIs, it had minimal effects on the loadings.
# Discussion
We utilized a multivariate, data-driven approach, in order to reveal a joint pattern of covarying features consisting of profiles of WM myelin integrity, cognitive performance, and demographic and disease factors in a cohort of RRMS subjects. The data fusion performed here is completely data-driven and uses simple linear weights in order to decompose the data sets into latent, maximally cross-correlated profiles. The data-driven nature of the analysis minimises a priori assumptions about potential interactions within and across data domains, while its multivariate nature allows the modeling of shared features across data sets. This is in contrast to univariate approaches where only local or distinct features can be examined. The power of MCCA analysis approach lies in its ability to naturally reveal cross-modality relationships and it suggests some interesting conclusions: 1) factors such as disease duration, gender and age predict cognitive performance in early MS more than myelin features (correlation between profiles of cognition and demographics r = 0.64, MWF vs cognition r = 0.37, and MWF vs demographics r = 0.31), 2) relative independent of lesion presence and exact lesion location, there is a robust association between myelin content in long-range intrahemispheric connections and the corpus callosum and cognitive performance.
Many cognitive domains can be profoundly affected by altered microstructural integrity as seen in MS. Processing speed and tasks requiring the ability to focus attention, scan quickly, discriminate and order information in order to process it, and working memory, are all commonly impaired in MS [bib_ref] Cognitive impairment in multiple sclerosis, Chiaravalloti [/bib_ref] [bib_ref] Cognitive dysfunction in multiple sclerosis, Guimarães [/bib_ref] [bib_ref] The effect of medial frontal and posterior parietal demyelinating lesions on Stroop..., Pujol [/bib_ref]. Complex attention involving alertness, selective/focused/divided attention, and vigilance rather than "simple" attention (e.g. repeating a series of digits) is also impaired [bib_ref] Cognitive impairment in multiple sclerosis, Chiaravalloti [/bib_ref] [bib_ref] Cognitive dysfunction in multiple sclerosis, Guimarães [/bib_ref]. Impairments in executive function, a set of abilities which facilitate goal-oriented behavior as well as adaption to environmental changes such as planning, shifting, and fluency, appears to impact the quality-of-life the most among the cognitive deficits seen in MS [bib_ref] Executive function in multiple sclerosis. The role of frontal lobe pathology, Foong [/bib_ref] [bib_ref] Fatigue, emotional functioning, and executive dysfunction in pediatric multiple sclerosis, Holland [/bib_ref] [bib_ref] The executive dysfunctions most commonly associated with multiple sclerosis and their impact..., Preston [/bib_ref]. Early research proposed that memory deficits in MS were caused by an inability to sustain or support effective information retrieval [bib_ref] Memory and "frontal lobe" dysfunction in multiple sclerosis, Beatty [/bib_ref] , however difficulty in acquiring new knowledge (i.e. memory encoding) might be a greater problem than information retrieval (i.e. memory retrieval) [bib_ref] Cognitive impairment in multiple sclerosis, Chiaravalloti [/bib_ref].
Several studies have attempted to establish the links between white matter changes and cognitive decline in MS. Demyelination, inferred by lesions in conventional MRI images in the medial frontal region, is associated with slow responses in an attention task [bib_ref] The effect of medial frontal and posterior parietal demyelinating lesions on Stroop..., Pujol [/bib_ref]. Studies utilizing DTI have suggested that processing speed deficits were related to reduced FA in the corpus callosum and superior longitudinal fasciculus, two major tracts connecting the two hemispheres and frontal, temporal, and parietal lobes [bib_ref] The relationship between executive functioning, processing speed, and white matter integrity in..., Genova [/bib_ref]. Compared to cognitively-preserved MS patients, cognitively-impaired patients evaluated in spatial and verbal memory, information processing speed, working memory, and verbal fluency spheres exhibited reduced FA in the corpus callosum, superior and inferior longitudinal fasciculus, corticospinal tracts, forceps major, cingulum, and fornices [bib_ref] Cognitive impairment in MS: Imapct of white matter integrity, gray matter volume,..., Hulst [/bib_ref].
While here we have shown a significant relation between cognition and MWF measures in RRMS, relations between MWF imaging and cognition have also been explored in the non-MS literature. In children (n = 108 children ages: 2.5 months -5.5 years), a significant positive association between myelination and cognitive and motor abilities can be shown [bib_ref] Characterizing longitudinal white matter development during early childhood, Dean [/bib_ref] [bib_ref] White matter maturation profiles through early childhood predict general cognitive ability, Deoni [/bib_ref]. Further, a relation was found between highly myelinated axons in the corpus callosum and the Wechsler Intelligence Scale for Children in five male children aged between 8 and 12 [bib_ref] Quantifying development: investigating highly myelinated voxels in preadolescent corpus callosum, Whitaker [/bib_ref]. A myelin water imaging study found positive relations between frontal lobe myelination and both age and years of education in controls (n = 27) but not in subjects with schizophrenia (n = 30) [bib_ref] Abnormalities of myelination in schizophrenia detected in vivo with MRI, and postmortem..., Flynn [/bib_ref]. A more recent study of young adults with ages ranging from 15 to 38 years found a positive relation between frontal lobe myelination and age, North American Adult Reading Test (NAART) IQ, and years of education [bib_ref] 48 echo T₂ myelin imaging of white matter in first-episode schizophrenia: evidence..., Lang [/bib_ref]. In older patients with mild cognitive impairments (MCI), substantial decreased myelin integrity has been observed; however, associations between decreased myelination and poor cognitive performance were not documented [bib_ref] Evidence of demyelination in mild cognitive impairment and dementia using a direct..., Bouhrara [/bib_ref]. Finally, a study of myelination in 61 healthy volunteers aged 18 to 84 years found a quadratic relation between MWF and age [bib_ref] Adult age differences in subcortical myelin content are consistent with protracted myelination..., Arshad [/bib_ref] emphasizing that age affects myelin differently across lifespan. Overall, these studies highlight the importance of often widely spatially distributed myelin profile integrity to cognitive function. However, the link between myelin integrity and cognitive abilities in MS is lacking. This study potentially overcomes the research gap of myelin-cognition relations in MS as we discovered multivariate relations between myelin and cognitive performance. Perhaps, the multivariate approach used in this study is a key factor to study human brain and behaviour.
As executive function profoundly affects the quality-of-life of people with MS, the original study design was to investigate the relations between imaging features, clinical variables, and primarily executive performance in MS. Therefore, the test battery for this study was based on widely-used tasks in assessing executive functioning and processing speed rather than commonly-used cognition screening tests such as the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) [bib_ref] Recommendations for a brief international cognitive assessment for multiple sclerosis (BICAMS), Langdon [/bib_ref]. Another neuropsychological battery that targets specific domains affected in MS is the Minimal Neuropsychological Assessment of MS Patients (MACFIMS) [bib_ref] Minimal neuropsychological assessment of MS patients: a consensus approach, Benedict [/bib_ref] , which provides comprehensive evaluation of cognitive function in MS. In fact, our test battery evaluates some domains that are also examined in MACFIMS such as working memory, processing speed, and executive function. Due to the fact that executive function and processing speed were our primary and secondary targets to evaluate, we specifically chose tests tailored to assess these domains, rather than the standardized test used in MS. Therefore, we decided to use sub-tests of WAIS IV to evaluate working memory and processing speed and executive function was also examined with TMT A/B and FAS. We note that the loadings across specific cognitive tests in the significant MCCA component were relatively uniform (note that for TMT A/B, longer times represent worsening performance, so the loadings in [fig_ref] Figure 3: Loadings of individual features per set when lesions were included in the... [/fig_ref] , lower left are reasonable since higher scores on TMT A/B are anticorrelated to the remaining cognitive tests where higher scores indicate better performance). We therefore do not believe that differing cognitive tests would alter our overall conclusions substantially.
We used MCCA to create profiles of WM myelin integrity, cognitive performance, and demographic and disease factors which were closely related to one another. Since WMI, PSI, and FAS together with gender and education all loaded positively on their respective profiles, it is implied that subjects with higher education and being female (females were coded 1, males were coded 0 in analysis) performed better in these cognitive tests. Most WM ROIs loaded positively on its profile, and the aforementioned cognitive tests loaded positively on their profile. This implies a positive relation between MWF and cognitive performance, education, and female gender. In contrast, the negative loadings of age, EDSS, and disease duration on the demographics profile, and the positive loadings of WMI, PSI, and FAS on the cognitive profile, suggest that age and disease progression have adverse effects on cognitive performance. Similarly, age, EDSS, and disease duration showed opposite loadings to that of WM ROIs on their respective profiles, indicating an inverse relation between MWF and age, and disease duration.
The cognitive tests TMT A/B loaded negatively on the cognitive profile, while in the demographics profile, age, EDSS (disease severity), and disease duration had negative loadings, and years of education had a positive loading. This indicates that age and a more severe disease state were associated with higher TMT A/B scores (i.e., slower times to complete the task, reflecting a worse performance), but years of education was not. Similarly, the mostly positive loadings of most WM ROIs in the myelin profile, suggest a worse performance in TMT A/B with low MWF, an imaging feature associated with more severe disease involvement.
Our results further support the notion that higher-order functions require multiple brain regions to coordinate together, especially the longitudinal fasciculus which connect frontal/parietal/occipital areas (i.e. long-range intrahemispheric connections) and the corpus callosum which links the two hemispheres (i.e. interhemispheric connections) [bib_ref] Contribution of callosal connections to the interhemispheric integration of visuomotor and cognitive..., Schulte [/bib_ref]. In the present study, white matter ROIs associated most with the imaging profile included the bilateral corticospinal tract, forceps major, forceps minor, bilateral inferior fronto-occipital fasciculus, bilateral inferior longitudinal fasciculus, bilateral superior longitudinal fasciculus, and left arcuate. These WM tracts connect geographically remote brain regions, as well as cortical and subcortical areas connecting regions for processing speed ability, higher-order cognitive functions, and motor function [bib_ref] White matter microstructure and cognitive function, Roberts [/bib_ref]. The forceps minor, forceps major, superior and inferior longitudinal fasciculus, and inferior fronto-occipital fasciculus have been reported to be involved in processing speed function, and influencing TMT performance in both older adults and MS subjects [bib_ref] The relationship between executive functioning, processing speed, and white matter integrity in..., Genova [/bib_ref] [bib_ref] Cognitive processing speed in older adults: relationship with white matter integrity, Kerchner [/bib_ref]. The inferior and superior longitudinal fasciculi have been shown to play a role in higher-order cognitive function as they connect association corticeskey regions for higherorder functions [bib_ref] The structural connectivity of higher order association cortices reflects human functional brain..., Jung [/bib_ref]. Indeed, the cognitive tests that are positively expressed in the cognitive profile are WMI, PSI, and FAS, tests that assess working memory, processing speed, and verbal fluency and are all impaired in MS [bib_ref] Cognitive impairment in multiple sclerosis, Chiaravalloti [/bib_ref] [bib_ref] Cognitive dysfunction in multiple sclerosis, Guimarães [/bib_ref] [bib_ref] The effect of medial frontal and posterior parietal demyelinating lesions on Stroop..., Pujol [/bib_ref]. On the other hand, the timed TMT A/B tests, where higher scores signify worse performance, are negatively associated with the cognitive profile, indicating an inverse relation between the imaging features and these particular tests.
The fact that the corticospinal tract WM ROI was also loading positively on the imaging profile is perhaps surprising. A DTI study suggested that the corticospinal tract has been related to motor performance but not higher-order cognitive functions [bib_ref] Changes in perceptual speed and white matter microstructure in the corticospinal tract..., Lövdén [/bib_ref] , as would be expected. While the quantitative measure of myelin and joint multivariate data analysis utilized here may be more sensitive, there may be other explanations. For example, the corticospinal tract is required to execute the processed information required in different cognitive tasks. One model parcellates executive function into three componentsinput (require long-range connections), core process (in the prefrontal cortex), and output (require coordination of motor and subcortical connections for taking actions) [bib_ref] An integrative theory of prefrontal cortex function, Miller [/bib_ref]. This model implies that the corticospinal tract might be involved in the output component to execute actions based on processed information. Therefore, as a resource to execute the information, it is reasonable that WM integrity in the corticospinal tract is involved in executive function. Further, there may be a correlative as opposed to a causal relation between corticospinal tract integrity and cognitive function: presumably people with worsening disease, and reduced WM integrity would be more likely to also have impairments in their motor system. Some of our results describing factors preserving executive function in MS are perhaps unsurprising. We found positive loadings of WMI, PSI, FAS, as well as a positive loading of education onto their respective profiles, suggesting a positive relation between these features. In the theory of cognitive reserve [bib_ref] What is cognitive reserve? Theory and research application of the reserve concept, Stern [/bib_ref] [bib_ref] Cognitive reserve in multiple sclerosis, Sumowski [/bib_ref] [bib_ref] The cognitive reserve hypothesis: a longitudinal examination of age-associated declines in reasoning..., Tucker-Drob [/bib_ref] , higher levels of education may alter synaptic organization and/or neuronal networks so individuals can still sustain damage and maintain adequate cognitive function [bib_ref] What is cognitive reserve? Theory and research application of the reserve concept, Stern [/bib_ref]. Also, we found that age, EDSS and disease duration loaded negatively on the demographic profile indicating a negative association with cognitive tests that loaded positively on the cognitive profile. Of note, EDSS and disease duration had the highest loadings in the demographics profile, indicating a central role of these measures' influence on both imaging and cognition sets. We also observed that better cognitive performance was associated with female gender. Although we speculate that such a pattern may support the purported neuroprotective effects of estrogen [bib_ref] Estrogen shapes dopamine-dependent cognitive processes: implications for Women's health, Jacobs [/bib_ref] [bib_ref] Gender differences in Parkinson's disease: clinical characteristics and cognition, Miller [/bib_ref] , more evidence is needed to draw such conclusion.
The cluster analysis produced a reasonable separation of mildly-and moderately-affected sub-groups across all three sets, further supporting a strong relation between MWF, cognitive abilities, and demographics. The additional comparison of an unrelated measure, namely cortical thickness, reinforces the utility of MCCA as well as strengthens our results of finding an association between MWF, cognitive performance, demographics and clinical variables. The groupings based on the MCCA results show a similar pattern of cortical thinning with the moderately affected sub-group having a decreased overall cortical thickness and are in line with previous research [bib_ref] Cortical atrophy patterns in multiple sclerosis are non-random and clinically relevant, Steenwijk [/bib_ref].
Importantly, our results were largely independent of exact lesion location. A multiple linear regression model was unable to detect a relation between lesion contribution per ROI and the MWF loadings we found suggesting that MWF loadings are independent of lesion contribution. Furthermore, when the MCCA analysis was performed with and without lesion tissue included in WM ROIs the results were largely unchanged. There has been a long-standing debate on local vs distributed representation of brain function [bib_ref] The Organization of Neocortex in mammals: implications for theories of brain function, Kaas [/bib_ref]. In MS there can be a very close association between lesion location and clinical effects, with optic neuritis being a prime example. However, with higher cognitive functions, such clinicopathological correlation between lesion location and behavioural effect is less clear, as distributed brain regions must be recruited to facilitate complex tasks. While we have shown a relationship between NAWM and some cognitive functions, we cannot discern, with the current analysis, whether or not this related to primary dysfunction in the NAWM, or secondary changes from focal lesions.
There are a number of limitations to our study. Since none of the MS subjects demonstrated overt cognitive impairment at the time of examination based on our previous report which investigated the same cohort [bib_ref] Cognitive performance in subjects with multiple sclerosis is robustly influenced by gender..., Lin [/bib_ref] , the results of this study reflect brain-behaviour associations at early stages of the disease. Further, the analyzed cohort was comprised solely from early stages of the RRMS subtype which was owed to the study design, extrapolations to different subtypes of MS should be taken with care. Due to the fact the analysis was based on a set of WM ROIs from a template, not all of the WM is necessarily covered, although we note that most major WM fibre bundles were included. This may have led to individual lesions not being accounted for in this analysis in case they did not overlap with any of the standard ROIs. In addition, the investigated cohort was in the relatively early stages of MS with low to moderate lesion burden such that the impact of focal demyelination of lesions within the ROIs was limited. Thus, the minor change in results of the MCCA when including and excluding lesions may be partly due to the fact that not all lesion had been considered. Finally, a complicating factor is that myelin as a function of age may follow a quadratic function [bib_ref] Adult age differences in subcortical myelin content are consistent with protracted myelination..., Arshad [/bib_ref] , so extrapolating to broader age ranges may pose a riskour results may therefore only be interpretable for the age range of our cohort.
To conclude, with quantitative WM myelin measures and a joint multivariate analysis, we found individual profiles of myelin integrity, cognition, and demographical features in MS that where highly similar. Higher myelin integrity supported better cognitive function and was positively related to education as well as female gender; while disease severity and aging were associated with worsening cognitive performance. In the future, a multimodal approach including functional measures gained from fMRI may be used to study the interplay between structure and function and use their complementary information to further prognosticate cognitive deficits in MS.
## Conflicts of interests
TRB, SJL, BK, IV, and AM have no conflicts of interest to declare. SK has received research funding from Sanofi Genzyme, F. Hoffmann La Roche. David Li has received research funding from the Canadian Institute of Health Research and Multiple Sclerosis Society of Canada. He is the Emeritus Director of the UBC MS/MRI Research Group which has been contracted to perform central analysis of MRI scans for therapeutic trials with Novartis, Perceptives, Roche and Sanofi-Aventis. The UBC MS/MRI Research Group has also received grant support for investigator-initiated independent studies from Genzyme, Merck-Serono, Novartis and Roche. He has acted as a consultant to Vertex Pharmaceuticals and served on the Data and Safety Advisory Board for Opexa Therapeutics and Scientific Advisory Boards for Adelphi Group, Celgene, Novartis and Roche. He has also given lectures which have been supported by non-restricted education grants from Academy of Health Care Learning, Biogen-Idec, Consortium of MS Centers, Novartis, Sanofi-Genzyme and Teva. Anthony Traboulsee has the following competing financial interests: Research funding from Chugai, Roche, Novartis, Genzyme, Biogen. Consultancy honoraria from Genzyme, Roche, Teva, Biogen, Serono. Martin McKeown has research funding from the Mottershead Foundation, the National Science and Engineering Research Council (NSERC) and has received honoraria from Abbvie Corporation and Allergan, Inc.
# Funding
MS Society of Canada.
[fig] Figure 2: Fig. 2. Correlations of the canonical variates or profiles. Top: A 3D representation of the correlation between canonical variates. Bottom: The pairwise correlations between canonical variates P are: 0.38 between set X (MWF) and Y (cognitive), 0.31 between set X and set Z (demographics and disease severity), and 0.64 between set Y and set Z. The overall significance of the component was p = 0.009, assessed with a permutation test. [/fig]
[fig] Figure 3: Loadings of individual features per set when lesions were included in the analysis. Top: displays loadings of the imaging set, lower left: shows the loadings of cognitive set, lower right: shows the loadings of demographic set. ant. Thal. radiation = anterior thalamic radiation, cingulum hipp. = cingulum hippocampus, IFOF = inferior fronto-occipital fasciculus, ILF = inferior longitudinal fasciculus, SLF = superior longitudinal fasciculus; DD = disease duration T.R. Baumeister, et al. NeuroImage: Clinical 24 (2019) 101926 3.2. Effect of lesions on MCCA A multiple regression model estimating the profile loadings of WM ROIs, as the dependent variable, with 'lesion percentage' and 'subject lesions', as the independent variables, was not significant (F (17,20) = 2.47, p = 0.114). The repeat MCCA analysis with lesion tissue removed resulted in a very similar correlation pattern among the three profiles with one significant component (p = 0.005) and pairwise correlations of r xy = 0.37, r xz = 0.31, and r yz = 0.64. Visualisations and tables with more details about the two MCCA runs can be found in the Supplementary Figs. F1-F4 and [/fig]
[fig] Figure 4: Scatter plots of canonical variates with color coding of different variables from set Y and set Z. Left: shows subjects with higher EDSS are clustered along the negative axes. Middle: color coding according to PSI scores where strongly performing subjects are clustered along the positive axes displaying an inverse pattern than plot on the left. Right: performance in TMT A scores color coded where poorly performing subjects cluster along the negative axes, showing a similar pattern to that of left figure which means that subjects with higher EDSS perform worse on TMT A. Size of markers reflects the distance from the point furthest back in P x -P y planeFig. 5. [/fig]
[fig] Figure 6: Using k-means on canonical variates of significant component looking for two clusters (one mildly (average EDSS = 2.09, average DD = 9.32 years) and one moderately (average EDSS = 3.80, average DD = 19.10 years) affected subgroup). The number of subjects per cluster are 36, and 10, respectively. [/fig]
[table] Table 1: Demographics, clinical, and cognitive measures. Displayed are averages and standard deviations. For clinical measures (EDSS and disease duration) the median and their respective ranges are shown. [/table]
[table] Table 2: Comparison of clinical and cognitive variables between cluster. Clinical, cognitive and cortical thickness measures with boldface p-values are significant at the 0.05 significant level. [/table]
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Antibody recognition of an immunogenic influenza hemagglutinin-human leukocyte antigen class II complex
The A/Japan/57 influenza hemagglutinin (HA) peptide HA 128-145, when bound by human histocompatibility leukocyte antigen-DRw11 cells, is recognized by the human CD4+ T cell clone V1. A rabbit antiserum has been raised against HA 128-145 which recognizes not only the free peptide, but also the HA 128-145/DRw11 complex on a solid matrix, in solution, or on the surface of viable cells. The detection of these complexes on viable cells was shown to be class II specific, DRw11 restricted, and commensurate with the level of DRw11 expression. The identity of DRw11 as the cell surface molecule binding HA 128-145 was confirmed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping. Using this antiserum, HA 128-145/DRw11 complexes could be detected on the cell surface as soon as 30 min after the peptide was added, and increased up to 24 h. Dissociation kinetics showed these complexes were long-lived, with a half-life of approximately 14 h. This anti-HA peptide antiserum represents the first direct means of studying antigenic peptide-human leukocyte antigen class II complexes on the surface ofliving cells without the addition of a nonamino acid moiety to the peptide. The properties of this antiserum thus provide the potential to study naturally processed antigenic peptides as well as the mechanism of processing itself in a physiologically relevant system .
T he immune response of CD4+ T cells is thought to be triggered by immunogenic MHC class II moleculepeptide complexes present on the surface of APC's [bib_ref] A hypothesis to relate the specificity of Tlymphocytes and the activity of..., Benacerraf [/bib_ref] [bib_ref] Antigen-presenting function of the macrophage, Unanue [/bib_ref] [bib_ref] High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR, Roche [/bib_ref]. Previous reports have demonstrated in vitro association of peptide antigen with purified class II molecules [bib_ref] Isolation of a functional antigen-la complex, Scrinivasan [/bib_ref] [bib_ref] Binding of immunogenic peptides to Ia histocompatibility molecules, Babbitt [/bib_ref] [bib_ref] Isolatio n and characterization of antigen-la complexes involved in Tcell recognition, Buus [/bib_ref] [bib_ref] Interaction of peptide antigens and class II major histocompatibility complex antigens, Guillet [/bib_ref] [bib_ref] Bindin g of photoreactive lysozyme peptides to murine histocompatibility class II molecules, Luescher [/bib_ref] , and proliferation of cloned T cells exposed to these complexes [bib_ref] Binding of antigen to Ia molecules on intact antigen presenting cells demonstrated..., Cousin [/bib_ref].
While antigen-specific T cells have been useful for qualitatively demonstrating the presence of such complexes, the low number of surface peptide-class II complexes needed to activate a T cell, and the all-or-none nature of the recognition event have made such assays less useful for studying the kinetics and nature ofpeptide-class II binding [bib_ref] Detectio n of peptide-MHC class II complexes on the surface of intact..., Busch [/bib_ref] [bib_ref] Quantitatio n of antigen-presenting cell MHC class II/peptide complexes necessary for Tcell..., Harding [/bib_ref] [bib_ref] The minimal number of class II MHC-antigen complexes needed for Tcell activation, Dematz [/bib_ref]. Studies of APC antigen processing and class II loading have been hampered by the inability to directly detect peptide bound in the antigen-binding groove ofthese molecules on the surface ofintact cells. Whole cell binding assays using radiolabelled peptide antigen have been plagued by nonspecific uptake of radiolabel into the cells . Those studies which promote meta-bolic antigen-processing have suffered from unacceptably high levels of nonspecific binding which have made it difficult to separately study the complexes of peptide antigen bound to surface class II molecules . Similar studies done under conditions which block APC intracellular antigen uptake and processing have demonstrated a high level of nonspecific cell surface binding and only a low level of binding to class II molecules [bib_ref] Identification of a peptide binding protein that plays a role in antigen..., Lakey [/bib_ref] [bib_ref] A peptide binding protein having a role in antigen presentation is a..., Van Buskirk [/bib_ref] [bib_ref] Tcell colonies recognize antigen in association with specific epitopes on la molecules, Clark [/bib_ref] [bib_ref] Major histocompatibility complex-controlled, antigenpresenting cell-expressed specificity of T cell antigen recognition, Hansburg [/bib_ref] [bib_ref] Macrophage processing of peptide antigens : identification of an antigen complex, Nairn [/bib_ref].
A series of rather elegant studies using biotinylated peptides and a fluorescent-avidin detection system has emerged from the laboratory of J.B. Rothbard, Immunologic Pharmaceutical Corporation (Palo Alto, CA) [bib_ref] Detectio n of peptide-MHC class II complexes on the surface of intact..., Busch [/bib_ref] [bib_ref] Interactions between immunogenic peptides and HLA-DR molecules, Rothbard [/bib_ref] , allowing valuable insights into the association of immunogenic peptides with cell surface class II molecules . However, the interpretation of these studies is complicated by the potential ofthe biotin moiety to alter the conformation of the peptide bearing the biotin, or to affect the binding of the peptide to the class II molecule when attached to an amino acid in the MHC-interacting region of the antigen.
An antibody reagent which could detect unmodified peptide-MHC class II complexes on the cell surface would clearly be advantageous, and also has the potential to detect naturally processed peptides derived from intact antigen. A series of studies in the late 1970's and early 1980's initially sought to raise such antisera [bib_ref] Nature of Tlymphocytes recognition of macrophage-associated antigens. I. Response of guinea pig..., Thomas [/bib_ref] [bib_ref] Antigen recognition by Tcells and B-cells: recognition of cross-reactivity between native and..., Chestnut [/bib_ref]. Although these studies did raise several antisera capable of binding to free peptide antigen, they were unsuccessful in obtaining binding of peptide to class II after the antisera had bound the peptide, or in demonstrating antisera binding to antigen after antigen uptake onto class II had occurred . Among the possibilities suggested to explain this failure were the denaturation or removal during antigen processing of epitopes on the peptide that are recognized by the antisera, masking of the epitope by the class II molecule, or alteration of the epitope upon binding of peptide to class II.
The human, cytolytic, CD4-positive T cell clone, V1, has been shown to recognize a fragment of hemagglutinin protein from the influenza strain A/Japan/57 (Brown, L.R ., N. Nygard, M.B. Graham, C. Bono, V.L . Braciale, J. Gorka, B.D. Schwartz, and T.J . Braciale, manuscript submitted for publication) . This hemagglutinin peptide includes the amino acid residues 128-145, and is recognized by V1 only in the context HLA of DRw11.
We report here the ability of a rabbit antiserum raised against this influenza hemagglutinin (HA)l peptide to directly detect the complex recognized by the human T cell clone V1, composed of an antigenic HA peptide without any artificial linkage element and bound to HLA-DRw11 on the surface of an APC.
# Materials and methods
Synthetic Peptides. All peptides used in this study were synthesized on an Applied Biosystems (Foster City, CA) automated solid phase peptide synthesizer. The photoprobe peptide was also made on the Applied Biosystems instrument by incorporating the photoreactive group 4-benzoylbenzoic acid on the e-amine of the N-terminal lysine according to the method of Gorka et al . [bib_ref] Automated synthesis of a C-terminus photoprobe using combined Fmoc and t-Boc synthesis..., Gorka [/bib_ref]. The sequences of all peptides used are given in .
Cell Lines and Clones. The T cell clone VI was generated and maintained as previously described [bib_ref] Influenza virus specific human cytotoxic Tcell clone: Heterogeneity in antigenic specificity and..., Kaplan [/bib_ref]. It is specific for the hemagglutinin peptide encompassing amino acid residues 128-145 (HA 128-145) from influenza strain A/Japan/57 when presented in the context of HLA-DRw11(Brown, L.R ., N. T Cell S'Cr Release Cytotoxicity Assay. The "Cr-release cytotoxicity assay was performed as previously described [bib_ref] Influenza virus specific human cytotoxic Tcell clone: Heterogeneity in antigenic specificity and..., Kaplan [/bib_ref].
ceHA Peptide Antiserum. ceHAP rabbit antiserum was prepared as in references 29 and 30 with the following modifications. 5 mg of HA 128-145 photoprobe peptide (HA 128-145 PP, was mixed with 4.5 mg of BSA or KLH and exposed to 362 nm UV light at a distance of 2 cm for 3 h at which time >65% of HA 128-145 PP was conjugated to the protein. 350 Ag of peptide-protein conjugate in 200 Al PBS was emulsified in 500 Al CFA, and used to immunize rabbits by s.c. injection at three sites. The rabbits were boosted at 2 and 4 wk by injection of 350 Pg of peptide-protein conjugate in 500 Al IFA, and were bled 1 wk thereafter. Negative control antiserum was prepared in an identical fashion with irrelevant DN10 peptide .
Mouse Monoclonal Antibodies. The hybridoma producing the anti-DR framework antibody L243 was obtained from the American Type Culture Collection (Rockville, MD). The L243 was affinity-purified using protein A-Sepharose, and used at a concentration of 0.4 mg/ml for whole cell lysate immunoprecipitation . FITC-conjugated L243 was purchased from Becton Dickinson & Co. (Mountain View, CA) for use in FACS analysis . OKDR (anti-DR framework mAb) was purchased from Ortho Diagnostic Systems (Raritan, NJ). SFR3-DR5 (31), an anti-DRw11 mAb was the gift of Dr. Susan Radka (Oncogene, Seattle, WA). Control antibodies used included MKD6 (BD #1360), a murine anti-IAd mAb, and G2CL, a murine anti H-2Kk mAb (BD #9051), both obtained from Becton Dickinson and Co. A511, a murine monoclonal IgG2a antibody made in our laboratory, was also used in some experiments as a negative control. DRw11 Class II Purification . DRw11 haplotype HLA class II protein was affinity-purified from Swei cellsby the method of Turkewitz et al . [bib_ref] Largescale purification of murine I-Ak and I-Ek antigens and characterization of the..., Turkewitz [/bib_ref] using L243 mAb.
allA Peptide Antiserum Enzyme-Linked Immunosorbent Assay.
Nunc #439454 microtiter plate wells (Nunc, Inc., Naperville, IL) were coated with peptide or protein overnight at 4°C and washed with PBS/1% BSA. Where indicated, the wells containing platebound protein were then incubated with 2 Rg peptide in 100 Al PBS for 3 h at room temperature and rewashed in PBS/1% BSA. 100 Al of rabbit serum diluted 1/100 in PBS/1% BSA were added to each well, the incubation continued for 45 min at room temperature, and the wells then washed. The second antibody added was 100 Al of a 1/1000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma Chemical Co., St . Louis, MO) followed by a 45 min incubation at room temperature . After washing, the ELISA was developed by addition to each plate well of 100 JAI of a 1 mg/ml substrate, p-nitrophenyl disodium phosphate, (Sigma #104-105 ; Sigma Chemical Co.) . Following a 1 h room temperature incubation, the OD was read at 410 nm on a Dynatech MR700 ELISA plate reader.
Fluorescence-Activated Cell Sorter Assay with ceHAR 106 indicated cells were incubated for 24 h at 37°C in 7% C02 atmosphere in 1 ml culture medium containing 5% dialyzed FCS and 21 AM of the indicated peptide. The cells were washed twice in PBS/1% FCS and resuspended at 5 x 107/ml in wash buffer. Equal aliquots of peptide-pulsed cells were incubated for 20 min at 4°C with 100 AI of 1/100 dilution of preimmune rabbit serum or immune rabbit otHA peptide antiserum, or the indicated mAb in PBS/1% FCS. After incubation the cell aliquots were washed in PBS/1% FCS, and then incubated in 100 Al of FITC-conjugated goat anti-rabbit IgG (Fisher Biotech #OB1400; Fisher Scientific, St . Louis, MO) for rabbit sera, 100 j.al of FITC-conjugated goat anti-mouse IgG (Fisher Biotech #OB1420-FITC) for unconjugated murine mAb, or no secondary reagent for FITC-conjugated mAb. After rewashing twice at 4°C in PBS/1% FCS, cells were fixed in 200 Fx1 1% formaldehyde and stored at 4°C until FRCS analysis. Cell pellets were brought up in 200 /.Al saline and run on a FACS® Analyzer (Becton Dickinson and Co.), counting 10,000 cells/plot. Immunoprecipitations of Radiolabeled HA (128-145)PP-Pulsed Swei Cell Lysates. 70 x 106 Swei cells were incubated in 35 ml culture medium containing 5% FCS and 250 Ag "'I-labeled HA 128-145 photoprobe peptide. The peptide was labeled by the iodobead method (Pierce Scientific, Rockford, IL) to a specific activity of 18 .7 uCi/Ag. After incubation for 24 h at 37°C, the cells were washed twice in cold PBS and exposed to 350 tun wavelength UV light at 4°C for 1 h. The cells were washed two more times in cold PBS and lysed in 1 ml cold PBS containing 1% NP-40, 50 ug/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 50 Ag/ml Nap-tosyl-L-lysine chloromethyl ketone (TLCK), and 200 Ag/ml PMSF. The lysate was centrifuged at 13,250 RPM for 15 min, and pre-cleared with 100 tt,l packed protein ASepharose (Pharmacia, Uppsala, Sweden) for 30 min at 4°C with agitation. After centrifugation, the supernatant was divided into equal aliquots, and 50 Fd of NRS, ciHAP, MKD6, OKDR, or L243 were added. Overnight incubation at 4°C was followed by addition of 100 ul packed protein A-Sepharose for 30 min at 4°C with agitation every 5 min. After centrifugation the pellets were washed three times with cold 0.25% NP-40, then boiled for 2 min in 165 141 2% mercaptoethanol SDS elution buffer and run on an 11% SDS-PAGE gel. After fixation and drying, the gel was subjected to autoradiography.
Trypsin Digestion and Peptide Mapping. The proteins to be compared were eluted from an excised piece of gel, subjected to trypsin digestion, and peptide mapped as previously described [bib_ref] Glycosaminoglycan modifications of membrane proteins, Giacoletto [/bib_ref]. . We modified this peptide by substituting tyrosine for valine at position 129 to allow I'll-radioiodination in some experiments, and for other experiments by adding a photoreactive side chain residue, 4-benzoylbenzoic acid to the e-amine of the N-terminal lysine . The recognition by clone Vl of the altered synthetic HA peptide (HA 128-145) was found to be identical to that of the native unmodified HA peptide when presented on DRw11 APC's . In addition, the HA 128-145 photoprobe peptide (PP) was recognized equally well by clone V1 when presented on HLA-DRw11 cells (data not shown) . Rabbit Antiserum oiHAP Recognizes Both Free HA Peptide and HA Peptide Bound to DRw11. When assayed by ELISA, rabbit antisera otHAP raised against HA 128-145 PP coupled to BSA or KLH did not react with the irrelevant peptide DN10 nor with affinity-purified DRw11 class II molecules alone . However, the antisera did react with plate-bound HA 128-145 at a titer >1 :200,000, as well as with complexes formed when plate-bound, affinitypurified HLADRw11 molecules were first exposed to soluble HA 128-145 . In contrast, these antisera did not recognize plate-bound DRw11 molecules were first exposed to soluble HA 128-145 . In contrast, these antisera did not recognize plate-bound DRw11 molecules exposed to 0 V1 Lysis with Altered HA Peptide . The homozygous DRw11 BLCL Swei was incubated with no peptide [fig_ref] Figure l: Sequences of peptides used in the study [/fig_ref] or with HA 128-145A, panel c) for 24 h, was reacted with allAP, stained with FITC-conjugated goat anti-rabbit immunoglobulin, and analyzed by FACS. A class II negative, bare lymphocyte syndrome BLCL, SJO, was similarly treated [fig_ref] Figure l: Sequences of peptides used in the study [/fig_ref]. A significant fluorescence shift was seen with otHAP for the HA peptide-pulsed, class II-positive Swei cells [fig_ref] Figure l: Sequences of peptides used in the study [/fig_ref] but not for the HA peptide-pulsed SJO cells, nor for the Swei cells not exposed to HA peptide . In addition otHAP did not react with Swei cells which had been exposed to theirrelevant DN10 peptide (data not shown) . These results suggest that HA peptide uptake is specific for class II-expressing cells, and that oeHAP can detect the peptide on such whole, living cells directly by FACS assay. The Surface Expression ofHA 128-145 on Antigen-Presenting Cells is DRwil-Restricted Homozygous BLCLs bearing DR1, DR2, DR3, or DR4, and one heterozygous (DR2,w11) BLCL (TBLCL) were incubated sequentially with non-photoprobe HA 128-145, with ciHAP, and with FITC-goat anti-rabbit antiserum . The marked fluorescence shift observed with Swei cells [fig_ref] Figure l: Sequences of peptides used in the study [/fig_ref] was not seen with SJO, nor with any of the other homozygous HLA class II-expressing lines studied [fig_ref] Figure l: Sequences of peptides used in the study [/fig_ref] , indicating allelic specificity in the formation of the DRw11-HA 128-145 complex . The DR2,w11-positive TBLCL showed approximately half the fluorescence obtained with Swei cells when both were stained with ciHAP [fig_ref] Figure l: Sequences of peptides used in the study [/fig_ref] and with the anti-DRw11 mAb SFR3-DR5 for DR expressionciHAP Immunoprecipitates DRw11-HA Complexes from DRw11 Cells Incubated with HA 128-145 PP. The previous results strongly suggested that otHAP was detecting HA 128-145/DRw11 complexes on the surface of Swei cells. To further demonstrate this association an immunoprecipitation experiment was performed. Swei cells were incubated with 125 1-labeled HA 128-145 PP for 24 h at 37°C. The cells were washed, exposed to UV light to form covalent class IIphotoprobe linkages, and were then detergent-lysed. HA 128-145 PP-conjugated molecular complexes were immunoprecipitated from whole cell lysates with ciHAP or anti-DR mAb OKDR and Sepharose-linked protein A, and were then analyzed on SDS-PAGE under reducing conditions. Although we had initially expected HA 128-145 PP to be associated with a number of cell components, autoradiographs demonstrated only two bands at -36 kD and 28 kD, corresponding to the expected approximate sizes of the -2 .3 kD HA 128-145 photoprobe peptide bound to DRw11 a (N34kD) and (3 (N26 kD) chains . The OKDR niAb immunoprecipitated two bands of identical size from the same Swei lysates . The absence of any other bands immunoprecipitated by otHAP from the DRw11-bearing cell lysate argues against the existence of any long-lived cell surface complexes formed between HA 128-145 and non-class II molecules.
# Results
## Irrelevant peptide
To further confirm the identity of these bands as DRw11 be and 0 chains, 3H-leucine labeled Swei cells were incubated in the presence or absence of HA 128-145 PP and were then UVlight exposed. After cell lysis the 3H-leucine labeled proteins photoconjugated to HA 128-145 PP were immunoprecipitated by otHAP from lysates of the cells incubated with HA 128-145 PP, and 3H-leucine labeled ci and a chains of class II were immunoprecipitated by anti-DR mAb L243 from lysates of the cells not incubated with HA 128-145 PP. After 248 Human Leukocyte Antigen Class 11-HA Peptide Complex SDS-PAGE, the corresponding bands (arrow heads) were excised, eluted, and subjected to trypsin digestion. Comparative tryptic peptide analysis indicated that the HA 128-145 PP-conjugated proteins were indeed the DRw11 a and (3 chains. DRwll-Binding ofHA 128-145 Inhibits L243 Binding FACS analysis of Swei cells incubated in the absence or presence of HA 128-145 before staining with FITC-conjugated L243 indicated that preincubation with HA 128-145 inhibited recognition of DRw11 by L243 and then protein A-Sepharose. This finding was confirmed by immunoprecipitation . When the lysate of Swei cells photoconjugated with 125 1-labeled HA 128-145 PP was reacted with L243,no bands were seen, despite the presence of bands with, and with OKDR. These results indicated that the epitope recognized by L243 is either located near the DRw11 antigen-binding site or is conformationally altered when HA 128-145 is bound.
The observation that uHAP was capable ofdirectly recognizing HA 128-145/DRwll complexes on the surface of living APC's indicated that ciHAP presented the most direct means to date of studying antigen-class 11 interaction in a human T cell/antigen/APC system and allowed functional studies of MHC-peptide interaction with viable APCs.
Kinetics of Association of HA 128-145 with HLA-DRw11. Swei cells were incubated with HA 128-145 for increasing time periods, then stained with FITC-goat anti-rabbit IgG, and analyzed by FACS. HA 128-145/DRw11 complexes were detectable within 30 min and increased for approximately 20-24 h with a 50% saturation time of 5 1/2 h . No further increase was seen at 48 h. Kinetics of Disappearance of HA 128-145/DRw11 Complexes. Swei cells were incubated with HA 128-145 for 24 h, placed in HA peptide-free media for increasing time periods, and then assayed by FACS for cell surface HA peptide. A prolonged half-life of approximately 14 h was seen for the surface HA 128-145/DRwll complexes , indicating that the complexes are relatively long-lived.
# Discussion
The immunogenic complex composed of the hemagglutinin peptide HA 128-145 and HLADRw11 is recognized by the cytotoxic CD4+ T cell clone V1. The experiments presented here provide direct biochemical evidence that the same cell surface immunogenic complex can be detected by an antiserum produced against the peptide . This antiserum thus provides the first direct means of studying MHC class II-peptide complexes in which the peptide has not been modified by the addition of any non-amino acid moiety. The absence of any such non-amino acid moiety on the peptide eliminates the possibilities that the affinity ofthe peptide for class II or the recognition of the complex by a T cell will in any way be altered .
Antibody recognition of cell surface complexes ofHA 128-145/DRw11 complexes was validated by demonstrating antibody binding to DRw11 positive B-LCL's exposed to HA 128-145, but not to DRw11 negative cells similarly exposed. Antibody also bound to DRw11 positive L cell transfectants incubated with HA 128-145, but not to parental untransfected L cells nor to L cells bearing a different class II molecule that were similarly incubated with peptide. In addition, the number ofcomplexes detected was always commensurate with the level of DR expression.
The complex could also be detected in lysates ofcells both when the peptide was unmodified, and after the DRw11 a and ß chain peptide interactions had been stabilized by photoconjugation with a photoreactive moiety attached to the peptide. The identity of the DRw11 molecule bound to the peptide under these conditions was confirmed through immunoprecipitation of the complexes by both ceHAP and the anti-DR mAb OKDR, by mobility on SDS-polyacrylamide gels, and by tryptic peptide mapping.
Our ability to detect unmodified peptide-class II complexes on the cell surface with anti-peptide antibody successfully for the first time most likely depends on at least three parameters which are optimized in our system . First, Swei cells have between 5 x 105 and 106 DRw11 surface molecules/cell (J. Pollack, Washington University, St. Louis, MO, unpublished observations), presenting a large number ofpotential binding sites for the HA peptide. Second, dose-response curves suggest a high affinity between HA 128-145 and DRw11 . Third, an epitope on HA 128-145 recognized by the ctHAP antiserum remains available to the antiserum when the peptide is bound in the DRw11 antigen-binding cleft . It is possible that in other systems these conditions were not coincident.
The ability of ctHAP to recognize the HLADRw11/HA 128-145 complex without the addition of non-amino acid moieties to the peptide allows delineation of the kinetics of formation and disappearance of complexes without concerns that the kinetics may be affected by the presence of a foreign element . In addition, because detection does not depend on the presence of a foreign element, the antiserum detection system for the first time presents the potential for following the kinetics of complex formation during intracellular uptake and antigen processing.
Initial kinetic characterization has been accomplished using the HA 128-145 peptide and detection of cell surface complexes by FACS. The association curve of HA 128-145/DRw11 complexes shows an appearance of the complex by 30 min that is consistent with previous reports [bib_ref] Binding of labelled influenza matrix peptide to HLA-DR in living B lymphoid..., Ceppellini [/bib_ref] [bib_ref] Kinetics of MHC-antigen complex formation on antigenpresenting cells, Roosneck [/bib_ref]. The continued complex accumulation to 24 h is longer than that previously reported by Ceppellini et al. [bib_ref] Binding of labelled influenza matrix peptide to HLA-DR in living B lymphoid..., Ceppellini [/bib_ref] for living APC's and is consistent with the report of Busch et al. [bib_ref] Detectio n of peptide-MHC class II complexes on the surface of intact..., Busch [/bib_ref] , who showed a lack of surface peptide,-class II saturation after 16 h of APC antigen incubation using a biotinylated HA 307-319 peptide on DR1-expressing cells. Studies of peptide saturation using solubilized, purified class II protein have also demonstrated a slow accumulation of the complex peaking at extended intervals of from 6 to 50 h [bib_ref] Isolatio n and characterization of antigen-la complexes involved in Tcell recognition, Buus [/bib_ref] [bib_ref] Binding of labelled influenza matrix peptide to HLA-DR in living B lymphoid..., Ceppellini [/bib_ref]. The very rapid plateau in binding of radiolabeled peptide on whole B-LCL's reported by may be a reflection of cell peptide internalization with the inability of the radiolabel assay to separate surface from internal peptide uptake .
The dissociation plot of indicates that the cell surface complex of HA 128-145 and DRw11 class II is very stable . The stability of these complexes has been shown previously with purified class 11 molecules [bib_ref] Isolatio n and characterization of antigen-la complexes involved in Tcell recognition, Buus [/bib_ref] [bib_ref] Detectio n of peptide-MHC class II complexes on the surface of intact..., Busch [/bib_ref] [bib_ref] Peptide binding to HLA-DRI: a peptide with most residues substituted to alanine..., Jardetsky [/bib_ref] , but the half-life of these complexes on intact cells has not been as well characterized [bib_ref] Detectio n of peptide-MHC class II complexes on the surface of intact..., Busch [/bib_ref] [bib_ref] Quantitatio n of antigen-presenting cell MHC class II/peptide complexes necessary for Tcell..., Harding [/bib_ref] [bib_ref] Binding of labelled influenza matrix peptide to HLA-DR in living B lymphoid..., Ceppellini [/bib_ref] [bib_ref] Peptide binding to HLA-DRI: a peptide with most residues substituted to alanine..., Jardetsky [/bib_ref].
The precise epitope(s) recognized by the ciHAP antiserum when the HA 128-145 peptide is bound in the DRw11 cleft has not yet been identified, and this identification may have implications for recognition of the naturally processed peptide. While the size of the naturally processed peptide bound References to a class I molecule has recently been determined [bib_ref] Isolation of an endogenously processed immunodominant viral peptide from the class I..., Van Bleck [/bib_ref] , no similar determination has yet been made for peptides bound to class II molecules. However, preliminary studies demonstrating that the aiHAP antiserum blocks V1 recognition of DRw11 positive target cells exposed to HA 128-145 (Mary Beth Graham, Washington University School of Medicine, St. Louis, MO, unpublished results) underscore the physiologic relevance ofthe antibody recognition, and suggest that cxHAP may well recognize the naturally processed peptide.
A number of technical difficulties have prevented demonstrating whether or not otHAP can recognize naturallyprocessed A/Japan/57 hemagglutinin on DRw11-expressing cells using cytofluorometry. The exposed position ofHA 128-145 on the A/Japan/57 virion allows aHAP to detect the intact virion . The inability to clear unprocessed virions either enzymatically or by natural turnover from the membranes of target cells pulsed with UVtreated virus has resulted in high levels of ciHAP staining by the FRCS assay even on non-DRw11 APC's. Intact hemagglutinin and a truncated 45 amino acid HA peptide containing the 128-145 epitope also bind nonspecifically to non-DRw11 cells and are also detected by c iHAP, again resulting in high background. Distinguishing the DRw11-associated HA peptide surface staining from this high background level has not been possible. Studies are currently underway using HA peptides coupled to transferrin to circumvent these difficulties.
The ability for the first time to directly detect peptide-MHC class II surface complexes on APC's with ciHA peptide antiserum allows a number of studies that previously have been difficult or impossible to perform . Indeed, it may be possible to characterize the naturally processed peptide and the intracellular compartments in which naturally processed peptide interacts with MHC class II antigens. These studies are currently underway, and should provide new opportunities for extending our knowledge of human MHC class II function.
[fig] Figure l: Sequences of peptides used in the study. (Top) native peptide sequence from hemagglutinin residues 128-145 of influenza strain A/Japan/57 . (Secondfrom top) altered nonphotoprobe hemagglutinin peptide (HA 128-145) showing tyrosine substitution at position 129. (Second from bottom) altered photoprobe peptide (HA 128-145 PP) showing photoreactive 4-benzoylbenzoic acid side chain on N-terminus lysine. (Bottom) irrelevant peptide DN10 . [/fig]
[fig] Figure 2, Figure 3: A comparison of VI T cell recognition of DRw11 positive target cells pulsed either with native or with altered nonphotoprobe HA 128-145. Recognition is measured on the ordinate as percent specific lysis over background of s'Cr-labeled, peptide-pulsed APC's exposed to Vl at indicated effector to target (E/T) ratios. Concentrations of HA peptide used in APC antigen-loading incubation are given on the abscissa.Native and Modified Forms of HA 128-145 Are Both Recognized by V1. The human CD4+ cytolytic T cell Rabbit antisera recognition of HA 128-145 by ELISA . (A) Rabbit sera (First antibody) were incubated with the indicated plate-bound peptide. (B) Indicated peptides were incubated over the various plate-bound proteins, then incubated with rabbit serum (First antibody). (C) SDS-PAGE Coomassie stain of the affinity-purified DRw11 class II preparation used in [/fig]
[fig] Figure 4: (A) HA 128-145 is specifically expressed on DRw11 positive BLCLs after 37°C incubation. Thick lines indicate initial staining in normal preimmune rabbit serum (NRS) . Thin lines indicate initial staining in rabbit anti-HAP antiserum ((MAP) . (a) SJO (class II negative) ; (b) Swei; (Drw11, wll) without HA peptide ; (c), (d), 0, (g), and (h) Swei (Drw11 wll) ; TBLCL (DRw11,2) ; 3104 (DR1,l) ; 3161 (DR2,2) ; 3098 (DR3,3) ; and 3164 (DR4,4) lymphoblastoid cell lines, respectively, incubated with HA peptide . Staining with cON10 resulted in FACS patterns indistinguishable from those obtained with NRS. (B) (t) Swei, and V) TBLCL, without HA peptide, stained with control mAb A511 (thin line) or for DRw11 expression with the anti-DRw11 mAb SFR3-DR5 (thick line) . [/fig]
[fig] Figure 5: (A) SDS-PAGE gel autoradiograph of immunoprecipitates of Swei whole cell lysates pulsed with 1251-labeled HA 128-145 PP (photoprobe peptide), and UVexposed before immunoprecipitation with normal rabbit serum (NRS), immune rabbit anti-hemagglutinin peptide antiserum (aHAP), or anti-DR mAb OKDR and protein A-Sepharose. Bands corresponding to the a and /3 chain proteins of DR class II are indicated. (B) SDS-PAGE autoradiograph of immunoprecipitates of whole cell lysates from 3H-leucine labelled Swei cells precipitated with NRS or ciHAP followed by protein A-Sepharose after HA 128-145 PP incubation and UV exposure (+HA 128-145 PP), or with MKD6 or L243 without HAP incubation (no peptide) . The positions of the indicated mol wt markers are shown on the left . The closed arrow heads mark the a chain protein bands of class II, and the open arrow heads the ,B chain protein bands . (C and D) HPLC of tryptic digests from bands inFig. 5B corresponding to a and /3 chain class II proteins, respectively, seen on SDS-PAGE of L243 and oeHAP immunoprecipitates.24 7 Nygard et al . [/fig]
[fig] Figure 6: (A) FACS analysis of Swei cells with (+HAP) and without (-HAP) preincubation in HA 128-145 stained with control mAb G2CL or with aDR mAb L243 . (B) Autoradiogram of SDS-PAGE analysis of whole cell lysates from Swei cells photoconjugated with 125 1-HA 128-145 photoprobe and immunoprecipitated with NRS, otHAP, anti-murine I-Ad mAb MKD6, and anti-HLADR mAb L243 followed by protein A-Sepharose . Mol wt markers are in the first lane.allAP [/fig]
[fig] Figure 7, Figure 8: Association plot of the percent of maximum increase in mean fluorescence above background occurring on Swei cells pulsed with HA 128-145 for progressively longer time periods prior to oiHAP staining and FACS Dissociation plot of the decay in percent mean fluorescence increase above background when Swei cells are pulsed with HA 124-145 for 24 h, washed, and then incubated for progressively longer time periods without HA peptide prior to oeHAP staining and FACS analysis .249 Nygard et al. [/fig]
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Study protocol of the Edinburgh and Lothian Virus Intervention Study in Kids: a randomised controlled trial of hypertonic saline nose drops in children with upper respiratory tract infections (ELVIS Kids)
[bib_ref] Rhinovirus infections in the first 2 years of life, Toivonen [/bib_ref] [bib_ref] Association of myeloperoxidase polymorphism (G463A) with cervix cancer, Castelão [/bib_ref] [bib_ref] The effect of a low concentration of hypochlorous acid on rhinovirus infection..., Yu [/bib_ref] [bib_ref] Cutaneous Na+ storage strengthens the antimicrobial barrier function of the skin and..., Jantsch [/bib_ref] [bib_ref] Association between infant swimming and rhinovirus-induced wheezing, Schuez-Havupalo [/bib_ref] [bib_ref] Incidence and severity of childhood pneumonia in the first year of life..., Le Roux [/bib_ref] [bib_ref] Antiviral innate immune response in non-myeloid cells is augmented by chloride ions..., Ramalingam [/bib_ref] [bib_ref] Antiviral innate immune response in non-myeloid cells is augmented by chloride ions..., Ramalingam [/bib_ref] [bib_ref] Effects of a low concentration hypochlorous acid nasal irrigation solution on bacteria,..., Kim [/bib_ref] [bib_ref] The effect of a low concentration of hypochlorous acid on rhinovirus infection..., Yu [/bib_ref] [bib_ref] Uses of inorganic hypochlorite (bleach) in health-care facilities, Rutala [/bib_ref] [bib_ref] Antiviral effectiveness of chlorine bleach in household laundry use, Jordan [/bib_ref] [bib_ref] Association of myeloperoxidase polymorphism (G463A) with cervix cancer, Castelão [/bib_ref] [bib_ref] Redox reactions and microbial killing in the neutrophil phagosome, Winterbourn [/bib_ref] [bib_ref] A specific and sensitive method for detection of hypochlorous acid for the..., Chen [/bib_ref] [bib_ref] 3% Hypertonic Saline Versus Normal Saline in Inpatient Bronchiolitis: A Randomized Controlled..., Silver [/bib_ref] [bib_ref] SABRE: a multicentre randomised control trial of nebulised hypertonic saline in infants..., Everard [/bib_ref] [bib_ref] Nebulised hypertonic saline in bronchiolitis: take it with a pinch of salt, Cunningham [/bib_ref] [bib_ref] The tale of 2 trials: disentangling contradictory evidence on hypertonic saline for..., Grewal [/bib_ref] [bib_ref] Hypertonic saline for the treatment of bronchiolitis in infants and young children:..., Baron [/bib_ref] [bib_ref] The tale of 2 trials: disentangling contradictory evidence on hypertonic saline for..., Grewal [/bib_ref] [bib_ref] Hypertonic saline for the treatment of bronchiolitis in infants and young children:..., Baron [/bib_ref] [bib_ref] A living systematic review of nebulized hypertonic saline for acute bronchiolitis in..., Badgett [/bib_ref] [bib_ref] Efficacy of isotonic nasal wash (seawater) in the treatment and prevention of..., Slapak [/bib_ref] [bib_ref] Comparison between the use of saline and seawater for nasal obstruction in..., Köksal [/bib_ref] [bib_ref] Saline nasal irrigation for acute upper respiratory tract infections, Kassel [/bib_ref] [bib_ref] A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation..., Ramalingam [/bib_ref] [bib_ref] A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation..., Ramalingam [/bib_ref]
## Methods and analysis study objectives primary objective
To investigate whether the use of parent/guardianinitiated HS nose drops administered to children with symptoms consistent with acute viral URTI reduces the duration of symptoms when compared with children managed using standard care.
## Secondary objectives
To determine the effect of HS nose drops on:
1. Severity of all symptoms. 2. Duration and severity of individual symptoms. 3. Contact with NHS 24, out of hours primary care (OOH), and primary care (GP).
4. Hospital attendance (ie, A&E attendance and/or hospital admission) and diagnosis. 5. Reduction in wheeze.. Over the counter medication use. 7. Duration, reduction or rate of reduction in viral shedding. 8. Transmission within the household. 9. Side effects associated with the use of saline nose drops. 10. Adverse events (AEs) associated with the use of saline nose drops. 11. Time off from school/nursery for child and workdays lost for parent/guardian. 12. Cost associated with illness (over the counter medication costs and NHS costs).
Study design and sample size ELVIS Kids is a parallel, open label, RCT of HS nose drops (~2.6% NaCl) versus standard care in children <7 years of age with symptoms of an URTI. The aim is to recruit a total of 480 children (240/arm). The study will run over ~42 months at participating sites in Scotland (sites are as listed on ClinicalTrials. gov). Children are recruited prior to, or within 48 hours of developing URTI symptoms by advertising in areas such as local schools, nurseries, health centres, hospitals, recreational facilities,workplaces public events and the community as well as local and social media. For the purposes of this study an URTI is defined as: at least two respiratory symptoms (nasal congestion, runny nose, cough, sore throat) or one respiratory symptoms and at least one systemic symptom (low energy/tired, muscle aches/pains, headache, fever ≥38°C). Willing parents/ guardians, will be directed by the study advertising to contact the research team at their local site) if they are interested in participating.
Children will be randomised to either a control arm of standard symptomatic care, or an intervention arm of three drops each nostril of HS at least four times a day and up to a maximum of 12 times a day until asymptomatic or maximum of 28 days. All parents/guardians will be requested to obtain a mid-turbinate nasal swab from the participant first thing in the morning (before nose drops in the intervention arm) for five consecutive days (unless the child is well before then), a daily diary (a global severity question, Canadian Acute Respiratory Illness and Flu Scale (CARIFS), 32 a validated illness measure in the UK, side effects and compliance with trial procedures) until they report the child as 'not unwell', an end of illness diary (infection in household contacts, ease of use and acceptability of intervention, medication and healthcare use, acceptability, time taken off usual activities, wheezing and whistling in the chest), a satisfaction questionnaire and AEs. Parents/guardians of the children allocated to the intervention arm will be taught how to prepare the HS (including sterilisation instructions for children under a year). Parents/guardians of children who are asymptomatic at recruitment are requested to inform their local research team when the child develops an URTI (within 48 hours) and follow the instructions already provided to them. On day 28, parents/guardians will be contacted to determine if their child suffered from wheezing or whistling in the chest either during the illness or at any point until day 28. Participation in the study will end on day 28.
## Eligibility and consent
Prescreening for eligibility to participate will be completed by a member of the research team at the clinical trials unit when parents/guardians phone to express interest in the study. If parents/guardians attend an appointment and take part in the study, the study number will be recorded on the screening log and details of eligibility will be recorded in the study database.
Inclusion criteria 1. Children between corrected gestational age of ≥40 weeks and <7 years of age. 2. Children without URTI OR ≤48 hours of URTI* starting. *An URTI being defined as at least two respiratory symptoms (nasal congestion (ie, stuffy nose), runny nose, cough, sore throat) or one respiratory symptom+at least one systemic symptom (low energy/tired, muscle aches/ pains, headache, fever 38°C).
## Exclusion criteria
1. Children needing immediate medical attention.
2. Children using saline drops/sprays at the time of randomisation. 3. Children on immunosuppressive medication, regular oral/inhaled steroids, regular antibiotics (use of antibiotics is allowed as long as the child does not need regular antibiotics). 4. Children with a known chronic illness (eg, cystic fibrosis, cardiac, renal, liver, lung, neurological conditions) apart from wheeze or asthma which are not exclusions if the child is otherwise well and not on regular steroids). 5. Children being followed up for developmental delay. 6. Children receiving the nasal influenza vaccine ≤14 days ago. 7. Children taking part in another interventional trial. 8. If parents/guardians indicating that they are unable to comply with the study protocol prior to randomisation. 9. If parents/guardians are unable to understand written or spoken English. 10. Children randomised to ELVIS KIDS on a previous episode of URTI. 11. Children with a concurrently participating sibling.
All ineligible and non-recruited participants will be recorded on the ELVIS Kids screening log with a reason given.
## Obtaining consent
Only trained and delegated members of the trial team will take consent-this will usually be the research nurse.
## Open access
The participant information sheet (PIS), which will explain the aims of the study and the potential risks and benefits of the study treatment, are provided to parents/ guardians when they meet the research team (also available online). A children's PIS will be available to discuss with older children attending the appointment with the option of giving their assent (online supplemental file 1).
If the parent/guardian wishes to participate in the study, then they will be asked to sign the informed consent form (ICF) (online supplemental file 1). Both the parent/ guardian and the person delegated to take consent will sign and personally date the ICF. The original signed ICF must be kept by the Investigator in the investigator site file, one copy is provided to the parent/guardian, one copy is placed in TRAK. The same would apply in the case of assent being given.
## Randomisation and treatment allocation
A member of the research team from the clinical research facility will perform the randomisation using a web-based randomisation service managed by the Edinburgh Clinical Trials Unit (ECTU). Children will be allocated to receive either HS nose drops or standard care in a 1:1 ratio using minimisation based on age (0-2, >2 years) and sex and allocated to receive the treatment which minimises the imbalance with a probability 0.8. The study is not blinded apart from those carrying out lab assessments of nasal swabs.
Sea salt will be provided by Cornish Sea Salt company in 225 g pots. They will be supplied to local pharmacies where they will be labelled and stored. A working stock will be issued to the research team. If a child is allocated to an intervention arm, the parent/guardian will be given instructions on the preparation and use of HS nose drops using instructional video, verbal and written information. Parents/guardians will be asked to add one level measure of sea salt to a fixed volume of freshly boiled water using the measuring spoon and clean glass jar provided. This provides a NaCl concentration of ~2.6% and the drops can be used once cooled. Two glass jars are provided so that the parent/guardian could use one and have a clean spare to prepare solution the next day. Two dropper bottles are provided with which nose drops can be applied (one in use, and a clean spare).
## Withdrawal of study participants
Parents/guardians are free to withdraw their child from the study at any point. If withdrawal occurs, the primary reason for withdrawal will be documented in the participant's case report form (CRF), if given. All data and swabs collected before withdrawal will be retained for analysis in cases where participants withdraw.
## Study assessments
The protocol is designed in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT). The trial overview of the study assessments is available as a SPIRIT figure [fig_ref] Table 1: Assessments and timepoints [/fig_ref]. At the appointment, a member of the research team will train the parent/guardian how to identify an URTI, how to measure temperature, how to complete the diaries and how to collect and return the mid-turbinate swabs. In addition, those in the intervention arm will be given instructions on how to prepare and apply nose drops. Baseline information on the child, contact details of parent/guardian and number of household members at the time of recruitment will be collected in the electronic CRF (eCRF). If recruited when symptomatic, parents will be instructed to start the study assessments the same day. If recruited when asymptomatic, and there are no changes to the child's medical information, parent/guardians will be asked to start the study assessments and to inform the study team the same day if possible or at least within 2 days of onset of illness. If recruited when asymptomatic, and there are changes to the child's medical information, parent/guardians will be asked to contact the study team to ensure the child still meets the eligibility criteria before starting the study procedures. If it is not suitable for the child to take part during this URTI (eg, received the influenza vaccine in the past 2 weeks) they will remain on study and be asked to contact the team at the onset of the next URTI.
Parents/guardians are requested to collect a nasal (mid-turbinate) swab as soon as possible on day 1 and first thing in the morning (and prior to HS nose drops being applied) on days 2-5 if children remain unwell. These samples are to be packed in the transport box provided, stored in the fridge and returned in the prepaid envelope or as soon as possible after completing collection. If samples are not received by day 10, a reminder will be sent to the parent/guardian. Parents/guardians will complete a daily diary (online supplemental file 1) which records any symptoms the child is experiencing, compliance to nasal swabs and HS drops, any side effects and use of healthcare services. Parents will be taught how to measure temperature with TempaDot. Parents are advised to measure the temperature only if they think the child has a fever. If the child has an axillary temperature of ≥38°C, it should be recorded as a fever in the daily diary. The diaries will be provided as an online form (unless parents cannot access this in which case a paper copy can be provided). If the online Daily Diary is not completed a reminder will be sent.
An end of Illness diary (online supplemental file 1) and satisfaction questionnaire (online supplemental file 1) will be completed by the parent/guardian once the child is asymptomatic for >24 hours or after a maximum of 28 days. On day 28, the parent/guardian will be contacted by email and sent a text message to ask if their child has experienced any wheeze since the end of illness diary was completed (online supplemental file 1).
Participants will be sent a £30 voucher by email as compensation for any inconvenience once they have returned the study data.
## Open access
Analysis and storage of samples Up to five nasal swabs will be collected and posted to the Department of Laboratory Medicine, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, EH16 4SA, where they will be stored and processed. Day 1 samples will be analysed by the respiratory panel and the cycle threshold (CT) of positive samples recorded. If the day one sample is missing, the first available sample will be tested to identify the virus. If an agent is identified, all samples (days 1-5) will be tested in parallel to estimate change in viral shedding and the CT recorded. If a sample is positive on day 1 and negative on subsequent days, they may be tested for human DNA to confirm a sample was collected. Log conversion of each positive result will be done using the following formula: (40-CT of specimen)/3.3 to estimate change in shedding. All nucleic acid extracts and remaining original samples will be stored in the Lothian NHS research Scotland BioResource biobank (REC reference 15/ES/0094) and can be used in future ethically approved studies.
## Outcomes/endpoints
Primary endpoint Duration of illness (measured as the number of days until the parent reports the child to be well).
## Open access
18. Number of days lost from work for parent/guardian. [bib_ref] Cutaneous Na+ storage strengthens the antimicrobial barrier function of the skin and..., Jantsch [/bib_ref]. Cost of over the counter medication used. 20. NHS costs associated with illness.
## Participant timeline
The participant pathways can be seen in figures 1 and 2. Participants will be active in the study for 28 days. There are no long-term follow-up assessments after day 28 of URTI developing.
## Data collection
Baseline data will be collected on the baseline eCRF by a member of the research team. Parents/guardians will record study data onto either an online form which will be saved into the eCRF. A paper CRF (pCRF) option is available if a parent/guardian prefers it. pCRF will be returned to the local research team and transcribed by a member of the research team into the database and crosschecked by another.
Virological results are downloaded (identifiable by study number) on a weekly basis on to a specific drive by the laboratory information management and technology team. These will be emailed to the ECTU on a monthly basis and uploaded into the study database.
The trial database will be created and maintained by ECTU. Trained and delegated members of the research team will be given password-protected logins to the database to complete data entry. Data completed online by parents/guardians will be transmitted into the study database. The data will be stored in a secure server in the University of Edinburgh for at least the archiving period.
## Open access
Adverse events Symptoms and side effects from the daily diary will be recorded in the CRF but will not be recorded as an AE or adverse reaction. Hospitalisation is a study outcome and is exempt from reporting to the sponsor as a serious AE.
Any other AEs identified between day 1 and 28 of the study will be recorded. Any events reaching seriousness criteria will be reported to the sponsor within 24 hours.
## Sample size calculation and statistical analysis
Sample size calculation is based on mean (SD) duration of illness values in a control population from Gruber et al of 13 [bib_ref] Incidence and severity of childhood pneumonia in the first year of life..., Le Roux [/bib_ref] days. To detect a 20% difference in mean duration, that is, 3 days, using a two sided, twosample test with 5% level of significance, 90% power and common SD of 9 days we will need a sample of 191 per treatment arm, without drop-outs. Hence, we will recruit 240 participants per arm to allow for up to 20% drop-outs.
Statistical analysis will be conducted according to the details specified in the prespecified statistical analysis plan. Differences in illness duration between treatment arms will be compared using a two-sample t-test or nonparametric equivalent, as appropriate. This method will also be employed to examine differences between treatment arms for other continuous outcome measures such as average symptom score, viral shedding between treatment arms. For binary categorical data, for example, the proportion of participants per arm attending their GP, attending hospital, etc, we will compare the treatment arms using a binomial test for the comparison of proportions. Where we have categorical data with more than two categories a χ 2 test will be used to examine the relationships between treatment arms. If the number of cases of individual viruses are sufficient, the above analysis will be repeated by virus type.
## Oversight arrangements
The study is cosponsored by Academic and Clinical Central Office for Research and Development, a partnership between the University of Edinburgh and NHS Lothian Health Board based at QMRI, 47 Little France Crescent, Edinburgh Email: enquiries@ accord. scot. The trial will be coordinated by a Project Management Group, consisting of the chief investigator, coinvestigators, trial manager, statistician and coordinating nurse. The trial manager will oversee the study and will be accountable to the chief investigator. ECTU is responsible for trial management and oversight of data collection. The Edinburgh Clinical Research Facility are responsible for the statistical analysis. A Delegation Log will be prepared, detailing the responsibilities of each member of staff working on the trial. A trial steering committee (TSC) has been established to oversee the conduct progress. As there will be no data monitoring committee for this project, the TSC will review safety information as part of their remit.
## Ethics and dissemination
The study will be conducted in accordance with the principles of the International Conference on Harmonisation Tripartite Guideline for Good Clinical Practice (ICH). The study has been approved by the West of Scotland Research Ethics Service (reference: 18/WS/0080). Any changes in research activity, except those necessary to remove an apparent, immediate hazard to the participant in the case of an urgent safety measure, will be reviewed and approved by the chief investigator. Amendments will be submitted to the sponsor for review and authorisation before being submitted in writing to the appropriate REC, and local research and development office for approval prior to participants being enrolled into an amended protocol. The findings will be disseminated through peerreviewed publications, conference presentations and on the study website.
## Confidentiality
All laboratory specimens, evaluation forms, reports, and other records must be identified in a manner designed to maintain participant confidentiality. All records must be kept in a secure storage area with limited access. Clinical information will not be released without the written permission of the participant's parent/guardian. The Investigator and study site staff involved with this study may not disclose or use for any purpose other than performance of the study, any data, record or other unpublished, confidential information disclosed to those individuals for the purpose of the study. Prior written agreement from the sponsor or its design must be obtained for the disclosure of any said confidential information to other parties.
All Investigators and study site staff involved with this study must comply with the requirements of general data protection regulations with regard to the collection, storage, processing and disclosure of personal information and will uphold the Act's core principles. Access to collated participant data will be restricted to individuals from the research team treating the participants, representatives of the sponsor(s) and representatives of regulatory authorities. Computers used to collate the data will have limited access measures via user names and passwords. Published results will not contain any personal data that could allow identification of individual participants.
## Patient and public involvement
Feedback was obtained from patient and public involvement (PPI) representatives on the study protocol, information sheets, diaries and consent forms and necessary modifications made prior to starting the study. A PPI representative is also invited to attend the trial steering committee meetings.
## Access to data
Ownership of the data arising from this study resides with the study team. On completion of the study, the study data Open access will be analysed and tabulated, and a clinical study report will be prepared in accordance with ICH guidelines.
Trial status This paper describes study protocol version V4 (06/01/2020). The trial opened on 2 November 2018. The first participant was recruited on 6 November 2018. The planned study end date is 30 November 2021. At the time of submission, study recruitment was suspended due to the COVID-19 pandemic.
# Discussion
The study is based on the recently discovered evidence that epithelial cells have an innate antiviral effect. [bib_ref] Antiviral innate immune response in non-myeloid cells is augmented by chloride ions..., Ramalingam [/bib_ref] This effect can be augmented by supplying the cells with chloride ion through NaCl. Saline, commonly used as a placebo cannot be used in that role here as it contains NaClthe substrate being tested. We are hence measuring viral shedding as an independent measure of any antiviral effect. The results from this trial will help determine if a simple and low-cost intervention could help to reduce the duration of symptoms of URTI in children. Changes to the duration of individual symptoms, wheeze, transmission within the household, over-the-counter medication use, need for further treatment, days lost and cost of illness are all secondary outcomes.
# Conclusion
Since numerous viruses can cause URTI and in the absence of an antiviral agent/vaccine against the vast majority of viruses, if successful, this low cost and easily accessible intervention can easily be rolled out globally.
[fig] Figure 1: ELVIS Kids Patient Pathway when the child has an URTI at recruitment. ELVIS, Edinburgh and Lothians' Viral Intervention Study; URTI, upper respiratory tract infection. [/fig]
[fig] Figure 2: ELVIS Kids Patient Pathway when the child does not have an URTI at recruitment. ELVIS, Edinburgh and Lothians' Viral Intervention Study; URTI, upper respiratory tract infection. [/fig]
[table] Table 1: Assessments and timepoints [/table]
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Sensory contribution to vocal emotion deficit in patients with cerebellar stroke
A B S T R A C TIn recent years, there has been increasing evidence of cerebellar involvement in emotion processing. Difficulties in the recognition of emotion from voices (i.e., emotional prosody) have been observed following cerebellar stroke. However, the interplay between sensory and higher-order cognitive dysfunction in these deficits, as well as possible hemispheric specialization for emotional prosody processing, has yet to be elucidated. We investigated the emotional prosody recognition performances of patients with right versus left cerebellar lesions, as well as of matched controls, entering the acoustic features of the stimuli in our statistical model. We also explored the cerebellar lesion-behavior relationship, using voxel-based lesion-symptom mapping. Results revealed impairment of vocal emotion recognition in both patient subgroups, particularly for neutral or negative prosody, with a higher number of misattributions in patients with right-hemispheric stroke. Voxel-based lesion-symptom mapping showed that some emotional misattributions correlated with lesions in the right Lobules VIIb and VIII and right Crus I and II. Furthermore, a significant proportion of the variance in this misattribution was explained by acoustic features such as pitch, loudness, and spectral aspects. These results point to bilateral posterior cerebellar involvement in both the sensory and cognitive processing of emotions.
# Introduction
Evidence from neuroimaging and clinical studies points to the functional involvement of the cerebellum in vocal emotion recognition (for a review, see. The vermian region, Lobule VI and Crus I in the lateral cerebellum have been identified as key regions that interact with the prefrontal cortex and temporal lobes, including the amygdala, via reciprocal connections [bib_ref] Functional topography in the human cerebellum: a meta-analysis of neuroimaging studies, Stoodley [/bib_ref]. Some studies among patients with cerebellar lesions have reported impaired recognition for all emotional prosody [bib_ref] Impairment of emotional facial expression and prosody discrimination due to ischemic cerebellar..., Adamaszek [/bib_ref] , whereas others have failed to find any deficit [bib_ref] Affective communication deficits associated with cerebellar degeneration, Heilman [/bib_ref]. The lack of agreement in the literature could be due to several factors, including type of task and methodology (for more details, see [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref]. In one previous study, the authors reported more fine-grained results, with patients giving erroneous ratings on the Surprise scale when they listened to fear stimuli. Using voxelbased lesion-symptom mapping (VLSM), they found that these emotional misattributions correlated with lesions in the right cerebellar hemisphere (Lobules VIIb, VIII and IX) [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref] , confirming the involvement of the cerebellum in vocal emotion processing, and also pointing to potential cerebellar hemispheric specialization in these processes.
Traditionally, the dentate-rubro-thalamo-cortical tract, identified as a feed-forward cerebellar pathway, is described as being composed of crossed fibers. Connections are mainly or even exclusively contralateral, suggesting a functional hemispheric asymmetry that mirrors that of cerebral function [bib_ref] Diffusion tensor imaging of the human cerebellar pathways and their interplay with..., Keser [/bib_ref]. Concerning emotion perception, fMRI studies suggest that the left cerebellum plays a critical role in the recognition of others' facial expressions [bib_ref] Neural substrates of the ability to recognize facial expressions: a voxel-based morphometry..., Uono [/bib_ref]. Based on these results and on the classic models of emotional prosody processing, more activation would be expected in the left cerebellar hemisphere during emotional prosody recognition. However, although fMRI studies investigating emotional prosody processing in healthy individuals have reported cerebellar activation, none of them has postulated that one of the cerebellar hemispheres is more specialized for this processing. Some studies have reported bilateral cerebellar activation [bib_ref] Vocal identification of speaker and emotion activates different brain regions, Imaizumi [/bib_ref] [bib_ref] Identification of emotional intonation evaluated by fMRI, Wildgruber [/bib_ref] , whereas others have found only left [bib_ref] Predicting vocal emotion expressions from the human brain, Kotz [/bib_ref] or right [bib_ref] The neural correlates of emotional prosody comprehension: disentangling simple from complex emotion, Alba-Ferrara [/bib_ref] activation. The basal ganglia (BG), which have very strong connections with the cerebellum [bib_ref] The basal ganglia and the cerebellum: nodes in an integrated network, Bostan [/bib_ref] , have been far more extensively studied in this domain. It has been suggested that the right BG [bib_ref] Vocal emotion decoding in the subthalamic nucleus: An intracranial ERP study in..., Péron [/bib_ref] [bib_ref] Hemispheric specialization of the basal ganglia during vocal emotion decoding: Evidence from..., Stirnimann [/bib_ref] are specifically involved in emotional prosody recognition. The question of the possible hemispherical specialization of the cerebellum in the recognition of emotional prosody therefore remains unanswered. Another critical question in this field of research concerns the involvement of low-level sensory processes in the processing of emotional prosody by the cerebellum.
Based on internal models theory (for reviews, see [bib_ref] Consensus paper: the cerebellum's role in movement and cognition, Koziol [/bib_ref] and the idea that both the BG and cerebellum participate in response selection and inhibition [bib_ref] Towards a computational neuropsychology of action, Krakauer [/bib_ref] , [bib_ref] Comparison of visual and auditory emotion recognition in patients with cerebellar and..., Adamaszek [/bib_ref] assumed that the cerebellum identifies and shapes appropriate reactions to a specific sensory state, while the BG compute the cost-benefit analysis and optimum resource allocation. It is key to identify the sensory contributions of both the cerebellum and BG to vocal emotion deficits. According to classic models of emotional prosody processing [bib_ref] Beyond the right hemisphere: brain mechanisms mediating vocal emotional processing, Schirmer [/bib_ref] [bib_ref] A cerebral network model of speech prosody comprehension, Wildgruber [/bib_ref] the first step of prosody processing consists of the extraction of suprasegmental acoustic information, predominantly subserved by right-sided primary and higher-order acoustic brain regions (including the middle part of the bilateral superior temporal sulcus and anterior insula). The BG have already been described as playing an important role in the extraction and integration of acoustic cues during emotion perception [bib_ref] Sensory contribution to vocal emotion deficit in Parkinson's disease after subthalamic stimulation, Péron [/bib_ref]. Concerning the cerebellum, studies of individuals with cerebellar disturbances have highlighted its contribution to timing and sensory acquisition, as well as to the prediction of the sensory consequences of a given action [bib_ref] Comparison of visual and auditory emotion recognition in patients with cerebellar and..., Adamaszek [/bib_ref]. Increased cerebellar activity has been reported during pitch discrimination tasks [bib_ref] Increased activation of the human cerebellum during pitch discrimination: a positron emission..., Petacchi [/bib_ref]. Patients with cerebellar disorder have difficulty comparing the durations of two successive time intervals [bib_ref] Timing functions of the cerebellum, Ivry [/bib_ref]. Moreover, according to [bib_ref] Affective and sensorimotor components of emotional prosody generation, Pichon [/bib_ref] , the cerebellar vermis modulates fundamental frequency (F0) in emotional speech production. A review of the literature on the perception and production of singing and speech [bib_ref] Speech and song: the role of the cerebellum, Callan [/bib_ref] highlighted left cerebellar hemispheric specialization for the processing of singing and, more particularly, its involvement in the analysis of low-frequency information (prosodic and melodic properties). By contrast, right hemispheric specialization for speech processing points to involvement in the analysis of high-pass filtered information (segmental properties). Interestingly, regarding the idea of cerebellar hemispheric sensitivity to different timescales, a recent studysuggested that temporo-cerebellar interactions are involved in the construction of internal auditory models. Reciprocal temporo-cerebellar connections thus contribute to the production of unitary temporally structures stimulus representations, and these internal representations fit cortical representations of the auditory input, to allow for the efficient integration of distinctive sound features. Thus, the cerebellum, in close collaboration with the BG, participates in the organization of sound processing and temporal predictions [bib_ref] Brain networks of emotional prosody processing, Grandjean [/bib_ref].
However, cerebellar hemispheric specialization for the processing of emotional information conveyed in a speaker's voice has not yet been established, and its contribution to acoustic feature processing during an emotional episode has never been explored. In this context, the purpose of the present study was to investigate i) cerebellar hemispheric specialization for the recognition of emotional prosody, and ii) the influence of acoustic features on this processing. To this end, we assessed the vocal emotion recognition performances of 13 patients with right cerebellar lesions (RCL) and 11 patients with left cerebellar lesions (LCL) due to ischemic stroke, comparing them with a group of 24 age-matched healthy control participants (HC). We used a previously validated methodology that has been shown to be sensitive enough to detect even slight emotional impairments [bib_ref] Major depressive disorder skews the recognition of emotional prosody, Péron [/bib_ref] [bib_ref] Recognition of emotional prosody is altered after subthalamic nucleus deep brain stimulation..., Péron [/bib_ref]. This relies on visual (continuous) analog scales, which do not induce biases, unlike categorization and forced-choice tasks (e.g., naming of emotional faces and emotional prosody). Thus, the results of this study would shed light on the participation of the cerebellum in the different sensory an cognitive processing steps described in current models of emotional prosody [bib_ref] Beyond the right hemisphere: brain mechanisms mediating vocal emotional processing, Schirmer [/bib_ref] [bib_ref] A cerebral network model of speech prosody comprehension, Wildgruber [/bib_ref]. They might also suggest hemispheric sensitivity to different temporal processing patterns (as already shown at the cortical level). This would strengthen evidence of temporo-cerebellar collaboration in prosody processing through internal models.
Regarding behavioral results, and on the basis of prior findings [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref] , we expected to observe a greater deficit in the recognition of vocal expressions (for all emotions, but not for neutral stimuli) in the patient group [bib_ref] Impairment of emotional facial expression and prosody discrimination due to ischemic cerebellar..., Adamaszek [/bib_ref] [bib_ref] Comparison of visual and auditory emotion recognition in patients with cerebellar and..., Adamaszek [/bib_ref] [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref] than in HC. We also postulated that patients with RCL would be more severely impaired, as a prior study [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref] had shown that the severity of emotion recognition disturbances is best be explained by lesions in the right posterior cerebellar lobe (Lobules VIIb, VIII and IX). Moreover, based on a previous hypothesis formulated by [bib_ref] Speech and song: the role of the cerebellum, Callan [/bib_ref] concerning the differential roles of the cerebellar hemispheres in speech and song processing, we predicted that the acoustic features of the emotional stimuli would have a differential impact on these emotional prosody disturbances, depending on the lateralization of the cerebellar lesion. We expected patients with LCL to have greater difficulty processing low-frequency information (prosodic, melodic properties), and patients with RCL to have greater difficulty processing high-pass filtered information (segmental properties) [bib_ref] Speech and song: the role of the cerebellum, Callan [/bib_ref]. However, even if acoustic feature processing significantly contributes to changes in emotional prosody recognition following cerebellar stroke, we predicted that any variance we observed would not be sufficient to explain all the emotionally biased results. As the cerebellum is involved in cognitive functions [bib_ref] The cerebellum and cognition, Schmahmann [/bib_ref] , it must also be involved in higher levels (cognitive evaluative judgments) of emotional prosody processing. [fig_ref] Table 1: Clinical, demographic, and neuropsychological data of the two subgroups of patients with... [/fig_ref] One group of 24 patients with first-ever cerebellar ischemic stroke (>3 months prior to enrolment, corresponding to chronic post-stroke phase) and one group of 24 HC took part in the study. The data of 12 of the 24 patients had already been acquired in a previous study [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref]. The patient group was divided into two subgroups: 13 patients with RCL, and 11 patients with LCL. The mean age of the RCL group was 61.4 years (SD = 12.33, range = 48-85), and the mean age of the LCL group was 62.4 years (SD = 10.15, range = 43-77). According to the criteria of the Edinburgh Handedness Inventory [bib_ref] The assessment and analysis of handedness: the Edinburgh inventory, Oldfield [/bib_ref] , 22 patients were right-handed and two were left-handed. The mean education level was 16 years (SD = 4.67, range = 9-22) for the RCL group, and 12.6 years (SD = 4.10, range = 7-20) for the LCL group. The two groups of patients were matched for sex (z = 0.64, p = 0.52), age (z = 0.61, p = 0.54), education level (z = -1.53, p = 0.12), and handedness (z = 0.03, p = 0.98). All patients were French speakers. Mean time since stroke was 29.2 months (SD = 34.16, range = 3-155). Exclusion criteria were 1) brainstem or occipital lesion (factor influencing clinical signs), 2) one or more other brain lesions, 3) diffuse and extensive white-matter disease, 4) other degenerative or inflammatory brain disease, 5) confusion or dementia, 6) major psychiatric disease, 7) the wearing of hearing aids or a history of tinnitus or a hearing impairment, as attested by the Montreal-Toulouse auditory agnosia battery (PEGA(mean total score = 28.4, SD = 2.0, range = 24-30), 8) age below 18 years, and 9) major language comprehension deficits precluding reliable testing. All the tasks described below were designed to be highly feasible even for patients in clinical settings. The participants in the HC group had no history of neurological disorders, head trauma, anoxia, stroke or major cognitive deterioration, as attested by their score on either the Mattis Dementia Rating Scale(mean score = 142, SD = 1.5, range = 139-144) or the French version of the modified telephone interview for cognitive status [bib_ref] Adaptation française d'un outil d'évaluation par téléphone des troubles mnésiques: le French..., Lacoste [/bib_ref] (mean score = 36.6, SD = 5.0, range = 34-43). They were all French speakers with a mean age of 60.4 years (SE = 8.9, range = 41-80). According to the Edinburgh Handedness Inventory criteria [bib_ref] The assessment and analysis of handedness: the Edinburgh inventory, Oldfield [/bib_ref] , all HC were right-handed, except for one who was left-handed. Their mean education level was 13.6 years (SE = 2.57, range = 9-18). As with the patient group, none of the HC wore hearing aids or had a history of tinnitus or a hearing impairment, as attested either by their PEGA score (mean = 27.75, SD = 1.3, range = 27-29) or by the results of a standard audiometric screening procedure (AT-II-B audiometric test) to measure tonal and vocal sensitivity.
# Materials and methods
## Participants
All participants gave their written informed consent, and the study was approved by the local ethics committee. The three groups were comparable for age (χ 2 = 0.70, p = 0.70) and education level (χ 2 = 2.87, p = 0.24) [fig_ref] Table 2: Statistical results of group [/fig_ref].
## Experimental protocol
Before patients performed the emotional prosody task, a motor assessment was carried out by a board-certified neurologist (FA) to quantify their cerebellar ataxia (Scale for the Assessment and Rating of Ataxia [bib_ref] Scale for the assessment and rating of ataxia: development of a new..., Schmitz-Hubsch [/bib_ref].
Neuropsychological and psychiatric tests were then administered to patients by either board-certified neuropsychologists (JP or AS) or a trainee neuropsychologist (MT or PV) supervised by the board-certified neuropsychologists. We administered the Montreal Cognitive Assessment (MOCA [bib_ref] Validation of Montreal Cognitive Assessment, MoCA, Alternate French Versions. Can, Nasreddine [/bib_ref] and a series of tests assessing executive functions: the Frontal Assessment Battery (FAB [bib_ref] The FAB: a frontal assessment battery at bedside, Dubois [/bib_ref] , categorical and literal fluency tasks [bib_ref] Formal and semantic lexical evocation in normal subjects. Performance and dynamics of..., Cardebat [/bib_ref] , and an action verb fluency task [bib_ref] Action (verb) fluency: Testretest reliability, normative standards, and construct validity, Woods [/bib_ref].
Depression was assessed with the Beck Depression Inventory (BDI-II [bib_ref] Action (verb) fluency: Testretest reliability, normative standards, and construct validity, Woods [/bib_ref] and alexithymia with the Toronto Alexithymia Scale (TAS-20 [bib_ref] The twenty-item Toronto Alexithymia Scale-I. Item selection and cross-validation of the factor..., Bagby [/bib_ref]. As apathy symptoms are commonly found in patients presenting cerebellar stroke [bib_ref] The cerebellum and neuropsychiatric disorders, Villanueva [/bib_ref] , we administered the Apathy Evaluation Scale (AES [bib_ref] Reliability and validity of the Apathy Evaluation Scale, Marin [/bib_ref].
Finally, participants performed the emotional prosody recognition task. The testing took around 90 min. To prevent the session from lasting too long and avoid the effects of fatigue, the self-report questionnaires (BDI-II and TAS-20) were given to participants at the end of the session for them to fill in at home.
## Vocal emotion recognition task
We used the same task as in a previous study [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref]. This task evaluating the recognition of emotional prosody has already been used with other clinical populations, such as patients with depression [bib_ref] Major depressive disorder skews the recognition of emotional prosody, Péron [/bib_ref] or Parkinson disease [bib_ref] Recognition of emotional prosody is altered after subthalamic nucleus deep brain stimulation..., Péron [/bib_ref]. Participants listen to meaningless speech (60 pseudowords) expressed in five different emotional prosodies (anger, fear, happiness, neutral, and sadness), played bilaterally through stereo headphones. For each pseudoword, they have to indicate the extent to which it expresses different emotions, by moving a cursor along a visual analog scale ranging from No emotion expressed to Emotion expressed with exceptional intensity. There is a scale for each emotion (happiness, anger, fear, and sadness), a neutral scale, and a scale to rate the surprise emotion (for more details concerning this task, see [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref].
## Extraction of acoustic features from the original stimuli
We extracted several relevant acoustic features from the original stimuli, using the openSmile toolkit [bib_ref] Recent developments in opensmile, the munich open-source multimedia feature extractor, Eyben [/bib_ref] , in order to study the proportions of variance they explained. The extended Geneva Minimalistic Acoustic Parameter Set (eGeMAPS) described by [bib_ref] The Geneva minimalistic acoustic parameter set (GeMAPS) for voice research and affective..., Eyben [/bib_ref] allowed us to obtain 88 features, divided into four groups: frequency-related features, energy-related features, spectral features, and temporal features (see SI 1 for the full list of acoustic features and their corresponding categories).
## Neuroradiological assessment
The brain images were acquired in a 1.5 T MRI scanner when each patient was admitted to hospital. All the lesions were mapped on diffusion-weighted (22 patients) or CT (2 patients) brain scans, using the Clusterize-toolbox (http://www.medezin.uni-tuebingen.de/kinder/ en/ research/neuroimaging/ software/).The resulting lesion map was then normalized to the Montreal Neurological Institute (MNI) single-subject template, with a resolution of 1 × 1 × 1 mm voxel size, using SPM12 software (http://www.fil.ion.ucl.ac.uk/spm/). In particular, we applied a deformation field estimated from a registered T2 (22 patients) or CT brain scan (2 patients) to each map. The mean time between stroke and image acquisition was 1.82 days (SE = 1.84, range = 0.5).
## . behavioral data analysis
Concerning vocal emotion recognition data, the exploratory analysis showed that the data were not normally distributed (abnormal proportion of 0). Consequently, we performed two levels of analysis, adopting a generalized linear mixed model (GLMM) approach. First, we compared the performances of the two patient subgroups (RCL and LCL) with that of the HC group. To do so, we ran a GLMM with a compound Poisson-Tweedie distribution, with emotion (5 levels) and scale (6 levels) as within-participants variables, group (HC, RCL and LCL) as the betweenparticipants variable, and participant as the random factor. The model following the Tweedie distribution presented a better fit of the data than the models following a Gaussian distribution did (Akaike information criterion; Tweedie: 93940; Gaussian: 160760). Second, we ran contrasts between the groups for each prosodic category and each scale, based on the GLMM model using the phia package in R. This type of statistical model allows for the control of random effects such as inter-individual variability, in addition to fixed effects. Each p value yielded by the contrasts was false discovery rate (FDR) corrected. However, as Tweedie analysis can be oversensitive to spurious effects, we performed a control analysis using the BayesFactor package. Bayesian t tests were performed between the groups, and significant Tweedie effects were selected if the Bayes factor (BF) exceeded 3. Although GLMM analysis provides an accurate estimation of effects of interest over confounding variables, the frequential approach it employs does not allow the null hypothesis (H0) to be tested. Nor does it provide any information about the strength of the observed effects. Thus, when this approach yields negative results, there is no way of determining whether these results stem from a lack of power and are indecisive, or whether they indicate the absence of an effect of the variable (i.e., indication of H0 validity). Furthermore, this approach is very sensitive to multiple comparisons error, and the methods used to resolve this issue are often so conservative that they can erase weak but real effects. It was to overcome these obstacles that we decided to adopt a Bayesian approach. Implemented using the Bayes-Factor package [bib_ref] Bayes factor approaches for testing interval null hypotheses, Morey [/bib_ref] or JASP software), this approach computes an index of the relative Bayesian likelihood of an alternative (H1) model compared with the H0 model. Consequently, the BF computed by this method allows H0 to be assessed. A BF > 3 is substantial proof of H1, while a BF < 1/3 is substantial proof of H0. Furthermore, this approach allows the strength of the proof to be directly assessed for either H1 or H0 (strong proof corresponds to a BF within the or [1/10-1/30] boundaries, while extreme proof corresponds to a BF > 100 or < 1/100). Finally, the comparative nature of the Bayesian GLMM renders it unsusceptible to multiple comparisons. We performed contrast analysis using the Bayesian t test in JASP software. A positive result was therefore considered to be significant when p < 0.05 in the classical GLMM analysis and BF exceeded 3 in the Bayesian analysis. A negative result was considered to be significant when p < 0.05 in the classical GLMM analysis and BF were below 1/3 in the Bayesian analysis. Any other scenario was deemed inconclusive.
Moreover, we looked for correlations between the clinical and emotional data for the patient group using Spearman's rank test as the distribution of the data was not normal. To avoid Type-I errors, we only included emotional variables that differed significantly between patients and HC, or between the two patient subgroups in the analyses.
Concerning the acoustic data we extracted, we first performed a principal component analysis and retained factors with an eigenvalue > 5. In the screeplot, we observed a strong decrease in variance contribution above the fifth factor, so we chose to retain five factors explaining 55.9% of the variance. After varimax rotation, we examined the factor loadings, and retained items that satisfied the following criterion: loading equal to or above 0.8 on the factor.
Second, we added the factorial score (of the five selected factors) as a covariate in our previous GLMM model, and performed contrast analyses (with FDR correction for each p value). Therefore, if the factor entered as a covariate explained a larger proportion of the variance for a given emotion and scale than the group difference, the previously significant results would become nonsignificant. Finally, to test each factor's contribution to the effects of interest, we calculated a model with the four-way interaction (three within-participants variables: emotion (5 levels), scale (6 levels) and factor; and one between-participants variable: group (HC, RCL and LCL)), as well as contrasts based on this GLMM model (with FDR correction for each p value).
## Cerebellar lesion-behavior relationship analysis
To evaluate the statistical relationship between lesion location and behavioral scores, we ran a VLSM analysis on the group level, using the t test statistic, with the absolute value of behavioral scores as a continuous, dependent variable. The behavioral scores were calculated by subtracting the mean of the control group. This algorithm distinguished individuals who had a lesion in a particular voxel from those who did not. We ran a t test on the dependent variable (i.e., ratings that differed significantly between the RCL and LCL groups) between the participants with a lesion versus those without. This procedure was repeated for every voxel (i.e., for each voxel, participants were arranged in a different constellation for the t test, depending on whether or not there was a lesion in that particular voxel). All the patients (RCL and LCL) were included in the same analysis. We decided not to flip the left lesions to the right in this analysis, in order to retain the ability to discern potential lateralization effects. Areas (minimum cluster size > 5 voxels) showing significant correlations with behavioral scores were identified using the FDR-corrected threshold of p = 0.001. The resulting statistics were mapped onto MNI standardized brain templates and color coded.
# Results
## Clinical examination
Comparisons between demographic and clinical screening scores of HC and each of the patients in the cerebellar stroke subgroups failed to reveal any significant differences (p > 0.05) [fig_ref] Table 2: Statistical results of group [/fig_ref]. We found a significant Group × Emotion × Scale three-way interaction, showing that group influenced the recognition of emotional prosody, χ2 (40) = 73.83, p < 0.0001.
## Vocal emotion recognition
Other main and interaction effects were as follows: emotion: χ2 (4) = 45.32, p < 0.0001; scale: χ2 (5) = 93.29, p < 0.0001; group: χ2 (2) = 1.38, p = 0.50; Group × Emotion: χ2 (8) = 2.73, p = 0.95; Group × Scale: χ2 (10) = 34.71, p < 0.001; and Emotion × Scale: χ2 (20) = 3875.9, p < 0.0001.
We performed contrasts for each vocal emotion and each rating scale, with FDR-corrected p values and controlled by the Bayesian t test analysis. We obtained the following results:
Anger. Anger stimuli on the Fear scale: contrasts revealed a significant difference between the LCL and HC groups, z(17186) = -2.32, p = 0.03 (BF = 5.38).
Neutral. Neutral stimuli on the Happiness scale: contrasts revealed a significant difference between the RCL and HC groups, z(17186) = -2.27, p = 0.04 (BF = 20.361), and between the RCL and LCL subgroups, z(17186) = -2.50, p = 0.02 (BF = 4.45) .
Neutral stimuli on the Fear scale: contrasts revealed a significant difference between the RCL and HC groups, z(17186) = -2.66, p = 0.01 (BF = 3.29).
Fear. Fear stimuli on the Happiness scale: contrasts revealed a significant difference between the RCL and LCL subgroups, z(17186) = 4.66, p < 0.001 (BF = 5.91).
Sadness. Sadness stimuli on the Surprise scale: contrasts revealed a significant difference between the RCL and HC groups, z(17186) = -3.46, p < 0.001 (BF = 20.43).
## Impact of clinical characteristics on vocal emotion recognition
We found a significant correlation between ratings on the Happiness scale for neutral prosody and scores on the AES [bib_ref] Reliability and validity of the Apathy Evaluation Scale, Marin [/bib_ref] (r = -0.54, p = 0.01). We also found a significant correlation between ratings on the Surprise scale for sadness prosody and the categorical fluency scores (r = -0.49, p = 0.01). All other correlations between clinical and emotional variables were nonsignificant (p > 0.05). In addition, no significant correlation was found between time since stroke and emotional variables. We entered the volume of the lesion in our statistical model to see if this variable affected the emotion ratings, taking relevant factors and interactions of interest into account. Results showed that lesion volume did not significantly affect ratings (χ 2 = 0.004, p = 0.95). We therefore calculated the Akaike information criterion (AIC) and Bayesian information criterion (BIC) to see whether the models containing the categorical fluency score or AES score variables presented a better fit of the data than the model that did not contain them. The lower the AIC or BIC value, the better the fit. Without the categorical fluency scores, the AIC was 46841.8 and the BIC was 47286.9. With the categorical fluency scores, the AIC was 46843.7 and the BIC was 47295.8. Accordingly, the prediction model was better if the categorical fluency scores were not included in it. The lack of verbal self-activation for the categorical fluency task did not, therefore, explain participants' judgments in the emotional prosody recognition task. However, with the AES scores, the AIC was 39247.6 and the BIC was 39688.2, suggesting that the prediction model was better if AES scores were included in it.
## Impact of acoustic features on vocal emotion recognition
Following a principal component analysis conducted on the 88 acoustic features extracted, analysis of the saturation matrix after varimax rotation (saturation cut-off applied = 0.80) allowed us to interpret the five extracted factors and identify a specific group of acoustic cues for each of them. Factors 1, 2 and 5 were related to spectral features, while Factor 3 was related to loudness features, and Factor 4 to F0. Factor 1 could be expressed as the ratio between high and low frequencies, Factor 3 as the variation in loudness, and Factor 4 as mean F0.
The GLMM we calculated, adding each of the five factors as a continuous predictor, revealed the following significant effects: Factor 1, z(1 1 3) = 44.875, p < 0.001; Factor 3, z(1 1 3) = 8.33, p = 0.004; and Factor 4, z(1 1 3) = 74.56, p < 0.001.
The contrasts, performed with one of the three significant factors identified above, revealed the following results . The differences between the RCL and HC groups for neutral stimuli on the Fear scale and between the RCL and LCL subgroups for fear stimuli on the Happiness scale remained significant after the addition of all the factors, so they were not explained by acoustic features. By contrast, the difference between the LCL and RCL subgroups for neutral stimuli on the Happiness scale ceased to be significant when Factors 1, 3 and 4 were added. Thus, a significant proportion of the variance was explained by Factor 1 (spectral features), Factor 3 (related to loudness), and Factor 4 (related to F0) and . Concerning the difference between the LCL and HC groups for anger stimuli on the Fear scale, a significant proportion of the variance was explained by Factors 1 and 4. Moreover, a significant proportion of the variance for the difference between the RCL and HC groups on the Surprise scale for sadness prosody was explained by Factor 4.
## Cerebellar lesion-behavior relationships
VLSM analysis, which was only performed on behavioral scores of interest (i.e., ratings that differed significantly between the RCL and LCL subgroups), revealed the following results:
The significant difference between the LCL and RCL subgroups on the Happiness scale for neutral stimuli correlated with lesions in the right Lobules VIIb and VIII and right Crus I and II (peak coordinates: x = 20, y = -75, z = − 42) . The significant difference between LCL and RCL on the Happiness scale for fear stimuli correlated with lesions in the same regions (peak coordinates: x = 28, y = -67, z = − 48).
# Discussion
The purpose of the present study was to investigate whether acoustic features have a differential impact on emotional prosody disturbances, depending on the location of the lesion. To this end, we quantified the misattributions made by patients with RCL or LCL, using a sensitive and validated behavioral methodology [bib_ref] Major depressive disorder skews the recognition of emotional prosody, Péron [/bib_ref] [bib_ref] Recognition of emotional prosody is altered after subthalamic nucleus deep brain stimulation..., Péron [/bib_ref] , as well as state-of-the-art statistical and neuroimaging analyses, in order to study Mean ratings and 95% confidence intervals (CI 95% ) on emotion scales in the emotional prosody recognition task for the LCL, RCL and HC groups.
## Lcl group (n ¼ 11)
Happiness the potential sensory contribution to emotion recognition deficits in patients with cerebellar stroke. Consequently, and as predicted, we confirmed impaired recognition of emotional prosody in patients and showed that these emotional misattributions are correlated with lesions in the right Lobules VIIb, VIII and IX. Moreover, we found for the first time, to our knowledge, that the pattern and number of errors differed according to the side of the cerebellar lesion, and that patients' misattributions were indeed partially explained by auditory sensory processing, with an interaction effect according to the hemispheric location of the lesion. The biased ratings displayed by LCL were explained by spectral and F0 features, whereas loudness, spectral and F0 features explained a significant proportion of the variance in the misattributions made by patients with RCL. First, as previously reported [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref] , we confirmed that patients' emotional misattributions were all made when listening to prosody with a neutral or negative valence. Cerebellar involvement in Mean ratings for neutral prosody on the Happiness scale for HC (blue), RCL (green), and LCL (purple) groups. (B) VLSM results. Misattributions of happiness to neutral stimuli correlated with lesions in right Lobules VIIb and VIII, and right Crus I and II (peak coordinates: x = 20, y = -75, z = − 42). The colors indicate t test values, ranging from black (nonsignificant) to red (maximum significance). The lesion-symptom maps were projected onto the ch2bet template of MRIcron®. (C) Differential impact of acoustic features on vocal emotion recognition between the HC, RCL and LCL groups. 1) Differential impact of the ratio between high and low frequencies (spectral features) on the Happiness scale when the stimulus was neutral between the HC (blue), RCL (green), and LCL (purple) groups. 2) Differential impact of loudness variation on the Happiness scale when the stimulus was neutral between the HC (blue), RCL (green), and LCL (purple) groups. 3. Differential impact of F0 on the Happiness scale when the stimulus was neutral between the HC (blue), RCL (green), and LCL (purple) groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) the processing of negative emotions has been mentioned several times in the literature [bib_ref] Cerebellum and processing of negative facial emotions: cerebellar transcranial DC stimulation specifically..., Ferrucci [/bib_ref] [bib_ref] Cerebral blood flow changes associated with attribution of emotional valence to pleasant,..., Paradiso [/bib_ref]. Authors have suggested that there is a complex cortico--cerebellar network specific to aversive stimuli, with functional connectivity between the cerebellum and cortical structures supposedly involved in the processing of negatively valenced emotions [bib_ref] Aversion-related circuitry in the cerebellum: responses to noxious heat and unpleasant images, Moulton [/bib_ref]. In this context, the postulate that the cerebellum is involved in affective communication seems to be confirmed. However, the question of its specific role in comparison with those of the cortex and other subcortical structures remains to be elucidated. It has recently been proposed that the so called limbic cerebellum modulates the amplitude of cortical oscillations based on prediction error feedback of the selected response relative to the given context [bib_ref] The cerebellum and cognition, Schmahmann [/bib_ref] [bib_ref] The role of the basal ganglia and cerebellum in language processing, Booth [/bib_ref]. Input to the cerebellum regarding the salience and motivational value of emotional stimuli guides internal models to determine how an emotional response benefits individuals in their current state, and therefore shapes how output from the cerebellum modifies the limbic response pattern. By continuously monitoring the performance of the individual in terms of prediction errors, the cerebellum ensures that large deviations from the expected response/ outcome are quickly corrected [bib_ref] The role of the human cerebellum in performance monitoring, Peterburs [/bib_ref]. In cases of cerebellar lesions, however, a lack of cerebellar amplification of emotional networks may lead to the blunted affect and decreased subjective experience of emotions that are often reported in patients.
As far as the comparison between RCL and LCL is concerned, congruent with our hypotheses, we observed that patients with RCL committed more misattributions in emotional prosody recognition than either patients with LCL or HC. RCL rated happiness significantly higher when they listened to neutral prosody than either patients with LCL or HC. Compared with HC, they also gave higher ratings on the Fear scale when they listened to neutral stimuli, and higher ratings on the Surprise scale when they listened to sadness prosody. Moreover, they gave significantly higher ratings on the Happiness scale than patients with LCL did when they listened to fear stimuli. This tendency to attribute significantly more positive emotions when listening to negative or neutral prosody, which had already observed in a previous study [bib_ref] Cerebellar contribution to vocal emotion decoding: Insights from stroke and neuroimaging, Thomasson [/bib_ref] , could be interpreted as reflecting a deficit in valence processing. In HC, increased BOLD signal intensity has been found in the right dorsal cerebellum as the degree of unpleasantness of the induced emotion increases [bib_ref] Neural systems subserving valence and arousal during the experience of induced emotions, Colibazzi [/bib_ref]. A possible functional segregation and specialization within the cerebellum in the processing of emotional dimensions has been suggested [bib_ref] Distinct cerebellar lobules process arousal, valence and their interaction in parallel following..., Styliadis [/bib_ref]. Authors have also postulated that the lateral zone of the right cerebellar hemisphere participates in the regulation of cognitive components of emotional task performance [bib_ref] Functional topography of primary emotion processing in the human cerebellum, Baumann [/bib_ref]. Interestingly, we observed that the patients who gave higher happiness ratings when they listened to neutral prosody had the lowest AES scores. Commonly seen as an affective disorder that can lead to inadequate appraisal (malfunctioning of conduciveness check) [bib_ref] Emotions are emergent processes: they require a dynamic computational architecture, Scherer [/bib_ref] , apathy could thus be related to a reduction in misattributions on the Happiness scale for neutral prosody among patients with RCL. Interestingly, and using VLSM analysis, we found that the difference between patients with RCL versus LCL correlated with lesions in the right Lobules VIIb and VIII and right Crus I and II. This finding partially confirms [bib_ref] fMRI activities in the emotional cerebellum: a preference for negative stimuli and..., Schraa-Tam [/bib_ref] 's fMRI results in HC [bib_ref] fMRI activities in the emotional cerebellum: a preference for negative stimuli and..., Schraa-Tam [/bib_ref] , showing activation of the posterior cerebellum (i.e., Crus II, hemispheric Lobules VI and VIIa, and vermal Lobules VIII and IX) during the processing of emotional facial expressions. Relations have been highlighted between Lobules VIIb and VIII and the task-positive and salience networks, suggesting that these two subregions are involved in the performance of attention-demanding cognitive tasks and the identification of salient stimuli to guide behavior. Moreover, we observed higher ratings on the Fear scale for angry stimuli among patients with LCL than among HC (whereas RCL did not differ significantly from HC). It has been suggested that there is a Misattributions of happiness to fearful stimuli correlated with lesions in the right Lobules VIIb and VIII, and right Crus I and II (peak coordinates: x = 28, y = -67, z = − 48). The colors indicate t test values, ranging from black (nonsignificant) to red (maximum significance). The lesion-symptom maps were projected onto the ch2bet template of MRIcron®. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) key hub in the left cerebellar hemisphere (more precisely, Crus I and II and Lobule VII), where emotional and cognitive information converges and is forwarded to cerebral (and more particularly prefrontal) areas [bib_ref] Influence of the cortical midline structures on moral emotion and motivation in..., Han [/bib_ref]. Moreover, we can assume that the amygdala participates in this process, given its important role in fear learning and its close links to the vermis and prefrontal areas [bib_ref] Cerebellar contributions to the Papez circuit, Snider [/bib_ref].
The second aim of our study was to investigate whether acoustic features have a differential impact on emotional prosody disturbances. We found that the biased ratings on the Fear scale when patients with LCL listened to anger prosody were explained by spectral (ratio between high and low frequency) and mean F0 features. This result is partially congruent with the speculative hypothesis concerning the role of the left cerebellar hemisphere in the processing of relatively low-frequency information (e.g., prosody, musical melodies). Interestingly, a study reported that fear ratings were predicted by spectral properties, whereas anger ratings were predicted by both spectral and pitch information [bib_ref] Perceptual cues in nonverbal vocal expressions of emotion, Sauter [/bib_ref]. Altered processing of these acoustic cues could explain patients' misattributions. Contrary to our hypotheses, we also observed that loudness, spectral and F0 features explained a significant proportion of the variance in the misattributions made by patients with RCL on the Happiness scale for neutral stimuli. Moreover, we found that biased ratings on the Surprise scale when patients with RCL listened to sadness stimuli were correlated with mean F0. Pitch is reportedly crucial for correctly judging surprise and sadness prosody [bib_ref] Perceptual cues in nonverbal vocal expressions of emotion, Sauter [/bib_ref]. However, this result was unexpected, regarding the differential temporal processing specialization of the cerebellar hemispheres, based on the cross-lateral interconnectivity hypothesis. The presence of contrasting cerebellar and cerebral hemispheric functional asymmetries has not always been supported. One study using a clinically applicable diffusion tensor imaging sequence highlighted both ipsilateral and contralateral connections [bib_ref] Ipsilateral and contralateral cerebro-cerebellar white matter connections: A diffusion tensor imaging study..., Karavasilis [/bib_ref]. Thus, as with the cerebral cortex, research investigating cerebellar hemisphere specialization for processing the emotional information conveyed by a speaker's voice has failed to yield consistent results. Our study also revealed that the biased ratings of patients with RCL on the Fear scale when they listened to neutral stimuli, or on the Happiness scale when they listened to fear stimuli, were not correlated with any acoustic features. Thus, these results revealed that patients' misattributions could not be explained solely by a deficit in sensory processing, suggesting the involvement of higher-order cognitive processes. The present results strengthen the idea that clinicians should assess patients with cerebellar dysfunction at the sensorimotor level, but also at the cognitive and emotional levels. The clinical community often tends to evaluate these patients less thoroughly than patients with lesions in the cerebral cortex, especially since they do not display gross clinical manifestations. Our patients successfully identified the target emotions, but nevertheless confused them with other emotions (which can be interpreted as an increase in noise in the decoding process). Small and underestimated neuropsychological deficits can potentially cascade into more severe deficits over time, as is the case for example with dysexecutive syndrome in traumatic brain injury. As emotional symptoms are sometimes the main complaint of patients with cerebellar injury, rehabilitation of emotion processing, focusing on decoding by low-level sensory processes, could be very beneficial.
Finally, in the context of existing physiological models of emotional prosody decoding, we suggest that in each processing subcomponent, the cerebellum detects and then adjusts the temporal pattern encoded for a given response, so that it is as close as possible to the expected one [bib_ref] The basal ganglia and the cerebellum in human emotion, Pierce [/bib_ref]. This ability to fine-tune and correct responses in sensorimotor analysis, acoustic integration, or cognitive evaluative judgment is above all made possible by the cerebellum's ability to analyze and process irregular temporal patterns [bib_ref] Double dissociation of single-interval and rhythmic temporal prediction in cerebellar degeneration and..., Breska [/bib_ref]. This work is done in tandem with the BG, which select and strengthen appropriate cortical responses using reward feedback [bib_ref] The basal ganglia and the cerebellum: nodes in an integrated network, Bostan [/bib_ref]. This is possible by the BG's ability to analyze and process regular temporal patterns [bib_ref] Double dissociation of single-interval and rhythmic temporal prediction in cerebellar degeneration and..., Breska [/bib_ref]. Regarding the hemispheric specialization of the cerebellum in this process, the present findings revealed cooperation between the two cerebellar hemispheres for sensory but also cognitive processes during the decoding of vocal emotional stimuli. Interestingly, a recent studyhighlighted bilateral and bidirectional connections between the cerebellum (Crus I, II and dentate nuclei) and the temporo-frontal region. The involvement of both cerebellar hemispheres in auditory spectrotemporal encoding, with hemispheric sensitivity for different timescales, would allow the construction of internal auditory models. More studies are needed to explore the precise nature and timing of this cooperation during emotional processing.
# Conclusion
For the first time to our knowledge, the present study tested how damage to either the left or right cerebellum impairs the recognition of emotional prosody and the sensory contribution to vocal affect processing. Deficits in both patient subgroups were observed, particularly for neutral or negative prosody, with a higher number of misattributions in patients with right-hemispheric stroke. VLSM revealed that some emotional misattributions correlated with lesions in the right Lobules VIIb and VIII and right Crus I and II. A significant proportion of the variance in this misattribution was explained by acoustic features such as pitch, loudness and spectral aspects, suggesting a sensory cerebellar contribution.
The present results seem to suggest two-step processing operating in Differential impact of the three significant factors identified on vocal emotion recognition between the LCL, RCL and HC groups. both cerebellar hemispheres, rather than hemispheric specialization per se: both right and left cerebellar hemispheres appear to process spectral and F0 features, but the right hemisphere is additionally involved in loudness processing during an emotional episode. Further studies are needed to better understand the processing of specific properties by left versus right cerebellar hemispheres, and integrate these results with recent suggestions that the cerebellum can refine the cortical/BG response and recalibrate the internal model by checking whether the state of the individual diverges from the expected state at any time during the emotion recognition process.
## Credit authorship contribution statement
## Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[fig] Figure 2: (A) Mean ratings for fear prosody on the Happiness scale for HC (blue), RCL (green), and LCL (purple) groups. (B) VLSM results. [/fig]
[table] Table 1: Clinical, demographic, and neuropsychological data of the two subgroups of patients with cerebellar stroke.Note. Dashes indicate missing values. Act. fluency: action verb fluency task; AES: Apathy Evaluation Scale; BDI-II: Beck Depression Inventory; Cat. fluency: categorical fluency task; FAB: Frontal Assessment Battery; MOCA: Montreal Cognitive Assessment; PEGA: Montreal-Toulouse auditory agnosia battery; SARA: Scale for the Assessment and Rating of Ataxia; TAS-20: Toronto Alexithymia Scale. # scores below the clinical threshold. [/table]
[table] Table 2: Statistical results of group (LCL, RCL and HC) comparisons for clinical, demographic, and neuropsychological data. Note. Stat. val.: statistical value; MOCA: Montreal Cognitive Assessment; FAB: Frontal Assessment Battery; AES: Apathy Evaluation Scale; PEGA: Montreal-Toulouse auditory agnosia battery; SARA: Scale for the Assessment and Rating of Ataxia; BDI-II: Beck Depression Inventory; TAS-20: Toronto Alexithymia Scale. [/table]
[table] Marine Thomasson: Conceptualization, Formal analysis, Investigation, Writing -original draft, Writing -review & editing. Damien Benis: Conceptualization, Formal analysis, Writing -review & editing. Arnaud Saj: Conceptualization, Formal analysis, Investigation, Writing -original draft, Writing -review & editing, Supervision. Philippe Voruz: Investigation, Writing -review & editing. Roberta Ronchi: Formal analysis, Writing -review & editing. Didier Grandjean: Conceptualization, Formal analysis, Writing -review & editing, Supervision. Frédéric Assal: Conceptualization, Investigation, Writing -review & editing, Supervision. Julie Péron: Conceptualization, Formal analysis, Investigation, Writing -original draft, Writing -review & editing, Supervision, Funding acquisition. [/table]
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Eye Movement Technique to Improve Executive Function in Patients With Stroke: A Randomized Controlled Trial
Objective: To investigate the efficacy of eye movement technique for the treatment of executive dysfunction of patients with stroke.Methods: This was a prospective, single-blinded, randomized, controlled, single-center clinical trial conducted from June 2018 to December 2019 in patients with stroke. The patients were randomized 1:1 to the routine (conventional management) and eye-move group (routine management plus eye movement technique: 5-min goal management training, 5-min computer-aided working memory, and 10 min of inhibitory control training and set conversion training). The intervention lasted 6 weeks, followed by a 4-week follow-up. The primary endpoint was the Behavioral Assessment of the Dysexecutive Syndrome (BADS) score. The secondary endpoints mainly included the Montreal Cognitive Assessment (MoCA), Wisconsin Card Sorting Test (WCST), and modified Barthel Index (MBI) scores.Results: Sixty-four patients were enrolled (32/group). After the 6-week intervention, the BADS and WCST scores of the eye-move group were significantly improved than those of the routine group (all P < 0.05), but the effects were attenuated in certain subscores after follow-up (all P > 0.05). The MoCA and MBI scores of the eye-move group were significantly higher, and the reaction time was significantly lower than those of the routine group at 4 weeks after the intervention (all P < 0.05). After follow-up, the MBI scores of the eye-move group were still higher than that of the routine group (P < 0.001), but there were no differences for MoCA scores and reaction time (both P > 0.05).Conclusion:The eye movement technique could improve the executive function of patients with stroke. These results have to be confirmed. This was a prospective, single-blinded, randomized, controlled, single-center clinical trial (ChiCTR2000036393). Clinical Trial Registration: [www.chictr.org.cn], identifier [ChiCTR2000036393].
# Introduction
A stroke is an episode of acute neurological dysfunction from either ischemic infarction or a collection of blood within the brain or ventricular system with a resultant focal injury of the central nervous system (CNS). Hemorrhagic strokes are typically due to intracerebral hemorrhage (ICH) or subarachnoid hemorrhage (SAH). The estimated global incidence of stroke is 2-3 per 1,000 person-years, with older patients and patients with carotid artery stenosis or atrial fibrillation having the highest risk.
With the development of medical treatments, the mortality rate of patients with stroke decreased. Nevertheless, many patients still have sequelae, such as motor disorders, speech disorders, and cognitive dysfunction. About 43-78% of patients will have cognitive dysfunction, including impairments of executive function (EF), thinking speed, and spatial orientation ability. A previous study showed that 66.6% of the patients with stroke still had EF dysfunction 2 years after stroke, mainly at the levels of choice, planning, decision-making, and control behavior disorder in daily life, seriously affecting the quality of life of the patients and increasing the risk of dementia . At present, most of the EF training methods are occupational therapy, computer-aided cognitive training, neurocognitive psychological rehabilitation, somatosensory interaction games, acupuncture, drug treatment, hyperbaric oxygen treatment, and transcranial magnetic stimulation. Nevertheless, the evidence for efficacy is limited, and those treatments require significant investments in manpower, material resources, and financial support, and the adverse reactions of some drugs may increase physical discomfort and reduce the quality of life of the patients. Therefore, there is a need for a training method that is simple, easy to operate, more acceptable for patients, and can always maintain a high degree of compliance and enthusiasm.
The visual system is the primary sensory system for maintaining a standing posture. Visual input provides information about the surrounding environment, posture, and head movements. Stroke patients show a variety of eye movement disorders, including reduced saccade and difficulty retaining spatial position, which are caused by damage to the muscles outside the eye, the cranial nerves that supply the eye muscles, or the neural pathways that control them. Eye movement disorders affect more than 70% of stroke victims. Up to 86% of symptomatic patients with stroke or non-traumatic acquired brain injury have eye movement disorders. In the general population of stroke victims, 7-55% have eye movement disorders at various stages of recovery. Visual field and visual accuracy can affect balance and motor ability, and people with impaired visual systems often have balance problems. Stroke patients with eye movement disorders have difficulty maintaining normal eye positions and moving their eyes appropriately (e.g., scanning, tracking, and staring), resulting in a loss of depth perception, reduced hand-eye coordination, and significant difficulty in approaching tasks and reading. A reduced ability to scan the visual environment may affect visual memory, recognition, and the ability to make plans and decisions, affecting EF. Therefore, improvement of visual attention is very important for the rehabilitation of stroke patients.
The eye movement technique is considered to be an effective method to improve postural control of stroke patients through visual feedback to regulate movement patterns in exercise training. The neurologic basis of eye training is to participate in the regulation of the vestibular reflex (VOR), which plays an important role in the perception of spatial position and balance of the human body. The generation and control of eye movements involve the cortex, basal ganglia, cerebellum, and brainstem, and signals from this complex network eventually converge on the eye motor neurons in the brainstem. During eye training, visual, auditory, vestibular, and proprioceptive receptors, as well as visuospatial perception, stimulate the central nervous system (CNS), enabling it to respond quickly and accurately to environmental changes. Visual and proprioceptive sensory information affecting balance control can improve agility and dynamic balance control in older adults. As a new way of human-computer interaction, the eye movement technique has higher efficiency and is closer to the natural behavior of humans compared with currently commonly used human-computer interaction devices such as a keyboard, mouse, and touch screen.
At present, studies on computer-based cognitive rehabilitation training have made some progress in China and abroad, showing effectiveness in improving cognitive functions such as memory and EF. Nevertheless, this technique is still seldom used in patients with stroke, and additional data are needed. The eye movement intervention strategy developed based on eye movement technology in this study focuses on the training of saccade, tracking, and gaze, alleviates eye movement disorders, activates the neural network of the brain, and promotes the improvement of EF and attention. The eye movement used in this study uses a restorative strategy, including those where there is direct training of the impaired function or repetitive stimulation of eye movement. In the past, it has been thought that restitutive approaches would have a limited effect on visual rehabilitation. However, treatments of convergent fusion and stereopsis through repetition training of the deficient function have been reported as effective (39). But few studies have reported the efficacy of the combination of routine rehabilitation and eye movement technique on executive function in patients with stroke. Therefore, this study investigated an EF rehabilitation strategy based on routine rehabilitation combined with the eye movement technique. This study should provide data on its effect of EF in patients with stroke and provide a reference for further discussion on EF rehabilitation treatment strategies.
# Methods
## Study design
This was a prospective, single-blinded, randomized, controlled, single-center clinical trial (ChiCTR2000036393).
## Ethics
This study was approved by the ethics committee of the Forth Shanghai Rehabilitation Hospital (SP2018002). All participants were informed of the study and signed the informed consent form prior to any study procedure. This study was performed according to the Declaration of Helsinki and Good Clinical Practices.
## Participants
The subjects were patients with stroke and cognitive dysfunction hospitalized in the rehabilitation ward of Shanghai Fourth Rehabilitation Hospital from June 2018 to December 2019. The inclusion criteria were: (1) diagnosis of stroke ; (2) the first stroke ever; (3) disease course ≤6 months; (4) no brain atrophy or leukoaraiosis with a moderate degree or above detected by imaging examination; (5) no visual field defect or visual space neglect; (6) right-handed; (7) Montreal Cognitive Assessment (MoCA) scale <26 points, which is defined as cognitive function impairment (if the education is <12 years, 1 point is added); (8) 50-75 years of age; (9) no consciousness disorder; and (10) no history of mental illness. The exclusion criteria were: (1) severe visual and hearing impairment, color blindness, color weakness, or aphasia; (2) patients with severe heart, liver, or kidney dysfunction, respiratory failure, malignant tumor, or other serious physical diseases; (3) drug abuse or alcohol dependence; (4) taking antidepressants or other psychoactive drugs; or (5) any other factors that could affect assessment or treatment.
## Sample size
According to the results of a preliminary study, the sample size was estimated according to the primary outcome of the Behavioral Assessment of the Dysexecutive Syndrome (BADS) as the indicator. With α = 0.05 and β = 0.1 (power = 0.9), a sample size of n = 25 was required, calculated using PASS 11.0. Considering a loss to follow-up of <20%, the total sample size of the two groups was estimated as 64.
## Randomization and blinding
The patients were randomized to the routine group and eye-move group using an MS Excel-generated randomization code, in a 1:1 ratio, 32 participants per group. The allocation was performed using sequential opaque envelopes. The envelopes were opened in turn. The outcome evaluators were blind to grouping.
## Intervention
The participants in the routine group received regular cognitive rehabilitation training, while the participants in the eyemove group received cognitive rehabilitation based on the eye movement technique. The study lasted 10 weeks. The first 6 weeks were the intervention period, and weeks 7-10 were the follow-up period.
The interventions in the two groups are detailed in. Four parts of the intervention were common to the two groups, while the last part was different. The rehabilitation intervention in the two groups was performed 6 days a week for 6 consecutive weeks. All patients were required to finish physical therapy, occupational therapy, and swallowing and speech function training, each of which lasted 20 min. Then, the patients were required to complete their group-specific intervention, i.e., routine cognitive training for the routine group and cognitive rehabilitation strategy module training for the eye-move group, each of which lasted 20 min. Hence, the training took 80 min and was required to be completed within 1 day, but it could be completed in multiple sessions, depending on the patient's physical condition.
A total of four investigators guided the rehabilitation training of the patients, including two primary and two intermediate rehabilitation specialists, who were all engaged in the field of neurological rehabilitation with a working experience of 5-10 years. Before the experiment, they all received unified training and guided the patients to carry out training in a unified language. The participants were assessed face-to-face by the same rehabilitation assessment therapist (blind to grouping) before and after the intervention as well as the end of the follow-up.
The termination criteria were: (1) intolerable participants during the experiment; (2) participants were unwilling to continue the clinical trials and actively proposed to suspend the trial, and the reasons would be recorded; (3) participants failed to be assessed on time.
## Assessment and data collection
Depression and anxiety of enrolled patients using the Self-Rating Depression Scale (SDS) (41) and the Self-rating Anxiety Scale (SAS)were recorded at baseline.
The following scales were used as evaluation tools to assess the ability of patients before and after the intervention as well as after the follow-up.
(1) MoCA (43): The MoCA assesses visual-spatial and EF, naming, memory, attention, language, abstraction, delayed recall, and orientation. The highest score is 30 points. The higher the score, the better the cognitive function of the subject. A score of <26 points indicates cognitive dysfunction.
(2) BADS (44): the BADS is the closest method to the scene of daily life for executive function assessment. It has the characteristics of short test time, easy cooperation, and comprehensive items. The test indexes are (1) rule conversion card test (RSCT), (2) action planning test (APT), (3) key finding test (KST), (4) time judgment test (TJT), (5) zoological map test (ZMT), and the revised six-element test (MSET). The BADS assesses a patient's rule switching, planning, problem-solving, and organizational and behavioral supervision. Each test is converted to a standard score through a preliminary score (the higher the error rate, the lower the score). The single standard scores range 0-4 points. The total standard score ranges of 0-24 points, and the lower the score, the worse the executive function.
(3) Wisconsin Card Sorting Test (WCST): The WCST is a traditional neuropsychological test and is a common tool to detect executive function. In this study, the computer version of the Jizhi rehabilitation diagnosis system was adopted. The test classification card has four stimulus cards and 128 classification cards, which are compiled according to color (yellow, red, blue, green), shape (pentacle, triangle, circle, cross), and number of figures. The subjects are asked to classify the 128 cards according to four stimulus cards within 5 min. The subjects are not informed of the classification principle but only know whether the result of each test is true or false. The indexes in the WCST are as follows. (1) Number of classifications completed:
## Routine group
Eye-move group
Physical therapy: Good position placement of the limb, conversion and transfer of body position, muscle strength and endurance training, range of motion exercises, balance, and coordination ability training, and gait training (20 min).
Occupational therapy: daily life activity training, including dressing, eating, brushing teeth, washing face, and auxiliary equipment training (20 min).
Swallowing and speech function training: oral and facial muscle training, eating training, articulating training, listening, speaking, and reading and writing training (20 min).
Drug intervention: for neurotrophy, circulation improvement, and control of risk factors of cerebrovascular disease.
Routine cognitive training (20 min): Card sorting, picture recognition, picture visual scanning, word retelling, short reading, picture memory, word pairing, simple calculation, discrimination, and classification.
## Endpoints
The primary endpoint was the BADS score after the intervention. The secondary endpoints were BADS scores after the follow-up, as well as the MoCA, WCST, reaction time, and MBI scores both after the intervention and at the end of follow-up.
## Adverse events
Respiration (times /min), pulse (times /min), and blood pressure (mmHg) were used as routine safety indexes. Any adverse reactions or other discomforts to the participants during the trial were recorded, including the name of the adverse event,
# Statistical analysis
Statistical analysis was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Categorical variables were presented as n (%) and were analyzed using the chi-square test or Fisher's exact test. Continuous variables were presented as means ± standard deviations (SD). The continuous variables were tested using the Shapiro-Wilk test to verify whether they met the normal distribution and using the homogeneity of variance test. If both conditions were met, the data were presented as means ± standard deviations (SD), and the independent samples t-test was used for inter-group comparison. Repeated measurement variance analysis was performed for comparison at different time points within each group, with the LSD post-hoc test. If the distribution was not normal or variance was not homogeneous, the data were expressed as median (range), and Mann-Whitney U-test was used for inter-group comparison, and the Friedman's rank-sum test was used for intra-group comparison. Two-tailed (except for the chi-square test) P < 0.05 were considered statistically significant.
# Results
## Baseline characteristics
There were 80 patients with stroke screened. Among them, 16 cases were not in accordance with the inclusion criteria, and 6 cases were unwilling to participate in the trial. At last, a total of 64 patients were eligible and randomly assigned to the routine group and eye-move group (1:1), and all participants completed the trial, as shown in. The demographic and clinical characteristics of the patients are shown in. There were no differences in years of education and depression and anxiety scores (all P > 0.05).
## Comparison of the bads scores between the two groups
As presented in, there were no significant differences in BADS total and subscores between the two groups before the intervention (P > 0.05). The BADS total scores of the eye-move group were significantly higher than those of the routine group after the intervention (median, 12 vs. 8, P < 0.001). However, after follow-up, the BADS scores of the eye-move group were only significantly different in the rule shift card test (RSCT) and temporal judgment test (TJT) subscores compared with those of the routine group (P < 0.05). In the routine group, the BADS total score improved after the intervention (median, 8 vs. 7, P < 0.05), but back to before after follow-up (P > 0.05). Similarly, there was a significant improvement in all subscores and total score of the eye-move group after the intervention (all P < 0.05), but there were no significant differences before treatment and after follow-up (all P > 0.05).
## Comparison of the wcst scores between the two groups
As shown in, there were no significant differences in WCST scores in each dimension between the two groups before intervention (all P > 0.05). After the intervention, subscores in dimensions of completed classification number (median, 4 vs. 4, P < 0.05) and correct response number (median, 68 vs. 55, P < 0.001) in the eye-move group were significantly higher than those of the routine group. And the wrong response number (median, 42 vs. 49, P < 0.05), persistent error number (11 vs. 18, P < 0.001), non-persistent error number (median, 23 vs. 26, P < 0.05) were much lower in the eye-move group than the routine group. Nonetheless, after follow-up, the effectiveness of eye movement technique treatment only existed in the eyemove group regarding the correct response number (median, 60 vs. 51, P < 0.001), wrong response number (median, 45 vs. 51, P < 0.05), and persistent error number (median, 12 vs. 18, P < 0.05), compared with the routine group. In the routine group, there were significant differences in all WCST subscores except for the completed classification number in each dimension before and after the intervention (all P < 0.05). After follow-up, there were no significant differences in the correct response number (P > 0.05), but there were still significant differences in wrong response number, persistent response number, non-persistent response number, and time score from learning to mastering before the intervention and after follow-up (P < 0.05). In the eye-move group, the WCST subscores in each dimension after intervention were significantly different from those before the intervention and after follow-up (all P < 0.05), except for the completed classification number between before intervention and after follow-up (P > 0.05;.
## Comparison of the moca, mbi, and reaction time scores between the two groups
The MoCA (17.3 ± 2.3 vs..0 ± 2.3, P = 0.03), and MBI scores (median, 65 vs. 61, P < 0.001) were significantly higher, and the reaction time (0.56 ± 0.07 vs. 0.60 ± 0.06, P = 0.022) was significantly lower in the eye-move group than those of the routine group after the intervention. However, after follow-up, there were no significant differences in MoCA scores (P = 0.482) and reaction time (P = 0.416) between the two groups, but MBI scores of the eye-move group were still higher than the routine group (median, 63 vs. 60, P < 0.001). Moreover, there were no significant differences in the MoCA scores of both groups before the intervention and after follow-up (both P > 0.05), but there were significant differences after the intervention (P < 0.001). The MBI and reaction time scores were all significantly different before the intervention, after the intervention, and after follow-up (P < 0.05;.
## Adverse events
No adverse events were observed during this trial.
# Discussion
Executive dysfunction is a feature of many patients who survived a stroke and greatly affects their quality of life. The available rehabilitation technique proposed so far have limited efficacy for EF rehabilitation. This study investigated the effect of the eye movement technique on the executive dysfunction of patients with stroke. The results suggest that the eye movement technique could improve the executive function of patients with stroke. Compared with the routine group in the same period: a P < 0.05; f P < 0.001; compared with before intervention: b P < 0.05; d P < 0.001; compared with after intervention: c P < 0.001; e P < 0.05; LSD post-hoc test was used. There are multiple cognitive impairments in patients after stroke, and various areas also affect each other. First, the form of the breakthrough game was adopted to improve the interest, flexibility, and compliance of patients to participate in training, and then improve their emotion. Second, the selection of appropriate rehabilitation interventions and enriched treatment environments are conducive to the reorganization of brain structure and function to a certain extent. Indeed, studies showed that colorful environmental stimulation and repeated intervention training could affect nerve cells, increase the dendrites, form new neural pathways, and promote the development of brain plasticity. Moreover, the eye movement technique is the first module of the rehabilitation strategy intervention and firstly focuses on the training of attention and spatial orientation. Patients with stroke often have decreased attention. If the patient's alertness is declined, they cannot effectively filter out the irrelevant stimulation in the procedure of information processing, which directly affects the follow-up processing. The intervention proposed here requires the patient's eyeball to follow the movement of the target. With the change of the target, the movement speed, scanning track, width, and direction of the eyeball change accordingly, which can activate other areas that are involved in the neural network controlling the eyeball movement, including the frontal and parietal lobes, which is conducive to the reorganization of the brain neural functions.
The results of this study showed that, compared with before the intervention and after 6 weeks of rehabilitation intervention, the scores of the BADS dimensions and WSCT dimensions (except "number of categories completed") were significantly improved in the eye-move group. In addition, the BADS scores of the intervention group were significantly higher than those of the routine group, and other sub-scores of the WCST in all dimensions were better than those of the routine group except "learning to mastering." These results suggest that cognitive rehabilitation training based on eye movement techniques could effectively improve the EF of stroke patients. In the cognitive rehabilitation training based on eye movement technology, the eye movement part includes tracking and staring training. The track ball and skiing training are eye movement visual tracking, while fruit cutting training, commodity purchasing training, and electronic organ training are eye movement visual staring. It is different from conventional cognitive rehabilitation training and visual training that includes only visual tracking components.
In the process of eye movement gaze, patients can change the direction of the gaze voluntarily and quickly through eye movement, which is consistent with the flexibility and inhibitory control ability involved in EF. This may be one of the reasons why eye movement technology can effectively improve the executive function of stroke patients. We also analyzed the index of "learning to mastering" of the WCST, which reflects the learning ability during the implementation process, but showed no significant difference after the intervention. The possible reason is that the participants were all elderly, and their learning ability to successfully apply past experience was generally decreased.
The results of this study showed that, after the intervention, the response time of the two groups was significantly shortened and the MOCA score was significantly improved, and the improvement was more obvious in the eye-move group than in the routine group. These results suggest that rehabilitation training based on eye movement technology can effectively improve the attention and cognitive function of the patients. Direct training of attention usually requires the patients to complete a series of repetitive exercises or exercises that enable them to respond to visual or auditory stimuli. We designed the eye movement rehabilitation on the basis of cognitive rehabilitation training modules, including auditory memory like listening to the words and listening to music for identification practice, all of which required to quickly respond to the patients through word and music recognition, which at the same time also could improve the patients' EF through auditory information extraction and auditory working memory ability.
This study also showed that the "rule switching card" in the BADS and the "correct response number, " "false response number, " and "persistent error number" in the WSCT were significantly improved after the intervention compared with the routine group. This suggests that the improvement of these indicators is through the improvement of inhibitory control ability in EF, which plays a certain positive role in the selection, allocation, and persistence of attention. Eye movement technology might also help patients obtain more information, improve their response time, and improve their alertness. The improvement in attention selection, distribution, and persistence in the process of attention represent the improvement of inhibition and control ability, which further contributes to the rehabilitation of executive function.
After 4 weeks of follow-up, the BADS, WSCT, and MOCA scores and response time of patients in both groups were decreased compared with those after the intervention. This suggests that although the intervention lasted 6 weeks and achieved some results immediately after the intervention, the training time might not be enough to stabilize these changes. Hence, the intervention time might need to be extended in the future, and the training intensity might need to be increased as well. Further studies are needed. EF is one of the most complex cognitive processes, and its recovery cannot be accomplished overnight and requires a period of accumulation and persistence. EF is a most complex cognitive process, the basis of human cognitive, emotional, and social skills, and a group of skills to independently complete purposeful and self-control behaviors. After a stroke, the patients need to carry out motor learning again, but this process requires individual selection, integration, organization, understanding, reasoning, and goals, and this process cannot be accomplished overnight but need time to be sustainable.
EF also emphasizes the importance of the interactions with the environment, requires the body to eliminate or suppress irrelevant information interference, selects necessary information input, extracts, compares, and integrates relevant information from long-term memory, and regulates daily activities or acquired activities. It is this regulatory role that makes daily activities and behaviors become coordinated, orderly, and purposeful. Therefore, EF is closely related to adaptive behavior in daily life. A previous study showed that the ADL of patients depends on cognitive function to some extent, and cognitive dysfunction could cause the decline of the self-care ability of the patients. In the multiple regression analysis conducted by Li et al., cognitive dysfunction was still an independent negative influence factor of ADL. Executive dysfunction could cause thinking rigidity, poor learning ability, low self-efficacy, and significantly reduced environmental adaptability in patients. Moreover, with natural aging, the EF decreases significantly, while after stroke, the EF decreases even more rapidly and more significantly.
This study has limitations. It was a single-center trial that might be biased by the local practice at the study hospital. There was no healthy control group. The patients were not tested for IQ, and questionnaires like the General Health Questionnaire were not used. Nevertheless, the aim of this study was not to determine the effect of eye movement training in the general population, but specifically in patients with stroke. Single treatment duration was used, and it is unknown whether the treatment effect might be consolidated using a longer treatment. The immediate and sustained effect of the intervention need to be further observed, and the neural plasticity basis of the treatment also needs to be further explored using electrophysiology, imaging, and other techniques.
In conclusion, this study indicates that rehabilitation training using the eye movement technique was beneficial to the recovery of EF and the improvement of quality of life in patients with stroke.
# Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
# Ethics statement
This study was approved by the ethics committee of the Forth Shanghai Rehabilitation Hospital (SP2018002). 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. |
Widespread metastatic breast cancer to the bowel: an unexpected finding during colonoscopy
Metastatic neoplasms to the gastrointestinal (GI) tract are an uncommon entity and in extremely rare cases originate from the breast. The clinical manifestations of metastatic breast cancer into the GI tract are frequently non-specific, and the interval between the diagnosis of lobular carcinoma and GI metastasis can often delay up to 30 years. Here, we present a 73year-old female with an unusual colonoscopy that revealed a submucosa nodular infiltrate throughout all the colon with a cobblestone-like appearance, which was later confirmed to be metastatic lobular carcinoma of the breast that was surgically removed 15 years early. A couple of months later, she developed malignant small bowel obstruction and laparotomy revealed extended small bowel and colonic metastatic involvement.
A 73-year-old female with a past medical history of breast cancer that was treated with bilateral mastectomy and adjuvant chemotherapy 15 years ago, after treatment there was no evidence of metastatic disease, she presented to the clinic with symptoms consistent with chronic colitis. A colonoscopy was performed and revealed a submucosa nodular infiltrate throughout all the colon with a cobblestone-like appearance, colonic biopsies showed diffuse metastatic lobular breast cancer with positive estrogen receptors, negative progesterone receptors and negative for HER2 overexpressionand c). After discussing medical treatment options (i.e. chemotherapy or hormonal therapy), the patient decided to forgo treatment. A couple of months later, she arrived to the emergency department with a proximal small bowel obstruction and surgical management was required, laparotomy revealed seven areas within the small bowel with either complete or partial malignant obstruction and infiltration of the ascending, transverse and descending colon. A proximal small bowel resection of the complete obstruction with a palliative ileostomy and mucous fistula was performed. No postoperative complications were reported.
Metastatic neoplasms to the gastrointestinal (GI) tract are rare that usually originate from skin, lung, kidney, prostate, ovary, stomach or hepatobiliary system and in extremely rare cases from the breast. Lobular carcinoma is the most common histopathological subtype of metastatic breast cancer that invades the GI tract [bib_ref] Secondary metastatic lesions to colon and rectum, Galanopoulos [/bib_ref] [bib_ref] Metastatic lobular carcinoma, of the breast masquerading as a primary rectal cancer, Matsuda [/bib_ref]. The interval between the diagnosis of lobular carcinoma and GI metastasis can be up to 30 years. Colonoscopy may reveal an abnormal lumen appearance with multiple polypoid lesion with a cobblestone-like appearance that might cause intraluminal obstruction. Diagnosis is made through histopathological examination. Although the majority of reports of metastatic breast cancer involves a single colonic segment, this is an abnormal presentation of metastatic breast cancer to the GI tract due to the extended involvement of several colonic segments and multiple regions of the small bowel, without evidence of extraintestinal metastasis [bib_ref] Secondary metastatic lesions to colon and rectum, Galanopoulos [/bib_ref] [bib_ref] An unusual clinical presentation of gastrointestinal metastasis from invasive lobular carcinoma of..., Balakrishnan [/bib_ref].
[fig] Figure 1: (A) Colonoscopy showing submucosal nodular infiltrate with a cobblestone-like appearance throughout all the circumference of the colon. (B) Microscopy of the colonic biopsies showing metastatic carcinoma infiltrating colonic mucosa with scattered residual glands. (C) Immunohistochemistry showing metastatic tumor cells with nuclear positivity for estrogen receptor [/fig]
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Management challenges associated with a pineal region chordoma: illustrative case
BACKGROUND Chordomas, which are rare malignant neoplasms arising from notochordal remnants, often cause gradually progressive clinical symptoms. Intradural cranial chordomas (ICCs) are extremely rare and generally have a favorable prognosis. However, the authors reported the case of a primary ICC originating in the pineal gland presenting with recurrent thalamic hemorrhage and displaying an aggressive postoperative clinical course.OBSERVATIONS A 41-year-old man arrived at the emergency department with morning headaches and recurrent syncope that had lasted several months. Computed tomography and magnetic resonance imaging (MRI) revealed a pineal gland mass causing obstructive hydrocephalus and a subacute hematoma in the right thalamus. Three weeks after an endoscopic third ventriculostomy was performed, recurrent hemorrhage was observed in the right thalamus. The tumor was promptly removed surgically. The yellowish-white tumor did not exhibit abundant bleeding. No evidence of intratumoral hemorrhage around the hematoma pocket was found. Histopathological examination revealed the characteristics of a chordoma with minimal vascularity. MRI performed 10 weeks postoperatively for worsening headaches revealed abnormal enhancement of multiple cranial nerves, suggesting leptomeningeal seeding (LMS) of the tumor.LESSONS Despite radiotherapy and intrathecal chemotherapy, the patient's neurological status worsened; he died 2 years postoperatively. A pineal ICC may cause recurrent thalamic hemorrhage and potentially fatal LMS, even in the early postoperative period.https://thejns.org/doi/abs/10.3171/CASE21110 ABBREVIATIONS CT 5 computed tomography; ETV 5 endoscopic third ventriculostomy; ICC 5 intradural cranial chordoma; ICV 5 internal cerebral vein; LMS 5 leptomeningeal seeding; MRI 5 magnetic resonance imaging; PET 5 positron emission tomography; WI 5 weighted imaging.
Chordomas are rare malignant neoplasms that arise from notochordal remnants along the axial skeleton 1 and usually present with gradually progressive clinical symptoms secondary to growth and bone destruction. [bib_ref] Giant petroclival primary intradural chordoma: case report and systematic review of the..., Otaibi [/bib_ref] Approximately 50% of all chordomas occur in the sacrococcygeal area, 30% occur in the skull base, and 20% occur in the mobile spine. [bib_ref] Diagnostic clues and treatment of intradural cranial chordoma, Zeng [/bib_ref] Intradural cranial chordomas (ICCs) are extremely rare and mostly located in the prepontine and parasellar areas. [bib_ref] Chordoma of the corpus callosum: case report, Rinaldo [/bib_ref] On the other hand, the prognosis of ICCs is generally better than those of typical chordomas because their sharply circumscribed margins allow total excision. [bib_ref] Intradural retroclival chordoma, Choo [/bib_ref] [bib_ref] Intradural retroclival chordoma without bone involvement: case report, Masui [/bib_ref] However, we report a rare case of a primary ICC originating in the pineal gland, presenting with recurrent thalamic hemorrhage and displaying an aggressive postoperative clinical course.
## Illustrative case clinical presentation
A 41-year-old man presented at the emergency department with morning headaches and recurrent syncope that had lasted several months. A computed tomography (CT) scan revealed a mass-like lesion with eggshell-like high density of the pineal gland extending to the right thalamus as a low-density lesion and resulting in obstructive hydrocephalus . Magnetic resonance imaging (MRI) revealed a tumor of the pineal gland, which appeared iso/ hypointense on T1-weighted imaging (WI) and hyperintense on T2-WI with heterogeneous contrast enhancement. The lesion in the right thalamus was identified as a subacute hematoma. A hypointense rim was also seen along the pineal tumor margin, especially along the anterior and right sides on T2-WI . Positron emission tomography (PET) with 2-deoxy-2-[fluorine-18]fluoro-D-glucose revealed a hypermetabolic lesion of the pineal gland. On the other hand, the right thalamic lesion was identified as a metabolic defect . Cerebrospinal fluid testing did not reveal any abnormal findings. Based on these findings, the initial diagnosis was a pineal parenchymal tumor with a rare tumor bleed.
## Surgical procedure
Initially, endoscopic third ventriculostomy (ETV) was performed to resolve the obstructive hydrocephalus. Subsequently, resection of the tumor was planned, and the patient was discharged. Three weeks after the ETV, the patient presented at the emergency department again with a sudden headache. A CT scan revealed a newly developed hematoma at the site of the previous hematoma in the right thalamus . Prompt surgery to remove the tumor was planned because the tumor seemed to be the cause of the recurrent bleeding. MRI was performed to aid in intraoperative navigation. Serial T2-WI revealed narrowing of the right internal cerebral vein (ICV) and an acute phase hematoma in the right thalamus .
The surgery was performed via an occipital transtentorial approach. The tumor was yellowish white, and bleeding from the tumor was not abundant. However, it was hard and tough and densely adhered to both ICVs, especially on the right side. Thus, it could not be easily removed either with a tumor forceps or with an ultrasonic aspirator. On the right side of the tumor, the hematoma pocket in the right thalamus was identified and evacuated. However, there was no evidence of bleeding in the tumor itself around the hematoma pocket. A subtotal resection was performed, leaving the anterosuperior part of the tumor intact to save the bilateral ICVs [fig_ref] FIG. 3: A subtotal resection was performed, leaving the anterosuperior part of the tumor... [/fig_ref].
## Histopathological findings
The gross histopathology revealed chords, sheets, and individual cells with bubbly cytoplasm (physaliphorous cells) arranged in FIG. 1. CT scan reveals a mass-like lesion with eggshell-like high density of the pineal gland extending to the right thalamus as a low-density lesion (A). MRI shows a tumor of the pineal gland that appears iso/ hypointense on T1-WI (B) and hyperintense on T2-WI (C) with heterogeneous contrast enhancement (D). The lesion in the right thalamus was identified as a subacute hematoma. E: PET with 2-deoxy-2-[fluorine-18]fluoro-D-glucose shows a hypermetabolic lesion of the pineal gland (arrow); the right thalamic lesion was identified as a metabolic defect.
## Fig. 2. a:
CT scan shows a newly developed hematoma at the site of the previous hematoma in the right thalamus. B and C: MRI was performed for intraoperative navigation; serial T2-WI shows narrowing of the lumen of the right ICV (yellow arrows) compared to that of the left ICV (red arrows) and an acute phase hematoma in the right thalamus. lobules set within a myxochondroid background. Increased cellularity and moderate nuclear polymorphism were also seen. Mitotic activity and necrosis were not definitively identified. The diagnosis of chordoma or chondrosarcoma was initially considered. Immunohistochemistry revealed that the tumor cells were reactive for both S100 and cytokeratin [fig_ref] FIG. 4: Hematoxylin and eosin staining [/fig_ref]. The final histopathological diagnosis was a chordoma with minimal vascularity and no definite evidence of intratumoral hemorrhage.
## Postoperative course
Proton beam radiotherapy was planned for treatment of the residual tumor, and the patient was discharged in the absence of any definite neurological abnormalities. Thereafter, the patient repeatedly visited the outpatient clinic or the emergency department for a progressively worsening headache 6 weeks after the tumor resection. A follow-up MRI performed 10 weeks after the surgery revealed interval growth of the residual tumor [fig_ref] FIG. 3: A subtotal resection was performed, leaving the anterosuperior part of the tumor... [/fig_ref] and abnormal enhancement of the cranial nerves, including the bilateral third nerves, trigeminal nerves, and facial nerves, suggesting leptomeningeal seeding (LMS) of the tumor [fig_ref] FIG. 3: A subtotal resection was performed, leaving the anterosuperior part of the tumor... [/fig_ref].
Whole-brain radiotherapy with a dose of 3,000 cGy was applied in 10 fractionations with an additional boost of 2,000 cGy of radiotherapy to the primary site. Next, the administration of intrathecal methotrexate was commenced. After two trials of intrathecal methotrexate, whole-spine radiotherapy with a total dose of 4,000 cGy was applied in 20 fractionations for the thoracolumbar area. A total of 31 trials of intrathecal methotrexate was performed in 2 years, and these procedures induced necrotizing leukoencephalopathy.
Despite the abovementioned treatments, including ventriculoperitoneal shunting for controlling intracranial pressure, the patient's neurological status worsened, and he died 2 years after the tumor resection.
# Discussion observations
## Pineal chordoma and recurrent bleeding
The key finding in this case was that a pineal ICC may cause recurrent thalamic hemorrhage and potentially fatal LMS, even in the early postoperative period. ICCs are rare, and to date, only approximately 60 cases have been reported. [bib_ref] Chordoma of the corpus callosum: case report, Rinaldo [/bib_ref] Moreover, ICCs of the pineal gland are extremely rare. Only one case of a chordoma of the pineal gland confirmed by autopsy has been reported, which was initially diagnosed as a germinoma. [bib_ref] Ectopic pineal chordoma, Figueiredo [/bib_ref] Before initiation of involution at approximately the 6th or 7th week of embryonic life, the notochord extends from the most caudal end of the vertebral column and terminates in what will develop into the dorsum sellae of the skull base. [bib_ref] Chordomas: a review, George [/bib_ref] Thus, the finding of a chordoma of the pineal gland is unexpected. Rests of notochordal cells have been found in the prepontine extradural space posterior to the clivus, [bib_ref] Retroclival ecchordosis physaliphora: MR imaging and review of the literature, Mehnert [/bib_ref] which is consistent with the spatial distribution of chordomas. [bib_ref] Chordomas: a review, George [/bib_ref] Further research is necessary to identify how far these notochordal vestiges can be found in the brain.
The symptoms of intracranial chordoma vary with the location of the lesion and its proximity to critical structures 9 and gradually progress because of tumor growth and bone destruction. However, although rare, cases of bleeding from a chordoma abruptly exacerbating neurological status have been reported. [bib_ref] Intracranial intradural chordoma presenting with intraventricular hemorrhage, Kim [/bib_ref] [bib_ref] Intradural chordoma presenting with intratumoral bleeding, Vellutini [/bib_ref] [bib_ref] Apoplexy in an intradural clival chordoma causing intraventricular bleed, Mohindra [/bib_ref] The cause of tumor hemorrhage in such cases remains to be elucidated. Intratumoral hemorrhage in chordomas may result from the rupture of thin-walled vessels, hemorrhagic necrosis due to rapid tumor growth, or destruction of the dural vessels by tumor invasion. [bib_ref] Intracranial intradural chordoma presenting with intraventricular hemorrhage, Kim [/bib_ref] [bib_ref] Apoplexy in an intradural clival chordoma causing intraventricular bleed, Mohindra [/bib_ref] However, we could not identify any evidence of tumor bleeding during surgery in this case, and there was no histopathological evidence of intratumoral bleeding. Failure of the venous drainage of the right thalamus could reasonably be assumed to have been the cause of recurrent bleeding in this case considering the narrowing or collapse of the right ICV by the firm and hard tumor.
## Lessons
## Lms of iccs
In previous case reports and small case series, ICCs were generally considered to have a more favorable prognosis and a different biological behavior relative to those of typical skull base chordomas. [bib_ref] Giant petroclival primary intradural chordoma: case report and systematic review of the..., Otaibi [/bib_ref] [bib_ref] Sudden death due to subarachnoid bleeding from ecchordosis physaliphora, Fracasso [/bib_ref] However, recent data suggest that the overall prognosis of ICCs may be comparable to those of typical chordomas, although gross-total resection may be more feasible for true intradural chordomas. [bib_ref] Giant petroclival primary intradural chordoma: case report and systematic review of the..., Otaibi [/bib_ref] Moreover, ICCs present an additional problem: chordomas with intradural extension are at risk of LMS along the subarachnoid space. [bib_ref] Chordomas: a review, George [/bib_ref] In fact, there have been reports of LMS or drop metastases of chordomas after surgery, as in the case reported in this paper. [bib_ref] Chordoma of the corpus callosum: case report, Rinaldo [/bib_ref] [bib_ref] Intradural cervical chordoma with diffuse spinal leptomeningeal spread: case report and review..., Zhang [/bib_ref] [bib_ref] Diffuse spinal spreading following previous intracranial intradural chordoma resection: a rare case..., Vellutini [/bib_ref] [bib_ref] Unusual intradural spinal metastasis of a cranial chordoma. Case report, Stough [/bib_ref] [bib_ref] Intradural drop metastasis of a clival chordoma, Martin [/bib_ref] [bib_ref] Primary intradural pontocerebellar chordoma metastasizing in the subarachnoid spinal canal, Korinth [/bib_ref] [bib_ref] Intradural spinal seeding and fatal progression of a sacrococcygeal chordoma: a case..., Ji [/bib_ref] [bib_ref] Chordoma radicular metastasis following cerebrospinal fluid dissemination. Article in French, Champeaux [/bib_ref] [bib_ref] Intradural spinal seeding of a clival chordoma, Asano [/bib_ref] The proximity of the lesions to the cerebrospinal fluid space and/or ventricular system and intraventricular exposure during surgery may increase the risk of LMS. [bib_ref] Chordoma of the corpus callosum: case report, Rinaldo [/bib_ref] Although LMS usually develops as an end-stage event several years after surgery, [bib_ref] Diffuse spinal spreading following previous intracranial intradural chordoma resection: a rare case..., Vellutini [/bib_ref] [bib_ref] Chordoma radicular metastasis following cerebrospinal fluid dissemination. Article in French, Champeaux [/bib_ref] it can occur in the early postoperative period, even within a week of surgery. [bib_ref] Intradural cervical chordoma with diffuse spinal leptomeningeal spread: case report and review..., Zhang [/bib_ref] In the present case, the headache that had ceased after surgery recurred within 6 weeks of the tumor resection. The follow-up MRI performed 10 weeks after surgery to identify the cause of the unresolved headaches confirmed LMS. In situations in which there is no clear evidence of whether radiotherapy will be effective in preventing LMS, as in this case, it is difficult to be sure if LMS could have been prevented had the adjuvant treatment commenced earlier. Therefore, it is important to bear in mind that LMS can occur even in the early postoperative period, and careful and timely follow-ups and optimal scheduling of the adjuvant treatment are necessary, especially in patients who exhibit postoperative neurological symptoms or unresolved headaches.
# Conclusions
ICCs are rare tumors that generally have a favorable prognosis. However, recurrent thalamic bleeding from a pineal ICC may occur as the result of the failure of venous drainage, and potentially fatal LMS can occur even in the early postoperative period. Therefore, careful and timely follow-ups and optimal scheduling of the adjuvant treatment are necessary, especially in patients who exhibit postoperative neurological symptoms or unresolved headaches.
[fig] FIG. 3: A subtotal resection was performed, leaving the anterosuperior part of the tumor intact to save the bilateral ICVs (A). Follow-up MRI performed 10 weeks after the surgery reveals interval growth of the residual tumor (B) and abnormal enhancement of the cranial nerves, including the bilateral third nerves (arrow in C) and trigeminal nerves (arrow in D), suggesting LMS of the tumor (C and D). [/fig]
[fig] FIG. 4: Hematoxylin and eosin staining (original magnification Â200) shows chords, sheets, and individual cells with bubbly cytoplasm (physaliphorous cells) arranged in lobules set within a myxochondroid background (A). Immunohistochemistry reveals that the tumor cells are reactive for both S100 (B) and cytokeratin (C). The final histopathological diagnosis is a chordoma. [/fig]
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Biologically-Based Mathematical Modeling of Tumor Vasculature and Angiogenesis via Time-Resolved Imaging Data
Simple Summary: The recruitment of new vasculature via angiogenesis is a critical component of tumor development, which fundamentally influences tumor growth and response to treatment. The characterization of tumor-induced angiogenesis via mathematical models could enable approaches to forecast tumor response and improve patient care. In this review, we discuss how time-resolved imaging data integrated with mathematical modeling can be used to systematically investigate angiogenesis from the cell to tissue scale and ultimately forecast response to therapy.Abstract: Tumor-associated vasculature is responsible for the delivery of nutrients, removal of waste, and allowing growth beyond 2-3 mm 3 . Additionally, the vascular network, which is changing in both space and time, fundamentally influences tumor response to both systemic and radiation therapy. Thus, a robust understanding of vascular dynamics is necessary to accurately predict tumor growth, as well as establish optimal treatment protocols to achieve optimal tumor control. Such a goal requires the intimate integration of both theory and experiment. Quantitative and time-resolved imaging methods have emerged as technologies able to visualize and characterize tumor vascular properties before and during therapy at the tissue and cell scale. Parallel to, but separate from those developments, mathematical modeling techniques have been developed to enable in silico investigations into theoretical tumor and vascular dynamics. In particular, recent efforts have sought to integrate both theory and experiment to enable data-driven mathematical modeling. Such mathematical models are calibrated by data obtained from individual tumor-vascular systems to predict future vascular growth, delivery of systemic agents, and response to radiotherapy. In this review, we discuss experimental techniques for visualizing and quantifying vascular dynamics including magnetic resonance imaging, microfluidic devices, and confocal microscopy. We then focus on the integration of these experimental measures with biologically based mathematical models to generate testable predictions.
# Introduction
In the early stages of tumor growth, a small population of tumor cells is supported by existing tissue vasculature and the diffusion of nutrients through the extravascular space. As this small population of tumor cells continues to grow, it may eventually reach a size where the diffusion of nutrients from existing vasculature is insufficient to support continued growth. Through the process of angiogenesis, new blood vessels are recruited from nearby vasculature to provide the crucial infrastructure needed to sustain further expansion of the tumor. These three key points inform the foundation of many mathematical models of angiogenesis and arose from the seminal work on tumor angiogenesis by Folkmanand othersover the past half a century. Additional studies on angiogenic signalingand vasculature propertieshave also greatly influenced the development of a mathematical theory on angiogenesis. One notable observation was that compared to healthy appearing vasculature, tumor-associated vasculature has substantial structural and functional abnormalities characterized by non-hierarchical vessel networks, heterogeneous blood flow, and heterogeneous permeability. These irregularities significantly influence the delivery of nutrients and removal of waste, while also having substantial implications on systemic and radiation therapy.
Tumor vasculature and the process of angiogenesis have a critical and complex role in the response of solid tumors to systemic therapies. First, the successful delivery of systemic agents is contingent on functional vasculature providing a homogenous delivery of therapeutics. Unfortunately, the abnormal structure and function of vessels associated with tumor-induced angiogenesis yields a heterogenous distribution of therapeutics (which can include chemotherapies as well as targeted and hormone therapies) throughout the tumor, contributing to varied efficacy within patient populations. For example, in breast cancer, clinical trials for neoadjuvant systemic therapies have resulted in only 6-26% of patients achieving a pathological complete response by the completion of treatment, which, in part, may be due to the abnormal structure and development of the vessels. Second, the vasculature itself can also be affected by targeted and non-specific systemic therapies that may hinder angiogenesis, eliminate vasculature, or normalize existing neovasculature. One perspective on cytotoxic agents inflicting damage to tumorassociated vasculature is that it is a positive outcome, which can prevent necessary nutrients from reaching the tumor and to induce necrosis. An opposing view, however, posits that efforts should be made to protect (and even normalize) the vessels to enhance the delivery of the therapeutics. Therefore, tumor-associated vasculature plays an important and evolving role in the effectiveness of systemic cancer treatment.
Radiotherapy is another primary treatment option for the majority of solid tumors and is capable of targeting unresectable or highly invasive disease. The efficacy of radiotherapy, however, is highly dependent on the structure and function of the tumor-associated vasculature. For over half a century, it has been well-known that tissue oxygenation influences the sensitivity of tumor cells to radiotherapy. Within tumors, tissue oxygenation is highly heterogeneous due to the structural and functional abnormalities of tumor vasculature, which can result in both acute and chronic hypoxic regions resistant to radiotherapy. Large hypoxic regions also occur from vascular injury or vascular occlusion-often downstream of increased mechanical pressures from increased tumor cellularity. It is generally presumed that tumor cells nearest to functioning vasculature and furthest from hypoxic regions are often the most responsive to radiotherapy. Thus, after radiotherapy, the remaining tumor is thought to be largely composed of poorly perfused and hypoxic tumor cells. However, radiotherapy itself also influences tumor-associated vasculature by promoting angiogenesis, the revascularization of the remaining tumor, and the reoxygenation of tumor tissue-thus improving the sensitivity of previously hypoxic cells to future doses of radiotherapy. To effectively control tumors via radiotherapy and identify optimal radiotherapy regimens, knowledge of the dynamic relationship that exists between tumor-associated vasculature and radiotherapy is required.
It is clear that tumor vasculature and angiogenesis significantly influence tumor growth and response to systemic and radiation therapies. To improve patient outcomes, therapeutic regimens need to be optimized while considering the structural and functional characteristics of an individual's tumor-associated vasculature. Achieving this goal requires a biophysical mathematical theory that accurately characterizes the relevant quantities of interest in the dynamic relationship between the tumor, vasculature, and therapy. Given such a theory, one could identify, through systematic, in silico evaluations, therapeutic regimens that are personalized to optimize treatment outcomes for each individual patient. While the literature is filled with numerous theoretical studies characterizing tumor-associated vasculature from the cell to tissue scale, there is a lack of research that explicitly links theory with quantitative experimental studies.
Quantitative and time-resolved imaging approaches, such as confocal imaging, photoacoustic imaging, and magnetic resonance imaging (MRI), could provide the necessary data to initialize, calibrate, and validate models of angiogenesis. Specifically, time-resolved imaging techniques of the vasculature have matured to the point where they can define or estimate subject-specific structural (e.g., vessel order and location) and functional (e.g., vessel permeability, blood flow) model parameters that would enable in silico investigations of tumor and vasculature dynamics. Non-invasive imaging techniques preserve the system under observation, allowing the state of the system to be assessed and quantified before, during, and after treatment, thereby capturing the evolution of both tumor and vasculature. This spatially and time-resolved data is a fundamental component of rigorous model development and validation that is required to translate modeling approaches to the clinic.
The mathematical modeling of tumor angiogenesis at the cell scale has developed into a rich literature over the last few decades. These models aim to give a rigorous mathematical description of tumor angiogenesis to enable the systematic investigation of the underlying biology that dictates vascular sprouting, perfusion, and response to therapy. By employing such models, it is possible to simulate and test scenarios in silico that are not easily tested experimentally. For example, comparing the limitless number of therapeutic regimens that can be constructed with varying dosing schedules and concentrations is experimentally intractable, but with a mathematical model these can be simulated and analyzed to select the optimal regimen. Recently, there has also been great interest in the modeling of tumor angiogenesis at the tissue scale. These efforts have often been motivated by the emerging availability of crucial vasculature properties in patient or animal data that previously could only be assessed through highly invasive means-such as angiogenesis and regression rates(i.e., a time scale of formation and regression of tumor-induced vasculature), interstitial pressure, and blood pressure along vessels. However, these models must be informed and validated by time-resolved, experimental data to initialize and calibrate key model parameters, or by modeling biologically based hypotheses and testing model output with experimental observables.
In this review, we identify promising approaches that integrate mathematical theory with experimental data from the in vitro cell scale to in vivo tissue scale, discuss opportunities for bridging cell and tissue scale models, and present future opportunities for applying these models to optimize therapeutic regimens and therefore improve patient care. To prepare this review, we first identified literature that integrated mathematical theory with experimental imaging data. We then identified reviews or landmark articles that provided the foundation for both the mathematical theory and experimental techniques.
## Overview of experimental techniques across scales
In this section, we discuss experimental techniques from the cell to tissue scales.summarizes the cell to tissue scale approaches for imaging experiments, whilelists the imaging techniques and the literature that integrates those techniques with mathematical theory at the cell and tissue scale. We note that the literature listed inis presented in detail in Sections 3.2 and 4.2 for the cell and tissue scale, respectively.
## Overview of experimental techniques across scales
In this section, we discuss experimental techniques from the cell to tissue scales.summarizes the cell to tissue scale approaches for imaging experiments, whilelists the imaging techniques and the literature that integrates those techniques with mathematical theory at the cell and tissue scale. We note that the literature listed inis presented in detail in Sections 3.2 and 4.2 for the cell and tissue scale, respectively.
## Figure 1.
Overview of cell to tissue scale imaging. Experimental platforms from the cell to tissue scales consist of cell culture (to investigate cell dynamics), microfluidics (a perfused cell culture platform to observe angiogenesis), skin fold window chambers (an in vivo platform for optical imaging), and small animal or human whole organ and body imaging (for in vivo studies). Imaging techniques (purple bars) vary across spatial and temporal scales. In vitro imaging consists primarily of the microscopies (e.g., confocal, multiphoton). In vivo imaging is achievable with all the imaging techniques shown above; however, there are limitations in the penetration depth for microscopy and photoacoustic imaging. Magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT) are primarily in vivo techniques capable of whole animal or human imaging. Whole animal or body imaging is feasible with microCT, though it is typically used for whole organ or ex vivo imaging.
## Quantitative techniques for observing tumor vasculature and angiogenesis at the cellular scale
At the cellular scale, microscopy is the dominant imaging technique for providing quantitative measurements of tumor vasculature with a spatial resolution on the order of microns. Confocal microscopy, combined with immunofluorescence staining, has been Overview of cell to tissue scale imaging. Experimental platforms from the cell to tissue scales consist of cell culture (to investigate cell dynamics), microfluidics (a perfused cell culture platform to observe angiogenesis), skin fold window chambers (an in vivo platform for optical imaging), and small animal or human whole organ and body imaging (for in vivo studies). Imaging techniques (purple bars) vary across spatial and temporal scales. In vitro imaging consists primarily of the microscopies (e.g., confocal, multiphoton). In vivo imaging is achievable with all the imaging techniques shown above; however, there are limitations in the penetration depth for microscopy and photoacoustic imaging. Magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT) are primarily in vivo techniques capable of whole animal or human imaging. Whole animal or body imaging is feasible with microCT, though it is typically used for whole organ or ex vivo imaging.
## Quantitative techniques for observing tumor vasculature and angiogenesis at the cellular scale
At the cellular scale, microscopy is the dominant imaging technique for providing quantitative measurements of tumor vasculature with a spatial resolution on the order of microns. Confocal microscopy, combined with immunofluorescence staining, has been used to acquire high resolution, temporally resolved images of vascular structure in angiogenic and vasculogenic assays. This technology allows for analyzing in vitro spatial distributions of fluorescently labeled cell lines and can be coupled with fluorescing microspheres to investigate vascular integrity, enabling the integration of quantitative fluorescence measurements with mathematical modeling. Furthermore, confocal microscopy has also been utilized in vivo to investigate functional microcirculationin tumor-associated vasculature, the effects of radiotherapyon neovasculature, and the oxygen distributionin dorsal skin fold chambers. While intravital microscopy provides a high-resolution longitudinal analysis in vivo, the chamber may alter the tumor-vasculature dynamics and it is fundamentally limited in the length of study (generally 2-3 weeks), and the number of imaging time points. Multiphoton microscopy, in comparison to confocal microscopy, has improved depth penetration and confines excitation to the focal plane of the lens, thereby decreasing the photodamage to the tissue. For many biological applications, tissue depths of~500 microns can be imaged over time.
In addition to microscopy, photoacoustic imaging offers high spatial resolution (10-100 microns) while also being able to reach tissue penetration depths of around 4-10 cm. Photoacoustic imaging probes the tissue of interest with pulses of light, creating changes in the pressure when the light is absorbed. These changes in pressure generate ultrasound waves that are detected at the tissue surface. The spatial and temporal resolution, imaging depth, and image contrast can be selected by utilizing different light sources, ultrasound wave detectors, and scanning methods to cater to the specific application under study, making photoacoustic imaging a promising emerging imaging modality at the cellular scale.
## Quantitative techniques for observing tumor vasculature and angiogenesis at the tissue scale
All the techniques presented in this section are suitable for small animal and human imaging. Ex vivo and in vivo imaging play a central role in understanding the morphology and function of tumor vasculature and angiogenesis. In particular, ex vivo imaging techniques, including histology imagingand micro-CT, can quantify tumor microvasculature and angiogenesis on excised tissue specimens and serve as the gold standard measurement. Less invasive observations of tumor-associated vasculature can now be achieved thanks to the development of in vivo imaging techniques, such as x-rayand computed tomography (CT), positron emission tomography (PET), MRI, and optical imaging. There are two main classes of imaging techniques applied to studying tumor-associated vasculature: (1) angiography, which is a technique used to visualize the vasculature structure, and (2) functional techniques used to quantify the properties of the tissue and vasculature. In conventional planar x-ray angiography, the patient is catheterized so that an iodinated contrast agent can be administered intravenously and then observed with fluoroscopy, thereby enabling the observation of the vascular architecture. CT angiography is an extension of x-ray angiography that enables the visualization of vessel structures in 3D. CT angiography has been commonly used to identify the location and anatomy of tumor-associated vessels (especially for pancreatic tumors), which plays a valuable role on diagnosis and the management of chemotherapy and surgery. Magnetic resonance angiography (MRA) is an alternative approach that does not use ionizing radiation and can be used to visualize blood vessels, especially large arteries and veins. MRA techniques aim at enhancing the contrast between blood vessels and the background tissue based on either the effects of blood flow on MR signal or the injection of exogenous contrast agents, thereby allowing for the quantification of several morphological characteristics of the vasculature, such as vessel tortuosity, density, diameter, and branching patterns as well as feeding and draining vessels.
While conventional angiography focuses on vascular morphology, functional imaging techniques enable the extraction of information regarding hemodynamics and pharmacokinetics. CT-based techniques have been developed to provide physio-pathological information of the vasculature beyond the anatomy. Functional or dynamic contrastenhanced (DCE-) CT can measure tumor vascular features including blood flow, blood volume, mean transit time, and permeability-surface area product. Functional CT could potentially be used to monitor the change of tumor perfusion in anti-angiogenic therapy. CT has also been combined with PET techniques for the staging and monitoring of multiple types of tumors via the evaluation of blood flow, such as melanoma, medullary thyroid cancer, hepatocellular carcinoma, and prostate carcinoma. Similar to the functional CT approach, DCE-MRI can also return estimates describing plasma volume fraction, extracellular extravascular volume fraction, and vessel permeability and perfusion. DCE-MRI Cancers 2021, 13, 3008 6 of 28 techniques with high temporal resolution (7 s per frame or even faster)further enable the extraction of information regarding hemodynamics and pharmacokinetics. Recent studies on the hybrid acquisition of MRA and DCE-MRI allow for the extraction of both morphological and functional features of tumor-associated vasculature, which have been shown to increase the diagnostic accuracy of suspicious tumors. Although in vivo imaging provides an observation of vasculature non-invasively, these technologies are significantly limited by the available spatial and temporal resolution and their signal-to-noise ratio. Thus, the common, clinically available angiography techniques cannot currently capture details of the microvasculature. To overcome this limitation, the development of window chamber models could be a promising approach, as this technology enables the combination of in vivo microscopy with MR imaging, thereby potentially enabling the validation of macroscopical measurements of microvasculature via MRI.
## Approaches for modeling tumor vasculature at the cell scale
In this section, we identify a few landmark mathematical, cell scale models of tumor vasculature and angiogenesis. We then describe the common quantitative methods for observing angiogenesis over time and conclude by discussing some efforts, both established and ongoing, to integrate mathematical models with experimental methods.
## Mathematical modeling of tumor vasculature and angiogenesis at the cell scale
Mathematical models of tumor angiogenesis vary in the extent of biological detail they characterize and can be summarized as discrete (treating endothelial cells and vasculature as individual objects), continuous (treating endothelial cells or vasculature as concentrations), or hybrid (combining methodology from both discrete and continuum theory) models. Discrete models may track all endothelial cells as individual agents, or simply tip endothelial cells or TECs (the cells responsible for directed migration in response to chemical stimuli). In discrete models, the vasculature changes through time based on sets of rules dictating cell behavior (e.g., whether a cell will divide or migrate). Continuum models are based on ordinary or partial differential equations (PDEs) that govern the behavior of the endothelial cells through time. Hybrid models couple these two theories by, for example, discretely characterizing the TECs and continuously modeling the overall vessel morphology through a PDE. We note that while hybrid models could refer to models that have a discrete and continuous component within the modeling framework (which would dictate a hybrid modeling approach), here, we define hybrid as utilizing both discrete and continuous methodologies specifically applied to model the vasculature. The reader is invited to refer to throughout this section as it shows examples of these three modeling approaches. All three modeling approaches are typically used to study the migration and development of tumor-associated vasculature in response to external stimuli (e.g., chemical, mechanical) in conjunction with a model of tumor growth. A simulation of the process of angiogenesis typically begins with the stimulation of endothelial cells by tumor angiogenic factors (TAF, a continuous field of pro-angiogenic proteins secreted by tumor cells) that are either explicitly coupled to a model of tumor cell growth or assumed to have a fixed initial distribution. Directed movement of endothelial cells is then influenced by chemical gradients (i.e., TAF), gradients in fibronectin or insoluble extra-cellular matrix (ECM) (i.e., haptotaxis), and mechanical cues (i.e., mechanotaxis).
could refer to models that have a discrete and continuous component within the modeling framework (which would dictate a hybrid modeling approach), here, we define hybrid as utilizing both discrete and continuous methodologies specifically applied to model the vasculature. The reader is invited to refer to throughout this section as it shows examples of these three modeling approaches. All three modeling approaches are typically used to study the migration and development of tumor-associated vasculature in response to external stimuli (e.g., chemical, mechanical) in conjunction with a model of tumor growth. A simulation of the process of angiogenesis typically begins with the stimulation of endothelial cells by tumor angiogenic factors (TAF, a continuous field of proangiogenic proteins secreted by tumor cells) that are either explicitly coupled to a model of tumor cell growth or assumed to have a fixed initial distribution. Directed movement of endothelial cells is then influenced by chemical gradients (i.e., TAF), gradients in fibronectin or insoluble extra-cellular matrix (ECM) (i.e., haptotaxis), and mechanical cues (i.e., mechanotaxis). Continuum models (panel c) describe this phenomenon in terms of endothelial cell densities and the concentration of TAF. Partial differential equations (PDEs) provide a continuous representation of endothelial densities and often describe the spatial and temporal evolution via diffusion, haptotaxis, and chemotaxis terms. Alternatively, discrete models (panel d) can be used to explicitly describe the movement and behavior of each individual endothelial cell. Hybrid models (panel e) generally combine both discrete and continuum approaches to model TEC movement and endothelial cell densities, respectively, in response to TAF.
## Continuum models
Continuum models (panel c in describe the spatial and temporal development of endothelial cells over time in terms of densities or volume fractions. These models are capable of capturing macroscopic features related to vasculature, TAF, and ECM but do not track individual cells or vessel segments. In continuum models, the spatial and temporal progression of these model components are described with a set of coupled PDEs. Anderson et al. developed a continuum model of tumor angiogenesis by considering the rate of change of endothelial cell density determined by the sum of the effects of Brownian motion (diffusion), chemical stimuli (chemotaxis), and mechanical forces (haptotaxis). The chemical stimuli considered was TAF, which caused a migration in endothelial cell density toward the TAF source. In the presence of angiogenic factors, the distribution of endothelial cells migrated across the domain. As Anderson et al.'s model system forms the foundation for numerous other models of angiogenesis,illustrates the proposed model. The same model can be conceptualized as a discrete model by considering the bulk changes in endothelial cell density as discrete events based on probabilities. The authors used a finite difference approximation of the continuous equation for endothelial cell density to determine the probability that endothelial cells move in a particular direction due to diffusion, chemotaxis, and haptotaxis. This work highlighted the potential of both discrete and continuum models to explore the same phenomena.
## Continuum models
Continuum models (panel c in describe the spatial and temporal development of endothelial cells over time in terms of densities or volume fractions. These models are capable of capturing macroscopic features related to vasculature, TAF, and ECM but do not track individual cells or vessel segments. In continuum models, the spatial and temporal progression of these model components are described with a set of coupled PDEs. Anderson et al. developed a continuum model of tumor angiogenesis by considering the rate of change of endothelial cell density determined by the sum of the effects of Brownian motion (diffusion), chemical stimuli (chemotaxis), and mechanical forces (haptotaxis). The chemical stimuli considered was TAF, which caused a migration in endothelial cell density toward the TAF source. In the presence of angiogenic factors, the distribution of endothelial cells migrated across the domain. As Anderson et al.'s model system forms the foundation for numerous other models of angiogenesis,illustrates the proposed model. The same model can be conceptualized as a discrete model by considering the bulk changes in endothelial cell density as discrete events based on probabilities. The authors used a finite difference approximation of the continuous equation for endothelial cell density to determine the probability that endothelial cells move in a particular direction due to diffusion, chemotaxis, and haptotaxis. This work highlighted the potential of both discrete and continuum models to explore the same phenomena.describes the spatial and temporal change in endothelial cell density (n) as the function of diffusion, chemotaxis along tumor angiogenic factor (c) gradients, and haptotaxis along fibronectin (f) gradients. Endothelial cell diffusion is characterized by a diffusion coefficient D, chemotaxis is characterized by chemotaxis coefficients 0 and k1, and haptotaxis is characterized by the haptotaxis coefficient 0 . In the presence of other cells endothelial cell movement via diffusion is directed away from high densities of n (white arrows in the illustration), otherwise the movement via diffusion is random. Both chemotaxis and haptotaxis result in endothelial cell movement towards higher concentration of c or f (black arrows in the illustration), respectively. The change in fibronectin distribution over time is the function of the production at rate ω by endothelial cells and the uptake at rate μ by endothelial cells. The change in tumor angiogenic factor distribution isdescribes the spatial and temporal change in endothelial cell density (n) as the function of diffusion, chemotaxis along tumor angiogenic factor (c) gradients, and haptotaxis along fibronectin (f ) gradients. Endothelial cell diffusion is characterized by a diffusion coefficient D, chemotaxis is characterized by chemotaxis coefficients χ 0 and k 1 , and haptotaxis is characterized by the haptotaxis coefficient ρ 0 . In the presence of other cells endothelial cell movement via diffusion is directed away from high densities of n (white arrows in the illustration), otherwise the movement via diffusion is random. Both chemotaxis and haptotaxis result in endothelial cell movement towards higher concentration of c or f (black arrows in the illustration), respectively. The change in fibronectin distribution over time is the function of the production at rate ω by endothelial cells and the uptake at rate µ by endothelial cells. The change in tumor angiogenic factor distribution is described by the uptake at rate λ by endothelial cells. The general formulation of the left-hand side of the equation expressing the rate of change of a quantity of interest, and the right-hand side describing all the ways it can change is frequently the over-arching guide for constructing such models.
## Discrete models
Discrete models (panel d in , however, specifically track individual endothelial cells rather than densities. Discrete models can be divided into two main categories: lattice-based and lattice-free. Lattice-based methods allow cells to migrate or divide according to a gridded system (i.e., the lattice), where each cell may occupy one or many lattice sites, while lattice-free methods (or agent-based methods) allow cells to freely migrate and divide in any direction. Lattice-based methods where a cell occupies one lattice site are called cellular automaton models, while models where cells occupy many lattice sites are called Cellular Potts models or CPMs. Cellular automaton models use a structured lattice where each cell occupies one lattice site and cells are updated through time as they move (from one lattice site to another), proliferate (cell divides and places a daughter cell in a neighboring site), or die (removal of a cell within a lattice site). A landmark cellular automaton model by Anderson et al. was extended in McDougall et al.to describe vessel formation, loop formation (anastomosis), and blood flow through the vasculature. They utilized a Poiseuille-like expression for blood flow that is dependent on vessel radius, where the radius adapts based on wall shear stress, intravascular pressure, and metabolic stimuli. These additions allow for the simulation of blood flow through dynamically remodeling vessels that subsequently affects the delivery of both nutrients and therapeutics. Owen et al.developed a multi-scale cellular automaton model to describe the evolution of vasculature through angiogenesis and vascular pruning due to low wall shear stress. A subcellular scale model describing cell cycle, apoptosis, and vascular endothelial growth factor (VEGF) secretion was coupled to a cellular scale model describing the movement and interaction between normal, tumor, and endothelial cells. Both the subcellular and cellular scale models were coupled to continuum models applied to diffusible species (e.g., VEGF and oxygen). In their approach, the level of tissue oxygenation drives normal cells to produce VEGF and stimulate endothelial sprouting. The authors applied their model to study angiogenesis and vascular remodeling under different initial vasculature networks, and observed that if the vasculature network was sparse the tumor would remain localized until new vessels are formed.
An alternative lattice-based approach is the CPM. In CPMs, cells may occupy several lattice sites, and each cell is identified by a unique lattice index. Therefore, lattice sites with different lattice indices are occupied by different cells. Neighboring cells form connections between each other and share an adhesive bond energy. CPMs are designed to minimize the energy of the system, where the effective energy is the sum of all the bond energies between cells and the differences between the volume of each cell and the target volume of a cell (this energy results from a cell's resistance to volumetric changes). The effective or total energy is captured by the Hamiltonian which is an operator that is the sum of energies describing the modeled biological processes (e.g., chemotactic energy, haptotactic energy, cell division energy). A typical CPM algorithm is as follows: (1) a random lattice site i is selected, (2) a neighboring lattice site j is selected and is changed to the same index as site i, (3) the Hamiltonian is calculated for this new configuration, and (4) if the energy decreases compared to the original configuration the site retains the new index otherwise it reverts to its original index. By changing the Hamiltonian describing biological systems, CPMs have become a mainstay in modeling tumor angiogenesis and endothelial cell arrangement. In Merks et al., the authors utilized a CPM to model vascular organization with and without contact inhibition between endothelial cells and displayed the ability of the model to recapitulate vessel networks with various morphologies. They included a term modeling chemical signaling based on the concentration of a generic chemoattractant (such as VEGF) around the endothelial cells, causing a shift in the energy to promote angiogenic sprouting. This energy formulation is coupled to a PDE of the chemoattractant describing its secretion by endothelial cells, its diffusion throughout the microenvironment, and its decay over time.
Lattice-free methods, or agent-based models, allow cells to migrate and divide in any direction and are not constrained by an underlying lattice. In Plank et al., an offlattice method is developed by considering TEC migration to be based on the turning rate of a cell (i.e., the rate at which a cell changes its orientation) and the preferred migratory direction along the gradient of TAF. They compared the resulting vasculature simulated from the lattice-free model with the results of several on-lattice models. Notably, the networks generated by the off-lattice model had a higher tendency to form anastomosis loops and had less orthogonal jumps, a common feature in lattice-based models. Phillips et al.developed an agent-based model of tumor-induced angiogenesis, where endothelial cells are activated by TAF, which is modeled as a continuous field through a PDE. The activated cells transition to TECs that migrate up the concentration gradient of TAF and cause neighboring cells to adopt a stalk phenotype, described by rapid proliferation to allow the extension of the angiogenic sprout. These cells interact through mechanical forces that establish lumen stability and allow for an angiogenic network to form. Additionally, the physical interaction between the tumor and the new vasculature network is included and allows the tumor to collapse vasculature segments and reduce nutrient delivery.
## Hybrid models
Hybrid models (panel e in combine discrete and continuum methodologies by (generally) describing the TECs as discrete agents that migrate chemotactically in the presence of a TAF gradient and a PDE model describing endothelial cell density. These models seek to take advantage of fast model computations when solving continuous PDE models, but also have a more robust description of specific cell actions. In Lima et al., TECs are modeled discretely and move according to the extracellular matrix conductivity, a chemotaxis constant, and the gradient of TAF, while the endothelial cell volume fraction is updated based on the movement of the TEC. In Vilanova et al., capillaries are modeled using a continuum approach describing the movement, proliferation, and apoptosis of the cells within the capillaries. TECs are identified within the field of capillaries based on the concentration of TAF and lateral inhibition (no TECs are within a distance threshold of the cell to be activated). The model is analyzed by considering scenarios of the growth phase of angiogenesis, chemical inhibition through therapeutics, and the reinitiating of vessel growth after removing chemical inhibition.
## Summary
The continuum, discrete, and hybrid modeling approaches above provide complementary information on angiogenesis, and the choice of modeling approach is dependent on the desired goal or quantity of interest from the model itself. The primary advantage of using a continuous representation of tumor vasculature is the low computational cost, and the ability to utilize sophisticated parallel solvers for continuum equations. However, a continuum approach lacks the ability to resolve local key features of the changing vasculature including, for example, the activation of TECs (the cell responsible for directed migration) and the competition for the TEC phenotype among other TECs and neighboring endothelial cells. Discrete models can resolve these local features but become computationally expensive as the number of cells increase. Hybrid models balance both approaches and produce robust and sophisticated vascular fields, but often require complex numerical schemes to solve them. All three modeling approaches have been shown to qualitatively describe the dynamics of tumor angiogenesis; however, many parameters in these models are often assigned values without any experimental validation. This leads to models matching qualitative properties of angiogenesis such as TEC activation, sprout elongation, formation of anastomosis, and establishing blood flow, but have difficulty predicting actual experimental outcomes, since parameters are freely assigned. Recent advances, though, indicate that time-resolved quantitative imaging can provide the data necessary to inform and calibrate model parameters specific to the vasculature network under investigation.
## Integrating theory and experimental data at the cellular scale
Integrating mathematical models and experimental data has the potential to yield a set of validated models that can then be used to make specific predictions in silico. These model predictions can then be rigorously tested experimentally. However, to date, there has been a paucity of published examples that rigorously calibrate mathematical models to experimental data of tumor angiogenesis at the cellular scale. This is due to complexities in both the computational and experimental efforts, and the difficulties in integrating the two. Computational complexities include the sophisticated numerical schemes that must be used to solve mathematical models of angiogenesis at the cell scale, (which can be very expensive to solve), the necessity of these numerical schemes to be fast enough to calibrate model parameters (which can take thousands of model runs), and ensuring that calibrated model parameters drive the system (since uncalibrated or free model parameters cannot be trusted to generate reliable model predictions). Experimental complexities include the necessity of reproducible, quantitative, high resolution, longitudinal imaging that can isolate processes critical to angiogenic sprouting and tumor vasculature. Microfluidic devices are one promising platform that enable the culturing of tumor and/or endothelial cells in 2D or 3D, while simultaneously incorporating biochemical gradients, fluid flow, and mechanical signaling. These devices can play a powerful role in the study of tumor angiogenesis and vasculature by providing a controlled, repeatable experimental platform in vitro that can isolate specific processes that are not easily studied individually in vivo. Many microfluidic devices are widely reproducible and allow for a systematic investigation of vasculogenesis, angiogenesis, and response to antiangiogenic therapies.
While computational advances in discrete, continuum, and hybrid modeling along with experimental advances in microscopy and microfluidic devices have largely bridged this gap, significant progress in the rigorous integration of mathematical models of angiogenesis and experimental observations have yet to be realized at the cell scale. We now highlight some promising approaches that integrate in vitro and in vivo experiments with mathematical theory.
Perfahl et al.extended a 2D multiscale model of vascular tumor growth, coupling blood flow, tumor-induced angiogenesis, and vascular remodeling in Owen et al.to 3D and initialized the model with vasculature imaged in an in vivo mouse model. To observe angiogenesis in vivo, a murine dorsal skin fold chamber was implanted with a 1 cm diameter glass coverslip and imaged after the mouse was inoculated with red fluorescing tumor cells and green fluorescing microvessels. The resulting vasculature network was imaged using multiphoton microscopy, with z-stacks (i.e., images acquired at different focal distances) acquired at 0.5 µm intervals. The z-stacks were then reconstructed to produce a 3D volume of vasculature, which was used to initialize the vasculature position in the mathematical model. Their angiogenesis model utilized a cellular automaton approach, where tumor cells release TAF that diffuses through the microenvironment and induces angiogenesis. Their approach was used to study how different initial vasculature networks influenced tumor growth dynamics.
Xu et al.developed a 3D hybrid model of tumor angiogenesis coupled to TAF, interstitial flow, and blood flow, which was initialized with photoacoustic imaging data. TAF dynamics were modeled with a reaction-diffusion PDE describing the secretion of TAF by hypoxic tumor cells, diffusion through the extracellular space, uptake by endothelial cells, and decay of TAF over time. Photoacoustic images were obtained fromin a murine xenograft with an imaged volume of 14 mm × 14 mm × 6 mm (depth) over 26 days. While the primary purpose of this experimental study is to investigate a novel photoacoustic contrast methodology, the authors yield longitudinal images of tumorassociated vasculature at depths approaching 10 mm and a spatial resolution of under 100 microns. Photoacoustic images were scaled between −1 and 1 to segment extravascular space and capillaries, respectively. The map of capillaries and extravascular space was then used as the initial vasculature network in the model by Xu et al.. While the model was not calibrated by time-resolved data, their image processing and modeling framework demonstrated an approach to readily utilize photoacoustic imaging data directly in the model without extensive image processing or manual adjustments.
In Stepanova et al., a multiscale cellular automaton model for angiogenesis was developed and compared to experimental data using the displacement, orientation, and directionality of endothelial cells across multiple concentrations of VEGF. The displacement, orientation, and directionality of endothelial cells was calculated in the model and compared with experimental values from longitudinal confocal microscopy images collected every 15 min for 36 h, under concentrations of 0 ng/mL, 5 ng/mL, and 50 ng/mL of VEGF. These estimates of the displacement, orientation, and directionality of endothelial cells were used to calibrate model parameters. The calibrated model parameters were used to simulate characteristic features of angiogenic sprouting such as branching, chemotactic sensing, the brush border effect, and cell mixing. Additional model validation was done by designing numerical simulations that recapitulate the experiments shown in Jakobsson et al.. Competition between wildtype endothelial and mutant endothelial cells (e.g., heterozygous for VEGF-1 and exposed to a Notch signaling inhibitor) for the TEC position was summarized by the percentage of time each cell line is in the lead cell position (acting as a TEC). In the experimental setup, wildtype and mutant cells are fluorescently labeled red and green, respectively, and time-lapse confocal microscopy of mosaic embryoid body cultures was done over periods of 1 to 4 days. Image segmentation and cell tracking ultimately provided the percentage of time each cell line acts as the TEC. Equivalent measurements from Stepanova et al.'s cellular automaton model were compared directly to the experimentally observed behavior and agreed with Jakobsson et al.'sstudies.
Phillips et al.have proposed integrating confocal microscopy data from an in vitro vascularized tumor platformwith an agent-based mathematical model of tumor angiogenesis. In their framework, time-resolved confocal measurements of individual angiogenic sprouts are used to calibrate and validate a multiscale agent-based model. The agent-based model captures the dynamics of endothelial cells. Each agent represents a single endothelial cell that can be in one of the following phenotypes: tip, stalk, or phalanx cell. Tumor cells release TAF, which is modeled by a reactiondiffusion equation and is responsible for guiding the movement and phenotypic transitions of endothelial cells. In their preliminary study, they calibrated the endothelial cell cycle duration and TEC velocity and used these parameters to estimate the total sprout length at the end of the imaging experiment. Phillips et al.observed a 12.5% error in sprout length between the model and the image measurement. Future efforts are aimed at improving the spatial agreement between the model and the measurements.summarizes the literature reviewed in this section and how the selected models are integrated with imaging data.
## Approaches for modeling tumor vasculature and angiogenesis at the tissue scale
In this section, we identify the current approaches to modeling tumor vasculature at the tissue scale. We then describe quantitative imaging techniques for observing vascular changes over time at this scale and conclude by discussing how these quantitative measures can be integrated with mathematical models to make testable predictions.
## Mathematical modeling of tumor vasculature and angiogenesis at the tissue scale
Similar to the cell scale approaches in Section 3.1, there are analogous continuous, discrete, and hybrid approaches that have been scaled up to describe angiogenesis and tumor-associated vasculature at the tissue scale. The choice of the modeling paradigm is highly influenced by the primary aim of the model and (potentially) the type of data used for validation. In this section, we identify four major areas of research at the tissue scale (shown inand discuss the modeling strategy or strategies applied to these areas. Broadly, these areas include: (1) representing the evolving geometry of the tumor's vascular network (panel a in
## Models of evolving tumor vascular network
The first areaof focus (panel a inbridges the cell to tissue scale by modeling the formation and evolution of tumor-induced angiogenic networks which are predominately modeled using a discrete (lattice-based and lattice-free), continuous, or hybrid strategy similar to those discussed in Section 3. Discrete approaches typically model individual TEC movement, while continuous approaches model the change in a spatially averaged, continuous variable (e.g., vasculature density or vascular volume fraction). Hybrid approaches combine the discrete and continuous approaches to provide a spatially resolved vasculature network, which can be mapped to a continuous domain to facilitate interaction with continuous elements of their mathematical modeling system (e.g., TAF or nutrients). One representative example by Frieboes et al.applies a hybrid approach to describe angiogenesis coupled to tumor growth. Frieboes et al. use a lattice-free description of angiogenesis to describe TEC motion due to chemotaxis in response to TAF gradients and haptotaxis in response to fibronectin gradients. Once anastomosis occurs between two vessel branches, it was assumed that the now connected vasculature network could act as a source of oxygen and nutrients. Then, the distribution and availability of oxygen and nutrients directly influences tumor cell dynamics. Additionally, the discretized vasculature is spatially averaged to facilitate coupling to continuous elements within the model (i.e., TAF and fibronectin). The simulated tumor-induced angiogenic network produced a spatially heterogeneous distribution of oxygen and nutrients that resulted in phenotypic heterogeneity of tumor cells within the tumor.
## Models of blood flow and blood-driven transport
The second area (panel b infocuses on estimating blood flow and transport within the vasculature and through the interstitial space. As described in Section 1, vascular flow has a profound influence on the dynamics of growth and therapeutic response of the tumor. The modeling of vascular flow usually includes a description of flow in the blood vessels, along with its coupling with flow in the tissue through a mass flux at the capillary walls or at the terminal ends of larger vessels. Similar to the cell-scale models reviewed in Section 3, these phenomena can be modeled by discrete, continuous, or hybridapproaches. In discrete vascular models both the pre-existing and the angiogenic vasculature are frequently approximated by a 1D network of connected straight cylinders with the flow in each cylinder simulated using the 1D Poiseuille law. In continuous vascular flow models the vasculature is described with a spatially averaged, continuous variable (e.g., vasculature density or vascular volume fraction), and the transport of the substance of interest (e.g., drug or nutrient) through the interstitial space is described with a reaction-diffusionadvection modeldescribing the delivery, diffusion, and the transport of that substance due to bulk fluid flow. Hybrid vascular flow modelscombine the discrete and continuum approaches; capillaries and smaller vessels are approximated with a continuum approach, whereas the large vessels are explicitly retained, and their flow is simulated as in discrete models. A formative example of blood flow and transport by D'Angelo et al.describes an approach to couple a 1D discrete model of tissue vasculature with a 3D continuum model of interstitial transport. Blood flow through the vessel network follows Poiseuille's law (which relates flow to vessel radius, pressure, and the viscosity of blood), and transport across the vascular walls is described by Starling's law (which relates the extravasation rate to vessel permeability and the pressure difference between the vessel and the tissue). Interstitial flow is dictated by Darcy's law, which relates flow to the pressure gradient and the hydraulic conductivity of the tissue. The approach by D'Angelo et al. allows the unique vasculature network structure to be preserved (and not reduced to a spatially averaged variable) while allowing for a coupling to a 3Dcontinuous model of the interstitial space.shows an illustration of these three foundational relations in modeling intravascular and interstitial flow. space.shows an illustration of these three foundational relations in modeling intravascular and interstitial flow.
## Models of tumor and vasculature growth and response to therapy
The third and fourth areas focus on describing the mechanisms underlying the complex interplay between tumor growth and vasculature in the absence of treatment (panel c inand during treatment (panel d in. Many of the same discrete, continuum, and hybrid models of angiogenesis and vasculature network mentioned in the previous two areas are also applied to study the interplay between the tumor and vasculature with an increased emphasis on modeling the tumors themselves, as they trigger the angiogenic cascade, influence the development of the neovasculature, and are the ultimate beneficiaries of the angiogenic blood supply. At the tissue scale, models of tumor cell dynamics are typically captured in a continuous fashion by means of a PDE system. This is most commonly achieved through either reaction-diffusion-advection equations or phase-field equations. Reaction-diffusion-advection equations describe the spatiotemporal dynamics of cell density (or tumor volume fraction) as a combination of random movement of cells via diffusion, directed movement of cells via advection, and reaction terms representing (for example) tumor cell proliferation, apoptosis, and cytotoxic effects due to treatments. For example, Hahnfeldt et al.developed a model of tumor volume dynamics as a function of the effective vascular support (or carrying capacity). The vascular influenced carrying capacity changes in response to stimulating effects (via tumor cells) and inhibitory effects. Illustration of a perfusion and transport model. Intravascular and interstitial flow is characterized by the laws of Poiseuille, Starling, and Darcy. Inset a illustrates Poiseuille's and Starling's law. Poiseuille's law relates intravascular flow (Q v , blue arrows in inset a) to the radius of the vessel (R), the dynamic viscosity of blood µ, and the gradient of the intravascular pressure p v . Starling's law relates the rate of extravasation (J v , red arrows in inset b) to the hydraulic conductivity of the vessel wall (L p ), the vascular surface area (S), the reflection coefficient (σ), the vascular oncotic pressure (π v ), and the interstitial oncotic pressure (π t ). Inset b shows an illustration of Darcy's law which relates the interstitial flow velocity (m t , blue arrows in inset b) to the interstitial tissue hydraulic conductivity (κ), and the gradient of interstitial pressure (p t ). These three relations are found throughout the literature on the physical modeling of tumor associated vascular flow and angiogenesis.
## Models of tumor and vasculature growth and response to therapy
The third and fourth areas focus on describing the mechanisms underlying the complex interplay between tumor growth and vasculature in the absence of treatment (panel c inand during treatment (panel d in. Many of the same discrete, continuum, and hybrid models of angiogenesis and vasculature network mentioned in the previous two areas are also applied to study the interplay between the tumor and vasculature with an increased emphasis on modeling the tumors themselves, as they trigger the angiogenic cascade, influence the development of the neovasculature, and are the ultimate beneficiaries of the angiogenic blood supply. At the tissue scale, models of tumor cell dynamics are typically captured in a continuous fashion by means of a PDE system. This is most commonly achieved through either reaction-diffusion-advection equations or phase-field equations. Reaction-diffusionadvection equations describe the spatiotemporal dynamics of cell density (or tumor volume fraction) as a combination of random movement of cells via diffusion, directed movement of cells via advection, and reaction terms representing (for example) tumor cell proliferation, apoptosis, and cytotoxic effects due to treatments. For example, Hahnfeldt et al.developed a model of tumor volume dynamics as a function of the effective vascular support (or carrying capacity). The vascular influenced carrying capacity changes in response to stimulating effects (via tumor cells) and inhibitory effects (via endogenous and exogenous factors). This modeling formulation allowed the investigation of different anti-angiogenic therapies. Alternatively, phase field models may be used to describe the coexistence of a number of phases representing different tissue types (e.g., tumor and normal tissue)and their interactions or transitions between each other.
The spatial and temporal evolution is dictated by a free energy potential, which restricts mixing and penalizes spatial variation in individual phases, and several source terms describing the growth, death, response to treatment, and transition from one species to another (e.g., a proliferative tumor cell may transition to hypoxic tumor cell in response to scarce nutrients). The PDE governing the dynamics of each cell species is obtained by combining the mass flux, which is defined in terms of the gradient of the free energy potential, and the aforementioned source terms. A detailed review of tumor growth modeling approaches can be found in.
An informative example of modeling the interplay between tumor and vasculature was proposed by Swanson et al., who employed a continuum approach to model the transition of tumor cells between different phenotypes as a result of the vasculature density. Specifically, three tumor cell phenotypes were considered: prolific, hypoxic, and necrotic. Prolific cells were considered to be proliferative and mobile tumor cells in a normoxic or oxygen sufficient state. Alternatively, hypoxic cells were considered to be mobile tumor cells in a hypoxic or oxygen deprived state and could not proliferate. Cells initially begin as prolific cells and then transition to hypoxic cells once the relative fraction of vasculature (used as a surrogate for oxygen supply) is insufficient to support all the prolific cells. If the vasculature remains insufficient to support prolific and hypoxic cells, they eventually transition to necrotic cells. Vasculature growth is stimulated via the release of angiogenic factors from prolific and hypoxic cells. Using this coupled PDE system, the authors were able to recapitulate histological features of malignant progression (such as increased cellularity, hypoxia-induced angiogenesis, and necrosis) as observed in vivo. This multispecies model was also applied to simulate tumor response to anti-angiogenic therapy.
Vavourakis et al.demonstrated an in silico method for modeling the influence of chemotherapies on tumor and vasculature dynamics using a model that characterizes tumor growth and therapeutic response, angiogenesis and vasculature remodeling, blood and interstitial flow, and the dynamics of key substances (e.g., TAF, oxygen, matrix degrading enzymes). The tumor-associated vasculature is modeled using a discrete approach, while the tumor is modeled using a continuum approach. The evolution of the concentration of cytotoxic drugs is modeled via continuous equations and accounts for several drug states (e.g., bound and unbound) as well as the different drug transport dynamics (e.g., advection and diffusion) in the bloodstream and the interstitial space. Using their comprehensive framework, the authors were able to investigate the influence of drug properties (e.g., size and affinity), vessel porosity, the normalization of vessels, and treatment schedule on tumor regression. They observed that time-of-treatment was an important factor for low-affinity cytotoxic drugs and that high-affinity cytotoxic agents resulted in a large vascular normalization window that might enhance the delivery of subsequent chemotherapy doses.
As noted in Section 3.1., the choice of continuum, discrete, or hybrid modeling approaches is dependent on the desired goal or quantity of interest from the modeling exercise itself. Continuum models of angiogenesis, that use a spatially averaged variable to describe the tumor-induced vasculature (e.g., neovasculature density or volume fraction), provide a computationally tractable approach to explore the interplay between the tumor and supporting vasculature at both greater length and time scales compared to discrete models. At the tissue scale, this is an important consideration as modeling efforts are often investigating tumor growth and treatment response on the time scale of months to years. The main advantage of discrete models is that they can capture the precise changes in the angiogenic network and blood flow, thereby providing a better description of the transport of nutrients and drugs to the tumor region. However, discrete models of angiogenesis may require the calibration of a large set of parameters as well as extensive computational resources to track both the existing and developing vasculature, and to couple the resolution of continuous and discrete phases in a multi-physics framework. Thus, discrete models are usually limited to small spatial scales and short time intervals (e.g., modeling the transition from avascular to vascular tumors). Hybrid models combine the advantages of both discrete and continuum approaches, as they retain the ability to represent large vessels via discrete methods resulting in a more accurate and patient-specific flow, while approximating the dynamics of tumor-induced capillaries through continuum approaches. Thus, hybrid models avoid explicitly tracking the evolution of every single branch in the angiogenic network independently, and therefore enable studying vascular tumor growth at various spatial and temporal scales. A final advantage for any of these approaches is the type of data available to calibrate or inform the model, discussed further in Section 2.2. Data that are able to resolve vessels may be more appropriate for discrete or hybrid modeling techniques, while imaging data that only return spatially averaged estimates of vascular volume are generally better suited for continuum modeling techniques.
## Integrating theory and experimental data at the tissue scale
Recent studies have proposed several promising approaches for integrating mathematical models with experimental imaging data at both the pre-clinical and clinical levels. In this section we identify approaches that focus on describing perfusion and delivery (Section 4.2.1) and treatment response (Section 4.2.2). The reader is referred tofor a summary of these approaches and the type of data used to inform the model.
## Applications to estimate perfusion and delivery
Recent studies have provided important foundations on integrating imaging measurements of tumor-associated vasculature with mathematical models, which can provide a means to rigorously understand and predict tumor blood flow, interstitial transport, and angiogenesis. For example, d'Esposito et al.performed fluorescence imaging to visualize tumor microvasculature in fixed tumor samples to inform a model of tumor perfusion. The segmented microvasculature was used to initialize the vasculature network for a computational fluid dynamic (CFD) model describing steady-state blood and interstitial flow. Using the CFD model, the authors estimated interstitial fluid pressure and velocity, blood flow and pressure, and the delivery of a widely used MRI contrast agent. Their CFD model predicted a heterogeneous spatial distribution of the contrast agent, which was validated against in vivo DCE-MRI. Similarly, Stamatelos et al.applied a CFD model to a whole tumor microvasculature network imaged with ex vivo micro-CT imaging. Stamatelos et al. applied their model to study intravascular oxygenation, hemodynamics, and vascular morphology across eight breast tumor xenografts. Through this modeling framework, the authors demonstrated that the unique microvasculature network in an individual tumor contributes to both the inter-and intra-tumor heterogeneity.
Adhikarla et al.developed a modeling workflow based on ordinary differential equations to simulate temporal changes in tumor vasculature and blood oxygenation. The microvasculature was initialized with micro-CT imaging, the tumor oxygenation status was calibrated with PET imaging data sensitive to hypoxia, and tumor growth was characterized by proliferation estimated from PET imaging data. These studies were able to use experimental data to provide physical conditions and domains for the mathematical modeling of tumor-related fluid dynamics. However, vasculature measurements from ex vivo imaging have limited clinical utility for diagnosis or prognosis because they require an invasive procedure that damages the system under investigation and, hence, cannot provide information on the remaining lesion or host tissue.
A non-invasive approach proposed by Wu et al.applies a CFD model to the clinically available MR data. Wu et al. established a rigorous framework for integrating multiparametric MRI with a mechanism-based, biophysical model enabling the characterization of the hemodynamics associated with breast cancer on a patient-specific basis. Specifically, pre-treatment quantitative MRI data, including DCE-MRI and diffusion-weighted MRI, were employed to identify the patient-specific tissue geometry (e.g., tumorous, adipose, and fibroglandular tissues, along with vasculature) and properties (e.g., vascular permeabil-ity, interstitial hydraulic conductivity). These data were used to constrain a CFD modeling system, which coupled 1D blood flow with 3D tissue flow, enabling the characterization of hemodynamic characteristics, including blood flow rates, fluid extraction rate, interstitial pressure, and flow velocity. Using this approach, the authors observed significant differences in tumor-associated interstitial flow velocity, blood pressure, and vascular extraction rate between malignant and benign lesions.
## Applications to treatment response
The treatment efficacy of systemic therapies administered intravenously relies on the delivery of drugs through the bloodstream, which is highly dependent on the vascular structure and associated perfusion. Additionally, multiple pre-clinical and clinical studies have shown that anti-VEGF therapy changes tumor vasculature towards a more "mature" or "normal" phenotype, thereby improving the delivery and efficacy of concomitant chemotherapies. Therefore, the use of data-driven modeling to evaluate angiogenesis is a promising means to assess and predict tumor response to therapies. The approach proposed by Titz et al.employed a continuum model to simulate tumor and vasculature responses to anti-angiogenic therapy. Pre-treatment PET measurements of cellular proliferation and hypoxia were used to initialize the simulation and estimate model parameters. In their simulations, hypoxic tumors released TAF or VEGF to stimulate endothelial cell proliferation and an increase in microvessel density. The estimated microvessel density was used to estimate the average voxel oxygenation. The model parameters describing cellular and vascular proliferation were adjusted to minimize the error between the measured oxygenation from PET and the model-estimated oxygenation. Using this modeling framework, the authors estimated the response to anti-angiogenic therapy and demonstrated that anti-angiogenic therapy could be personalized based on the initial levels of VEGF within the tumor. The influence of vasculature on tumor response to radiotherapy was considered by Hormuth et al., who used a coupled PDE-based model of tumor growth and angiogenesis in a murine model of glioma. Quantitative MRI collected before and after radiation therapy were used to initialize estimates of tumor cellularity (from diffusionweighted MRIand blood volume fraction (from DCE-MRI) as well as to calibrate model parameters. The two PDEs were coupled by assuming the blood volume fraction was linearly related to the maximum amount of tumor cells that could be supported in a given voxel as determined in a previous study in the absence of treatment. Similarly, a previous study assessing the validity of 39 models of tumor growth and radiotherapy responsewas used to guide modeling of tumor and vasculature response to radiotherapy. When response to radiotherapy was considered, Hormuth et al. observed that spatially varying the efficacy of radiotherapy as a function of local blood volume fraction also improved predictions of tumor response.
A similar approach by Jarrett et al.modeled the action of neoadjuvant therapy on breast cancer in a patient-specific setting. Jarrett et al. extended the PDE-based model of breast cancer response to neoadjuvant therapy proposed byby including the effects of drug delivery. The tumor response model was initialized with patient-specific diffusionweighted MRI data and drug delivery estimated using DCE-MRI data. The literature estimates of the drug concentration in the plasma were coupled to patient-specific estimates of vessel permeability and perfusion to simulate the intra-tumor distribution of neoadjuvant therapies. This study demonstrated the plausibility of using DCE-MRI data as a means to estimate drug delivery on a patient-specific basis in predictive models and represents a pivotal step towards the goal of achieving individualized prediction of tumor response to therapy. Additionally, this work has been extended by calibrating the model with follow-up images collected during neoadjuvant therapy. The extended model enables a rigorous prediction of patient-specific response to the prescribed treatment, thereby providing novel opportunities to identify alternative treatment regimens for patients with inadequate response to standard-of-care treatments.
## Opportunities for multiscale modeling of angiogenesis
The formation of blood vessels during tumor growth is a process that spans multiple spatial and temporal scales. For instance, signaling pathways activated in endothelial cells in response to the binding of TAFs to its receptor occur at the subcellular scale, the movement of TECs as well as cell-cell and cell-extracellular matrix interactions happen at the cellular scale, and blood flow along with the delivery of nutrients and therapeutics occurs at the tissue scale. Therefore, each scale provides a complementary picture of the formation of the tumor vasculature. Additionally, while signaling pathways and TEC motion may feature fast mechanisms on the order of milliseconds to seconds, the formation of fully functioning new vessels may take days and the vascular-induced changes in tumor growth may occur over weeks. Thus, to fully characterize the complexity of angiogenesis, multiscale mathematical models that combine the description of biological processes underlying the formation of tumor-induced neovasculature at multiple scales are needed. Some models of angiogenesis already include a multiscale component. For example, Vilanova et al.modeled TEC motion along with capillary formation, which occur at the cell and tissue scale, respectively. Furthermore, Vavourakis et al. have proposed a multiscale model including interstitial and vascular transport, ECM degradation, explicit vessel formation and remodeling, tumor-induced tissue deformation, and the dynamics of drug distribution, binding, and internalization. Ultimately, these models constitute a promising approach to precisely predict tumor vascularization, vascular-induced changes in tumor dynamics, and therapeutic outcome. For example, by modeling the delivery of drugs in the vasculature and interstitial space, their interaction with tumor cells at the cellular scale, and the specific action of the drugs on signaling pathways at the subcellular scale, multiscale models could enable the exploration of the cascade of effects of different treatment strategies.
It is important to acknowledge that while the use of high-performance computing techniques is becoming more common, solving multiscale models of angiogenesis is still computationally intensive and one of the fundamental challenges in model development. Multiscale models are generally hybrid models that combine systems of ordinary differential equations (e.g., signaling pathways) and PDEs (e.g., blood flow, drug delivery, and tissue heterogeneity) with discrete models (e.g., cell-cell iteration, TEC movement). The coupling of these models, while considering the stochastic nature and different time scales of many angiogenesis processes, contributes to the challenge in developing computationally tractable numerical solvers to perform computer simulations. There is also an abundance of plausible models that can be applied to represent mechanisms at each scale. Thus, selecting the most appropriate model is a great challenge and techniques are needed to systematically evaluate the validity of models. While scale-specific model selection has already been investigated, the selection and combination of models at different scales is yet to be explored. Finally, due to the model complexity and large number of parameters in multiscale formulations, there is a fundamental challenge to obtain sufficient data to calibrate and validate these models. While one can still draw useful conclusions from qualitative experiments, the model parameters must be initialized and constrained with patient-specific data to make clinically relevant predictions. However, even with the advances in medical imaging, with the current clinically available data it is impossible to assign values to every parameter in multiscale models.
## Future directions
The recent convergence of time-resolved imaging and mathematical modeling is beginning to enable in silico investigations into the spatial-temporal evolution of vasculature structure and function that can then be tested in the in vitro and in vivo settings. There are several promising avenues for future research to further develop image-driven biologically based models of angiogenesis. First, there is an abundance of imaging techniques at the tissue scale that can quantify tumor vasculature (Section 2.2). Several of these techniques are routinely collected in the standard-of-care setting, but the quantitative analysis of these data is less common outside of the research setting. To enable the widespread use of tissuescale models of angiogenesis, these imaging analysis techniques need be translated into the clinic. Additionally, acquisition and analysis protocols to reduce uncertainty in the imaging measurements need to be developed. Weand othershave begun to demonstrate that quantitative imaging techniques to quantify tissue vascularity can be performed with high accuracy and precision. Furthermore, we have shown that certain MRI measures can be collected with high quality in the community setting (and not in a research or academic setting) using widely available hardware.
Second, the modeling of angiogenesis at the cell scale has been predominantly validated by experiments in a retrospective manner, rather than first informing or calibrating the model with longitudinal, time-resolved, data and then performing a prospective validation. However, there are limitations in both the experimental and computational techniques needed to effectively calibrate these models. For the microscopy-based approaches, phototoxicity or limitations in the number of fluorescent markers (or assays) may limit the duration of experiments and reduce the number of observed species, respectively. In addition, stochasticity in both observed endothelial cell movement and model implementations (e.g., discrete or hybrid models of angiogenesis) of endothelial cell movement results in an additional challenge in parameter estimation.
Finally, as the structure and function of vasculature fundamentally influences the efficacy of systemic and radiation therapies, and therefore patient outcomes, a practical understanding of a patient's vasculature dynamics could be leveraged to identify improved therapeutic regimens. More specifically, we posit that image-driven modeling frameworks could be used to investigate systemic drug delivery, radiotherapy efficacy, and the identification of optimal therapeutic regimens. The current standard-of-care treatment regimens are the result of large, expensive, and time-consuming clinical trials designed to assess treatment efficacy in a population of patients rather than identifying the optimal regimen for an individual patient. An in silico trial system may enable systematic evaluations of therapeutic regimens for individual patients based on a "digital twin"of a patient's unique tumor and vasculature network. Several promising modeling approaches have investigated optimizing chemotherapy based on imagingor genomic data. Preliminary efforts by Jarrett et al.and Wu et al., which include information about drug delivery, vasculature function, and tumor cell distribution in their modeling framework, were able to identify protocols that outperform a standardized dosing regimen. These modeling techniques could be integrated with optimal control theoryto provide a systematic approach to personalizing therapeutic regimens that improve therapeutic efficacy as well as reducing side-effect toxicity. This is particularly important for novel therapeutics and immunotherapy where there are substantial efforts at developing the mathematical theoryto characterize treatment response, but limited longitudinal imaging studies of the effects on the tumor and associated vasculature. One challenge for applying this image-driven framework is the parameterization of the effect of these novel therapeutics on a patient's tumor or vasculature to determine the optimal regimen. Thus, without the pre-requisite data we are only able to hypothesize treatment effects. By combining experimental time-resolved imaging data with practical, validated, models of tumor growth and angiogenesis, there is a promising opportunity for precise, clinically relevant forecasts of patient-specific therapeutic response, which, in turn, may fundamentally shift (and improve) how patient care is delivered.
# Conclusions
In summary, the integration of biologically-based mathematical modeling of tumor vasculature and angiogenesis with time-resolved experimental data promises to enable further understandings of angiogenesis from the cell to tissue scales. Models validated by experimental data, could then be used to generate testable hypotheses or predict the spatial-temporal evolution of the tumor and its associated vasculature. Furthermore, at the clinical level mathematical models initialized and constrained by quantitative imaging techniques could produce timely and actionable forecasts of tumor growth and response that could help guide clinical decisions and fundamentally improve patient care.
## Conflicts of interest:
The authors declare no conflict of interest.
## Abbreviations
MRI, magnetic resonance imaging; CT, computed tomography; PET, positron emission tomography; MRA, magnetic resonance angiography; DCE-dynamic contrast-enhanced; PDE, partial differential equations; TAF, tumor angiogenic factors; TEC, tip endothelial cell; ECM, extra-cellular matrix; CPM, cellular Potts models; VEGF, vascular endothelial growth factor; DW, diffusion weighted; CFD, computational fluid dynamic; ODE, ordinary differential equation. |
Disseminated Juvenile Xanthogranuloma with a Novel MYH9-FLT3 Fusion Presenting as a Blueberry Muffin Rash in a Neonate
[bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] [bib_ref] Juvenile xanthogranuloma: forms of systemic disease and their clinical implications, Freyer [/bib_ref] [bib_ref] The various clinical spectra of juvenile xanthogranuloma: imaging for two case reports..., Höck [/bib_ref] [bib_ref] Neonatal violaceous skin lesions: expanding the differential of the "blueberry muffin baby, Holland [/bib_ref] [bib_ref] A 3-week-old with an isolated "blueberry muffin" rash, Darby [/bib_ref] [bib_ref] Newborn who has a rash, Niemi [/bib_ref]
## Case description
A 3,015-g male infant was born at 37 weeks' gestation via spontaneous vaginal delivery to a 26-year-old gravida 3, para 3 mother who received good prenatal care. Pregnancy was uncomplicated and maternal serologies were significant for a borderline rubella titer. Infant had Apgar scores of 8 and 9 at 1 and 5 minutes, respectively. At the time of delivery, infant was noted to have multiple petechiae, non-blanching purple nodules, and macules ranging from 1 mm to 2 cm in diameter (►Fig. 1). These lesions were scattered across his face, torso, and extremities. A thorough physical examination also revealed hepatosplenomegaly. A complete blood cell count, obtained at the birth hospital, was significant for a platelet count of 18,000/mm 3 and a complete metabolic panel was significant for direct hyperbilirubinemia of 1 mg/dL. The infant received intramuscular vitamin K, erythromycin eye prophylaxis, and hepatitis B vaccine and was then transferred to a tertiary care facility for further evaluation of his rash, petechiae, thrombocytopenia, and direct hyperbilirubinemia.
Upon admission, a head ultrasound to rule out an intracranial bleed and an abdominal ultrasound to rule out an intra-abdominal mass were obtained and were normal. Liver was noted to be of normal homogenous echogenicity, and the gallbladder showed an echogenic nonshadowing structure compatible with biliary sludge. The infant was transfused 15 mL/kg of platelets (►Fig. 2) and pediatric hematology and infectious disease specialists were consulted. Workup for infectious etiologies including a blood culture, qualitative cytomegalovirus (CMV) urine polymerase chain reaction (PCR), rubella immunoglobulin M (IgM), toxoplasmosis IgM, herpes simplex virus blood PCR, rapid plasma reagin, severe acute respiratory syndrome coronavirus 2 nasopharyngeal PCR, and histoplasma antibody titers (due to family ownership of chickens) were obtained and negative.
However, qualitative CMV blood PCR was positive. Due to the discrepancy between the qualitative urine and blood CMV PCR results, a quantitative blood CMV PCR was obtained, and the infant was started on intravenous ganciclovir, which was later switched to oral valganciclovir, when he started tolerating enteral feeds. Infant passed an auditory brainstem response test, and an ophthalmologic examination was normal, but a brain magnetic resonance imaging (MRI) showed scattered tiny foci of restricted diffusion throughout the supratentorial white matter, left basal ganglia, left thalamus, and scattered tiny areas of susceptibility artifact in the cerebellum that were thought to be areas of hemorrhage.
Neonatal alloimmune thrombocytopenia testing was negative and platelet counts did not respond to intravenous immunoglobulin treatment on two separate occasions (►Fig. 2). He also developed severe neutropenia which was thought to be secondary to valganciclovir treatment and it remained persistent despite holding a few doses, so he received granulocyte colony stimulating factor (G-CSF) treatment. Once the absolute neutrophil count improved above 1,500/mm 3 , G-CSF was stopped. Urine homovanillic acid and vanillylmandelic acid levels were normal, ruling out neuroblastoma.
At this time, decision was made to biopsy a large 3-cm diameter lesion on the right distal thigh (►Fig. 3) which revealed the diagnosis of congenital disseminated JXG. The biopsy showed a monomorphic histiocytic infiltrate, which in areas had a vacuolated cytoplasm, with scattered multinucleated giant cells. Notable atypical features included a deep infiltrative growth pattern and increased proliferation, but no atypical mitosis or high-grade morphologic features were seen. Immunostaining showed diffuse expression of factor XIIIa, CD163, and CD68 stains, but the lesion was negative for CD1a stain (►Fig. 4). Additionally, anaplastic lymphoma kinase (ALK) immunostain and BRAF mutant-specific immunostain (BRAF V600E) were also negative. CMV immunostain was negative for viral inclusions and the quantitative blood CMV PCR was negative, so valganciclovir was stopped. Due to the unique presentation of JXG in this patient, somatic mutation analysis of the tumor was done which showed a novel MYH9-FLT3 fusion. A bone marrow biopsy was also performed at around 2 weeks of age, which showed no clonal proliferation with trilineage hematopoiesis. The erythroid and myeloid maturation was intact without any dysplasia; however, there was an increase in the megakaryocytic precursors by 50% and there was also evidence of dysmegakaryopoiesis.
During the hospital stay, the lesions remained stable in size and the platelet count remained low, but stable, despite multiple platelet transfusions (►Fig. 2). The infant also received multiple packed red blood cell transfusions due to severe anemia secondary to multiple laboratory draws. Direct hyperbilirubinemia gradually improved and an MRI prior to discharge showed stable scattered foci of hemosid-erin deposition which were similar to the previous scan with no areas of restricted diffusion. At the time of discharge, around 1 month of age, the infant was direct breast feeding with good weight gain. He was followed closely by a hematologist oncologist outpatient and his thrombocytopenia gradually improved by 4 months of age (►Fig. 2).
The lesions slowly started fading in color and their growth was negligible compared with the relative growth of the patient (►Fig. 3). A bone marrow biopsy repeated around 1 year of age showed normal morphology and trilineage maturation. Flow cytometry did not show any abnormal immunophenotypes and bone marrow sequencing did not show the MYH9-FLT3 fusion present in the JXG lesions. The patient was noted to have normal neurodevelopmental milestones at 18 months of age.
# Discussion
JXG is the most common non-Langerhans cell histiocytosis (LCH) 1 and presents mostly as a single skin lesion in early childhood. However, it can also present with extensive systemic involvement with lesions disseminated in the deep soft tissues, liver, spleen, lungs, bone, bone marrow, or brain. 3 Disseminated JXG has some striking similarities to LCH in that they are both histiocytic disorders that present in the neonatal period and can be associated with significant morbidity and mortality 9 ; however, the distinguishing clinical feature of JXG is its greater potential for spontaneous resolution, usually in 1 to 5 years. [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] The histologic appearance of JXG depends on its location and age of presentation.Early cutaneous JXG is characterized by a dense mononuclear histiocytic infiltrate, similar to LCH. As time progresses, this is replaced by more vacuolated histiocytes and Touton giant cells, which were seen in our case (►Fig. 4). Touton giant cells are multinucleated cells with a foamy cytoplasm due to their high lipid content. However, these cells may be absent in extracutaneous JXG. [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] Eventually transitional or regressing JXG shows progressive fibrosis. [bib_ref] Juvenile xanthogranuloma, Hernandez-Martin [/bib_ref] In the presence of considerable clinical and histologic overlap between disseminated JXG and LCH in neonates, immunohistochemistry is vital in distinguishing these entities. JXG is consistently positive for factor XIIIa, CD163, and CD68 1 but negative for S100 and CD1a stains, which are specific to LCH.JXG has also been described in association with neurofibromatosis type 1 and juvenile myelomonocytic leukemia. [bib_ref] Juvenile xanthogranuloma: diverse presentations of noncutaneous disease, Murphy [/bib_ref] While genetic mutations are not commonly seen in solitary lesions of JXG, a significant number of genetic alterations have been described with disseminated systemic JXG, namely ALK translocation, BRAF V600E mutation, and mutations in the genes involved in the MAPK pathway. [bib_ref] Systemic juvenile xanthogranuloma has a higher frequency of ALK translocations than BRAFV600E..., Xu [/bib_ref] [bib_ref] Diverse and targetable kinase alterations drive histiocytic neoplasms, Diamond [/bib_ref] Genomic analysis of the lesion in our patient was negative for these mutations but showed a novel MYH9-FLT3 fusion. FLT3 rearrangements have been reported with myeloid and lymphoid neoplasms with eosinophilia, with a high sensitivity in vitro to tyrosine kinase inhibitors, 14 but the specific partner (MYH9) identified in this case has not been previously reported to be rearranged with FLT3 in any other neoplasm.
The treatment of JXG is dependent on the involved sites. Majority of JXG involving cutaneous, subcutaneous, and soft tissues is self-limiting and regresses spontaneously without scarring; however, some larger lesions may develop atrophic scarring. [bib_ref] Histiocytic syndromes: a review, Gianotti [/bib_ref] Systemic disseminated JXG may need treatment with excision or a combination of radiotherapy and chemotherapy. [bib_ref] Treatment of juvenile xanthogranuloma, Stover [/bib_ref] Standard-of-care chemotherapeutic agents for LCH (vinblastine, prednisone, and mercaptopurine) are usually used for treating systemic JXG. [bib_ref] Treatment of juvenile xanthogranuloma, Stover [/bib_ref] There have also been reports of the successful use of cladribine and cytarabine for the treatment of central nervous system JXG. [bib_ref] Successful treatment of central nervous system juvenile xanthogranulomatosis with cladribine, Rajendra [/bib_ref] [bib_ref] Successful treatment of systemic juvenile xanthogranulomatosis with cytarabine and 2-chlorodeoxyadenosine: case report..., Maintz [/bib_ref] Recently, disseminated JXG with specific genetic mutations have been targeted with tyrosine kinase inhibitors. [bib_ref] Dasatinib induces a dramatic response in a child with refractory juvenile xanthogranuloma..., Eissa [/bib_ref] The overall prognosis of JXG is good but a high degree of morbidity and mortality is associated with disseminated JXG in infants with extensive visceral involvement.Our patient showed gradual spontaneous regression of lesions and their growth was negligible compared with the relative growth of the patient, by 6 months of age (►Fig. 3). A bone marrow biopsy repeated around 1 year of age showed normal morphology and trilineage maturation. Flow cytometry did not show any abnormal immunophenotypes and bone marrow sequencing did not show the novel MYH9-FLT3 fusion present in the JXG lesions. The patient was noted to have normal neurodevelopmental milestones at 18 months of age.
# Conclusion
Disseminated JXG should be considered in the differentials of a blueberry muffin rash in a neonate, once infectious and hematological etiologies have been ruled out. While JXG is mostly a benign condition with spontaneous resolution expected in most cases, disseminated JXG can be associated with significant morbidity and mortality in neonates. In the future, routine genomic analysis and targeted chemotherapy may be used to effectively treat and improve outcomes associated with disseminated JXG.
[fig] Figure 2: Platelet counts from birth to 4 months of life. T-(n) represents platelet transfusions and IVIG-(n) represents intravenous immunoglobulin. [/fig]
[fig] Figure 1: Photo panel representing appearance of lesions at birth. American Journal of Perinatology Reports Vol. 13 No. 1/2023 © 2023. The Author(s). [/fig]
[fig] Figure 3: Top panel represents appearance of hand and foot lesions at 2 months of age and bottom panel represents appearance of lesions at 6 months of age. Arrow points to the site of the biopsy of a large 3-cm diameter lesion on the right distal thigh which revealed the diagnosis. [/fig]
[fig] Figure 4: A) High power view with hematoxylin and eosin (H&E) stain demonstrating dense infiltrate of mononuclear histiocytic cells, some with a vacuolated cytoplasm, with extension into subcutaneous fat (black arrow). (B) Lesion was uniformly positive for CD163 immunostain confirming its histiocytic origin. The lesion was also positive for factor XIIIa and CD68 stains (not displayed). [/fig]
[table] Table 1: Differential diagnosis of blueberry muffin rash in newborn[6][7][8] [/table]
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Maize harvester gearbox design modification for improved fatigue life
The gearbox has the advantage of being able to change the torque and rotational speed according to the gear ratio and has high power transmission efficiency by transmitting power through the contact of the gear pair. When evaluating the strength and fatigue life of a gearbox using a design load or an equivalent load, there is a possibility that the results will be very different from the actual ones. Therefore, in this study, the load duration distribution (LDD) constructed based on the actual workload was used to evaluate the strength and fatigue life of the gearbox reliably. As a result of evaluating the strength and fatigue life of the gearbox using LDD, it was confirmed that the existing gearbox did not satisfy the target lifespan in the operating environment. Therefore, the reasons for these results were analyzed, and design modification was performed based on the analyzed results. As a result of design modification, shaft deflection decreased by rearrangement of the bearings, from an overhung type to a straddle type, thereby improving the fatigue life of gears and bearings. Finally, the load distribution acting on the gear tooth surface was improved through micro-geometry modification of the gears.A gear is a machine element in power transmission that is widely used in various fields 1 . A gearbox is a power transmission system that consists of gears, shafts, bearings, and housings; and the power input to the shaft is transmitted to the driven gear (herein, gear) through the driving gear (herein, pinion). Additionally, when power is transmitted using a gear-pair, since the gear ratio changes the rotation speed and torque, the advantage of controlling the rotation speed and torque by changing the gear ratio arises. The performance of a gearbox can be evaluated by parameters such as fatigue life, noise, vibration, and power transmission efficiency. In the case of fatigue life, since it determines whether the gearbox operates or not, it needs to reliably predict and evaluate the life of the gearbox 2 .For reliably predicting and evaluating the performance of the gearbox, it is necessary to define the load acting on the gearbox accurately. The load magnitude of the load acting on the gearbox, duration under the load, and the fluctuation range of the load are determined according to the purpose and environment of use of the gearbox. However, it is challenging to numerically define the load acting on the gearbox. Therefore, many researchers used the cumulative fatigue damage theory based on the Palmgren-Miner rule and predicted and evaluated the gearbox performance under equivalent load conditions using the concept of averages 3,4 . While using the equivalent load in evaluating the gearbox's performance can shorten the calculation time, there is the disadvantage of being unable to consider the effect of the load fluctuation and peak load acting on the gearbox. Additionally, the fatigue damage exponent used to derive the equivalent load is a value that varies depending on the failure mode of each element constituting the gearbox. At the design stage, the fatigue damage exponent cannot be accurately determined because the key failure modes of the gearbox are not available in advance 5-7 .Dong et al. 8 conducted a study on the effect of fluctuating wind speed on the gear contact fatigue of a wind turbine gearbox. The gear contact fatigue was analyzed using a total of 11 different wind speeds-available in literature-for implementing the wind speed fluctuations. However, since this analysis did not reflect the practical environment wherein the wind turbine gearbox is actually used, there was a limit to the reliability of the OPEN
The gearbox has the advantage of being able to change the torque and rotational speed according to the gear ratio and has high power transmission efficiency by transmitting power through the contact of the gear pair. When evaluating the strength and fatigue life of a gearbox using a design load or an equivalent load, there is a possibility that the results will be very different from the actual ones. Therefore, in this study, the load duration distribution (LDD) constructed based on the actual workload was used to evaluate the strength and fatigue life of the gearbox reliably. As a result of evaluating the strength and fatigue life of the gearbox using LDD, it was confirmed that the existing gearbox did not satisfy the target lifespan in the operating environment. Therefore, the reasons for these results were analyzed, and design modification was performed based on the analyzed results. As a result of design modification, shaft deflection decreased by rearrangement of the bearings, from an overhung type to a straddle type, thereby improving the fatigue life of gears and bearings. Finally, the load distribution acting on the gear tooth surface was improved through micro-geometry modification of the gears.
A gear is a machine element in power transmission that is widely used in various fields. A gearbox is a power transmission system that consists of gears, shafts, bearings, and housings; and the power input to the shaft is transmitted to the driven gear (herein, gear) through the driving gear (herein, pinion). Additionally, when power is transmitted using a gear-pair, since the gear ratio changes the rotation speed and torque, the advantage of controlling the rotation speed and torque by changing the gear ratio arises. The performance of a gearbox can be evaluated by parameters such as fatigue life, noise, vibration, and power transmission efficiency. In the case of fatigue life, since it determines whether the gearbox operates or not, it needs to reliably predict and evaluate the life of the gearbox 2 .
For reliably predicting and evaluating the performance of the gearbox, it is necessary to define the load acting on the gearbox accurately. The load magnitude of the load acting on the gearbox, duration under the load, and the fluctuation range of the load are determined according to the purpose and environment of use of the gearbox. However, it is challenging to numerically define the load acting on the gearbox. Therefore, many researchers used the cumulative fatigue damage theory based on the Palmgren-Miner rule and predicted and evaluated the gearbox performance under equivalent load conditions using the concept of averages [bib_ref] Design of 2-speed transmission for electric commercial vehicle, Shin [/bib_ref] [bib_ref] Strength analysis of mechanical transmission using equivalent torque of plow tillage of..., Kim [/bib_ref]. While using the equivalent load in evaluating the gearbox's performance can shorten the calculation time, there is the disadvantage of being unable to consider the effect of the load fluctuation and peak load acting on the gearbox. Additionally, the fatigue damage exponent used to derive the equivalent load is a value that varies depending on the failure mode of each element constituting the gearbox. At the design stage, the fatigue damage exponent cannot be accurately determined because the key failure modes of the gearbox are not available in advance 5-7 .
Dong et al. [bib_ref] Time domain-based gear contact fatigue analysis of a wind turbine drivetrain under..., Dong [/bib_ref] conducted a study on the effect of fluctuating wind speed on the gear contact fatigue of a wind turbine gearbox. The gear contact fatigue was analyzed using a total of 11 different wind speeds-available in literature-for implementing the wind speed fluctuations. However, since this analysis did not reflect the practical environment wherein the wind turbine gearbox is actually used, there was a limit to the reliability of the analysis results. Patel and Joshi 9 performed a design and fatigue analysis of the gearbox carrier and confirmed that its fatigue life changed depending on its material and shape. However, the analysis suffered from the same limitation as that of the previous study, along with the additional limitation of being performed under only one load condition. Du et al. [bib_ref] Fatigue life prediction of the gear box in tracked vehicles based on..., Du [/bib_ref] conducted a study to predict the fatigue life of the gearbox of a tracked vehicle using a running simulation test. The environment wherein the gearbox operated was simulated, and the load acting on the gearbox was derived using the simulation results. Additionally, the fatigue life of the gearbox was evaluated using the derived load. However, since the derived load was not validated, there was a limit to the reliability of the simulation results. Kim et al. [bib_ref] Fatigue life simulation of tractor spiral bevel gear according to major agricultural..., Kim [/bib_ref] built a transmission simulation model of a tractor using commercial software and developed a model that could evaluate the fatigue life of spiral bevel gears. Additionally, the load generated in the operating environment was measured, and the fatigue life of the spiral gear was predicted by constructing a load spectrum based on the measured data. The load duration distribution (LDD) method was intended for predicting the performance of the gears and bearings 12 ; this study incorrectly predicted their performance with the load spectrum using the rainflow-counting algorithm. Similarly, in most studies conducted in various fields that predicted and evaluated gearbox performance, the definition of the operating environment was insufficient. Wang et al. [bib_ref] On design, modeling, and analysis of a 10-MW medium-speed drivetrain for offshore..., Wang [/bib_ref] conducted research on the design, modeling, and analysis of offshore wind turbine drivetrains. An iterative design procedure was presented to minimize the weight and volume when designing the drivetrain of wind turbine, and the model was validated by comparing the designed multibody simulation model with the previously developed model. However, there is a limitation in that the design load rather than the actual environment load was used when designing and validating the drivetrain of wind turbine. Yoo et al. [bib_ref] Application of flexible pin for planetary gear set of wind turbine gearbox, Yoo [/bib_ref] developed a simulation model of the wind turbine gearbox to confirm the performance of the planetary gear set to which the flexible pin was applied. The simulation was performed using commercial software. As a result of the study, it was confirmed that load sharing and load distribution among planet gears were improved when flexible pins were applied to the planetary gear set. However, there is a limitation in that the environment in which the wind turbine gearbox is operated was assumed in performing the performance of the planetary gear set.
To solve this problem, Kim et al. [bib_ref] Determination of design loads of maize harvester using actual working load, Kim [/bib_ref] performed an actual harvesting operation using a maize harvester developed by Kang et al. [bib_ref] Performance evaluation and design of an edible fresh corn harvesting machine, Kang [/bib_ref]. A sensor that could measure torque and rotational speed was attached to the tractor power take-off (PTO), and the actual workload generated during maize harvesting was measured using the sensor. Additionally, using the measured real workload, a load duration distribution that could evaluate machine elements that transmitted or supported a load through contact, such as gearbox components like gears and bearings, was constructed.
In this study, the gearbox simulation model of the maize harvester introduced by Kang et al. [bib_ref] Performance evaluation and design of an edible fresh corn harvesting machine, Kang [/bib_ref] was developed using the commercial software Romax Nexus 17 . Additionally, the strength and fatigue life of the gears and bearings in the gearbox were evaluated using the gearbox model and the LDD constructed by Kim et al. [bib_ref] Determination of design loads of maize harvester using actual working load, Kim [/bib_ref]. The evaluation revealed that the gearbox did not satisfy the target fatigue life of the maize harvester; the target fatigue life was satisfied by modifying the bearing arrangement and shaft length, which are design variables of the gearbox. Finally, by performing gear micro-geometry modification, the load distribution acting on the gear tooth surface was improved.
# Methods
All methods were carried out in accordance with relevant guidelines and regulations and obtained permission from the Research Institute of Agriculture and Life Science, Rural Development Administration for collecting maize.
Load duration distribution. Fatigue failure occurs when machine elements are subjected to fluctuating loads of varying magnitudes for many cycles. To check the safety of machine elements against fatigue failure, the external load acting on the element should be measured under the actual load condition. The load should then be processed according to the results of the safety evaluation. Among gearbox components, machine elements that transmit or support loads through contacts, such as gears and bearings, can constitute a load spectrum with the load magnitude, speed, and duration under the load. [fig_ref] Figure 1: Sample data for explaining LDD method [/fig_ref] shows the sample data used for explaining the LDD method. The interval is divided into arbitrary load bins after the minimum and maximum values are checked in the measured torque data. In the sample data, the interval between the minimum and maximum torques of 500 and 670 Nm, respectively, is divided into two sections with an interval of 100 Nm. The magnitude of the load in the ith section of the sample data is obtained as an average of torque values from 500 to 600 Nm. The time data of the ith section is t 1 + t 2 + t 3 , which is the total time of exposure to the torque. Finally, the speed data of the ith section is obtained as an arithmetic average of the rotational speeds belonging to the time data corresponding to the section. In LDD, the magnitude, duration, and speed of the load are expressed through the following equations: where i is the bin number, T i is the ith average torque in bin, T i,j is the ith jth torque in bin, n is the ith data in the bin, t is the time interval of the measurement data, ω i is the ith average speed of the bin, and ω i,j is the ith is the speed of the bin. [fig_ref] Table 1: LDD for gear rating of maize harvester gearbox15 [/fig_ref] shows the details of the LDD method Kim et al. [bib_ref] Determination of design loads of maize harvester using actual working load, Kim [/bib_ref] used for maize harvesting.
[formula] (1) T i = n j=1 T i,j n ,(2)t i = t · n,(3)ω i = n j=1 ω i,j n , [/formula]
Maize harvester gearbox model. The maize harvester consists of a maize harvester gearbox, which is used for harvesting, wherein maize stalks are transferred and peeled; the first multiplier transmission (gear ratio: 0.645), which receives power directly from the tractor PTO; and the second multiplier transmission (belt-pulley ratio: 0.714), which transmits the power of the first multiplier transmission (gear ratio: 0.645) to the maize harvester gearbox. [fig_ref] Figure 2: Configuration of power transmission system for maize harvester15 [/fig_ref] shows the power transmission from the tractor PTO to the maize harvester gearbox. [fig_ref] Figure 3: Gearbox simulation model of maize harvester [/fig_ref] shows the simulation model of the maize harvester gearbox that was developed using Romax Nexus 17 . S1, the input shaft of the maize harvester gearbox, transmits power to S2 and S3, the output shaft of the detach part, and S4, the output shaft of the transport part. A bevel gear set (BGS), a machine element that can transmit power vertically, is used between S1 and S2 to transmit power to S2 and S3, which are perpendicular to S1. Additionally, the power transmitted to S2 through the BGS is transmitted to S3 through the spur gear set (SGS) 1, which is a parallel shaft power transmission element. Finally, SGS 2 is used between S1 and S4 to transmit power to the transfer unit. www.nature.com/scientificreports/ In the gearbox model, the spur and bevel gears are defined with non-linear contact stiffness and represented by macro-geometric parameters (modules, number of teeth, center distance, pressure angle, face width, among others). The gear mesh misalignment and non-linear tooth stiffness were considered for contact analysis of the gear. Since the gear mesh force is influenced by the contact position of the tooth flank, we performed modeling considered and analyzed all the gear meshing points, load distributions, and boundary conditions. To analyze the contact of the gear, the slice model, assuming that each slice is operating as a spur gear and independent, was used. The non-linear stiffness model of rolling element bearing was defined as internal detail parameters (curvature of the raceways, internal clearance, roller profile, etc.). Shafts were also modeled as flexible 1D beam elements. The gear specifications used in the maize harvester gearbox are shown in [fig_ref] Table 2: Specification of bevel gear set [/fig_ref] , and FAG 6207 was used for all rolling bearings.
# Results and discussion
Gearbox evaluation using a simulation model and actual load data. In this study, the gear rating and bearing fatigue life were evaluated using the LDD generated based on the developed simulation model of the maize harvester gearbox and the actual workload measured during maize harvesting. The ratings for the spur gear and bevel gear were given based on ISO 6336 6 and ISO 10300, respectively. Additionally, the fatigue life of the bearings was evaluated using ISO 281. [fig_ref] Table 4: Gear safety factors of maize harvester gearbox using LDD [/fig_ref] shows the rating results for the gear simulation.
The gear rating results in [fig_ref] Table 4: Gear safety factors of maize harvester gearbox using LDD [/fig_ref] revealed the gear with the highest failure probability and that the failure mode for it was gear surface pitting caused by the contact stress of SGS 1 (pinion and gear). Therefore, to confirm that the face load distribution had a dominant influence on the safety factor for the contact stress, the face load distribution was analyzed using the finite element model and non-linear contact model of Romax Nexus. The finite element model and non-linear contact model analyzed the face load distribution using the following four theories and calculated the face load factor ( K Hβ ) using the analysis results 17 : [fig_ref] Table 5: Face load factor of SGS 1 according to load level in LDD [/fig_ref] shows the maximum load per unit length and face load factor of SGS 1, and [fig_ref] Figure 4: Face load distribution of SGS 1 on load level 8 [/fig_ref] shows the face load distribution at load level 8 of SGS 1. [fig_ref] Figure 4: Face load distribution of SGS 1 on load level 8 [/fig_ref] , it was confirmed that the contact pattern of SGS 1 was extremely skewed to the left. As a result, the tooth surface area that transmitted the load was reduced, and it was confirmed that the safety factor for the contact stress was low owing to the induced high contact stress. [fig_ref] Table 6: Lifetime of bearings in maize harvester gearbox in LDD [/fig_ref] show the fatigue life evaluation results of radial and axial loads of all the bearings acting on each bearing at load level 8. [fig_ref] Table 6: Lifetime of bearings in maize harvester gearbox in LDD [/fig_ref] confirms that the bearings B3 and B4 located in S2 did not satisfy the target fatigue life of 4800 h for the maize harvester gearbox, and [fig_ref] Table 7: Reaction force of bearings on load level 8 in LDD [/fig_ref] confirms that the load acting on these bearings is very large.
Design modification of maize harvester gearbox. From the results of the gearbox simulation, it was confirmed that the weak parts of the maize harvester gearbox were B3 and B4, located at SGS 1 and S2, respec- www.nature.com/scientificreports/ tively. In this study, it was determined that the cause of the occurrence of the weak part of the gearbox was as follows.
1. The arrangement of B3 and B4 in S2 as an overhung type was unfavorable to moment support. www.nature.com/scientificreports/ 2. The moment was generated in S2 due to the gear mesh force of BGS and SGS 1 that led to the deflection of S2. 3. Gear mesh misalignment occurred in SGS 1 owing to the deflection of S2. 4. The Increased K Hβ and reduced safety factor for contact stress was due to gear mesh misalignment.
To solve the above problem, the shaft length between the bevel gear and the spur gear of S2 was increased by 20 mm, as shown in [fig_ref] Figure 5: Change in bearing arrangement according to design modification of gearbox [/fig_ref]. Additionally, by positioning B4 between the bevel gear and the spur gear, B3 and B4 were arranged like a straddle, an arrangement advantageous for moment support. Finally, the shaft length of S3 was increased by 20 mm to position B6 at the top together with B4. [fig_ref] Figure 5: Change in bearing arrangement according to design modification of gearbox [/fig_ref] shows the bearing arrangement before and after the design modification, and [fig_ref] Figure 6: Modified gearbox simulation model of maize harvester [/fig_ref] shows the modified simulation model.
As performed for the model before design modification, the gear rating and bearing fatigue life of the designmodified simulation model were evaluated using LDD, and the results are shown in As shown in , it was confirmed that the safety factor for the contact stress of SGS 1, a weak component of the existing gearbox, increased by approximately 1.9 times owing to the design modification. According to the load level, the maximum load per unit length decreased by 3.74 times on average, and the K Hβ decreased by 3.82 times on average. As shown in [fig_ref] Figure 7: Face load distribution of SGS 1 on load level 8 for modified... [/fig_ref] , the decrease in the safety factor for the contact stress and maximum load per unit length was assumed to be due to the relatively even distribution of the contact pattern of www.nature.com/scientificreports/ SGS 1 compared to that of the existing gearbox. Also, as shown in [fig_ref] Table 1: LDD for gear rating of maize harvester gearbox15 [/fig_ref] , the load applied to B3 and B4 was significantly reduced through the design modification. Accordingly, the lifespans of B3 and B4, which did not meet the target fatigue life in the existing gearbox, were 1.6x10 8 and 8672 h, respectively, confirming that the target fatigue life was satisfied.
Micro-geometry modification of SGS 1 in maize harvester gearbox. Although the safety factor of SGS 1 increased through the design modification of the gearbox, since the face load distribution acting on SGS 1 was still skewed to the left of gear tooth surface, it caused high contact stress and shortened the fatigue life of the [bib_ref] Analysis of load distribution and sharing on the planetary reducer for wind..., Park [/bib_ref]. Therefore, in this study, we performed a micro-geometry modification of the gear on SGS 1 to improve the face load distribution. This modification was performed with a lead crown and lead slope, and a parameter study was performed for a total of 121 cases, under which the crown was increased by 1 μm from 0 to 10 μm and the slope was increased by 2 μm from 0 to 20 μm. The parameter study was performed to calculate the K Hβ at each of the 8 load levels and derive the combination of the lead crown and lead slope with the smallest sum of [fig_ref] Table 1: LDD for gear rating of maize harvester gearbox15 [/fig_ref] show the results of the strength evaluation of the gears after the micro-geometry modification. [fig_ref] Figure 8: Face load distribution of SGS 1 on load level 8 after micro-geometry... [/fig_ref] shows the face load distribution at load level 8 of SGS 1, which was subjected to the microgeometry modification. The micro-geometry design modification increased the safety factor for the contact stress of SGS 1, which was the weak component of the initial design model, by about 2.55 times. Additionally, the maximum load per unit length according to the load level decreased by 7.14 times on average compared to that of the initial design model, and the K Hβ decreased by 6.27 times on average compared to that of the initial design model.
# Conclusions
The purpose of this study is to evaluate the previously developed maize harvester gearbox based on the simulation model and the actual workload. This gearbox was modeled using Romax Nexus, and the actual workload was based on pre-existing research results. The evaluation revealed that the previously developed gearbox did not satisfy the target fatigue life. This was attributed to the shaft deflection that occurred due to the bearing misarrangement and gear mesh misalignment from the internal clearance of bearing and uneven load distribution of the gear tooth surface. The required target fatigue life of the maize harvester was satisfied by calculating the shaft deflection, gear mesh misalignment, and uneven load distribution of the gear teeth. www.nature.com/scientificreports/ not satisfied in B3 and B4 of S2, and the safety factor for the contact stress of SGS 1 was derived as 1.00, confirming that the gearbox required improvement. 2. Since B3 and B4 of the existing gearbox were arranged overhung over S2, a moment was generated by the gear of BGS located in S2 and the pinion of SGS 1. It was determined that deflection occurred in S2 owing to the moment, which resulted in a shortened bearing life and increased mesh misalignment for SGS 1. To solve this problem, design modification was performed to change the overhung arrangement of B3 and B4 to a straddle one. After the modification, both B3 and B4 satisfied the target fatigue life of the gearbox for the maize harvester, and it was confirmed that the safety factor for the contact stress of SGS 1 increased to an average of 1. 9. Furthermore, for the face load distribution of SGS 1, the maximum load per unit length decreased by an average of 3.77 times. However, the face load factor, which indicated the face load distribution, was as large as 1.8-2.1, confirming that further improvement was required. 3. A micro-geometry modification was performed to improve the face load distribution of SGS 1. The modification was performed on the lead crown and lead slope, and the smallest sum of the face load factors was derived at all the load levels through a parameter study. As a result, the maximum load per unit length of SGS 1 was reduced by approximately 7.14 times compared to that of the existing SGS 1, and the face load factor was found to be 1.0-1.3, which decreased by about 6.27 times on average. 4. Finally, for performing the gear strength and bearing life evaluations for the gearbox, (1) a high-precision simulation model that could accurately simulate the actual gearbox and (2) an actual load-based LDD were essential. (3) It was confirmed that the gearbox should be evaluated and design modifications should be applied, based on (1) and (2).
## Data availability
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Received: 17 June 2022; Accepted: 7 September 2022
[fig] Figure 1: Sample data for explaining LDD method. [/fig]
[fig] Figure 2: Configuration of power transmission system for maize harvester15 . [/fig]
[fig] Figure 3: Gearbox simulation model of maize harvester. Bending based on Mindlin plate theory; 2. Compression based on Timoshenko beam theory; 3. Root rotation based on an empirical theory; 4. Root shear based on an empirical theory. [/fig]
[fig] Figure 4: Face load distribution of SGS 1 on load level 8: (a) Pinion contact pattern and (b) gear contact pattern. Scientific Reports | (2022) 12:15576 | https://doi.org/10.1038/s41598-022-19982-z [/fig]
[fig] Figure 5: Change in bearing arrangement according to design modification of gearbox: (a) Before and (b) after design modification. Scientific Reports | (2022) 12:15576 | https://doi.org/10.1038/s41598-022-19982-z [/fig]
[fig] Figure 6: Modified gearbox simulation model of maize harvester. [/fig]
[fig] Figure 7: Face load distribution of SGS 1 on load level 8 for modified gearbox: (a) Pinion contact pattern and (b) gear contact pattern. [/fig]
[fig] 1: The strength and fatigue life analyses of the gears and bearings in the gearbox of a maize harvester were performed using the maize harvest LDD and simulation model. The target fatigue life of the gearbox was [/fig]
[fig] Figure 8: Face load distribution of SGS 1 on load level 8 after micro-geometry modification: (a) Pinion contact pattern and (b) gear contact pattern. [/fig]
[table] Table 2: Specification of bevel gear set. [/table]
[table] Table 3: Specification of spur gear sets 1 and 2. [/table]
[table] Table 4: Gear safety factors of maize harvester gearbox using LDD. [/table]
[table] Table 5: Face load factor of SGS 1 according to load level in LDD. [/table]
[table] Table 6: Lifetime of bearings in maize harvester gearbox in LDD. [/table]
[table] Table 7: Reaction force of bearings on load level 8 in LDD. [/table]
[table] Table 8 Table 9: Gear safety factor of modified gearbox in LDD. Face load factor of SGS 1 in modified gearbox according to load level in LDD. [/table]
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The clinical and experimental significance of blinking behavior
Evaluations of tear functions frequently involve some form of voluntary control over blink behaviour. To the degree that voluntary control of blinking risks departure from normalrange spontaneous blinking, the tear function findings from such studies may be confounded. Even subject awareness that blinking is being assessed may influence findings if such awareness results in any degree of voluntary control. Ideally, the influence on blink rate and tear functions induced by therapeutic or experimental interventions could be measured against a normal-range baseline spontaneous blink rate in order that any differences found could be validly attributed to those interventions. Sometimes pre-intervention 'rest-related' baseline blink rates have been incorrectly described as 'basal' blink rates without specification of pre-intervention conditions of 'rest' or consideration of any contributions from voluntary control. Also, studies which use only blink rates to measure blink efficiency ignore the critically important contribution of incomplete blinking to blink inefficiency. This review finds that the assessment of normalrange spontaneous blink rates depends on measurement conditions which have frequently been ignored previously. For example, normal-range spontaneous blink rates appear more likely to occur with fixation targets which have a disengaged affect and an associated neutral influence on and from dopamine activity. Ideally, fixation targets should also involve minimal cognitive loading and vision demands. In addition, normal-range (symptom free) spontaneous blink rates are more likely to be assessed in a comfortable ambient environment without subject awareness that blink behaviour is being assessed and when voluntary blinking is not involved. Voluntario; Reflejo; Funciones de la lágrimaSIGNIFICADO CLÍNICO Y EXPERIMENTAL DEL COMPORTAMIENTO DEL PARPADEOResumen Las valoraciones de las funciones de la lágrima incluyen alguna forma de control voluntario del parpadeo. Hasta el punto de que el control voluntario del parpadeo puede tener su origen en el parpadeo espontáneo de rango normal, los hallazgos sobre función de la lágrima de dichos estudios pueden resultar confusos. Incluso la concienciación del sujeto acerca de que se está evaluando el parpadeo puede influir en los hallazgos, cuando dicha concienciación deriva en cualquier grado de control voluntario. De forma ideal, la influencia sobre las tasas de parpadeo y las funciones de la lágrima inducidas por intervenciones terapéuticas o experimentales podría medirse frente a una tasa de parpadeo espontáneo basal de rango normal, a fin de poder atribuir válidamente cualesquiera diferencias a dichas intervenciones. A veces, las tasas de parpadeo de referenciarelacionadas con el descansopre-intervención se han descrito incorrectamente como tasas de parpadeobasal, sin especificar las condicionesde descansopre-intervención, o la consideración de cualquier contribución del control voluntario. De igual modo, los estudios que utilizan únicamente tasas de parpadeo para medir la eficiencia del parpadeo, ignoran la contribución críticamente importante del parpadeo incompleto sobre la ineficiencia del mismo. Esta revisión encuentra que la valoración de las tasas de parpadeo espontáneo de rango normal depende de las condiciones de medición, que con frecuencia han sido ignoradas previamente. Por ejemplo, es más probable que se produzcan tasas de parpadeo espontáneo de rango normal con objetivos de fijación, que tienen un efecto de desactivación y una influencia neutra asociada sobre la actividad de la dopamina. De manera ideal, los objetivos de fijación deberían implicar también una carga cognitiva mínima y unas demandas de visión. Además, es más probable que las tasas de parpadeo espontáneo de rango normal (con ausencia de síntomas) se valoren en un entorno confortable, sin que el sujeto sea consciente de que se está valorando el parpadeo, y cuando el parpadeo voluntario no se ve implicado.
Blinking plays an important role in the maintenance of the integrity of the ocular surface because it contributes to the maintenance of ocular surface humidity and favours tear drainage, with expression and dispersion of lipids from meibomian glands. [bib_ref] The influence of eye solutions on blinking and ocular discomfort at rest..., Acosta [/bib_ref] Spontaneous blinks are usually the most common form of blinking and have a major role in maintaining the integrity of the ocular surface by spreading and restoring the tear film thickness 2 as well as maintaining tear layer stability as the basis for clear vision. [bib_ref] Spontaneous blink activity, Cruz [/bib_ref] Spontaneous blinking is an unconscious brief eyelid closure of both upper eyelids which occurs in the absence of any evident stimulus. [bib_ref] Spontaneous blink activity, Cruz [/bib_ref] Given that the mechanisms for the control of spontaneous blinking are not completely understood, [bib_ref] Spontaneous blink activity, Cruz [/bib_ref] the conditions of 'rest' under which spontaneous blink rates (SBRs) approach normal-range physiological levels cannot be accurately specified. Usually 'basal' is a descriptive for the lowest physiological level but as discussed further below, in the case of blink rates (BRs), factors such as dopamine activity and complex visual and/or cognitive demand can reduce BR to below normal-range SBR levels that might be associated with 'rest'. As conditions for a state of 'rest' vary so might the associated SBRs be expected to vary. Reflex and voluntarily controlled blinking are the antithesis of spontaneous blinking but being able to confidently eliminate reflex and voluntary influences on BRs may sometimes not be possible. The rate and degree of tear film evaporation and cooling, or extent of tear film break up (TFBU) and the associated threshold level for increased osmolarity and dry eye symptom influence on BR, do not appear to be known. For example, threshold levels for symptoms due to hyperosmolarity to stimulate reflex blinking may vary with ocular surface sensitivity and the area of any TFBU for example.
## Spontaneous or reflex or voluntary blinks
Symptoms of dry eye disease (DED) are known to increase BRs., [bib_ref] Quantitative videographic analysis of blinking in normal subjects and patients with dry..., Tsubota [/bib_ref] [bib_ref] The effects of increasing ocular surface stimulation on blinking and sensation, Wu [/bib_ref] Apparently, at some stage of DED progression, stimuli related to evaporative cooling and/or increased hyperosmolarity increase toward a conscious level and blinking becomes reflex. The demand for blink-related ocular surface maintenance becomes more critical according to the development of one or more age-and/or pathology-related tear dysfunctions as well as in response to adverse ambient conditions and influences from environmental factors such as increased cognitive and visual demands. Under most normal-range circumstances and conditions, BRs not only vary according to a wide variety of such factors but also in response to the methods used to measure them. Examples of clinical and experimental methods for blink and/or tear function assessments which have involved various forms of voluntary control of blinking are shown in . For example, just before tear meniscus measurements, Qiu and co-authors asked each participant ''to hold their blink during the acquisition of the optical coherence tomography scan and then they were instructed to blink normally''. [bib_ref] Age-related variations of human tear meniscus and diagnosis of dry eye with..., Qiu [/bib_ref] An instruction to blink normally may result in voluntary blinking 1. Just before tear meniscus measurements, the participant was asked "to hold their blink during the acquisition of the scan and then instructed to blink normally". 6 2. Subjects were instructed to ''blink and then hold their eyes open for five seconds''. 7 3. Subjects were instructed to ''blink normally and then to refrain from blinking''. 8 4. Following instillation of fluorescein, the patient was instructed to ''blink several times''. 9 5. During each trial, subjects were asked to ''hold their eyes wide open as long as possible''. 10 6. Prior to an examination of correlations between tests of tear dysfunction subjects were asked to "blink gently and completely three times". [bib_ref] Correlation of lipid layer thickness measurements with fluorescein tear film break-up time..., Isreb [/bib_ref] which alters tear meniscus volume for example. The characteristics of the blink prior to a scan, and any effect it has on tear meniscus volume is uncertain and, as a result, repeated scans involving voluntary blinks may not represent normal-range values. Compared to conditions for SBRs, other characteristics of blinks may vary widely under voluntary control. Accordingly, voluntary control could be invasive with respect to normalrange spontaneous blink performance; perhaps especially when effort is made to be sure that a full blink is achieved. For example, voluntary blinks may be more forceful than spontaneous blinks and so more likely to promote lipid secretion and increase tear layer thickness and tear meniscus volume. Voluntary blinks involving greater force could also promote faster tear drainage or create tear surface irregularities.
The evidence that BR increases as a function of time on task is compelling but apart from the effects of fatigue, for example, other environmental variables influencing BR include the complexity and associated acuity demands of concurrent visual activity as well as cognitive factors. [bib_ref] Blink rate: a possible measure of fatigue, Stern [/bib_ref] For example, cognitive factors include for instance, the perceptual demand of the concurrent activity such as blink timing (interblink intervals) with respect to information processing requirements. [bib_ref] Blink rate: a possible measure of fatigue, Stern [/bib_ref] Acosta and co-authors showed how activities such as listening and talking can increase BRs while tasks involving visual information processing such as reading, can reduce BRs. 1 Some other influences on BR have been broadly described as attention, level of concentration, as well as stress and anxiety. [bib_ref] Relation between tear break up time and spontaneous blink rate, Al-Abdulmunem [/bib_ref] A videotape recording of BR while subjects were either resting quietly, reading aloud or conversing, led to the conclusion that cognitive processes were the chief influence on BR. 14 Some of the variable conditions for 'resting quietly' in relation to an associated BR are elaborated below. BR inhibition during visual tasks has been assumed to preserve input information processing, thus reflecting attentional engagement. [bib_ref] Cardiac responses associated with affective processing of unpleasant film stimuli, Palomba [/bib_ref] For example, visual activities involving a sequence of increasing degrees of attentional engagement such as looking straight ahead, watching a movie, identifying rapidly changing letters and playing a computer game, were found to progressively reduce BRs and elevate incomplete blinking rates in both dry DED and normal subjects. [bib_ref] Blinking and tear break-up during four visual tasks, Himebaugh [/bib_ref]
## Blink efficiency
Blink efficiency is a measure of the ocular surface protective functions of blinking and is determined by the degree that blinking supports tear functions and so contributes to the health of the ocular surface. For example, both incomplete blinking and low rates of complete blinking reduce blink efficiency. Spontaneous blink efficiency is commonly assessed by only the measurement of BR, [bib_ref] Relation between tear break up time and spontaneous blink rate, Al-Abdulmunem [/bib_ref] [bib_ref] Analysis of blink rate patterns in normal subjects, Bentivoglio [/bib_ref] [bib_ref] The (b)link between creativity and dopamine: spontaneous eye blink rates predict and..., Chermahini [/bib_ref] [bib_ref] Diurnal variation in spontaneous blink rate, Barbato [/bib_ref] but several other features of blink behaviour appear likely to also contribute to blink efficiency. Variations in interblink intervals and different forms of incomplete blinking such as variations in blink amplitude (or degree of closure), the frequency of partial closures such as twitch blinks involving a very minor (aborted) degree of closure, flurries which are a series of rapidly repeated twitches, as well as differences in the speed of closure and opening phases for example, [bib_ref] A tentative mechanism for inferior punctate keratopathy, Abelson [/bib_ref] [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] Variation in interblink intervals may be an indication of changing cognitive processing demands during assessment of BRs. [bib_ref] Blink rate: a possible measure of fatigue, Stern [/bib_ref] Tear osmolarity increases with tear evaporation during an interblink interval. BR and completeness must be maintained at rates which prevents the formation of areas of significant evaporation and tear hyperosmolarity which have the potential to cause symptoms, tissue damage and/or a reduction in vision 20 with associated reflex blinking.
## Current knowledge of blink control
Knowledge about the control of spontaneous blinking is incomplete [bib_ref] Spontaneous blink activity, Cruz [/bib_ref] with little known about its neural basis. [bib_ref] Characterizing the spontaneous blink generator: an animal model, Kaminer [/bib_ref] A simple part-explanation for complex blink behaviour is that the spinal trigeminal complex is a critical element in the generation of spontaneous blinks. [bib_ref] Characterizing the spontaneous blink generator: an animal model, Kaminer [/bib_ref] However, BRs are otherwise believed to reflect a complex interaction between peripheral influences mediated by the ocular surface and the level of central dopamine activity, 3 both of which are discussed further below. In order that an evaluation of tear functions is able to produce evidence that is representative of a patient's customary blink behaviour, such evaluations should ideally involve a blinking phase which is representative of normal-range spontaneous blink activity. In this way the findings are more likely to have relevance to typical levels of blink efficiency for example. Rather than representing 'background noise' or being a randomly occurring phenomenon, variations in blood pressure have been shown to be the result of complex interactions between extrinsic environmental and behavioural factors and intrinsic cardiovascular regulatory mechanisms. [bib_ref] Assessment and management of blood-pressure variability, Parati [/bib_ref] To limit short-term influences on findings, suggested recommendations for the assessment of baseline blood pressure include rest in a comfortable physical position in a calm mental state for at least 5 min and no smoking or alcohol/caffeine intake for at least 30 min prior to assessment for example. [bib_ref] Assessment and management of blood-pressure variability, Parati [/bib_ref] Such measurements could provide a level of blood pressure against which the influence of an intervention could be compared. Similarly, the influence of an experimental intervention on blink activity would ideally be measured against normal-range baseline spontaneous blink performance. For example, assessment of tear film break up time (TFBUT) following voluntary complete blinking appears likely to thicken tear layers and give findings which are not representative of TFBUT during normal-range spontaneous blink activity. [bib_ref] Reducing the invasive nature of tear stability assessments, Mcmonnies [/bib_ref] This review examines how those kinds of evaluations might and might not be validly achieved. PubMed searches (17th March 2019) using the terms spontaneous blink, voluntary blink, and reflex blink yielded 424, 190, and 4667 potentially useful publications respectively. Selections of those which were found to be the most relevant and representative of a balanced account of this topic, as well as some selected reports referenced in those publications, were included in this review.
## Some differences between spontaneous, voluntary and reflex blinking
Spontaneous blinks are entirely sub-cortical while voluntary blinks are preceded by pre-motor cortical readiness. [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] Voluntary blinks can differ from spontaneous blinks in regard to factors such as rate or interblink interval variability, degree of completeness, duration of closure, and the force involved, because they are associated with motor function. [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] Consequently, the influences on tear functions can be very different according to the type of blink. The neural pathways involved in ocular surface controls over reflex blinking arise in ocular surface sensory input through the trigeminal nerve that projects to the motor neuron of the seventh cranial nerve. [bib_ref] The effects of increasing ocular surface stimulation on blinking and sensation, Wu [/bib_ref] In this way, stimulation of the ocular surface, such as the symptoms associated with DED, increase both BR and regularity. [bib_ref] The effects of increasing ocular surface stimulation on blinking and sensation, Wu [/bib_ref] Thus, when driven by symptoms, blinking in DED appears to be reflex rather than spontaneous. When task concentration levels were 'controlled', pneumatic stimulation of the ocular surface was found to increase BR and regularity in healthy human subjects. [bib_ref] The effects of increasing ocular surface stimulation on blinking and sensation, Wu [/bib_ref] Apparently, the pneumatic stimulation and associated faster evaporation induced an artificial state similar to one that could be found in DED. The relationship between pneumatic stimulation and BR was linear suggesting a linear dose-related response relationship between ocular surface input and blinking. [bib_ref] The effects of increasing ocular surface stimulation on blinking and sensation, Wu [/bib_ref] Even a barely noticeable air stimulus to the ocular surface, using a small electric fan at an average distance of 50 cm, produced an increased frequency and regularity of blinking. [bib_ref] The effects of mild ocular surface stimulation and concentration on spontaneous blink..., Wu [/bib_ref] Apart from air movement, any other aspect of ambient conditions such as temperature and humidity could influence BR by altering the rate of tear evaporation. The input for triggering ocular surface-stimulated blinks depends on the integrity of the surface nerves. [bib_ref] The effects of increasing ocular surface stimulation on blinking and sensation, Wu [/bib_ref] Naase and co-authors found that in a sample of 40 males without significant ocular surface disease, 37/40 (92.5%) showed a reduction in BR with topical ocular anaesthesia. For the 37 who showed a reduction, the mean reduction in BR was 37.4%. [bib_ref] An assessment of the pattern of spontaneous eyeblink activity under the influence..., Naase [/bib_ref] Loss of corneal sensitivity with age could be expected to influence BR. [bib_ref] Relation between tear break up time and spontaneous blink rate, Al-Abdulmunem [/bib_ref] However, Bentivoglio and co-authors did not find any correlation between BR and age, [bib_ref] Analysis of blink rate patterns in normal subjects, Bentivoglio [/bib_ref] and Sun and coauthors found a modest increase in BR with age. [bib_ref] Age-related changes in human blinks, Sun [/bib_ref] However, apart from corneal sensitivity, many other factors may confound the relationship between age, and BRs. It remains possible that increased BRs due to age-related DED could sometimes be moderated by concurrent reduced corneal sensitivity. The possibility that any sensory message sent by ocular surface cold receptors during evaporation also contributes to the modulation of blinking, is a functional alternative that deserves experimental scrutiny. [bib_ref] Cold thermoreceptors, unexpected players in tear production and ocular dryness sensations, Belmonte [/bib_ref] Is it possible that normal-range evaporative increases in osmolarity during an interblink interval, could also provide stimuli for spontaneous blinking in healthy eyes? Such peripheral controls could occur in the absence of any apparent stimulus and could be independent of any central control mechanism. However, increased BR in DED 4,5 may be due to faster evaporation and an associated more rapid rate of tear cooling and osmolarity elevation with resulting greater stimulation of the ocular surface. Even in healthy young females, a significant correlation between TFBUT and BR was found. 13
## Other influences on blink efficiency
Drew reported that BR varied inversely with task difficulty for all subjects that were studied. [bib_ref] Variations in reflex blink-rate during visual-motor tasks, Drew [/bib_ref] Variability of interblink intervals in normal subjects suggests an ability to vary BR and even suppress blinking according to variable perceptual/cognitive needs over time. [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] Usually, TFBU does not occur during interblink intervals unless one or more factors such as incomplete blinking, [bib_ref] Incomplete blinking: exposure keratopathy, lid wiper epitheliopathy, dry eye, refractive surgery, and..., Mcmonnies [/bib_ref] tear layer instability, adverse ambient conditions, high visual acuity demand and/or elevated cognitive task loading are present. A dry eye subject's need for more frequent blinking supersedes the requirements of a reading task for example. [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] Again, such blinking is not spontaneous to the extent that factors influencing BR, such as comfort levels, are outside normal ranges. For individual subjects, BRs are specific to the circumstances under which they are measured and their individual responses to them. For example, the BR for 67.3% of healthy volunteers was greatest during conversation compared with while 'resting quietly', but then it was slower again when reading. 14 However, for 22.7% of the same sample of healthy volunteers, compared to conversing the rates were fastest for resting although still slowest for reading. 14 These findings suggest significant variation in individual normal responses. The level of cognitive activity during all three tasks for example may vary widely between individuals, and explain some of the differences in response, even during a socalled 'resting' period. The conditions for resting were not specified in that study. 14 For example, thoughts may be dominated by rest disturbing stresses and anxieties, or some subjects may be familiar with yogic or other relaxation techniques and take the opportunity to achieve a deep level of relaxation during rest. The level of rest achieved may also vary with, for example, uncontrolled influences from visual activity and levels of body comfort.
## Basal or baseline blink rate?
BRs measured by observations from a video-recording after a 10-minute period of adaptation to unspecified experimental conditions of 'rest' and prior to exposure to experimental interventions, were described as basal BRs. [bib_ref] Psychological resistance factors as predictors of general health status and physical symptom..., Ebert [/bib_ref] Basal BR suggests a rest-related physiological SBR but, given that there are many factors that might be influencing BR during a period of 'rest', blinking prior to intervention might be more appropriately described as baseline rather than basal. Conditions such as subjects being completely rested before and during assessment and freedom from emotional stress, such as measurement-related apprehension, 31 may be important. For example, ideally subjects would be unaware that they were being filmed or videotaped during a study of blink behaviour 32 so that any related apprehension, anticipatory thoughts or other potential influences on BR, such as voluntary control of blinking, are avoided. Bentivoglio and co-authors described BRs recorded by videotaping during quiet 'rest' as the basal BRs when compared to those recorded during the experimental conditions involving reading and conversation. 14 In that study, subjects were sitting and had agreed to being videotaped but were not aware of the purpose of the study. 14 No other conditions of quiet rest were specified for that study and again, rather than basal, baseline might have been a more accurate description of the pre-intervention BR. To the extent that spontaneous blinks occur in the absence of any apparent stimulus, 3 specification of measurement conditions would better inform baseline BR findings and their relationship to spontaneous-range BRs. Lack of awareness that blink behaviour is being examined might be a key feature of patient/subject ability to avoid voluntary blinking control. It is unknown to what extent would being physically uncomfortable and/or being exposed to unfamiliar measurement methods or equipment help to determine BR. For example, estimates of BR by observation during a biomicroscopic examination, which includes a potentially uncomfortable unnatural posture and mental state as well as abnormal lighting conditions, appear unlikely to be relevant to most real-world conditions.
## Consequences of other aspects of methods of measurement
Cruz and co-authors reviewed BR assessment methods which included procedures such as lever arms being attached to the lid to detect lid motion, electrooculography, electromyography, wearing lid movement detecting spectacles as well as evaluation of high-speed videotapes. 3 A magnetic search coil method was described as the gold standard method. [bib_ref] Spontaneous blink activity, Cruz [/bib_ref] However, that method requires that a small coil be taped to the upper lid and for the subject to be placed inside a magnetic field produced by a surrounding cubic frame. [bib_ref] Spontaneous blink activity, Cruz [/bib_ref] The high potential for invasiveness of these kinds of circumstances may reduce the possibility of capturing blink performance that is relevant to normal range real-world conditions.
## Dopamine influences on blinking
Dopamine is a hormone functioning as a neurotransmitter in several distinct systems within the brain. [bib_ref] The (b)link between creativity and dopamine: spontaneous eye blink rates predict and..., Chermahini [/bib_ref] One such system plays a major role in reward-motivated behaviour with most types of reward increasing the level of dopamine in the brain. [bib_ref] The (b)link between creativity and dopamine: spontaneous eye blink rates predict and..., Chermahini [/bib_ref] BR is affected by central dopamine level 5 such that, in healthy adults, SBR is regarded as a marker of the level of central dopamine functioning [bib_ref] Dopamine and inhibitory action control: evidence from spontaneous eye blink rates, Colzato [/bib_ref] and the daily pattern of SBR is a peripheral measure of central dopamine activity. [bib_ref] Diurnal variation in spontaneous blink rate, Barbato [/bib_ref] The validity of these relationships is conditional on the degree that measured BR is influenced by factors other than dopamine. Blinks are only an indirect measure of dopamine level which appears to have an important role in cognitive control [bib_ref] Blinking predicts enhanced cognitive control, Van Bochove [/bib_ref] which, in turn, has a reciprocal influence on BR. [bib_ref] Blink rate: a possible measure of fatigue, Stern [/bib_ref] The idea of an effective modulation of cognitive control through altered dopamine activity converges with common theories that suggest links between positive affect, dopamine and cognitive control. [bib_ref] Dopamine and cognitive control: the influence of spontaneous eyeblink rate and dopamine..., Dreisbach [/bib_ref] It has been assumed that the cognitive effects of positive affect are modulated by increased brain dopamine levels in the prefrontal areas. [bib_ref] Dopamine and cognitive control: the influence of spontaneous eyeblink rate and dopamine..., Dreisbach [/bib_ref] The nature of the visual activity during blink behaviour evaluation may have profound influences on affective state and cognitive demand. For example, difficulty seeing may cause frustration and an associated emotional response. Manipulation and monitoring of affective state are far from simple processes [bib_ref] Manipulating affective state using extended picture presentations, Sutton [/bib_ref] and so appear to be outside the scope of attempts to control affective state during routine BR measurements. For example, cognitive tasks given to subjects such as counting or detecting particular features in a target display may create confusion, frustration or even reward and satisfaction and might be best avoided during blink behaviour assessment. Ideally BR would be measured under conditions of a state of stable dopamine activity and neutral affect with the avoidance of assessment conditions which could increase apprehension for example. It is possible that a neutral affective state with minimal cognitive loading may be approached during measurements of SBR by presenting subjects with a meaningless and visually undemanding colour kaleidoscopic film target which undergoes constant colour and pattern changes, as is described further below.
## How some previous studies may have failed to provide satisfactory conditions for valid tear function assessment
Al-Abdulmunem measured BR by direct observation and secretly counting of blinks for subjects who were attending a lecture. [bib_ref] Relation between tear break up time and spontaneous blink rate, Al-Abdulmunem [/bib_ref] Lecture content such as varying intellectual and visual demands associated with oral and slide presentations may have confounded such measures of SBR which would otherwise be irrelevant to a wider range of circumstances. After subjects were informed that their voluntary blinking was going to be assessed and were asked to blink as normally as possible, Kwon and co-authors videotaped their blink behaviour. [bib_ref] High-speed camera characterization of voluntary eye blinking kinematics, Kwon [/bib_ref] However, attempts to blink normally may have the opposite effect to that required so that the results obtained could not represent normal-range SBR according to the levels of voluntary blink control that were involved.
# Discussion
In summary, some of the many factors which are known to influence BR include mental and/or physical fatigue, visual and cognitive tasking and environmental conditions, tear quality and quantity influences on ocular surface health and TFBUT, corneal sensitivity, symptoms of discomfort and tearrelated reductions in vision. [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] A complete understanding of the interaction between these influences has not been achieved. [bib_ref] Blink: characteristics, controls, and relation to dry eyes, Rodriguez [/bib_ref] Spontaneous blinking is the most common type of blinking and so makes a dominant contribution to ocular surface health. However, blink performance is commonly assessed by only measuring BR [bib_ref] The influence of eye solutions on blinking and ocular discomfort at rest..., Acosta [/bib_ref] [bib_ref] Analysis of blink rate patterns in normal subjects, Bentivoglio [/bib_ref] [bib_ref] Blinking and tear break-up during four visual tasks, Himebaugh [/bib_ref] although incomplete blinking can be another major factor in relation to blink efficiency. [bib_ref] Incomplete blinking: exposure keratopathy, lid wiper epitheliopathy, dry eye, refractive surgery, and..., Mcmonnies [/bib_ref] Incomplete blinks approximately double the exposure of the inferior ocular surface [bib_ref] Incomplete blinking: exposure keratopathy, lid wiper epitheliopathy, dry eye, refractive surgery, and..., Mcmonnies [/bib_ref] and have much greater significance when BRs are low and interblink intervals are longer, and especially if incomplete blinks occur successively.
The results of assessments of blink behaviour appear to be specific to the conditions of measurement, for which the results may or may not be representative of a subject's or patient's normal-range SBR according to the degree that voluntary or reflex influences are involved. Optimum methods for appropriate baseline spontaneous blink behaviour intended to be relevant to normal-range 'rest-related' blink behaviour would appear to need to create the least disturbance to the subject's physical and mental comfort. Informing patients/subjects that their blinking is being assessed might cause them to become conscious of their blinking so that some degree of voluntary control is exercised. That prospect is assured if subjects/patients are asked to blink normally. For example, for patients/subjects who are asked to breathe normally, the chances appear to be low for them to be able to match the depth and rate of breathing which occurs spontaneously under the same conditions. Similarly, there are multiple potential influences on blinking which are altered with voluntary control, when BR, blink completeness, interval rate and regularity as well as force of closure may vary from those features which occur during spontaneous blinking. SBRs can be influenced during tests involving higher levels of attention [bib_ref] Diurnal variation in spontaneous blink rate, Barbato [/bib_ref] and cognitive activity 12 so that an undemanding (neutral) visual/cognitive conditions appear to be required for a baseline spontaneous blink performance to be assessed. In the same manner, operational memory and visual imagination may share components with the visual perceptual systems, and inhibition of blinking may be involved in the protection of these vulnerable processes. [bib_ref] Blinking and thinking, Holland [/bib_ref] Avoiding making demands on subjects' and patients' memories or their imaginations, as well as not placing demands on their attention, such as by providing them with a counting or a letter identification task for example, may contribute to having a better chance of measuring normal-range rest-related baseline SBRs. Apart from neutral ambient conditions to limit reflex blinking, another important factor determining the validity of blink assessment findings might be that the subject is unaware of their blink performance being assessed. For example, sham advice that pupil reactions are being monitored could be helpful in avoiding awareness of blink behaviour. Ideally subjects would also not even be aware that they are being videotaped for example. [bib_ref] Interactions of eyelids and tears in corneal wetting and the dynamics of..., Doane [/bib_ref] Emotionally significant information appears to reflect motivational importance. [bib_ref] Opposing influences of affective state valence on visual cortical encoding, Schmitz [/bib_ref] That rewardmotivated behaviour is associated with changes to the level of dopamine in the brain [bib_ref] The (b)link between creativity and dopamine: spontaneous eye blink rates predict and..., Chermahini [/bib_ref] suggests that ideally, vision experiences during evaluation of spontaneous blink behaviour should be neutral for emotion, motivation and reward. Such conditions could provide an indication of a baseline SBR against which other influences on blink efficiency might be more usefully compared. Assessment of SBR provides only a limited indication of blink efficiency. Ideally assessment of features of blink behaviour other than BR, such as the frequency and amplitude of incomplete blinking as well as the degree of variation in interblink intervals, which may also have a profound impact on blink efficiency, could also be achieved. However, to the extent that such detailed analysis appears to be dependent on evaluation of videotape records, it does not appear to be suitable for clinical application.
Lack of repeatability in clinical evaluations of tear functions 9 may at least partly be due to variable influences from blinking prior to or during those evaluations. [bib_ref] Reducing the invasive nature of tear stability assessments, Mcmonnies [/bib_ref] Ideally, the method of assessment should not influence the findings. Providing subjects with the visual task of watching a kaleidoscopic colour pattern video on a television monitor during blink behaviour evaluation might create a minimally cognitive demand with neutral emotional content and influence on central dopamine activity. For example, this type of task might not have a significant potential to provide a rewarding or emotional experience that could contribute to dopamine activity. The 'Splendor of Color Kaleidoscope' video versions v1.1 to v1 3: 108p YouTube http://hdcolors.com present a continuously varying series of coloured kaleidoscopic patterns, and may be suitable for this purpose. Because the kaleidoscopic pattern changes are continuous, rather than periodic, there appears to be less chance that any associated recurring influences on blink regulation would occur with this type of target. The kaleidoscopic target also places low demand on visual clarity because the pattern and colour changes are still evident when blurred.
Normal-range (symptom free) spontaneous blink rates are more likely to be assessed in a comfortable ambient environment without subject awareness that blink behaviour is being assessed so that voluntary blinking is not involved. Blink behaviour in such clinical or experimental conditions could provide a reference baseline performance against which the influence of particular interventions could be assessed. For example, any influences on blink behaviour due to symptoms of ocular discomfort, contact lens wear or treatment for dry eye disease may be detected. However, blink behaviour in those clinical or experimental conditions are unlikely to represent the performance found in real-world circumstances which involve more complex interactions between variable ambient conditions, cognitive activity, dopamine levels, visual demands and degrees of mental and physical fatigue for example. Failure to recognise how non-spontaneous blinking, which is induced by conditions of assessment, could alter tear layer characteristics such as thickness and regularity, as well as lipid content and drainage rate in an unpredictable manner, could undermine the significance of subsequent evaluations of tear functions.
## Conflicts of interest
There are no conflicts of interest or financial interests to declare in relation to this review.
AcknowledgementThe author is grateful for discussions with and useful suggestions from Professor Jose Gonzalez-Meijhome. |
Intra-Arterial Delivery of Idarubicin in Two Patients with Glioblastoma
There is no effective treatment for recurrent glioblastoma (GB) when temozolomide-based radiochemotherapy fails. In theory, intra-arterial (IA) delivery of cytotoxic agents could achieve higher drug concentrations in tumors compared to intravenous injection. Moreover, choosing a highly lipid-soluble drug could make the most of the first-pass effect. Here, we evaluated idarubicin (IDA), a lipophilic anthracycline, in an in vitro assay using four human GB cell lines and compared it with 11 other drugs previously used for the IA treatment of brain tumors. Despite impressive in vitro cytotoxicity, IA IDA did not produce a beneficial effect in 2 patients with recurrent GB.
# Introduction
No effective treatment exists against recurrent glioblastoma (GB) when the standard first-line treatment using a temozolomide-based radiochemotherapy fails. Bevacizumab and lomustine increased progression-free survival, but their impact on overall survival is controversial [bib_ref] Single-agent bevacizumab or lomustine versus a combination of bevacizumab plus lomustine in..., Taal [/bib_ref]. There is no defined third-line treatment. Intra-arterial (IA) delivery of cytotoxic drugs has been explored over decades, but with no significant results to date [for review, . IA studies commonly use the same drugs as for i.v. delivery (melphalan, carboplatin, methotrexate, and temozolomide). Drugs are not specifically selected in order to exploit the concentration/antitumor effect anticipated by using IA delivery. Drugs have to be rapidly and irreversibly taken up during their first pass through the tissue circulation [bib_ref] Arterial drug infusion: pharmacokinetic problems and pitfalls, Dedrick [/bib_ref]. Moreover, the selected drug should be highly cytotoxic against targeted tumor cells. Here, we evaluated the in vitro cytotoxicity of idarubicin (IDA), a lipophilic anthracycline, using four human GB cell lines. Encouraging in vitro data led us to evaluate IDA by the IA route in patients with recurrent GB.
# Results
# In vitro results
IDA cytotoxicity was compared to 11 other antineoplastic drugs previously used for IA chemotherapy in GB or brain metastases in 4 human GB cell lines [fig_ref] Figure 1: In vitro colorimetric assay of 12 chemotherapeutic agents using four human GB... [/fig_ref]. Briefly, U87MG, CGL-1 CGL-3 or CGL-8 cells were seeded in complete culture medium and grown for 48 h in cell-culture plates. U87-MG cells were obtained from American Type Culture Collection (Manassas, Va., USA). The CGL-1, CGL-3 and CGL-9 cell lines were established from resected GB from 3 different patients and kindly donated by Dr. Philipe Genne (Oncodesign, Dijon, France). Subconfluent cells were incubated for 30 min with antitumor drugs at one selected concentration, then washed and grown again for 7 days in drug-free culture medium. The selected drug concentration was calculated assuming an unilateral carotid blood flow of 0.33 liter/min (10 liters in 30 min) and taking into account the dose usually given i.v. in patients with various tumors (table 1). The 30-min duration of in vitro exposure was selected since it was tolerated by patients receiving intracarotid infusion. A colorimetric assay was used to quantify drug-induced cell death. Surviving cells adherent to the bottom of the well were fixed with pure ethanol and stained with crystal violet dye. The dye was eluted with 33% acetic acid. Absorbance was determined by spectrophotometry (UVM 340; Bioserv) at 570 nm wavelength. Percent cell survival was calculated as optical density in treated wells/ optical density in control wells ×100.
Ninety percent or more of the cells from the 4 human GB cell lines were killed after a 30min exposure to 3 μg/ml IDA. This is an achievable concentration in the brain blood flow for a 30-mg dose of IDA. Other drugs were active less consistently.
## Case presentations
The impressive in vitro results led us to design a phase 1 study of IDA administration via intracarotid delivery (IDACAR) to treat recurrent GB. The IDACAR trial was approved on January 12, 2012, by the ethics committee (Comité de Protection des Personnes Nord-Ouest 2) and registered under EUDRACT No. 2011-004176-11. A dose increase was planned. After femoral puncture under local anesthesia, the catheter was placed into a carotid branch (S1, A1 or M 1, depending on the tumor localization) and above the ophthalmic artery in order to minimize ocular complications. Whole perfusion of the tumor was checked by angiography. IDA (1 mg/ml in saline solution) was infused in the carotid blood flow by an electric pump over 30 min. Patients were kept conscious and regularly asked to speak and move their limbs.
## Patient 1
A 56-year-old female had a history of resected colon adenocarcinoma in 2004. She was referred for behavioral disorders. A right frontal tumor with cystic contingent was evidenced by MRI. Partial resection of a GB was done in October 2010, followed by 60-Gy radiotherapy with concomitant and adjuvant temozolomide for 6 months. Disease recurred in November 2011 and progressed on MRI despite 3 months of bevacizumab. WHO status was 2 at inclusion with a mild left hemiparesis. The catheter was placed into the superficial branch of the middle cerebral artery that mainly irrigated the tumor. No adverse effects were observed during the IA injection of 22 mg IDA (12 mg/m 2 ) over 30 min. Anisocoria, worsening of the left hemiparesis, and severe cognitive impairment occurred at day 3 after IA infusion. The neurological degradation was considered to be a case of irreversible and severe toxicity, although tumor progression could not be completely ruled out considering the MRI at day 28 [fig_ref] Figure 2: Gadolinium-enhanced T1-weighted MRI before and 28 days after the IA delivery of... [/fig_ref]. A grade III leuconeutropenia occurred at day 7 despite granulocyte colonystimulating factor (G-CSF). Grade I anemia and thrombocytopenia were also recorded. The patient died 42 days after IA IDA due to disease progression.
## Patient 2
A 57-year-old female had an expansive sustentorial left occipital tumor. Complete resection of a GB in January 2009 was followed by 60-Gy radiotherapy with concomitant and adjuvant temozolomide for 6 months. Disease progressed in November 2011, and the patient received bevacizumab until March 2012. WHO status was 1 at inclusion. A catheter was placed into the posterior cerebral artery, and 21 mg IDA were infused IA over 30 min. No adverse effects occurred during the IA injection. Grade III leuconeutropenia was registered at day 7 nadir despite G-CSF. The neurological status and WHO status remained stable for 30 days. MRI evaluation on day 28 showed tumor stability according to the Response Assessment in Neuro-Oncology (RANO) Criteria. The patient refused a second IA injection and died 26 weeks after treatment due to disease progression.
Considering the possible grade IV neurological toxicity in the first patient and the lack of clinical benefit in both patients, the Independent Data Monitoring Committee (IDMC) recommended to stop the trial.
# Discussion
We reported here for the first time the IA delivery of IDA in 2 patients with recurrent GB. Our in vitro results were in agreement with previous papers. Schott and Robert [bib_ref] Comparative cytotoxicity, DNA synthesis inhibition and drug incorporation of eight anthracyclines in..., Schott [/bib_ref] compared growth inhibition, DNA synthesis inhibition, and cell incorporation of eight anthracyclines in a model of doxorubicin-sensitive and -resistant rat C6 GB cells. They found that new anthracyclines, including IDA, were more potent than the reference drugs (daunorubicin and doxorubicin) in rat GB cells. They observed reduced cross-resistance of IDA, pirarubicin and 4′-deoxy-4′-iododoxorubicin in a doxorubicin-resistant cell line. Among these anthracyclines, only IDA still remains available for clinical treatment of acute myeloid leukemias. Kuffel et al. [bib_ref] Anthracyclines and their C-13 alcohol metabolites: growth inhibition and DNA damage following..., Kuffel [/bib_ref] evaluated the growth-inhibitory and DNA-damaging activities of IDA, daunorubicin, doxorubicin, and epirubicin against human tumor cell lines, including the U87-MG human GB cell line. IDA was 2-5 times more potent than the other three anthracycline analogs. Considering that IDA is more lipophilic than other anthracyclines and more cytotoxic in glioma cell lines, Boogerd et al. [bib_ref] Penetration of idarubicin into malignant brain tumor tissue, Boogerd [/bib_ref] found a significant uptake of IDA and its major metabolite idarubicinol by the brain tumor tissue from 1 patient with a brain metastasis from breast cancer and from 4 patients with malignant glioma after a single oral dose of IDA (25-45 mg/m 2 ). Based on these preclinical data, a phase II trial was performed in children with progressive brain tumors [bib_ref] Pediatric Oncology Group: Phase 2 study of idarubicin in pediatric brain tumors:..., Dreyer [/bib_ref]. i.v. IDA (18 mg/m 2 ) was followed by G-CSF. Most patients developed progressive disease. Grade III/IV hematopoietic toxicities were common. The authors concluded that it is unlikely that IDA will be useful for the treatment of primary central nervous system tumors by the i.v. route.
In parallel to GB, we performed an experimental study to select the best candidate drug for transarterial chemoembolization of hepatocellular carcinoma (HCC). The SNU-398, HepG2, and SNU-449 human HCC cell lines were exposed to doxorubicin, epirubicin, IDA, mitoxantrone, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, gemcitabine, mitomycin C, or paclitaxel for 30 min. IDA was the most active drug against all three HCC cell lines [bib_ref] Screening of anticancer drugs for chemoembolization of hepatocellular carcinoma, Boulin [/bib_ref]. A phase I trial of IDA loaded on eluting beads for chemoembolization produced encouraging results in 21 patients with HCC and liver cirrhosis (response rate of 52% and 28% of complete responses) [bib_ref] Idarubicinloaded beads for chemoembolisation of hepatocellular carcinoma: results of the IDASPHERE phase..., Boulin [/bib_ref].
Considering the experimental data from our study and others about the strong in vitro antineoplasic activity of IDA and arguing that IA delivery could theoretically increase the tumor drug concentration by a factor of 5-10, we designed the IDACAR trial in recurrent GB. In contrast to HCC, clinical results were disappointing in our 2 GB patients. Neither of the 2 patients displayed a response on MRI, and irreversible grade IV neurological toxicity was suspected in the first patient. Many hypotheses can be proposed to explain the discrepancy between the in vitro and the clinical results. Selecting a drug that is potent on GB cells in vitro is not enough to improve the antitumor effect in patients. Heterogeneity of perfusion into the complex angioarchitecture of GB, reduced drug penetration due to the residual blood-brain barrier, and drug streaming in the locally accelerated blood flow and a number of other technical and pharmacological pitfalls remain to be solved in order to improve the IA treatment of brain tumors [bib_ref] Reassessing the role of intraarterial drug delivery for glioblastoma multiforme treatment, Ellis [/bib_ref]. Moreover, the lack of experimental models of GB in big animals greatly hinders progress. Blood flow in the selected carotid artery was estimated to be at 0.33 ml/min (10 liters in 30 min). The selected drug doses were in the range of those used by i.v. route for various tumors.
[fig] Figure 1: In vitro colorimetric assay of 12 chemotherapeutic agents using four human GB cell lines. Bars indicate the mean of 3 wells (standard deviation <10%). [/fig]
[fig] Figure 2: Gadolinium-enhanced T1-weighted MRI before and 28 days after the IA delivery of IDA in Patient 1. [/fig]
[table] Table 1: Calculations of drug concentrations for the in vitro cytotoxicity assay [/table]
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In Vitro Synergy of Isavuconazole Combined With Colistin Against Common Candida Species
Interactions of isavuconazole and colistin were evaluated against 57 common Candida strains belonging to the species Candida albicans (n = 10), Candida glabrata (n = 10), Candida kefyr (n = 8), Candida krusei (n = 10), Candida parapsilosis (n = 9), and Candida tropicalis (n = 10) by a broth microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methodology for antifungal susceptibility testing. Results were analyzed with the fractional inhibitory concentration index and by the response surface analysis. Interpretation by the fractional inhibitory concentration index showed synergy for 50%, 80%, 90%, and 90% of the C. kefyr, C. krusei, C. glabrata, and C. tropicalis strains, respectively. Combination of isavuconazole with colistin against C. albicans and C. parapsilosis exhibited only indifference for 100% and 90% of the strains, respectively. The results were confirmed by response surface analysis for all species except for C. glabrata, for which an indifferent interaction was found for the majority of strains. Antagonistic interaction was never seen regardless of the interpretation model was used.
# Introduction
Candidemia is a severe and life-threatening infection caused by different Candida species. In Germany, candidemia ranks as the 6th most common bloodstream infection after Escherichia coli, Staphylococcus aureus, coagulase-negative staphylococci, Klebsiella pneumoniae, and enterococci [bib_ref] The Epidemiology of Bloodstream Infections and Antimicrobial Susceptibility Patterns in Thuringia, Germany:..., Schöneweck [/bib_ref]. From the bloodstream, Candida can disseminate to multiple organs involving most commonly the liver, spleen, kidney, myocardium, or eyes and less frequently the brain. Invasive candidiasis is associated with a high mortality rate of about 40%. While many different Candida species can be responsible for invasive diseases, 95% of the infections are caused by the five species Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis [bib_ref] Epidemiology and Risk Factors for Invasive Candidiasis, Yapar [/bib_ref]. Although C. albicans is still responsible for the majority of infections in Germany [bib_ref] Epidemiology of Candidemia and Impact of Infectious Disease Consultation on Survival and..., Mohr [/bib_ref] [bib_ref] Epidemiology, Clinical Characteristics, and Outcome of Candidemia in Critically Ill Patients in..., Schroeder [/bib_ref] , non-albicans Candida species can represent up to 60% of the cases of candidemia in other parts of the world like France [bib_ref] Epidemiology of Candidemia in NICE Area, France: A Five-Year Study of Antifungal..., Vannini [/bib_ref] , Italy [bib_ref] Epidemiology of Invasive Fungal Infections in the Intensive Care Unit: Results of..., Montagna [/bib_ref] , or the United States [bib_ref] Epidemiology and Outcomes of Candidemia in 3648 Patients: Data From the Prospective..., Pfaller [/bib_ref]. Compiled data from the ARTEMIS DISK registry including data from 147 medical centers in 41 countries all over the world for the years 1997-2007 indicated that despite a decrease in frequency, C. albicans remained the most frequent species worldwide, while the frequency of C. glabrata and C. krusei was stable, and the frequency of C. parapsilosis and C. tropicalis increased [bib_ref] Results From the ARTEMIS DISK Global Antifungal Surveillance Study 1997 to 2007:..., Pfaller [/bib_ref]. Echinocandins are recommended as first-line therapies for invasive candidiasis, and azoles can be used, as step-down therapies, but decreased susceptibility and even resistance to antifungals can occur. Decreased susceptibility or drug resistance in Candida species can be intrinsic, like decreased caspofungin susceptibility in C. parapsilosis [bib_ref] In Vitro Susceptibilities of Candida spp. To Caspofungin: Four Years of Global..., Pfaller [/bib_ref] or fluconazole resistance in C. krusei [bib_ref] Mechanism of Fluconazole Resistance in Candida Krusei, Orozco [/bib_ref] , or can be acquired like echinocandin resistance in C. glabrata [bib_ref] Emergence of Resistant Candida Glabrata in Germany, Aldejohann [/bib_ref]. The high mortality rate, the shift towards more difficult to treat Candida species, and the lack of efficacy in monotherapy for some difficult-to-treat infections make alternative approaches necessary [bib_ref] Role of Antifungal Combinations in Difficult to Treat Candida Infections, Vitale [/bib_ref]. It has been shown in vitro [bib_ref] Combination of Amphotericin B With Flucytosine is Active In Vitro Against Flucytosine-Resistant..., Schwarz [/bib_ref] , in vivo [bib_ref] Efficacy of Amphotericin B in Combination With Flucytosine Against Flucytosine-Susceptible or Flucytosine-Resistant..., Schwarz [/bib_ref] , and in patients [bib_ref] Combination Antifungal Therapy for Cryptococcal Meningitis, Day [/bib_ref] that a combination of two antifungals can increase their potency and decrease resistance. However, against invasive candidiasis, no favorable combination has been found [bib_ref] Combination Antifungal Therapy, Johnson [/bib_ref]. Apart from the combination of two antifungals, combinations of antifungal and other molecules can also lead to favorable interactions [bib_ref] Antifungal Combinations in Mucorales: A Microbiological Perspective, Schwarz [/bib_ref]. The antibiotic colistin has shown in vitro synergy in combination with amphotericin B against Candida species [bib_ref] In Vitro Activity of Amphotericin B in Combination With Colistin Against Fungi..., Schwarz [/bib_ref] , but routine use in critically ill patients is limited due to the nephrotoxicity of the molecules. Isavuconazole is a broadspectrum azole with favorable tolerability [bib_ref] Isavuconazole: A New Broad-Spectrum Azole. Part 2: Pharmacokinetics and Clinical Activity, Ledoux [/bib_ref] , which makes it an interesting partner for combinations of molecules. In vitro combination of isavuconazole with colistin has been shown to be synergistic against Aspergillus nidulans, Aspergillus niger [bib_ref] Colistin and Isavuconazole Interact Synergistically, Schwarz [/bib_ref] , and Candida aurisand could thus be also an interesting combination against Candida species. We therefore explored the combination of isavuconazole with colistin against common Candida species by a checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology for antifungal susceptibility testing.
# Materials and methods
## Strains
In this study, a total of 57 clinical Candida strains belonging to 6 common Candida species were included. Candida species included 10 C. albicans, 10 C. glabrata (teleomorph in the Nakaseomyces clade), 8 Candida kefyr (teleomorph Kluyveromyces marxianus), 10 C. krusei (teleomorph Pichia kudriavzevii), 9 C. parapsilosis, and 10 C. tropicalis. Candida strains were mainly obtained from the Department of Microbiology of the University Hospital Marburg (n = 53). Additional isolates (n = 4) belonged to the collections of the American Type Culture Collection (ATCC) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM). The complete Internal Transcribed Spacer (ITS)1-5.8S-ITS2 region of the non-collection strains was sequenced as described elsewhere [bib_ref] Molecular Identification of Zygomycetes From Culture and Experimentally Infected Tissues, Schwarz [/bib_ref] to obtain molecular identification of the strains to the species level. Sequences were deposited at GenBank under the accession numbers OL351325 to OL351356 [bib_ref] In Vitro Activity of Amphotericin B in Combination With Colistin Against Fungi..., Schwarz [/bib_ref] and under OM859334 to OM859357. Only C. krusei strain U2106778 was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) due to ITS region sequence heterogeneity [bib_ref] Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical..., Zhao [/bib_ref]. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality controls in each batch of microplates.
## Drugs
Isavuconazole (Pfizer, Berlin, Germany) stock solution was prepared at 3,200 µg/ml in dimethyl sulfoxide (DMSO). A stock solution of colistin (Merck, Darmstadt, Germany) at 12,800 µg/ml was prepared in sterile, distilled water. Stock solutions were kept at −25°C until use.
## Medium preparation
For this study, Roswell Park Memorial Institute 1640 (RPMI) medium (with L-glutamine, with pH indicator, but without bicarbonate) (Merck) prepared in double strength was used as the test medium. It contained 2% (w/v) of D-glucose and was buffered with 3-(N-morpholino)propanesulfonic acid (Merck) at a final concentration of 0.165 mol/L. The final pH of 7.0 was adjusted with 2 molar NaOH. The medium was sterilized through a 0.22-µm pore size filter by vacuum filtration (Merck).
## Microplate preparation
An antifungal susceptibility testing protocol, modified for broth microdilution checkerboard procedures, based on the EUCAST guidelines was used in this study. Combination experiments were carried out in Nunclon ™ delta surface 96-well microtiter plates for adherent cells (Thermo Fisher Scientific, Darmstadt, Germany). The combination of isavuconazole with colistin was studied on a two-dimensional checkerboard [bib_ref] Techniques for the Assessment of In Vitro and In Vivo Antifungal Combinations, Bidaud [/bib_ref]. Twofold serial dilutions of each drug were done in the double strength test medium. Final concentrations for isavuconazole ranged from 0.0001 to 0.03 µg/ ml for C. albicans, C. kefyr, C. parapsilosis, and C. tropicalis or 0.002-0.5 µg/ml for C. glabrata and C. krusei. Colistin concentrations ranged from 1 to 64 µg/ml for all tested species. Before the addition of the inoculum, each well contained 100 µl of double-strength RPMI medium with 1% (v/v) of DMSO.
## Inoculum preparation and inoculation of microplates
Before the experiments, Candida strains were cultured on Sabouraud dextrose agar slants supplemented with chloramphenicol and gentamicin (Bio-Rad Laboratories, Feldkirchen, Germany) at 35°C and 95% humidity for 24 h. By using inoculation loops, fungi were transferred from the agar slants to sterile tubes containing pure water. After the cells were counted in a hemocytometer, the suspensions were adjusted to the final inoculum size of 2 × 10 5 colony forming units (CFU)/ ml. One hundred microliters of the inoculum was distributed into each well of the microplates using Eppendorf Xplorer plus (Eppendorf, Hamburg, Germany) electric multichannel pipettes. After incubation at 35°C and 95% humidity for 24 h, optical densities were read spectrophotometrically at a wavelength of 530 nm using a MultiSkan FC spectrometer (Thermo Fisher Scientific). Before the reading, microplates were shaken for 2 min at 1,100 rpm with a PMS-1000 Microplate Shaker (Grant Instruments, Shepreth, UK). Before the analysis of the results, the optical density values of a blank plate, each well inoculated with 100 µl of sterile distilled water and incubated under the same conditions as mentioned above, were subtracted from the values of the microplates inoculated with yeast cells. The final inoculum was further diluted at 1:10, and 50 µl were spread once on Sabouraud dextrose agar plates with a sterile Drigalski spatula. After 24 h of incubation, CFU were counted to ensure inoculum size. Combination experiments were run in duplicate.
## Interpretation of the results by fractional inhibitory concentration index
Optical density values from the microplates were transformed into a percentage of growth compared to the growth control. Fifty percent of inhibition was chosen as an endpoint for the determination of the minimum inhibitory concentrations (MICs) alone for both drugs and in combination. High offscale MICs were converted to the next log 2 dilution. If the lowest fractional inhibitory concentration index (FICI) on the microplate was ≤0.5 or >0.5 to 4, synergy or indifference was assumed, respectively. For FICIs higher than 4.0, antagonism was concluded [bib_ref] Synergy, Antagonism, and What the Chequerboard Puts Between Them, Odds [/bib_ref].
## Interpretation of the results by response surface analysis
Compared to the FICI, response surface analysis allows the evaluation of drug interactions without using an inhibition endpoint and is therefore independent of MICs. It enables the determination (and visualization) of the interaction for all tested concentrations and not only for the MICs in combination. Based on the growth rates in the wells of the molecules alone (in this study isavuconazole and colistin), dose-response curves for the drugs alone are generated. According to the chosen theoretical model, using the dose-response curves of the two drugs alone, an indifferent dose-response surface is calculated. In this study, the Bliss independence model was chosen, which is based on the hypothesis that drugs act independently from each other. To determine the interaction of the molecules, the (experimentally) observed combination dose-response surface is compared to the predicted (calculated) indifferent dose-response surface. A combination effect is defined as synergistic if the observed effect lies below the predicted indifferent dose-response surface (corresponds to less growth on the microplate and a greater effect of the combination) and antagonistic when the observed effect lies above (corresponds to more growth on the microplate and a weaker effect of the combination). To quantitatively assess the interaction of the drugs, the SUM-SYN-ANT metric is calculated, which is defined as the sum of all effects greater than the predicted indifferent effect (SYN-SUM), minus the sum of all effects weaker than the predicted indifferent effect (ANT-SUM). The intrinsic variability of the broth microdilution technique necessitates the definition of a threshold, for which the interaction of the two drugs is defined as indifferent. This threshold is determined experimentally by combining the active molecules with themselves. Therefore, the combination of isavuconazole with itself was tested on the two-dimensional checkerboard with twofold serial dilutions as described above. The highest concentration of isavuconazole was 0.12 µg/ml in the x-and y-axes. For the determination of the threshold, C. krusei ATCC 6258 was tested in triplicate on these plates. Based on the results of the experimental plates, synergy was assumed when the SUM-SYN-ANT was ≥56.0%, and antagonism was assumed when ≤−56.0%. Between −56.0% and 56.0%, indifference was concluded.
To determine the SUM-SYN-ANT metric of the different tested strains, the results of both runs were combined. All calculations were done by the Combenefit software (http:// sourceforge.net/projects/combenefit/) (Di [bib_ref] Combenefit: An Interactive Platform for the Analysis and Visualization of Drug Combinations, Veroli [/bib_ref].
# Results
The interactions of isavuconazole with colistin were evaluated by checkerboard and interpretation of the results by FICI or by response surface analysis against strains from six common Candida species as presented in [fig_ref] TABLE 1 |: Interaction of isavuconazole with colistin against common Candida species by checkerboard and... [/fig_ref]. A summary of the results is presented in . [fig_ref] FIGURE 1 |: Synergy distribution for the combination of isavuconazole with colistin against Candida albicans... [/fig_ref] shows the synergy distributions for the combination of isavuconazole with colistin against a representative isolate of each tested species.
The 57 Candida strains exhibited MICs for isavuconazole ranging from 0.0001 to 0.5 mg/ml [fig_ref] TABLE 1 |: Interaction of isavuconazole with colistin against common Candida species by checkerboard and... [/fig_ref] with MIC50, MIC90, and geometric mean MIC of 0.008, 0.125, and 0.018 mg/ml, respectively. Isavuconazole MICs ranged from 0.0005 to 0.004, 0.125 to 0.5, 0.03 to 0.25, 0.008 to 0.03, 0.008 to 0.06, and 0.0001 to 0.008 µg/ml for C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, and C. kefyr, respectively. When tested alone, colistin exhibited MICs ranging from 16 to 128 µg/ml for the different species (128 mg/ml being the high-off scale MIC) with MIC50, MIC90, and geometric mean MIC of 64, 128, and 63.23 mg/ml, respectively. The best activity of colistin was seen against C. tropicalis with MICs ranging from 16 to 32 µg/ml. Significant activity was also seen for C. kefyr with a geometric mean MIC of 32 µg/ml. Against C. krusei, colistin MICs were high with a geometric mean MIC of 59.71 µg/ml. Colistin showed almost no activity against C. albicans, C. glabrata, and C. parapsilosis with geometric mean MICs of 119.43, 119.43, and 109.73 µg/ml, respectively. The geometric mean MIC for colistin in combination with the synergistic strains was 9.93 µg/ml. Between experiments, isavuconazole and colistin MICs were within ±1 log 2 dilutions in 99.12% of the cases for all Candida species tested (data not shown). The interaction was synergistic for 58% of the strains with FICIs ranging from 0.25 to 0.5 with a geometric mean FICI of 0.4. Synergy was obtained for 0%, 33%, 50%, 80%, 90%, and 90% of C. albicans, C. parapsilosis, C. kefyr, C. krusei, C. glabrata, and C. tropicalis, respectively . All other interactions were indifferent. The geometric mean FICI for all strains was 0.48. Analysis of the checkerboard data of the 57 Candida strains by the response surface approach led to similar results compared to the FICI results. Although synergy was less frequently obtained than by FICI analysis, the tendency if the combination is synergistic, or for a certain species indifferent (at least 50% of the strains for both interpretation models), was correct in 5/6 cases . The SUM-SYN-ANT metric for the synergistic strains ranged from 59.37 to 122.67, with a mean of 78.22. For C. glabrata, the mean of the SUM-SYN-ANT metric of all strains was 40.42. Synergy was obtained for 0%, 0%, 10%, 50%, 50%, and 67% of C. albicans, C. parapsilosis, C. glabrata, C. krusei, C. tropicalis, and C. kefyr, respectively. When comparing the results of the FICI with the response surface approach, synergy was obtained for at least 50% of the strains for both interpretation models for C. kefyr, C. krusei, and C. tropicalis. One difference between the interpretation techniques was that interaction against C. glabrata was synergistic (9 of 10 strains) by FICI and indifferent by response surface analysis (9 of 10 strains).
# Discussion
Advantages of drug repurposing are the use of de-risked compounds, potentially lower development costs, and shorter development timelines [bib_ref] Drug Repurposing: Progress, Challenges and Recommendations, Pushpakom [/bib_ref]. Repurposed drugs against Candida belong to the antihelmintics (quinacrine), cytotoxic agents (doxorubicin and daunorubicin), or 5a- reductase inhibitors (finasteride) and inhibit filamentation [bib_ref] Drug Repurposing for the Treatment of Bacterial and Fungal Infections, Miro-Canturri [/bib_ref]. Colistin is an antibiotic with activity against gram-negative bacteria [bib_ref] Resurgence of Colistin: A Review of Resistance, Toxicity, Pharmacodynamics, and Dosing, Lim [/bib_ref] , but it has been shown to be able to damage the membrane in C. albicansand increase its permeability [bib_ref] Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality, Bibi [/bib_ref] , which makes the molecule an interesting partner to test combinations with antifungals. Compared to other studies, the MICs of isavuconazole for the different Candida species in this study were in the same range as previously reported [bib_ref] Isavuconazole MIC Distribution of 29 Yeast Species Responsible for Invasive Infections, Desnos-Ollivier [/bib_ref] [bib_ref] EUCAST Susceptibility Testing of Isavuconazole: MIC Data for Contemporary Clinical Mold and..., Jorgensen [/bib_ref]. MICs for colistin were comparable to those of another study, but apart from C. albicans, only one strain of each species has been tested [bib_ref] Synergy of the Antibiotic Colistin With Echinocandin Antifungals in Candida Species, Zeidler [/bib_ref]. Compared to our recently published study on the combination of amphotericin B and colistin, colistin MICs were similar [bib_ref] In Vitro Activity of Amphotericin B in Combination With Colistin Against Fungi..., Schwarz [/bib_ref].
Combination MICs of colistin ranged from 1 to 32 mg/ml, with a geometric mean MIC for the synergistic strains of 9.93 mg/ ml. Peak serum levels of 13 to 32 have been reported in patients with cystic fibrosis [bib_ref] The Pharmacokinetics of Colistin in Patients With Cystic Fibrosis, Reed [/bib_ref] , which would be sufficient against the strains tested in this study, but colistin use in patients is limited due to its nephrotoxicity [bib_ref] A Review on Colistin Nephrotoxicity, Javan [/bib_ref]. However, in patients with cryptococcosis, it has been shown that synergy can be achieved with lower serum levels than those tested in vitro [bib_ref] Calcineurin Inhibitor Agents Interact Synergistically With Antifungal Agents In Vitro Against Cryptococcus..., Kontoyiannis [/bib_ref].
Against common Candida species, in vitro synergy of combinations including colistin has been reported for echinocandins [bib_ref] Synergy of the Antibiotic Colistin With Echinocandin Antifungals in Candida Species, Zeidler [/bib_ref] and for amphotericin B [bib_ref] In Vitro Activity of Amphotericin B in Combination With Colistin Against Fungi..., Schwarz [/bib_ref]. Combinations of colistin with azoles were evaluated by two studies. One study found indifference for the combinations of colistin with either fluconazole or itraconazole against C. albicans, but both combinations were only tested against one strain. Our study is in accordance with these results. We found indifference for the combination of isavuconazole with colistin by checkerboard and interpretation of the results by FICI and response surface analysis. Another study also evaluated the combination of colistin with fluconazole by checkerboard against one C. albicans strain and found synergy in vitro and in vivo in a Galleria mellonella model of invasive candidiasis [bib_ref] Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality, Bibi [/bib_ref]. This discrepancy compared to our results might be strain specific, as only one strain was tested or might be related to the different azole used.
For the other species tested in this study, we found synergy for the majority, or at least for half of the tested strains, for C. glabrata, C. krusei, C. tropicalis, and C. kefyr. Only C. parapsilosis indifference was found for the majority of the strains. Although synergy was less frequently seen, when the checkboard data were evaluated by response surface analysis, at least 50% of the strains exhibited synergy regardless of the interpretation model used for C. kefyr, C. krusei, and C. tropicalis. For C. parapsilosis, both interpretation models evaluated indifference. Between the two interpretation techniques, one difference was found for C. glabrata. Interpretation by the FICI showed synergy for 90% of the strains, while interpretation by response surface analysis exhibited indifference for 90% of the strains. Nevertheless, despite the formal indifference of the C. glabrata results by response surface analysis, the mean of the SUM-SYN-ANT metric of all strains was quite high (40.42). The discrepancy could indeed be related to the stringent threshold (56.0) used in the present work compared to previous studies [bib_ref] In Vitro Activity of Amphotericin B in Combination With Colistin Against Fungi..., Schwarz [/bib_ref].
In conclusion, we found in vitro synergy of the combination of isavuconazole with colistin against C. glabrata, C. krusei, C. tropicalis, and C. kefyr. Against C. albicans and C. parapsilosis, the combination only exhibited indifference. Antagonism was never seen regardless of interpretation model used. These results warrant further animal experiments.
# Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/genbank/, OM859334 to OM859357 https://www. ncbi.nlm.nih.gov/genbank/, OL351325 to OL351356.
# Author contributions
[fig] FIGURE 1 |: Synergy distribution for the combination of isavuconazole with colistin against Candida albicans V2105529, Candida glabrata U2106602, Candida kefyr V2108462, Candida krusei N2102435, Candida parapsilosis V2105056, and Candida tropicalis N2102715. The mode of interaction was defined based on the SUM-SYN-ANT metric. IND, indifference; SYN, synergy. [/fig]
[fig] 2 |FICI: Summary of the in vitro interactions of isavuconazole with colistin against common Candida species evaluated by EUCAST broth microdilution checkerboard methodology and interpretation by fractional inhibitory concentration index and response surface analysis. , fractional inhibitory concentration index; RSA, response surface analysis; EUCAST, European Committee on Antimicrobial Susceptibility Testing. [/fig]
[fig] FUNDING: Open Access funding provided by the Open Acess Publication Fund of Philipps-Universität Marburg with support of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). This study was supported by internal funding. [/fig]
[table] TABLE 1 |: Interaction of isavuconazole with colistin against common Candida species by checkerboard and interpretation by fractional inhibitory concentration index and response surface analysis. [/table]
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Dietary Factors Associated with Dental Erosion: A Meta-Analysis
Background: Some diet factors are risk factors for dental erosion.Methods: We performed computer searches of PubMed, Cochrane Library, EBSCO, CALIS, et al., to search for studies investigating risk factors for dental erosion. For risk factors investigated in a comparative way, we computed pooled odds ratios (ORs) using the Mantel and Haenszel method.Results: A total of 9 studies met the inclusion criteria, and 6 risk factors were considered, including soft drinks, sports drinks, juice, vitamin C, milk, and yoghourt. The following associations were found for soft drinks (OR = 2.41, 95%CI = 2.03-2.85) and vitamin C (OR = 1.16, 95%CI = 1.10-1.22). While juice (OR = 0.90, 95%CI = 0.25-3.24), sports drinks (OR = 1.58, 95%CI = 0.88-2.85), milk (OR = 0.67, 95%CI = 0.11-4.01), and yoghourt products (OR = 1.05, 95%CI = 0.28-3.96) were not associated with dental erosion.Conclusions: This meta-analysis provides comprehensive evidence-based assessment of diet-related factors for dental erosion. Preventive strategies should be taken to reduce dental erosion.
# Introduction
Dental erosion is clinically defined as the progressive and irreversible loss of dental hard tissue caused by a chemical process of acid dissolution that does not involve bacteria [bib_ref] Definition of erosion and links to tooth wear, Ganss [/bib_ref]. According to recent studies, there is some evidence that the presence of dental erosion is steadily increasing [bib_ref] Systematic review of the prevalence of tooth wear in children and adolescents, Kreulen [/bib_ref]. Dental erosion can have extrinsic or intrinsic causes. Extrinsic factors include demineralizing acidic foods-such as citrus fruits and acidic beverages and some medicines-such as effervescent vitamin C preparations, chewable vitamin C tablets [bib_ref] The role of diet in the aetiology of dental erosion, Lussi [/bib_ref] [bib_ref] Erosion due to vitamin C tablets, Meurman [/bib_ref]. Intrinsic causes of erosion include recurrent vomiting as a result of psychological disorders, e.g., in anorexia and bulimia or regurgitation of gastric contents because of some abnormality in the gastrointestinal tract [bib_ref] Gastric reflux is a significant causative factor of tooth erosion, Holbrook [/bib_ref]. One important additional factor in dental erosion is low salivary flow, which, naturally, results in inadequate rinsing and buffering of demineralizing acids on tooth surfaces [bib_ref] The effect of salivary factors on dental erosion in various age groups..., Piangprach [/bib_ref].
There is growing evidence of a considerable increase in consumption of potentially erosive drinks. There have been significant associations shown between soft drink consumption and dental erosion [bib_ref] Dental erosion and soft drink consumption in Swedish children and adolescents and..., Hasselkvist [/bib_ref]. Many previous reports have found dental erosion to be significantly associated with the diet factors although some have not [bib_ref] Diet and dental erosion in young people in southeast Brazil, Waterhouse [/bib_ref] [bib_ref] A multifactorial analysis of factors associated with dental erosion, Dugmore [/bib_ref] [bib_ref] Epidemiological studies of tooth wear and dental erosion in 14-year old children..., Milosevic [/bib_ref] [bib_ref] Maxillary incisor palatal erosion: no correlation with dietary variables?, Chadwick [/bib_ref] [bib_ref] The prevalence of tooth wear in a cluster sample of adolescent schoolchildren..., Bartlett [/bib_ref] [bib_ref] The prevalence of dental erosion in preschool children in China, Luo [/bib_ref] [bib_ref] Dental erosion among 12-14 year old school children in Khartoum: a pilot..., Karim [/bib_ref] [bib_ref] Prevalence and associated factors of dental erosion in children and adolescents of..., Nahás Pires Corrêa [/bib_ref] [bib_ref] Factors associated with the incidence of erosive wear in upper incisors and..., Aidi [/bib_ref] [bib_ref] Risk indicators for tooth wear in Sri Lankan adolescents, Ratnayake [/bib_ref] [bib_ref] Prevalence of tooth erosion of 5-year-old and 12-year-old children in Xuzhou city, Chen [/bib_ref] [bib_ref] Diet and dental erosion in young people in south-east Brazil, Waterhouse [/bib_ref] [bib_ref] A multifactorial analysis of factors associated with dental erosion, Gurgel [/bib_ref] [bib_ref] Epidemiological studies of tooth wear and dental erosion in 14-year old children..., Milosevic [/bib_ref] [bib_ref] Tooth wear in the deciduous dentition of 5-7-year-old children: risk factors, Gatou [/bib_ref] [bib_ref] Risk factors associated with tooth wear in teenagers: a case control study, Milosevic [/bib_ref]. However, the contributions of the associated diet factors to dental erosion are still unclear. Therefore, the aim of this meta-analysis study was to review dietary factors associated with dental erosion, with emphasis on estimation of the quantitative contributions.
# Methods
## Search strategy and selection criteria
As the basis for our analysis, we performed a computer searches of PubMed, Cochrane Library, EBSCO, CALIS, Wanfang Database, China National Knowledge Infrastructure (CNKI) and Weipu Database for the literature form 1992 to December 2011. The search themes were combined as ''(carbonated or soft drink or soft drinks) and (water or milk or juice or fruits or diet) and (tooth or teeth or dental) not vitro''. In addition, we performed a Google Scholar search.
Two investigators (YZ, HL) independently reviewed titles and abstracts, and selected articles addressing diet and dental erosion. Disagreements were resolved by discussion and consensus. On a second sift; we selected original studies on diet and dental erosion with the following inclusion criteria :(1)Addressing diet and dental erosion, [bib_ref] Systematic review of the prevalence of tooth wear in children and adolescents, Kreulen [/bib_ref] The study should provide sample size, (3)The study should provide associated factors with odds ratio with interval. Studies were exclude if one of the following existed: (1) Not related to diet and dental erosion, (2) Animal studies, (3) odds ratios or frequency were not reported, and (4) reviews and abstracts.
The computer search produced 417 citations. Records after duplicates removed resulted in 417 citations. Review of titles and abstracts resulted in the selection of 12 papers, among which 9 met the inclusion criteria.
## Data extraction
For each study included, the full text was retrieved and the following data were extracted: location, year of publication, size and mean age of the sample. For each associated factors, we extracted the Odds Ratios(ORs) together with confidence intervals (CIs).
Two investigators performed the extraction of the data in duplicate to avoid errors. Multivariate estimates were always selected when available; otherwise the unadjusted results were recorded. We pooled studies that present ORs.
Given the high number of potential associated factors, we restricted our analysis to those for which the risk was assessed by at least 4 studies. Each associated factor was analyzed separately.
# Statistical analysis
We used SAS (r) Software 9.2 to analyze data. We assessed statistical heterogeneity among studies using the x 2 test and I 2 statistics was calculated to quantify the proportion of the total variation due to heterogeneity. We estimated ORs and 95% CIs using Mantel and Haenszel method [bib_ref] Meta-analysis in clinical trials, Dersimonian [/bib_ref] using generic inverse variance methodwhen the P value .0.05 for the Q test which indicated a lack of heterogeneity among the studies. Forest plots were created, which show the effect estimate, level of variability around that estimate for each study and the weight given to each study in the meta-analysis along with the overall pooled result. Statistical significance was set at P,0.05 and 95% confidence intervals are quoted throughout. The two authors inputted the data in the statistic software SAS (r) Software 9.2 to perform the statistical analysis in dependently and got the same results.
Forest plots were created, which show the effect estimate [fig_ref] Figure 2: Meta-analysis for the association between soft drink risk and dental erosion [/fig_ref] , 5, 6, 7). [fig_ref] Figure 1: Flow chat of selection of studies for inclusion in meta-analysis [/fig_ref] presents the flow of papers through the study selection process. The literature search produced 417 citations. Review of titles and abstracts resulted in the selection of 12 papers, among which 9 met the inclusion criteria. Selected characteristics of the 9 included articles were reported individually in . Most studies were conducted in UK and Asia.
# Results
When interpreting the result from this meta-analysis, one must consider the issue of heterogeneity among studies. In the process of meta-analysis of 6 associated factors, the P value of the heterogeneity test in the overall analysis was .0.10, since a Mantel and Haenszel method was used. In most instances, however, results were fairly consistent in the direction of the effect (ie, pointing toward an increase or decrease in risk), even though studies differed in the estimation of the effect size.
# Discussion
Although many studies have investigated associated factors for dental erosion, a comprehensive and quantitative summary has been lacking and several studies presented crude ORs only despite the possible confounders on the association between each factor and the risk of dental erosion. This review underscores factors that associated to dental erosion, and although most associations are not large, these factors are common for the association with dental erosion. Soft drink was associated with about 2.4 fold risk of dental erosion in this meta-analysis. Soft drinks, excluding milk and water could cause damage to the teeth for two reasons: Firstly, the low pH and high titratable acidity. Secondly the sugars in drinks are metabolized by plaque microorganisms to generate organic acids that bring about demineralization. Erosion is due to the loss of the outermost surface of enamel and occurs when the surface pH falls below 5.5 [bib_ref] Etiology of dental erosion-extrinsic factors, Zero [/bib_ref]. For instance, the mean pH of Coca Cola samples analyzed was 2.30, while the mean calcium and fluoride ion concentration were 0.58 and 0.066 respectively. The low pH as well as the low calcium and fluoride ion concentration indicate the high erosive potential [bib_ref] Influence of beverage composition on the results of erosive potential measurement by..., Jager [/bib_ref]. So intervention measures should be taken to prevent or reduce dental erosion from diet factors. Dental professionals should educate the patient about the consequences of frequent soft drink consumption and provide positive suggestions to minimize the risk. Public health providers should guide the people especially teenagers and children to limit the intake of soft drinks. Oral health educators should reinforce important practices to soft drink users such as decreasing the time that the soft drink remains in the mouth.
Chewing vitamin C tablets were significantly associated with the development of tooth wear. Vitamin C (ascorbic acid) has low pH and high titratable acidity. In the light of World Health Organization's recommendations to consume at least 400 g fruit per day to prevent the onset of chronic conditions, people should be guided by public health providers and oral health educators to develop reasonable oral health behavior.
Juice, sports drink, milk and yoghourt products were not found to be associated with dental erosion in this present meta-analysis. Milk and Yogurt provides an important source of dietary calcium, phosphate and casein, all of which are known to protect enamel [bib_ref] Effect of CPP-ACP paste on mechanical properties of bovine enamel as determined..., Yamaguchi [/bib_ref]. Since the significant risk factors included soft drinks, the frequent consumption of milk could be considered as a substitute way in diet behavior to prevent dental erosion.
The present meta-analysis had several limitations that must be taken into account. First, the number of available studies that could be included in this meta-analysis was moderate. Therefore, the results could be influenced by the factors like random error. Second, though we have searched as many publications as we could by means of various searching approaches, these results may be biased by the relatively small number of subjects.
This meta-analysis provides the first comprehensive evidencebased assessment of relevant factors on dental erosion and intends to drive more attention to the necessity of further community education concerning the loss of the hard tooth substances due to erosion. Erosions are mainly caused by excessive consumption of erosive food and drinks especially among children. In addition, considering the limitation of this study, further larger studies should be considered and investigated to validate the current meta-analysis.
# Author contributions
[fig] Figure 1: Flow chat of selection of studies for inclusion in meta-analysis. doi:10.1371/journal.pone.0042626.g001 [/fig]
[fig] Figure 2: Meta-analysis for the association between soft drink risk and dental erosion. doi:10.1371/journal.pone. [/fig]
[fig] Figure 3: Meta-analysis for the association between juice risk and dental erosion. doi:10.1371/journal.pone.0042626.g003 [/fig]
[fig] Figure 4: Meta-analysis for the association between sports drink risk and dental erosion. doi:10.1371/journal.pone.0042626.g004 Dietary Factors and Dental Erosion PLOS ONE | www.plosone.org [/fig]
[fig] Figure 5: Meta-analysis for the association between milk risk and dental erosion. doi:10.1371/journal.pone.0042626.g005 [/fig]
[fig] Figure 6: Meta-analysis for the association between vitamin C risk and dental erosion. doi:10.1371/journal.pone.0042626.g006 [/fig]
[fig] Figure 7: Meta-analysis for the association between yoghourt products risk and dental erosion. doi:10.1371/journal.pone. [/fig]
[table] Table 1 Table 2: Summary of studies for dietary factors associated with dental erosion. NR indicates not reported. doi:10.1371/journal.pone.0042626.t001 Associated diet factors for dental erosion. [/table]
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Analysis of Anaphylactic Shock Caused by 17 Types of Traditional Chinese Medicine Injections Used to Treat Cardiovascular and Cerebrovascular Diseases
Several reports describing anaphylactic shock following treatment of cardiovascular and cerebrovascular diseases with Chinese herbal injections were described. Our analysis of these reports showed that anaphylactic shock caused by traditional Chinese medicine (TCM) injections for the treatment of cardiovascular and cerebrovascular diseases is common but also sometimes fatal. Therefore, we proposed the following four suggestions for improving the clinical safety of delivering Chinese herbal injections and reducing the occurrence of allergic shock. First, patients with cardiovascular and cerebrovascular diseases are at high risk, so they should only be given TCM injections after a doctor's diagnosis and approval. Second, people in allergic groups can suffer anaphylactic shock, so vigilance is important in the treatment of all age groups, although even more caution should be exercised when treating children or elderly people. In fact, TCM injections may not be appropriate for those age groups, so that they should be carefully considered before treatment. Third, no significant gender differences have been noted in patients with anaphylactic shock, so all patients should be carefully monitored, irrespective of gender. Fourth, the timeframe in which different drugs cause anaphylactic shock varies; thus, patients should be observed as long as possible.
# Introduction
In recent years, traditional Chinese medicine (TCM) injections have been widely used in the clinic for the treatment of many conditions, including hypertension, coronary heart disease, diabetes, nephrosis syndrome, rheumatoid arthritis, fracture, and cervical degenerative disease. However, a new dosage form of TCM injections has changed the traditional route of administration, while still retaining the characteristics of TCM. This new form works more quickly and effectively in treating certain disease [bib_ref] Analysis of the adverse reaction of traditional Chinese medicine injections activating blood..., Liu [/bib_ref] , especially for cardiovascular and cerebrovascular disease, digestive system disease, respiratory system disease, tumor, and so on. It is found that compound Danshen, Honghua, Shuxuetong, Ginkgo biloba, ligustrazine, Erigeron breviscapus, Ciwujia, Mailuoning, Ge Gensu, and Dan Hong injection have obvious advantages in the treatment of coronary heart disease, angina, and acute cerebral infarction [bib_ref] Application of traditional Chinese medicine injection in the cardiovascular and cerebrovascular disease, Liu [/bib_ref]. Recently, with the increased incidence of cardiovascular and cerebrovascular diseases, the usage of TCM injections has increased each year due to its clinical efficacy. However, despite the effectiveness of TCM injections in treating some diseases, its toxicity has been of concern as it can induce adverse drug reactions (ADRs), such as allergy including anaphylactic shock, a common side effect. Therefore, the toxicity of TCM injections should be recognized and, as such, be carefully utilized.
Allergy occurs when the immune system develops specific antibodies against an exogenous antigen (allergen) that results in an exaggerated response toward a substance that is normally harmless, thereby causing tissue damage. Anaphylactic shock is a severe, systemic allergic reaction to a specific allergen that occurs due to acute peripheral circulatory failure. It is rapid in onset and can be fatal if not treated quickly. The clinical manifestations of anaphylactic shock include heart palpitations, chest tightness, laryngeal obstruction, dyspnea, pale or cyanotic complexion, chills, sweating, cold perception in the limbs, weak pulse, drop in blood pressure, loss of consciousness, coma, convulsions, incontinence, and even sudden cardiac death. China's National Center for Adverse Drug Reaction listed the top 10 TCM injections that caused serious side effects. Of these, seven types were responsible for the most cases of anaphylactic shock and included Shenmai injection, Xuesaitong injection, Salvia miltiorrhiza injection, compound Danshen injection, Shengmai injection, Xueshuantong injection, and Mailuoning injection. Mailuoning injection caused 64 cases of anaphylactic shock. In addition, between January 1, 2011 and December 31, 2011, the National Drug Adverse Reaction Monitoring Center received a total of 1500 ADR case reports from Mailuoning injection, of which 189 cases were severe. Compound Danshen injection caused 53 cases of anaphylactic shock. On , the Ministry of Health and China's State Food and Drug Administration jointly issued an urgent notice requesting the immediate termination of the usage, selling, and production of compound Danshen injection from Taizhou Tianrui Pharmaceutical Company. Ciwujia injection caused 33 cases of anaphylactic shock, with the most severe cases occurring after . Subsequent investigation showed that these serious ADRs were caused by drug contamination.
Since these drugs have similar pharmacological effects, their common characteristics may cause ADRs. However, these injections also have specificity, as they have distinct chemical compositions [bib_ref] Analysis of the adverse reaction of traditional Chinese medicine injections activating blood..., Liu [/bib_ref]. In 2009, the article has reported serious anaphylaxis caused by nine Chinese herbal injections used to treat common colds and upper respiratory tract infections [bib_ref] Comments on serious anaphylaxis caused by nine Chinese herbal injections used to..., Ji [/bib_ref]. Also, another article has reported anaphylactic shock and lethal anaphylaxis caused by Houttuynia cordata injection for antibacterial and antiviral therapy [bib_ref] Anaphylactic shock and lethal anaphylaxis caused by Houttuynia Cordata injection, a herbal..., Ji [/bib_ref]. So far, there is no study on anaphylactic shock caused by traditional Chinese medicine injections used to treat cardiovascular and cerebrovascular diseases. In order to further explore the characteristics underlying the ADRs to TCM injections and to provide evidence that may ensure the safe and effective clinical usage of TCM injections, we studied the general pattern and characteristics of anaphylactic shock caused by TCM injections.
## Cases
In this report we analyzed 316 articles, collected from medical journals published in China between 1980 and 2013 that described cases of anaphylactic shock caused by herbal injections for cardiovascular and cerebrovascular diseases. A total of 17 different types of herbal injections were described in these reports. Collectively, 350 episodes of anaphylactic shock and 10 deaths were reported (summarized in [fig_ref] Table 1: Patients who suffered anaphylactic shock after being treated with 17 types of... [/fig_ref]. All 10 lethal anaphylaxis incidences occurred following intravenous injection (IVI). Among the 350 anaphylactic shock cases, 4 patients (1.14%) were administered intramuscular injections (IMI), and 346 patients (98.86%) were given IVI. Patient age ranged from 9 to 97 years. Of the 350 patients who suffered anaphylactic shock, 5 were children under the age of 18, 108 were between 19 and 45 years of age, 104 were between 46 and 59 years of age, 132 were over 60 years of age, and 1 patient was a pregnant woman. The studies included a total of 169 females and 181 males. The interval between herbal injection and time of anaphylactic shock ranged from 3 s to 2.5 h. The patients in these studies were those who were hospitalized for cardiovascular and cerebrovascular diseases. When allergic reactions to herbal injections occurred, all patients were immediately taken to the emergency department [fig_ref] Table 1: Patients who suffered anaphylactic shock after being treated with 17 types of... [/fig_ref].
# Analysis
3.1. Shenmai Injection. Shenmai injection can be used for the treatment of shock, coronary heart disease, viral myocarditis, chronic cor pulmonale, and neutrophils to reduce. It also can improve the immune function of tumor patients and reduce the adverse reaction caused by chemotherapy. Shenmai injection mainly contains ginseng, Ophiopogon japonicus, the active components of ginseng saponin, flavones, and trace ginseng polysaccharides. These components can stimulate the body to produce antibodies, resulting in adverse ADRs. In addition, the improper use of red ginseng can cause severe ADRs, such as psychiatric and neurological symptoms, arrhythmia, gastrointestinal bleeding, or even death [bib_ref] Pharmacokinetics of Shengmai and Shenmai injection in healthy volunteers, Li [/bib_ref]. It is possible that the pathogenesis of red ginseng is due to component structural changes and modifications that occur after preparation of red ginseng and Ophiopogon japonicas; however, further studies are needed to validate this theory. The content of Ophiopogon japonicus in Shenmai injection is significantly lower than that of Panax species, so more studies have focused on ginseng (species of the genus Panax). Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, and Ophiopogon in D are the active and main components of Shenmai injection. Test results after Shenmai injection showed that the blood concentration of ginsenoside Rb1 was relatively high, but those of ginsenoside Rg1 and ginsenoside Re were low. Ginsenosides Rg1 and Re were rapidly distributed and eliminated in vivo, but ginsenoside Rb1 was slowly metabolized in vivo. The efficacy of ginsenoside Rb1 may be related to the fact that it has a long half-life of up to 47 h [bib_ref] Analysis of the adverse reaction of salvia miltiorrhiza and its preparations, Zhong [/bib_ref] , so the anaphylactic shock caused by Shenmai injection may be mainly due to ginsenoside Rb1.
## Ciwujia injection.
Ciwujia injection contains several active components, including Eleutheroside, isofraxidin glycosides, Ding Xiangdai, Hyperoside, and Acanthopanax senticosus polysaccharide. These components can dilate blood vessels, increase coronary blood flow, increase myocardial oxygen consumption, improve blood circulation, and increase appetite. It has been difficult to determine the main component that causes anaphylactic shock, and it may be caused by the active components themselves or impurities in the drug preparation.
As aforementioned, anaphylactic shock is a serious, potentially life-threatening allergic response. Ciwujia injection contains various active polymers, and once administered directly into the blood stream intravenously, this exogenous antigen stimulates the immune system and causes an allergic reaction. No significant relationship has been found between Type I allergic reactions and drug concentration and dosage, indicating that allergic reactions are associated with drug quality and the active components or Distributions of onset: within 1 min/between 1 min to 10 mins/between 11 mins to 60 mins/between 61 mins to 120 mins/over 120 mins.
IVI: intravenous injection. IMI: intramuscular injection.
The references of the cases are in the support material.
impurities in the drug preparation [bib_ref] Allergic reaction induced by Ciwujia injection, Yan [/bib_ref]. It has been reported that patients with drug allergies are more susceptible to anaphylactic shock from Ciwujia injection; thus, before treatment, patients should be asked to detail their history of drug sensitivity [bib_ref] Analysis of 2 cases of allergic reaction induced by Ciwujia injection, Deng [/bib_ref]. In addition, patients with idiosyncratic reactions should be banned from taking this drug [bib_ref] One case of serious allergic shock induced by refined Ciwujia injection, Zhou [/bib_ref].
## Salvia miltiorrhiza (danshen)
Injection. Salvia miltiorrhiza can dilate coronary artery, increase the blood volume, improve myocardial ischemia, promote myocardial ischemia or injury recovery, and reduce the myocardial infarction scope. It also can fight against thrombosis, regulate blood lipid, inhibit the formation of atherosclerotic plaques, and protect liver cells and gastric mucosa. It also has antiinflammatory and antiallergic effect. The mechanism underlying the allergic reaction to Salvia miltiorrhiza injection is unclear; however, there are two possibilities. First, tanshinone and acid crystals in the Salvia miltiorrhiza injection may bind to plasma proteins and have sufficient immunogenicity to cause an allergic reaction. Second, the active components of Salvia miltiorrhiza injection are caffeic acid ester derivatives that polymerize to form orthodihydroxy compounds. It is similar to the structure of tannin and also binds to plasma proteins [bib_ref] One case of Danhong injection induced serious adverse reaction, Yuan [/bib_ref]. It has been reported that the anaphylactic shock caused by Salvia miltiorrhiza injection was related to the drug concentration. Specifically, two cases [bib_ref] One case of Salvia miltiorrhiza injection intravenous drip too fast induced allergic..., Zhang [/bib_ref] of anaphylaxis occurred upon administration of 24 g Salvia miltiorrhiza using 250 mL low molecular weight dextran for an intravenous drip at 30 drops/min. However, when the Salvia miltiorrhiza concentration decreased to 10 g, no cases of anaphylactic shock occurred. Therefore, it appears that allergic shock was caused by the high density of the drug. Salvia miltiorrhiza can slow the heart rate and dilate blood vessels. At high concentrations, excessive doses, and fast drip rates, its concentration in the bloodstream increases rapidly to dilate small blood vessels and decrease blood pressure. Therefore, in the clinic, input concentration, velocity, and dose of Salvia miltiorrhiza should be tightly controlled, particularly in patients with bradycardia, to avoid the occurrence of severe ADRs [bib_ref] One case of high concentration Salvia miltiorrhiza injection induced hypovolemic shock, Jiang [/bib_ref]. In addition, Salvia miltiorrhiza combined with low molecular weight dextran has a larger probability of causing anaphylactic shock. Low molecular weight dextran functions as a blood volume expander but also has mild anticoagulant effects. At the same time, Salvia miltiorrhiza can promote blood circulation to remove blood stasis and increase the number of mast cells. After combining Salvia miltiorrhiza with low molecular weight dextran, the extracellular fluid moves from the blood vessels into the tissue, and the mast cells release chemical mediators such as histamine and serotonin. These chemical mediators can cause muscle spasms and increase vascular permeability. Furthermore, the antigenicity of dextran can stimulate the body to produce antibodies, resulting in anaphylactic shock. The residual ethanol in the Danshen injection can undermine the dextran precipitates and destroy colloidal solution, causing drug degeneration. Thus, Salvia miltiorrhiza should not be combined with low molecular weight dextran for intravenous administration [bib_ref] Analysis of adverse reaction induced by Tanshinone II a sodium sulfonate injection, Kong [/bib_ref]. The same to Danhong injection and Compound Danshen injection those contain Danshen.
## Tanshinone iia sodium sulfonate injection.
Tanshinone IIA is derived by sulfonation of the water-soluble substance Tanshinone IIA sulfonate. Tanshinone IIA is derived from phenanthrene-quinone, which is isolated from Salvia miltiorrhiza. Tanshinone IIA sodium sulfonate can increase coronary flow, inhibit platelet aggregation and antithrombotic ischemia, reduce myocardial infarct size, and so on [bib_ref] Analysis of one case of adverse reactioninduced by Tanshinone II a sodium..., Li [/bib_ref]. Tanshinone IIA increases water solubility by sulfonation, so Tanshinone IIA sodium sulfonate has high solubility in water. However, because Tanshinone IIA sodium sulfonate is susceptible to hydrolysis, the soluble components are separated from the insoluble ones; therefore, Tanshinone IIA sodium sulfonate injection has slight precipitate particles during long-term storage that can cause allergic reactions in the body. In fact, reports have shown that anaphylactic shock is caused by the small amount of impurities and resin remnants that remain after the extraction process.
## Danhong injection.
Danhong injection can be used for antiatherosclerosis, inhibit platelet aggregation, and protect vascular endothelial cells. The main components of Danhong injection are Salvia miltiorrhiza and safflower. The mechanism by which Danhong injection induces anaphylactic shock is similar to that of Salvia miltiorrhiza. Safflower injection may contain pollen protein, which can cause anaphylactic shock [bib_ref] One case of Danhong injection induced serious adverse reaction, Yuan [/bib_ref]. Because Danhong injection contains Salvia miltiorrhiza, it should also not be combined with low molecular weight dextran. This was proven when a patient in one case report suddenly succumbed to anaphylaxis after being administered an intravenous drip of Danhong combined with 250 mL low molecular weight dextran [bib_ref] Analysis of 11 cases of adverse reaction caused by Erigeron breviscapus injection, Hua [/bib_ref]. Danhong injection has certain drip specifications. For example, patients with heart conditions can receive Danhong injection at a rate of ∼30-40 drops/min, but for other adults, an injection rate of 60 drops/min is suitable; the injection rate for children varies according to age and physical circumstances. The intravenous drip rate can greatly affect the incidence of ADRs, so the doctor should carefully control drip speed and closely observe the patient's reaction. Danhong injection has a safe medication dose range and must be used in accordance with the drug dosage given in the instructions. Patients can take Danghong injection at ∼20-30 drops/min for 1-2 times per day. Danghong should be diluted with 5% glucose (100-500 mL) for injection. Some patients with allergic shock may be related with solvent. Yuan and Zhao [bib_ref] One case of Danhong injection induced serious adverse reaction, Yuan [/bib_ref] reported that one patient, aged 83, due to the eyes of ischemic optic nerve disease in hospital, with a history of hypertension, cerebral infarction, and myocardial infarction, with no history of adverse drug reaction, suffered anaphylactic shock when he/she accepted Dan Hong injection 20 mL with 0.9% NS 250 mL intravenous drip on day 8. Therefore, except special condition, the user of Danhong injection should be strictly in accordance with the drug instructions.
3.6. Breviscapine Injection. Breviscapine injection contains flavonoids, scutellarin, and the Erigeron 60 hormone, although scutellarin is the main component. Scutellarin can inhibit the intrinsic coagulation system, promote the activity of plasmin, reduce platelet count, and inhibit platelet aggregation. Flavonoids in acidic environments are easy to precipitate; therefore, breviscapine is stable in 0.9% NaCl or 5% glucose for injection. In addition, the pH should not be less than 4.2 or it will crystallize, leading to an increase in the number of particles and subsequent allergic reactions. Breviscapine injection is a pure TCM preparation with a complex composition.
## Erigeron injection. erigeron injection is composed of
Erigeron breviscapus extracted with sterile water solution made of phenolic compounds. Erigeron injection esters mainly contain scutellarin and coffee. It is commonly used for the treatment of ischemic stroke, coronary heart disease, and angina pectoris due to the fact that it promotes circulation and removes stasis. The literature has reported that Erigeron breviscapus solution is weakly alkaline (pH 7.0-7.5). When the pH is low, Erigeron breviscapus solution can easily crystallize, thereby increasing the number of insoluble particles. In addition, when glucose is used as the solvent for Erigeron breviscapus injection, there is an increase in the number of insoluble particles, whereas the number of particles is much lower when 0.9% NaCl is used as the solvent. In these studies, 5% glucose was used as the solvent in most patients, although the instructions stated that the solvent should be 0.9% NaCl. He and Lei [bib_ref] Three cases of allergic reaction induced by Erigeron injection, He [/bib_ref] [bib_ref] Three cases of allergic reaction induced by Erigeron injection, Lei [/bib_ref] reported that three patients with the use of 10% glucose or 5% glucose as solvent showed the symptoms of anaphylactic shock. The insoluble particles may be the main cause of allergic shock, so 0.9% NaCl should be used as the solvent in the clinic. Although there did not appear to be a significant correlation between drug dose and anaphylactic shock, some patients were injected with up to 100 mL Erigeron breviscapus, which is two times more than the instructions specified. In particular, elderly people had poor tolerance and were more likely to suffer from ADRs.
## Compound danshen injection.
Compound Danshen injection is composed of Danshen extract and Lignum dalbergiae odoriferae extract. Salvia miltiorrhiza can scavenge free radicals and resist lipid peroxidation; Dalbergia can reduce lipid peroxidation injury. Dalbergia and Salvia miltiorrhiza have synergistic effects and can decrease heart rate, act as a sedative, and cause hypnotic and transient hypotension. The mechanism underlying allergic reactions induced by Salvia miltiorrhiza is similar to that of compound Danshen. Long-term toxicity studies of Salvia miltiorrhiza have shown that if it is infused too quickly, a fall in blood pressure will occur, which can easily cause anaphylactic shock [bib_ref] Study on clinical adverse effects of Salvia miltiorrhiza and its preparation, Li [/bib_ref]. Compound Danshen has an injection pH range of 4 to 6.5. The number of insoluble particles greatly increased when compound Danshen was mixed with 0.9% NaCl for injection at an alkaline pH. This increases the chance that a patient will suffer from an ADR. Most patients infused at a drip speed of 85 drops/min suffered from anaphylactic shock, which often occurred as quickly as a few seconds after injection. Thus, aninfusion rate of 30 drops/min is more appropriate. Importantly, patients should be closely observed for at least 15 min after injection, as patients cannot adjust the drip speed themselves. If the patient suffers from an ADR, the nurse should immediately close the valve and report to the physician. Compound Danshen causes mild vasodilatation; if the concentration is too high or if it is infused too quickly, the blood concentration of the drug will increase rapidly in a short period of time, causing blood vessel dilatation due to small decreases in blood pressure and bradycardia, which can lead to anaphylactic shock. Therefore, the input velocity and drug concentration must be tightly controlled with compound Danshen injection [bib_ref] Description of adverse reactions Puerarin injection, Lv [/bib_ref].
## Puerarin injection.
Puerarin injection can dilate coronary and cerebral blood vessels, improve myocardial systolic function, inhibit platelet aggregation, reduce blood viscosity, and improve microcirculation. Anaphylactic shock mostly occurred in patients who were continuously using Puerarin, which belongs to I type allergic reaction. The patients recovered a few days after drug treatment was terminated and they received antiallergy medication. Anaphylactic shock may have been caused from impure substances in Puerarin resulting from drug preparation, which induced antigenicity [bib_ref] To explore the adverse reactions induced by puerarin injection, Shao [/bib_ref].
Additionally, Puerarin itself or its byproduct may have decomposed by drug metabolism and then may have combined with a protein carrier to form a hapten carrier complex, resulting in an immune response. Puerarin injection mainly contains flavonoid glycosides but also contains small amounts of daidzein, daidzin, and other effective components of TCM. These components or impurities in the formulation can serve as antigens or haptens and cause anaphylactic shock. Puerarin is an isoflavone compound with low solubility. Isoflavones as strong planarity molecular are arranged closely between the molecule and another one, so it is difficult to dissolve in water. In order to increase the solubility of Puerarin, 50% propylene glycol was added to the injection as a solvent. Propylene glycol can degrade to produce pyruvic acid, lactic acid, acetic acid, and other allergens in certain conditions, which can cause vascular stimulation in patients and symptoms of fever. In addition, propylene glycol can directly cause the dissolution of red blood cells. Studies have shown that the number of particles in TCM injections is significantly greater than that in Western medicine intravenous injections. Puerarin injection, as a TCM preparation, may produce insoluble particles that cannot be metabolized in the body, resulting in allergic reactions [bib_ref] Analysis of 63 cases of adverse reactions induced by Puerarin injection, Quan [/bib_ref].
If Puerarin injection is used as a treatment over a long period of time, toxic effects can be caused by drug accumulation. Most cases of anaphylactic shock resulted from the continuous use of Puerarin, unlike the anaphylactic shock caused by penicillin. Most people who suffered from an allergic reaction had no history of drug allergies; however, doctors should still ask patients to detail their personal and family histories of drug allergies before treatment, and patients with drug allergies should use Puerarin carefully. Patients who suffered from an ADR following Puerarin injection recovered within a few days of drug withdrawal and antiallergy medication. Before allergic shock occurred, patients had adverse reactions such as fever and chest tightness. When this occurs, doctors should immediately terminate the drug and treat the patients' symptoms.
The use of Puerarin should be strictly controlled, especially in elderly patients. Patients' blood routine, liver, and kidney function should be monitored, and treatment should not be for too long as the intermittent use of Puerarin can easily induce the production of antibodies. Large doses of medication can trigger immune complexes to cause tissue injury, and subsequent use of the medication will cause memory cells to respond quickly, leading to serious ADRs. Sun [bib_ref] Adverse reaction of Puerarin injection, Sun [/bib_ref] reported that one patient, aged 58, with coronary heart disease, showed suddenly palpitations, shortness of breath, sweating, cold extremities, and other symptoms of anaphylactic shock 30 minutes after the intravenous drip of Puerarin on day 8. Therefore, we suggest a treatment schedule of 1 course for 1 week, which should be repeated after 1 week. This course of treatment will significantly reduce the risk of ADRs. Since Puerarin is a vasodilator, the patient's full medical history should be taken into account before it is administered [bib_ref] Investigation and analysis of 11 cases of adverse reactions of Safflower injection, Xu [/bib_ref].
## Safflower injection.
Safflower injection is a TCM injection of safflower extract. It has many functions, such as scavenging free oxygen radicals, inhibiting platelet aggregation, and reducing vascular resistance and dilation of the coronary artery. Previous studies indicated that the excessive use of safflower injection can increase the number of insoluble particles, which can result in anaphylactic shock. Excessive use can also increase the liquid concentration, such that, at the same drip speed, patients are more susceptible to ADR due to the increase in pharmacological effects. The drug instructions recommend using 5% and 10% glucose (∼250-500 mL) as the dilution solvent. Lu and Ye [bib_ref] Nursing experience of one case of allergic shock due to Safflower injection, Lu [/bib_ref] reported that one patient, aged 63, suffering lumbar disc herniation, with history of cephalosporin allergy, appeared dizziness, chest tightness, shortness of breath, clammy skin, and other symptoms of anaphylactic shock when accepting intravenous injection of safflower injection +0.9% Sodium Chloride Injection 10 minutes later. In the first medication, compatibility and stability tests showed that as the pH of the solvent increased, the number of insoluble particles significantly increased as well. Thus, it is better to not combine safflower with 0.9% NaCl for injection [bib_ref] Analysis of 34 cases of adverse reactions induced by safflower injection, Zeng [/bib_ref].
ADRs induced by safflower injection involve multiple systems of the human body. Foreign antigens combine with antibodies in the material, leading to an abnormal immune reaction that may be due to the safflower injection or due to patients' specific allergies. The elements of safflower injection are complex and contain Safflor yellow and safflower glucoside. Antigens or haptens injected or transfused directly into the bloodstream can cause allergic reactions. In particular, when yellow pigment combines with sugar in the body, it forms a semiantigen. Hapten combines with the material in the red blood cell surface to form an antigen. Then, the antigen acts on the mast cells of target cells (laryngeal, tracheal, and bronchial) to produce IgE antibodies to cause allergic reactions. Some patients previously used safflower and produced specific antibodies in their bodies. In those cases, the intravenous drip of safflower injection acted as an allergen that activated the intracellular enzyme release of active substances, such as histamine like, causing an allergic reaction [bib_ref] One case of immmediate allergic reaction induced by Safflower injection, Han [/bib_ref].
Safflower injection may contain pollen protein, which can cause anaphylactic shock. Therefore, we advised manufacturers to improve the purification process of safflower injection and try to remove impurities and pollen protein [bib_ref] Analysis of 16 cases of allergic reactions induced by Safflower injection, Ge [/bib_ref].
## Mailuoning injection.
Mailuoning injection is a compound preparation that contains Achyranthes root, Radix Scrophulariae, Dendrobium, and honeysuckle. Mailuoning functions to clear away heat, nourish Yin, promote blood circulation, and remove blood stasis. The main components of honeysuckle are chlorogenic acid and isochlorogenic acid. Chlorogenic acid is an allergen that can cause allergic reactions, although it does not cause allergic reactions when being orally prepared [bib_ref] Analysis of the adverse reaction induced by Mailuoning injection, Zhang [/bib_ref]. Achyranthes, Radix Scrophulariae, Dendrobium, and honeysuckle in Mailuoning injection are from nature. People who had contact with or used these drugs previously will be sensitive to them. Therefore, the majority of allergic reactions will occur on first drug use [bib_ref] Analysis of the cases of allergic shock induced by Shengmai injection, Lei [/bib_ref]. Mailuoning injection is prepared by chemical extraction; however, due to extraction methods, its purity is not guaranteed. In addition, during the preparation process, manufacturers add solubilizing agent, stabilizer, and other additives to improve the solubility and stability of the active components of Mailuoning injection. These additives can cause allergic reactions. For example, the use of polysorbate 80 as a solubilizing agent for TCM injections correlated with the occurrence and severity of ADRs.
## Shengmai injection.
Shengmai injection contains ginseng, Ophiopogon japonicus, and Schisandra chinensis. Red ginseng is a type of ginseng, with ginsenoside being as the active component. Ginsenoside can adjust blood pressure, improve circulation, and promote metabolism and protein synthesis. The active component of Ophiopogon japonicus is Maidong saponin. The active component of Fructus Schisandra is Schisandrin. The effect of Shengmai injection is determined by the interaction of the 3 components, and there are many reasons that it can lead to ADRs.
Some possible reasons that Shengmai injection may cause anaphylactic shock include the fact that the treating physician may not fully understand the indications of Shengmai injection. For example, fracture of diaphragm fibers can be caused by Shengmai injection. Second, the physician may not ask patients about their personal and family history of drug allergies before administering the drug. This will cause some patients with drug allergies, food allergies, or chronic bronchial asthma to receive Shengmai injection, leading to ADRs. Third, some doctors may give patients an excessive dose of medication. The usage and dosage of Shengmai injection are specified as follows: 1 intravenous drip of 20-60 mL diluted with 5% glucose (250-500 mL). However, in some cases, the doctors gave an intravenous drip of 80-100 mL, which is more than the prescribed maximum dose of 60 mL. Fourth, the doctor may choose an inappropriate infusion solvent, which could lead to anaphylactic shock, although this theory needs to be further researched. Lastly, if Shengmai is infused too quickly, anaphylactic shock can occur [bib_ref] Twenty-three cases of allergic shock induced by Shenmai injection, Lei [/bib_ref]. Zhang [bib_ref] Report of 3 cases of allergic reaction caused by Shengmai injection, Zhang [/bib_ref] reported that 3 patients by intravenous of Shengmai injection 100 mL + 0.9% Sodium Chloride Solution 250 mL showed allergic shock symptoms in 5 minutes.
Since the majority of patients suffered allergic shock for the first time after Shengmai injection, and the majority of cases (13/16, 81.25%) occurred within 10 min after administration, we can conclude that allergic shock induced by Shengmai injection leads to an immediate allergic reaction. However, the components that cause anaphylactic shock are still unclear, although we speculate that ginsenoside may be the underlying culprit [bib_ref] Adverse reaction of Shuxuetong injection, Song [/bib_ref]. Only red ginseng Shengmai injection, all the effective components were determined. The effective components of Ophiopogon japonicus, Schisandra, have no quantitative determination. The quality of TCM injection cannot be guaranteed due to imperfect quality standards. Thus, additional studies are needed to strengthen the quality standard and to establish a method for determining the active components of specific drugs. This would most likely reduce the occurrence of ADRs.
## Shuxuetong injection.
Extracts of the leech and earthworm are the active components of Shuxuetong injection. These extracts contain hirudin, earthworms, earthworm enzyme, and other antithrombotic substances. Hirudin is found so far the strongest thrombin specific inhibitor. It can reduce the activity of thrombin, block the formation of fibrin, prevent hemostatic response and platelet activation response induced by thrombin, and reduce platelet activity, exerting its anticoagulant effect. This enzymatic system expresses the tissue type plasminogen activator, which has strong fibrinolytic activity. Thus, ADRs may be associated with these protein components [bib_ref] To observe the extraction process and quality control of traditional Chinese medicine..., Yu [/bib_ref]. To prepare Shuxuetong injection, large molecules, such as amino acids, peptides, and mucopolysaccharides, are removed using a filter [bib_ref] Analysis of adverse drug reactions induced by Shuxuetong injection, Kong [/bib_ref]. The molecular weight of the remaining polysaccharides, polypeptides, and amino acids with physiological activity is about 5800 Da and should be less than 1% of the Shuxuetong injection material. However, small amounts of residual polymer protein may cause ADRs [bib_ref] Two cases of adverse reaction induced by Xingnaojing injection, Qian [/bib_ref]. Although the mechanism by which ADRs occur following Shuxuetong injection remains unclear, there are two characteristic possibilities. First, the protein constituents contained in leech and earthworm may have sensitizing effects. Second, Shuxuetong injection has anticoagulant and antithrombotic pharmacological activities and improves blood rheology. Improvements in technology may lead to improvements in drug purity, thereby reducing the occurrence of ADRs [bib_ref] Analysis of one case of allergic shock caused by Shuxuetong injection, Li [/bib_ref].
3.14. Xingnaojing Injection. Xingnaojing injection has detoxicating, cooling blood, and restoring consciousness effect. Xingnaojing injection is composed of musk, borneol, turmeric, and gardenia. Protein, fatty acids, and other molecules can be used as antigens in the body to stimulate the immune system to produce antibodies. The antibody attaches to mast cells, which then become sensitized. When mast cells are exposed to the same antigen, the antigen reacts with antibodies on the surface of mast cells, after which the mast cells release particles into the surrounding media [bib_ref] Sixty-three cases of adverse reaction of Safflower yellow injection, Chen [/bib_ref]. Stimulation of the immune system can lead to an allergic reaction. The blood capillaries expand and their permeability increases, and the respiratory system, cardiovascular system, and skin and mucous membranes change to induce anaphylactic shock. The possibility of allergies induced by musk is great, possibly because musk is animal drugs [bib_ref] One case of allergic reaction caused by Xingnaojing injection, Yin [/bib_ref].
## Xuebijing injection.
Xuebijing injection mainly contains safflower, chuanxiong, and Salvia. Xuebijing injection can improve circulation, reduce infection and injuries induced by bacterial endotoxin, reduce the inflammatory response, and inhibit the formation of granuloma. During the process of extraction, there are many problems such as low purity and a large amount of residue. At the same time, the active components of safflor yellow A can enter the bloodstream and stimulate the body to produce antibodies or sensitized lymphocytes. Thus, when the allergen enters the body again, an allergic reaction will occur [bib_ref] One case of allergic purpura caused by Xuesaitong, Wu [/bib_ref].
Manufacturers need to further improve the production process and quality of their products to strengthen the quality control of Xuebijing injection, which will reduce the occurrence of ADRs. The body is in a state of stress after an operation. The number of peripheral blood phagocytes increases, enhancing the immune system. This makes the body susceptible to allergic reactions.
## Xuesaitong injection.
Xuesaitong injection is composed of panaxnoto ginseng extracted from Panax pseudoginseng. It can dilate cerebral vessels, inhibit platelet aggregation, and have antiatherosclerosis, antithrombosis, and antiarrhythmia activities. The elements of Panax notoginseng are complex, containing more than 20 kinds of saponin-active material and 17 kinds of trace elements, protein, vitamins, and polysaccharides.
There are four other possible causes of allergic reactions due to Xuesaitong injection. First, Panax notoginseng is the main component of Xuesaitong. It is not stable in aqueous solution, easily precipitates when stored, and directly affects the quality of the drug. Second, the dilution solvent for Xuesaitong is ethanol, so patients with alcohol allergies are banned from taking Xuesaitong injection. Third, Xuesaitong mixed with NaCl forms insoluble particles. Fourth, the occurrence of allergic shock correlates with the condition of the patient [bib_ref] Analysis of 86 cases of adverse reaction induced by Xueshuantong injection, Chen [/bib_ref].
3.17. Xueshuantong Injection. Similar to Xuesaitong injection, Xueshuantong injection is composed of Panax notoginseng extracted from Panax pseudoginseng, with the main ingredients of ginsenoside Rg1 and Rb1. It can promote blood circulation, remove blood stasis, expand blood vessels, and improve microcirculation. The ADRs caused by Xueshuantong injection may be due to some factors. First, patients with personal or family histories of allergies are more susceptible to ADRs. Second, Panax notoginseng is the active component of Xueshuantong injection and can stimulate the immune system.
# Discussion
The cases of anaphylactic shock caused by different drugs varied greatly. The abovementioned data and examples showed that doctors should pay more attention to the drug that frequently causes anaphylactic shock. The first reason that anaphylactic shock occurs is because TCM injection components are complex. They are composed of organic compounds, such as pigment, tannin, starch, protein, and other ingredients in colloidal form, which can stimulate the body's immune system and produce antibody-sensitized T lymphocytes to induce hypersensitivity [bib_ref] Analysis of 159 cases of adverse reaction induced by traditional Chinese medicine..., Liang [/bib_ref]. Second, allergic shock correlates with the temperature and humidity of transportation and storage. In addition, TCM injection is unstable; if storage conditions do not meet certain requirements, the amounts of harmful components and unstable particles will increase, causing anaphylactic shock [bib_ref] Analysis of the adverse reaction induced by Mailuoning injection, Chen [/bib_ref]. Third, the TCM injection with the cosolvent, stabilizer, and possible allergens may activate the H1 receptor of the skin tissue, causing histamine release and an increase in body reactivity [bib_ref] Analysisi of 19 cases of adverse reaction induced by Xueshuantong injection, Zhang [/bib_ref]. The poor drug quality and insoluble particles are the main reasons underlying the occurrence of ADRs, so nurses must observe the medicine to ensure that it has no liquid crystals, turbidity, or sedimentation.
People of any age can suffer anaphylactic shock induced by TCM injection. The age distribution in studies was such that 5 cases of allergic shock appeared in patients under the age of 18, 108 cases were in patients aged 19 to 45, 106 cases were in patients aged 46 to 59, and 132 cases were in patients over 60 years old. ADRs occurred in patients of all ages, but the largest number of cases (350, 67.43%) was in patients above 45 years old. According to the data, Tanshinone IIA sodium sulfonate, Erigeron injection, Shengmai injection, and Xuesaitong injection (over 50% of those treated) were more likely to cause the elderly (>60 years old) to suffer anaphylactic shock. This may be because the elderly are susceptible to a variety of cardiovascular and cerebrovascular diseases with frequent usage of the injection. The more drug they were administered, the more likely they were to suffer an ADR. In addition, individual doses in elderly patients cause different degrees of decline in organ function. Compared to youth, the elderly have different sensitivities and drug tolerance and are thus prone to drug accumulation. However, to a surprising extent, there was a significant proportion of young people who suffered ADRs. This occurred for two main reasons. First, young people often have a good physique and are thus easily ignored by doctors. Second, youth take medications more frequently than other ages because of physical maturity. Otherwise, persons under the age of 18 are relatively fewer, mainly because this group accounted for a small proportion of total population taking TCM injection. Doctors should exercise precaution when treating minors as they are still in the growth stage, and their liver and kidney functions and some enzyme systems have not matured. In conclusion, before selecting TCM injections for treatment, doctors should ask about patient to detail their personal and family medical and drug allergy history. In addition, doctors should exercise caution when treating elderly people, children, and other special groups, such as patients with a history of drug allergies.
In general, there were no significant differences in gender with regard to anaphylactic shock; therefore, for the most part, doctors can equally consider the treatment of male and female patients with cardiovascular disease with TCM injection. However, some drugs did have gender-specific effects. For example, females were more prone than males to suffer anaphylactic shock after Shenmai injection and Xuesaitong injection, whereas males were more susceptible to anaphylactic shock after taking Breviscapine injection, Puerarin injection, and Xingnaojing injection.
Among the 350 people in the described studies, only 4 took TCM injection by IMI; the other patients were treated by IVI. At the same time, the ten death cases were received intravenously. Although IVI has a fast curative effect, treatment is more difficult when anaphylactic shock occurs. To reduce the incidence of anaphylactic shock, doctors should fully understand the drug indications, usage, dosage regimen, and route of drug administration. The drug should be administered orally, followed by IMI, with the final choice of drug administration by IVI or infusion. Use of Chinese herbal injections should be restricted to treatment of severe diseases or critical cases [bib_ref] Analysis of the adverse reaction of traditional Chinese medicine injections activating blood..., Liu [/bib_ref]. In addition, the patient should be prohibited from blindly taking a large dose and undergoing treatment for a long period of time, especially with IVI.
Together, the data showed that anaphylactic shock usually occurred between 3 sand 2.5 h, although there were some differences, depending upon a patient's fitness level, pathological state, genetic makeup, and drug sensitivity. In addition, TCM injections can more easily cause allergies when they are given as macromolecules; thus, this is the form that makes patients more susceptible to ADRs. Based on the abovementioned two reasons, the time of occurrence of anaphylactic shock differed between patients. Thus, the length of time that a patient should be monitored should be adjusted to suit that individual and in accordance with the type of drug administered. For example, patients who were administered Shenmai injection, Salvia miltiorrhiza injection, Dan Hong injection, compound Danshen injection, Mailuoning injection, Shengmai injection, or Xingnaojing injection suffered anaphylactic shock within 1 min. So, doctors need to pay close attention to patients who are treated with these drugs that cause immediate hypersensitivity. In addition, the appropriate emergency measures need to be immediately taken when serious ADRs occur. Patients who were administered Ciwujia injection, Breviscapine injection, compound Danshen injection, and Puerarin injection suffered anaphylactic shock after more than 2 h; thus, with these drugs, patients need to be observed for an extended period time. During the course of treatment, the drip rate should be strictly controlled, and the patient should be closely observed. Once an ADR occurs, treatment should be terminated, and the appropriate measures should be taken. I suggest that the patients should be observed more than two hours. The patients may leave after two hours, if they have no discomfort.
# Conclusions
Based on the analyses of the aforementioned studies, we suggest the following. The first suggestion is for pharmaceutical enterprises. Chinese herbal injections should be approved by the SFDA according to the results of double-blind randomized controlled clinical trials [bib_ref] Analysis of the adverse reaction of traditional Chinese medicine injections activating blood..., Liu [/bib_ref]. Although TCM injections can cause allergic shock and other serious ADRs, instructions on how to use these drugs are rarely mentioned. In addition, some TCM injection instructions are not standardized, are poorly written, and lack information and warnings about possible clinical toxicities and side effects [bib_ref] Discuss the causes and counter measures of adverse reactions of TCM injections, Li [/bib_ref]. Therefore, pharmaceutical companies should equip TCM with detailed instructions, including possible ADRs and contraindications.
The second suggestion is for pharmaceutical supervisory and administrative departments. The ADRs caused by TCM injection are multifaceted, with diverse clinical manifestations. The production of TCM injection is extracted according to the theory of TCM and a precise process. The essence of TCM is Bianzhenglunzhi. According to Bian zheng lun zhi, the patient's health situation can be treated holistically. Holistic approaches may have their merits, especially in terms of promoting optimum health [bib_ref] Comments on serious anaphylaxis caused by nine Chinese herbal injections used to..., Ji [/bib_ref]. Therefore, the use of Chinese medicines should be guided by TCM theory and, in particular, should be according to the syndrome differentiation treatment principle. Doctors should not simply and blindly follow instructions, without allowing for differences between individual patients. They should pay attention to different diseases that have the same symptoms or one disease that has different symptoms, thereby achieving the goal of reasonably applying the principle to TCM injections [bib_ref] Discuss the causes and counter measures of adverse reactions of TCM injections, Li [/bib_ref]. This requires doctors to strictly master the application method of the drug and to build upon their knowledge of ADRs caused by TCM injections. In order to improve public safety, doctors should take measures to avoid the occurrence of ADRs to the extent possible.
The third suggestion is for the pharmacy department and pharmacists. Pharmacists must check prescriptions, especially those for TCM injections, to reduce the occurrence of repeated drug use, drug overuse, and irrational drug use. In addition, information on TCM injections, contraindications, and ADRs should be obtained and collected. Any observed ADRs should be reported to the hospital in a timely manner. Drug consultation services should be accessible that will provide accurate medical information to patients, thereby ensuring the reasonable, effective, and safe use of medication.
In addition, clinical pharmacists should do ward rounds of the wards together with physicians for the purpose of strengthening the clinical supervisory activities and guiding the proper clinical use of the medications [bib_ref] Analysis of 64 cases of adverse reaction induced by traditional Chinese medicine..., Li [/bib_ref].
Fourth, it is important for patients to detail their personal and family history of allergies before being administered TCM injection. Patients who are allergic to medication should be closely observed after injection, and if any ADR occurs, doctors should immediately terminate drug administration [bib_ref] Analysis of adverse reaction induced by Tanshinone II a sodium sulfonate injection, Kong [/bib_ref].
All things considered, TCM injection has a unique role in medical practice, as it not only has the common advantages of injection, but also retains the characteristics of TCM. As a new dosage form of TCM, TCM injection has the advantages of being effective and accurately dosed compared with other TCM dosage forms, especially in treating the seriously ill. Importantly, TCM injection has overcome the inconvenience and difficulty in boiling process. The naysayers of this drug have brought great attention to the ADRs caused by TCM injection due to its complex composition, incomplete preparation process, and lack of rigorous evaluation of clinical efficacy. In order to maximize the effectiveness of this treatment and to reduce the associated ADRs, regulators are seeking to more strictly monitor and reevaluating these drugs and improve the relevant laws and regulations. Medical personnel should also cooperate with regulatory authorities to monitor and report ADRs and to take active preventative measures to reduce or avoid the occurrence of severe ADRs, thereby ensuring drug safety to the public [bib_ref] Discuss the causes and counter measures of adverse reactions of TCM injections, Li [/bib_ref].
[table] Table 1: Patients who suffered anaphylactic shock after being treated with 17 types of traditional Chinese medicine injections for cardiovascular and cerebrovascular diseases. [/table]
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ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR modulator therapies
# Introduction
Small-molecule cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for cystic fibrosis are the first therapies since the disease was initially described by FANCONI et al.in 1936 to target and partially restore the function of the CFTR Cl − channel. CFTR modulator therapy is expected to have significant clinical benefits for many, but it does not result in a cure and is not appropriate or available for all patients with cystic fibrosis. In this review, evidence is described suggesting that inhibiting the epithelial Na + channel (ENaC) responsible for the Na + /fluid absorption that contributes to airway surface dehydration and impaired mucociliary clearance (MCC) observed in cystic fibrosis airways may significantly improve clinical outcomes irrespective of the CFTR genotype, and may synergise with currently approved CFTR modulators to further improve clinical outcomes.
## The cftr modulator landscape
Significant progress has been made in the treatment of patients with cystic fibrosis with the introduction of CFTR modulator therapies, which consist of CFTR correctors that improve folding and trafficking of the common F508del-CFTR mutation and potentiators that improve the open probability of mutant CFTR channels at the apical cell membrane . Current CFTR modulator drugs vary in efficacy in improving CFTR function and clinical outcomes. The potentiator ivacaftor was the first approved CFTR modulator for cystic fibrosis patients with at least one G551D-CFTR gating mutation and was shown to rescue mutant CFTR function to ∼50% of wild-type levels, which was associated with an improvement in mean absolute percentage predicted forced expiratory volume in 1 s ( ppFEV 1 ) of ∼11% in the pivotal trial . Subsequently developed corrector-potentiator combinations for patients homozygous for the common F508del-CFTR allele (lumacaftor/ivacaftor and tezacaftor/ivacaftor) or patients with one F508del allele and a residual function allele (tezacaftor/ivacaftor) showed smaller improvements in CFTR function (to ∼10-20% of wild-type levels), which were associated with more modest improvements in ppFEV 1 of ∼3-4% in F508del homozygous patients and ∼7% in patients with one F508del allele in combination with a residual function allele. In addition, it has been shown that bacterial counts and inflammatory markers are reduced in sputum of patients treated with ivacaftor, but that bacteria are not eradicated over time. A triple-agent CFTR modulator drug (elexacaftor/tezacaftor/ivacaftor) has recently been approved in patients with a single F508del-CFTR allele. Elexacaftor, like tezacaftor, is a CFTR corrector, but acts additively at a second site in the F508del-CFTR protein to improve multiple folding defects. Consistent with this additive effect at the molecular level, this triple-combination CFTR modulator therapy resulted in greater improvement in lung function and other clinical outcomes (an approximately 11-14% improvement in ppFEV 1 compared with control) in patients homozygous for F508del or compound heterozygous with a minimal function alleleand is predicted to become the "gold standard" for the treatment of up to 90% of patients with cystic fibrosis (those with at least one F508del allele). However, for most patients with chronic cystic fibrosis lung disease, this improvement in pulmonary function is not necessarily a return to the normal range, and patients continue to have exacerbations, albeit at much reduced rates. Improving clinical outcomes further in patients receiving elexacaftor/tezacaftor/ ivacaftor has the potential to have a significant impact on the cystic fibrosis population (figure 1). In addition, the approximately 10% of cystic fibrosis patients for which CFTR modulator therapy is ineffective (those who do not carry at least one F508del allele) could benefit from a mutation-agnostic approach. Finally, while long-term safety of treatment with elexacaftor/tezacaftor/ivacaftor remains unknown, other therapeutic avenues should be considered.
ENaC: role in healthy and cystic fibrosis airways, and potential therapeutic target
In the conducting airways, ENaC is expressed at the apical membrane of airway epithelial cells and provides the limiting pathway for transepithelial Na + absorption that drives absorption of Cl − and water through the paracellular shunt pathway (figure 2). In healthy airways, coordinated Cl − secretion by CFTR and other Cl − channels and Na + absorption by ENaC is essential for proper volume regulation of airway surface liquid (ASL), which comprises the periciliary layer (PCL) and the overlying mucus layer. ENaC is regulated by several intracellular and extracellular mechanisms. Intracellular mechanisms include activation by convertase-type proteases, such as furin, and inhibition by CFTR. Extracellular activation mechanisms include proteolytic cleavage by neutrophil elastase and other proteases released from neutrophils and other inflammatory cells in the airways, as well as by bacterial proteases released in airway infection. Therefore, ongoing inflammation and infection in cystic fibrosis patients with established lung disease, including those treated with CFTR modulators, is likely to cause ongoing proteolytic cleavage and activation of ENaC in the airways.
Evidence suggests that ENaC is hyperactivated in cystic fibrosis as a result of CFTR dysfunction and/or proteolytic activation by host-and bacteria-derived proteases. Hyperactivated ENaC in the airways of cystic fibrosis patients results in markedly increased Na + absorption, leading to increased paracellular Cl − and water absorption from the airway lumen, ASL volume depletion, hyperconcentration of mucus, reduced height of the PCL with compressed cilia, reduced MCC, mucus accumulation, airway plugging, bacterial colonisation, inflammation, progressive tissue damage and decline in lung function . The pathogenic role of increased ENaC function has been supported by the lung phenotype of mice with airway-specific overexpression of the β-subunit of ENaC (βENaC-Tg) that phenocopy cysticfibrosis-like airway surface dehydration/mucus hyperconcentration and develop cystic-fibrosis-like lung disease. Collectively, these results support ENaC inhibition in the airways as an attractive target for cystic fibrosis therapy. In this context, it is noteworthy that patients with pseudohypoaldosteronism with loss-of-function mutations in the αand β-subunits of ENaC have increased ASL volume and MCC rates, and cystic fibrosis patients with a mutation in the δ-subunit of ENaC causing reduced ENaC activity were found to have slow progression of lung disease.
Inhibition of ENaC was first demonstrated with amiloride, and in the kidney, this is an effective diuretic drug. In βENaC-Tg mice, which display a cystic-fibrosis-like lung phenotype, intrapulmonary treatment with amiloride was effective, leading to a reduction in mucus plugging, airway inflammation and pulmonary mortality when started as preventive therapy immediately after birth, i.e. when the lungs were structurally normal; however, no beneficial effects were observed in adult βENaC-Tg mice with established cystic-fibrosis-like lung disease. In cystic fibrosis patients, probably due to amiloride's short half-life and limited potency, studies of the effect of inhaled amiloride on MCC and lung function resulted in only moderate and inconsistent improvements, even with multiple dosesIn healthy airways, a balance between cystic fibrosis transmembrane conductance regulator (CFTR)-mediated secretion and epithelial Na + channel (ENaC)-mediated absorption of NaCl and H 2 O facilitates proper hydration of airway surfaces essential for effective mucociliary clearance (a). In cystic fibrosis airways, deficient CFTR-mediated Cl − /fluid secretion and increased ENaC-mediated Na + /fluid absorption leads to airway surface dehydration (reduced PCL and hyperconcentrated mucus), flattened cilia, impaired mucociliary clearance, bacterial colonisation and neutrophilic inflammation (b). Partial restoration of these pathological features by rescue of mutant CFTR function with CFTR modulators (correctors and potentiators) (c) or ENaC inhibition (d). Hypothesised synergy between CFTR modulation and ENaC inhibition, resulting in further improvement in airway surface hydration and reduction in pathological features in cystic fibrosis airways (e). Arrows indicate direction and magnitude of ion and water movement. -ve: negative regulation; PCL: periciliary layer.
GS-9411, whilst 100-times more potent than amiloride and with a longer duration of action, failed Phase I trials as an inhaled ENaC blocker due to ENaC inhibition in the kidney, resulting in hyperkalaemia, which may have cardiac and neurological safety implications. These studies led to the hypothesis that if the poor pharmacodynamics and safety observed with amiloride and GS-9411 could be overcome with a newer-generation compound, ENaC inhibition would be a viable therapy to improve airway surface hydration and pulmonary outcomes in patients with cystic fibrosis, irrespective of CFTR genotype (i.e. a mutation-agnostic therapy), particularly in those with rare CFTR mutations that have no currently approved CFTR modulator therapy. Further, more recently, mucus hyperconcentration has also been implicated in the pathogenesis of a spectrum of other muco-obstructive lung diseases including chronic bronchitis and non-cystic fibrosis bronchiectasis, suggesting that ENaC inhibition may be beneficial far beyond cystic fibrosis.
Importantly, ENaC inhibition may act synergistically with CFTR modulators (figure 2e). CFTR can secrete or absorb Cl − across epithelial surfaces depending on the electrochemical driving force that is determined by i) the intra-and extracellular Cl − concentrations that are tightly regulated and result in a reversal potential for Cl − (i.e. the membrane potential at which there is no net flow of Cl − from one side of the membrane to the other, also known as the Nernst potential) in the range of -30 mV in cystic fibrosis airway epithelial cells, and ii) the membrane potential of the cell that is set by the relative conductances of Cl − , Na + and K + channels and respective intra-and extracellular concentrations of these ions. It is therefore predicted that if ENaC is inactive or not expressed in the same cell, cAMP-mediated stimulation will lead to a concomitant activation of apical CFTR and basolateral K + channels (reversal potential for K + approximately -90 mV) that drives the membrane potential more negative than the reversal potential of Cl − , and thus generate a driving force for CFTR-mediated Cl − secretion; however, if ENaC is active in the same cell (reversal potential for Na + approximately +60 mV), this will depolarise the membrane potential, reduce the driving force for Cl − secretion, and may even result in CFTR-mediated Cl − absorption as observed in the sweat duct. Regarding the combined use of CFTR modulators and ENaC inhibitors in patients with cystic fibrosis, this implies that inhibition of hyperactive ENaC in the apical membrane of airway epithelial cells may not only block ENaC-mediated Na + /fluid absorption, but will also hyperpolarise the apical cell membrane and thus increase the driving force for Cl − /fluid secretion via mutant CFTR channels that have been rescued and inserted into the apical plasma membrane by CFTR modulators. Whether this is reflected in synergistic effects on airway surface hydration, MCC and pulmonary outcomes in patients with cystic fibrosis and other muco-obstructive lung diseases needs to be assessed in clinical trials.
## Clinical development of new enac inhibitors
Several new compounds designed for ENaC inhibition are currently in active preclinical development. These new compounds employ different modes of action ranging from highly potent and durable inhibition of the ENaC channel pore, inhibition of ENaC-activating proteases, to inhibition of ENaC expression by antisense oligonucleotides or small interfering RNA. Of these, the new small-molecule ENaC inhibitor BI 1265162 is the only compound currently in Phase II development. It has demonstrated efficacy in a preclinical investigation, with a markedly higher potency than amiloride (a 30-70-fold lower half maximal inhibitory concentration), and no effects on serum K + and plasma electrolytes have been observed. In addition, BI 1265162 has shown safety in Phase I volunteer studies.
Importantly, preclinical work supports the potential mutation-agnostic property of BI 1265162. Using highly differentiated human airway epithelial cell cultures grown at an air-liquid interface, it was found that transepithelial fluid absorption from the apical surface to the basolateral compartment was reduced by BI 1265162, with or without CFTR modulators, in both cystic fibrosis and normal airway cultures. This work also supports the hypothesis of a potential synergistic effect with CFTR modulators. Addition of lumacaftor/ivacaftor alone to F508del/F508del cystic fibrosis cultures partially restored mucus transport velocity to approximately 50% of that observed in normal airway cultures; however, addition of BI 1265162 to lumacaftor/ivacaftor further improved mucus transport velocity in cystic fibrosis cultures to a level similar to normal airway cultures. Taken together, the above evidence suggests that BI 1265162 is a promising candidate as a monotherapy for cystic fibrosis patients for whom CFTR modulators are ineffective and in combination with CFTR modulators providing synergistic effects. However, no ENaC inhibitor development, so far, has translated into clinical successand several clinical development programmes of ENaC inhibitors for patients with cystic fibrosis (VX-371; QBW276; Novartis and SPX-101; Spyryx (an indirect inhibitor)) have recently been terminated (table 1). The clinical development of VX-371 for primary ciliary dyskinesia (NCT02871778) has also recently been terminated (clinicaltrials. gov NCT02871778). Failure of ENaC inhibitors to progress in clinical development may be due to inadequate dosing and/or deposition by inhalation in cystic fibrosis patients with chronic lung disease characterised by heterogeneous airway mucus plugging. These ENaC inhibitors demonstrated https://doi.org/10.1183/13993003.00946-2020 improvement of MCC in the sheep model, but lack of translation of this preclinical model to patients with cystic fibrosis may be related to the fact that the sheep do not have airway mucus plugging and structural lung damage commonly present in patients with cystic fibrosis. Therefore, the dose of inhaled ENaC inhibitor that improves MCC in the sheep model may not be sufficient to achieve therapeutically active ENaC inhibition in cystic fibrosis patients. To address this issue in more detail, more recent cystic fibrosis animal models such as the cystic fibrosis pig and cystic fibrosis ferret, which feature cystic-fibrosis-like lung disease including heterogeneous mucus plugging, chronic airway infection, inflammation and bronchiectasis, may be utilised to better understand issues related to target engagement and the potential role of ENaC inhibitors as a therapeutic option in cystic fibrosis lung disease. Whether the hurdle of sufficient delivery of inhaled BI 1265162 to mucus-obstructed airways can be overcome in patients with cystic fibrosis remains to be seen in clinical trials. In addition, systemic side effects such as hyperkalaemia, short study duration, non-study-related exacerbations and lack of sensitivity of traditional endpoints such as ppFEV 1 to detect treatment benefits may impede the clinical development of an inhaled ENaC inhibitor therapy.
How could these hurdles be overcome in the clinical development of a new ENaC inhibitor therapy for cystic fibrosis? First, a successful ENaC inhibitor will have to be highly potent, delivered to the airways in a sufficient dose and have a long duration of action to provide effective ENaC inhibition and maximal treatment effect. Second, the new ENaC blocker should result in minimal off-target effects and systemic exposure. In this context, the following translational challenges that have ended the clinical programmes of earlier ENaC inhibitors have been addressed in order to minimise BI 1265162's risk of failure in Phase II studies. The risk of underdosing was minimised by basing the dose for the Phase II trial in cystic fibrosis patients on fluid absorption data in the rat model (in which BI 1265162 was instilled into the trachea) in addition to MCC data in the sheep model (in which BI 1265162 was nebulised), factoring in the expected lung deposition using the Respimat device in humans. Of note, the improvements in MCC observed with BI 1265162 in the sheep modelwere comparable to the improvement obtained in cystic fibrosis patients with the G551D gating mutation after starting CFTR modulator therapy with ivacaftor. These data suggest that inhaled BI 1265162, if delivered successfully to the cystic fibrosis lung, has the potential to provide similar benefits on MCC in cystic fibrosis patients with genotypes that do not respond to CFTR modulators. In the subsequent Phase I studies, BI 1265162 did not result in drug-related hyperkalaemia and was well tolerated when administered as single or multiple doses up to 1200 µg daily. Moving into Phase II, lung clearance index (LCI) derived from multiple breath washout has been included as an additional endpoint; LCI reflects ventilation inhomogeneity in the lung, correlates with mucus plugging and other morphological changes of the airwaysand is more sensitive than spirometry ( ppFEV 1 ) to detect functional abnormalities in the small airways typically affected in cystic fibrosis. Crucially, BI 1265162 is an ENaC inhibitor that is considerably more potent than amiloride and has, at least preclinically, been shown to work as a mutation-agnostic monotherapy and in synergy with CFTR modulation.
# Conclusion
Despite the exciting breakthroughs in the development of CFTR modulator therapy, this therapy is not effective for all patients with cystic fibrosis, as only patients with at least one F508del allele can benefit. Additionally, functional restoration of the underlying ion/fluid transport defect and clinical outcomes are still suboptimal in those patients who are eligible for CFTR modulator therapy. Whereas systemic delivery of CFTR modulators can improve CFTR function in multiple affected organs, potential benefits of inhaled ENaC blockers are likely limited to the lungs. However, ENaC inhibition may become a viable option for patients for whom existing CFTR modulator therapy is ineffective, and it has the potential to act synergistically with CFTR modulation in those patients for whom it is. ENaC inhibition is therefore a promising strategy to optimise therapeutic benefit. Clinical trials with novel, long-acting ENaC inhibitors such as BI 1265162 will now have to demonstrate that ENaC inhibition is safe and translates into clinical benefit in people with cystic fibrosis. |
Sodium–glucose cotransporter‐2 inhibitors induced euglycemic diabetic ketoacidosis: Two case reports and a review of the literature
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.AbstractIf not detected early, euglycemic diabetic ketoacidosis can be a serious adverse effect of sodium-glucose cotransporter-2 (SGLT2) inhibitors. Unfortunately, euglycemic diabetic ketoacidosis is underreported in recent trials and missed because of normal blood sugar levels and nonspecific symptoms on presentation. We present two patients with type 2 diabetes mellitus who developed dapagliflozinassociated euglycemic diabetic ketoacidosis followed by hyperglycemic ketoacidosis. The second patient had euglycemic ketoacidosis twice despite instructions to stop using the medication dapagliflozin.
# | introduction
Current treatments for type 2 diabetes have centered on increasing insulin availability (either through direct insulin administration or through agents that promote insulin secretion), improving insulin sensitivity, delaying the delivery and absorption of carbohydrates from the gastrointestinal tract, or increasing urinary glucose excretion.Sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce blood glucose by increasing urinary glucose excretion. 1 SGLT2 inhibitors are used as second-line therapy in type 2 diabetes combined with other oral hypoglycemic drugs, such as dipeptidyl peptidase-4 (DPP-4) inhibitors, metformin, sulfonylurea, or basal insulin, in an attempt to achieve good glycemic control, which is based on HbA1c levels. [bib_ref] Pharmacologic approaches to glycemic treatment of type 2 diabetes: synopsis of the..., Doyle-Delgado [/bib_ref] As a serious complication of DM, diabetic ketoacidosis (DKA) should be recognized early, especially in the face of normal blood sugar levels; otherwise, DKA will become life-threatening. The underlying mechanism of DKA development is a deficiency in insulin activity in the body. [bib_ref] Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors, Ogawa [/bib_ref] DKA is frequently seen in individuals with poorly controlled type 1 diabetes or those with type 2 diabetes with infections or injuries or in a prolonged fasting. Hyperglycemia is a main feature of DKA, but DKA can occur in the setting of normal blood glucose, especially in patients taking SGLT2 inhibitors. [bib_ref] Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors, Ogawa [/bib_ref] [bib_ref] Dapagliflozin-induced severe ketoacidosis requiring hemodialysis, Maadarani [/bib_ref] Euglycemic or normoglycemic DKA is defined as DKA that occurs in the presence of a blood glucose concentration of <11 mmol/L (200 mg/dl). [bib_ref] Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors, Ogawa [/bib_ref] Euglycemic DKA develops mostly in individuals with type 1 diabetes and rarely occurs in those with type 2 diabetes. [bib_ref] Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors, Ogawa [/bib_ref] We present the cases of two patients with euglycemic DKA with no obvious precipitating factor except that they were on SGLT2 inhibitors.
## | case 1
A 64-year-old woman with known diabetes and an oral hypoglycemic agent and glargine basal insulin was recently diagnosed with triple vessel disease and underwent CABG 4 weeks earlier; she was discharged on Glucophage/dapagliflozin, aspirin, and atorvastatin. She visited the emergency department in the cardiac center because of headaches and breathlessness. The patient did not report chest pain, fever, cough, dysuria, or hemoptysis. The patient's blood pressure was 140/70; she was hyperventilating, but her chest sounds were clear. Her blood sugar level was 5 mmol/L. She was reassured that her condition was satisfactory, and she was sent home. Twelve hours later, her level of consciousness deteriorated, and she was brought to the emergency department.
Physical examination showed tachycardia (124 beats/ minute), tachypnea (RR 32/minute), low blood pressure (70/40 mmHg), and dry oral mucosa. She was unconscious with shallow breathing, with a Glasgow Coma Scale score of 7. Emergency physicians successfully intubated her. Her oxygen saturation was 98% on room air. Chest examination was normal, with a clean healed sternal wound, and the remainder of the examination findings were unremarkable. The blood glucose level was 34 mmol/L (normal 4-6.2 mmol/), and kidney function was normal (eGFR 103-estimated by the CKD-EPI). She also had severe metabolic acidosis, with a pH of 6.6 (normal 7.35-7.45), an anion gap of 38 (normal 8-16), and a serum bicarbonate level of 5 mmol/L (normal 22-28) . Her lactate level was normal, and her hydroxybutyrate level was 9 mmol/L (normal 0.4-0.5 mmol/L). Serum sodium was 143 mmol/L, serum potassium was 5.5 mmol/L, and serum osmolality was 295 mosm/kg. Urine analysis showed no evidence of infection, and urine electrolytes were within normal limits. Urine osmolality was 150 mosm/kg. In the complete blood count, the WBC count was 18.000 cells/mcl. C-reactive protein was within the normal range (5 mg/L). Test results for toxic alcohols, including methanol, ethylene glycol, and diethylene glycol, were negative. There was no osmolar gap. Given her severe anion gap metabolic acidosis and ketoacidosis with a euglycemic-hyperglycemic profile, she was admitted to the ICU and started on infusions of intravenous fluid, insulin (up to 10 units/hour), and dextrose to prevent hypoglycemia (glucose was maintained at approximately 7 mmol/L), and bicarbonate. After a few hours of treatment, laboratory test results showed slow improvement with the dextrose bicarbonate infusion.
Based on the finding of high anion gap metabolic acidosis, with no obvious precipitating factor of ketoacidosis, such as infection or starvation, euglycemic-hyperglycemic ketoacidosis induced by dapagliflozin was considered. Insulin treatment was continued until the ketones were cleared, and the anion gap was closed in 48 hours while serum glucose levels were maintained. The patient was drowsy and confused the next day, but she improved later and was extubated. CT of the brain was normal. She was discharged on glargine basal insulin, insulin aspart, before meals, and Glucophage. At the 1-month follow-up, the patient was stable, with no neurological deficits and wellcontrolled blood sugar levels.
## | case 2
The patient, a 56-year-old male, was an ex-smoker who had suffered from diabetes mellitus since 2007. He had no documented retinopathy, nephropathy, or neuropathy. He had been on insulin aspart before each meal, insulin degludec (ultralong-acting basal insulin), and liraglutide (a long-acting glucagon-like peptide-1 agonist) injections. However, 2 months prior to admission, he had been on semaglutide once weekly. His last HBA1C was 8.8. Three days before admission, the patient did not feel well and was thirsty, losing appetite. On the day of admission, he was weak and got out of bed with the help of his family members but felt dizzy. His son brought him to the ER. He vomited once in the emergency room. He had no fever, chills, rigors, urinary symptoms, headache, skin rash, abdominal pain, or dyspnea. He had not started any new medications, nor did he have a history of illicit drug intake.
His pulse rate was 112 per minute, his respiratory rate was 20/minute, his blood pressure was 120/70 mmHg, and his oxygen saturation was 98% in room air. He also had dry skin.
Laboratory tests showed a pH of 6.99 (normal 7.35-7.45), and HCO3-of 8.8 mmol/L (normal 22-28), a high anion gap (normal 8-16), a hydroxybutyrate of 6.9 mmol/L (normal 0.4-0.5 mmol/L), and urine ketones +++++. Test results for toxic alcohols, including methanol, ethylene glycol, and diethylene glycol, were negative. There was no osmolar gap. The above picture of normal blood sugar levels and a high anion gap with elevated ketone bodies suggested a diagnosis of euglycemic metabolic acidosis. He was treated with intravenous dextrose fluid with normal saline and insulin infusion in two different intravenous lines, with a bolus of Na2HCO3 to keep the pH above 7. The next morning, the anion gap closed, and he felt better. He was provided hydration therapy and was discharged home. His discharge medications were insulin aspart before each meal, insulin degludec, and onceweekly semaglutide.
The patient ran out of acute rapid insulin 3 weeks later and resumed dapagliflozin-metformin 5 mg/1000 mg tablet. Unfortunately, he was readmitted 10 days later with general fatigue and ketonuria, high blood ketone and blood sugar levels (5 mmol/L), a pH of 7.2, and an HCO3of 12 mmol/L. He was managed again with the same protocol and discharged home with extensive counseling to never use this combination of medications again. He was seen 4 weeks later in the diabetic clinic with a fasting blood sugar level of 7 mmol/L and was placed on insulin aspart before each meal and daily insulin degludec.
# | discussion
Recently, SGLT2 inhibitors were evaluated in many studies to determine the levels of improvement in renal and cardiovascular outcomes in diabetic and nondiabetic patients. Unfortunately, these studies did not show an increased incidence of DKA compared to placebo. The results of the Canagliflozin and Cardiovascular and Renal Events in Type 2 Diabetes (CANVAS Program Collaborative Group) trial revealed that only a small number of diabetic ketoacidosis events were observed with canagliflozin and placebo (0.6% vs. 0.3% participants with an event per 1000 patient-years; hazard ratio, 2.33; 95% CI, 0.76-7.17). [bib_ref] Canagliflozin and cardiovascular and renal events in type 2 diabetes, Neal [/bib_ref] In the Cardiovascular and Renal Outcomes with Empagliflozin in Heart Failure study (EMPEROR-Reduced Trial), there were no reported cases of diabetic ketoacidosis among 1863 patients who received the drug. [bib_ref] Cardiovascular and renal outcomes with empagliflozin in heart failure, Packer [/bib_ref] In the Dapagliflozin in Patients with Heart Failure and Reduced Ejection Fraction (DAPA-HF) trial, only 4/2368 (0.2%) participants who received dapagliflozin developed DKA, compared with 4/2368 (0.2%) participants who received placebo. [bib_ref] Dapagliflozin in patients with heart failure and reduced ejection fraction, Mcmurray [/bib_ref] In the EMPEROR-Preserved Trial, serious adverse events occurred in 1436 patients (47.9%) in the empagliflozin group and 1543 patients (51.6%) in the placebo group. Adverse events led to treatment discontinuation in 571 patients (19.1%) in the empagliflozin group and 551 (18.4%) in the placebo group. DKA was reported in four patients in the empagliflozin group and five in the placebo group. In the SCORED (Sotagliflozin in Patients with Diabetes and Chronic Kidney Disease) trial, DKA occurred in 30 (0.6%) patients in the sotagliflozin group (N = 5291) and 14 (0.3%) patients in the placebo group (N = 5286; p value 0.02).
All previously mentioned studies falsely reassured cardiologists and nephrologists that DKA incidence does not differ between a treatment drug and a placebo. In March 2015, the US Food and Drug Administration (FDA) issued a warning for SGLT2 inhibitor-associated diabetic ketoacidosis after 20 cases had been reported . In December 2015, the FDA released another statement regarding 73 cases of diabetic ketoacidosis in which the patients required hospitalization or emergency department presentation.Fifty percent of cases were associated with precipitating events, including acute illness (e.g., infection and surgery), reduced oral intake, and reduced insulin dose.In an analysis of 487 cases of ketoacidosis from the WHO pharmacovigilance database, ketoacidosis was more frequently reported with gliflozins than with other glucose-lowering drugs (adjusted reporting odds ratio 15.5 [95% confidence interval 12.8 to 18.7]). [bib_ref] SGLT-2 inhibitors and ketoacidosis: a disproportionality analysis in the World Health organization's..., Moumouni [/bib_ref] High-quality evidence from a systematic review and meta-analysis (39 RCTs, 60,580 patients) suggested an increased risk of diabetic ketoacidosis with SGLT2 inhibitors in type 2 diabetes compared with placebo or other antidiabetic drugs (relative risk 2.13 [1.38-3.27]), with an absolute rate of 3 events per 1000 patient-years. [bib_ref] Sodium-glucose co-transporter-2 inhibitors and the risk of diabetic ketoacidosis in patients with..., Liu [/bib_ref] Euglycemic diabetic ketoacidosis occurs in relatively low insulin levels in the face of acute illness or reduced caloric intake. SGLT2 inhibitors lower blood glucose levels by increasing urinary glucose excretion, leading to decreased plasma glucose and hence decreased insulin release in the face of an increased counterregulatory response by increased glucagon levels. [bib_ref] Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors, Ogawa [/bib_ref] The decline in circulating insulin levels results in a lowering of the antilipolytic activity of insulin and consequent stimulation of the production of free fatty acids, which are converted to ketone bodies by βoxidation in the liver. [bib_ref] Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors, Ogawa [/bib_ref] Another possible mechanism of the development of ketosis is that SGLT2 is expressed on the alpha cells of pancreatic islets, and an increase in glucagon serum levels after the administration of dapagliflozin and empagliflozin could explain the ketogenic effects of direct pancreatic stimulation by the drugs. [bib_ref] SGLT2 inhibitors may predispose to ketoacidosis, Taylor [/bib_ref] Important risk factors for precipitating EGDKA are vomiting, dehydration, acute infection, surgery, vigorous or prolonged exercise, and insulin pump or infusion site failure. [bib_ref] Risks associated with SGLT2 inhibitors: an overview, Singh [/bib_ref] The first case presented here demonstrates the importance of recognizing the side effects early in view of normal blood sugar levels before DKA becomes a life-threatening condition with severe hyperglycemia and metabolic derangement. The second case confirms the importance of immediately stopping treatment with SGLT2 inhibitors if diabetic ketoacidosis is suspected or confirmed. Do not restart treatment if another clear precipitating factor for the condition is not identified and resolved as recommended by the European Medicines Agency.In conclusion, following the introduction of the SGLT2 inhibitors in therapeutic practice for type 2 DM treatment and cardiovascular disease, the amount of euglycemic diabetic ketoacidosis increased. DKA caused by SGLT2 inhibitors is a serious complication, and physicians should be aware of early symptoms, check serum and urine ketone levels, and stop the medication before DKA becomes a life-threatening complication. Do not restart SGLT2 inhibitors if clear precipitating factor for the condition is not identified.
## Acknowledgement
None.
## Conflict of interest
The author(s) declare no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
# Author contributions
ZB wrote the article, ZB, OM, and FA shared in the discussion and with WE, KT, Mj, and ME shared in the management and in collecting the data and revision of the manuscript. Our working website is www.kockw.com (Kuwait Oil Company, Ahmadi Hospital).
## Consent
Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.
# Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request. |
Comparative Mapping of Seed Dormancy Loci Between Tropical and Temperate Ecotypes of Weedy Rice (Oryza sativa L.)
Genotypic variation at multiple loci for seed dormancy (SD) contributes to plant adaptation to diverse ecosystems. Weedy rice (Oryza sativa) was used as a model to address the similarity of SD genes between distinct ecotypes. A total of 12 quantitative trait loci (QTL) for SD were identified in one primary and two advanced backcross (BC) populations derived from a temperate ecotype of weedy rice (34.3°N Lat.). Nine (75%) of the 12 loci were mapped to the same positions as those identified from a tropical ecotype of weedy rice (7.1°N Lat.). The high similarity suggested that the majority of SD genes were conserved during the ecotype differentiation. These common loci are largely those collocated/linked with the awn, hull color, pericarp color, or plant height loci. Phenotypic correlations observed in the populations support the notion that indirect selections for the wild-type morphological characteristics, together with direct selections for germination time, were major factors influencing allelic distributions of SD genes across ecotypes. Indirect selections for crop-mimic traits (e.g., plant height and flowering time) could also alter allelic frequencies for some SD genes in agroecosystems. In addition, 3 of the 12 loci were collocated with segregation distortion loci, indicating that some gametophyte development genes could also influence the genetic equilibria of SD loci in hybrid populations. The SD genes with a major effect on germination across ecotypes could be used as silencing targets to develop transgene mitigation (TM) strategies to reduce the risk of gene flow from genetically modified crops into weed/wild relatives. KEYWORDS seed dormancy weed comparative genomics quantitative trait locus segregation distortion Weeds are unwanted plants that have adapted to agroecosystems and compete with crop cultivars (Harlan 1965; Booth et al. 2003). Seed dormancy (SD) plays a critical role in the adaptation. Weed seeds, usually dormant upon maturation, may survive in the soil for months to years, depending on genotypes and environments. Presumably, the genotypic differentiation of SD in a species occurred at multiple loci during evolution before specific populations adapted to agroecosystems as weeds, given the relatively short history of domestication for major crops (,9000 yr; Chopra and Prakash 2002). Thus, it is important to know about the degree of similarity in SD genes between distinct ecotypes and factors influencing their genotypic/allelic frequencies. This information may help design new weed management strategies. We selected weedy rice as a model system to address the ecological genetic issues in this research. Weedy rice refers to various forms of plants that belong to the Oryza genus and infest rice fields from tropical to temperate areas(Oka 1988;Delouche et al. 2007). The rice Oryza sativa was domesticated from the wild ancestor (O. rufipogon Griff.) and differentiated into the indica and japonica subspecies that are distributed across tropical/subtropical and temperate areas, respectively(Khush and Brar 2002). The origin of the conspecific weedy rice was associated with the domestication and subspeciation processes. For example, weedy rice populations can be indicaor japonica-like. The indica-like populations in tropical areas (tropical ecotypes) could originate from natural variants of the wild ancestor, or
from hybrids between wild and cultivated rice. The japonica-like populations in temperate areas (temperate ecotypes) that are historically absent of wild rice may originate from old/extinct cultivars, or hybrids between indica and japonica cultivars [bib_ref] Genetic characterization of weedy rice (Oryza sativa L.) based on morphophysiology, isozymes..., Suh [/bib_ref] [bib_ref] Genetic characterization of weedy rices and the inference on their origins, Tang [/bib_ref]. Despite the ecotype differentiation, weedy rice populations, particularly those adapted to an ecosystem for a long period, usually have strong SD and some other wild-type characteristics [bib_ref] Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a..., Oka [/bib_ref]. The phenotypic similarity between distinct ecotypes could arise from the same or different sets of genes, depending on the relatedness of weed populations and the coevolutionary relationship between SD and the other adaptive or domestication-related traits in local ecosystems. We used a comparative mapping approach to infer the differentiation at QTL for SD (qSD) between temperate and tropical ecotypes of weedy rice.
Wild and weedy rice are divergent from cultivated rice in SD, as evaluated under controlled environment conditions [bib_ref] Variation in the loss of seed dormancy during after-ripening of wild and..., Veasey [/bib_ref] [bib_ref] Dormancy imposed by covering tissues interrelated with seed shattering and morphological characteristics..., Gu [/bib_ref]. Several lines of wild (O. rufipogan or O. nivara)/weedy rice were crossed with cultivars to identify QTL associated with domestication-related traits, including SD or germination capacity [bib_ref] Genomic regions affecting seed shattering and seed dormancy in rice, Cai [/bib_ref] [bib_ref] Mapping quantitative trait loci for yield, yield components and morphological traits in..., Thomson [/bib_ref] [bib_ref] Genetic analysis of adaptive syndromes interrelated with seed dormancy in weedy rice..., Gu [/bib_ref] [bib_ref] Identification of QTLs for domestication-related and agronomic traits in an Oryza sativa..., Lee [/bib_ref] [bib_ref] Genetic analysis of rice domestication syndrome with the wild annual species, Li [/bib_ref] [bib_ref] Mapping QTL for seed dormancy in weedy rice, Jing [/bib_ref] [bib_ref] Genetic architecture of seed dormancy in U.S. weedy rice in different genetics..., Subudhi [/bib_ref] [bib_ref] Quantitative trait locus and haplotype analyses of wild and crop-mimic traits in..., Mispan [/bib_ref]. The number of reported SD QTL varied with mapping populations or environments (years) from a few to $20. Some of them remain to be confirmed because several factors in a distant cross, such as partial sterility or low seed set, segregation distortion, and seed shattering, could have a negative impact on the QTL analysis [bib_ref] Genomic regions affecting seed shattering and seed dormancy in rice, Cai [/bib_ref]. In the previous research, we identified 10 SD QTL in a primary and advanced BC populations derived from a tropical ecotype of weedy rice [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref] [bib_ref] New seed dormancy loci detected from weedy rice-derived advanced populations with major..., Ye [/bib_ref]. All of these 10 loci have been confirmed, and some of them have been cloned. The objectives of this research were to: (1) identify SD QTL for a temperate ecotype of weedy rice and (2) compare these loci with those mapped for the tropical ecotype to infer shared genetic and evolutionary mechanisms underlying the adaptive trait.
# Materials and methods
## Parental lines and mapping populations
Two ecotypes of weedy rice: The pure lines, LD and SS18-2, were selected from the previous research [bib_ref] Dormancy imposed by covering tissues interrelated with seed shattering and morphological characteristics..., Gu [/bib_ref] to represent the temperate and tropical ecotypes of weedy rice, respectively. LD was purified from "LüDao" (in Chinese), a population of volunteer rice historically present in the Lianyungang area (34.33-34.46°N Lat.) of East China [bib_ref] A study on LüDao from Liangyungang, Jiang [/bib_ref]. This population was similar to some local landraces (O. sativa ssp. japonica) in plant type and seed (spikelet) morphology (black hull and red pericarp colors, long awn, and medium grain), but different from the old landraces in seed shattering and dormancy [bib_ref] A study on LüDao from Liangyungang, Jiang [/bib_ref]. SS18-2 was purified from SS18, a population of weedy rice from the Songkla (7.18°N Lat.) area of Southern Thailand [bib_ref] Genetic characterization of weedy rices and the inference on their origins, Tang [/bib_ref] , and is similar to LD in seed morphology and dormancy (Supplemental Material, in File S1). Despite the phenotypic similarity, there was no direct relationship Figure 1 A framework linkage map and positions of seed dormancy QTL identified in this and previous research. The map was marked with RM loci segregating in the BC 1 F 1 EM93-1//EM93-1/LD population. Asterisks indicate segregation distortion loci in favor of the allele from EM93-1 ( Ã ) or LD ( ÃÃ ). Black bars indicate C.I. (equivalent to one-LOD support lengths) of qSD, qAn, or qHC detected in this BC 1 F 1 population [fig_ref] Figure 2: Genome-wide scan for seed dormancy QTL in the BC 1 F 1... [/fig_ref]. Five-point stars indicate qSDs detected in the BC 2 F 1 (9) and/or BC 2 F 1 (139) , but not in this BC 1 F 1 population. Ovals indicate approximate positions of qSDs previously detected in the BC 1 F 1 EM93-1//EM93-1/SS18-2 population or its advanced generations [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref] [bib_ref] New seed dormancy loci detected from weedy rice-derived advanced populations with major..., Ye [/bib_ref]. LD and SS18-2 are temperate and tropical ecotypes of weedy rice, respectively. Ch, chromosome; LD, pure line LüDao; LOD, logarithm of the odds; qAn, QTL for awn; qHC, QTL for hull color; qSD, QTL for seed dormancy; QTL, quantitative trait loci; RM, rice microsatellite.
in origin between these two geographically isolated weed populations. Based on diagnostic characteristics and isozyme markers, LD and SS18 were classified into the japonica-and indica-like groups of weedy rice, respectively [bib_ref] Genetic characterization of weedy rices and the inference on their origins, Tang [/bib_ref].
Recurrent parent and BC populations: EM93-1, an early maturation semidwarf indica line [bib_ref] Genetic and physiological characterization of two clusters of quantitative trait loci associated..., Ye [/bib_ref] , was used as the female, recurrent parent to develop BC populations. The BC 1 F 1 "EM93-1//EM93-1/LD" population, which had been previously evaluated for phenotypic correlations between seed-related traits [bib_ref] Dormancy imposed by covering tissues interrelated with seed shattering and morphological characteristics..., Gu [/bib_ref] , was used to scan for QTL along the LD genome. In addition, two BC 1 F 1 plants (#9 and 139), which were similar to EM93-1 in flowering time, were selected to develop the BC 2 F 1 (9) "EM93-1/BC 1 F 1 plant #9" and BC 2 F 1 (139) "EM93-1/BC 1 F 1 plant #139" populations. The BC 2 F 1 (9) and (139) populations were used to confirm the detected QTL and to identify additional loci whose effects on germination may have been masked by some major genes segregating in the BC 1 F 1 population [bib_ref] New seed dormancy loci detected from weedy rice-derived advanced populations with major..., Ye [/bib_ref].
## Plant cultivation, and seed harvesting and storage
The BC 1 F 1 and BC 2 F 1 populations were grown in greenhouses in different years. To capture all available genotypes in a mapping population, hybrid seeds were air-dried to break dormancy, germinated at 30°a nd 100% relative humidity for 5 d, and cultured with a nutrient solutionfor 2 wk. Seedlings were transplanted into pots (28 cm diameter · 25 cm height), with one plant per pot, and filled with a mixture of clay soil and Sunshine #1 medium (Sun Gro Horticulture). Greenhouse temperatures were set at 29/21°for day/night, and the day-lengths were natural, except from the 6th to 8th wk when a 10-hr (8:00-18:00) short-day treatment was used to synchronize flowering. Plants were tagged for flowering dates when the first panicle in a plant emerged from the leaf sheath. Panicles were covered with white pollination bags at $10 d after flowering and the bags fixed to bamboo poles to prevent shattering due to brushing or shaking the plant. Seeds were harvested at 40 d after flowering, air-dried in the greenhouse for 3 d, and stored in a freezer (220°) to maintain the status of dormancy developed on the plant (i.e., primary dormancy).
## Phenotypic identifications for sd and morphologies
SD: The primary dormancy was evaluated by germination percentage for both seeds and caryopses from the BC 1 F 1 and for seeds from the BC 2 F 1 populations. A "seed" in grass species usually refers to a dispersal unit, which consists of the seed component (embryo, endosperm, and testa) and covering (pericarp and hull, or lemma and palea) tissues, whereas a caryopsis is a hull-removed seed enclosed by the pericarp. To evaluate seed germination, after-ripening (AR) treatments were used to release part of the primary dormancy to better display genotypic variation on the percentage scale. Briefly, seeds from each plant were allocated into three or four sets and stored in a lab room (24-25°) for a series of 7 or 10 d intervals to obtain various degrees of partially AR samples. Caryopses were prepared by hand removal of the hull from non-AR seeds. About 50 seeds/caryopses were distributed in a 9 cm petri dish, which was lined with a filter paper and wetted with 8 ml deionized water. A germination experiment was replicated three times in an incubator set for 30°, 100% relative humidity, and dark conditions. Germinated seeds (radicle protrusion . 3 mm) were counted at day 7.
Awn: The BC 2 F 1 populations were evaluated for the morphological traits awn, hull color, and pericarp color, to confirm their correlations with SD in the BC 1 F 1 "EM93-1//EM93-1/LD" population [bib_ref] Dormancy imposed by covering tissues interrelated with seed shattering and morphological characteristics..., Gu [/bib_ref]. An awn is a needle-like appendage extended from the terminal end of a lemma and functions in aiding seed dispersal or movement into wet soil. The awn trait varies in length with plants, as well as with seeds on a panicle, in a segregating population. Thus, the trait was quantified by the mean awn length, and the percentage of seeds with an awn, in a random sample of .50 seeds from a BC 2 F 1 plant.
Hull color: This trait was measured with the ChromaMeter Minolta CR310, which transfers reflectance spectra into the L à , a à , and b à readings to quantify blackness, redness, and yellowness, respectively. The L à readings range from 0 to 100, with 0 and 100 indicating completely nonreflective (black) and perfectly reflective (white), respectively. The a à readings vary from 2100 to 100, with negative and positive values indicating greenness and redness, respectively. The b à readings also vary from 2100 to 100, with negative and positive values indicating blueness and yellowness, respectively. The reflectance spectra were measured using $100 seeds in a 6 cm petri dish on a dark background, and means of three independent readings for each of the spectra used for data analysis.
Pericarp color: This trait was visually scored as red/brown (1) or white (0) for correlation analysis. This was partly because most BC 2 F 1 plants had an insufficient amount of seeds to prepare caryopses for the reflection spectrum measurement after the replicated germination tests. In addition, the pigment trait is controlled by the gene Rc encoding a bHLH familiar transcription factor in rice [bib_ref] Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in..., Sweeney [/bib_ref] [bib_ref] The Rc and Rd genes are involved in proanthocyanidin synthesis in rice..., Furukawa [/bib_ref]. This regulatory gene is also one of the QTL for SD (i.e., qSD7-1) and its functional alleles are present in tropical and temperate ecotypes of weedy "red" rice, including LD and SS18-2, to control maternal tissue-imposed dormancy [bib_ref] Association between seed dormancy and pericarp color is controlled by a pleiotropic..., Gu [/bib_ref] n Summary of segregation distortion loci linked to seed dormancy QTL in the BC 1 F 1 (EM93-1//EM93-1/LD) and BC 2 F 1 populations
## Locus (qtl)
Chr Population a Significance of the deviation from the 1:1 expectation at probability levels of à P , 0.05, Ãà P , 0.01, or ÃÃà P , 0.0001.
## Number of plants
b
This population was segregating for a short segment containing qSD7-1/ RM21197 on Chr 7 that may not encompass the segregation distortion locus.
## Marker genotyping and map construction
Fresh leaves were used to prepare genomic DNA samples for marker genotyping. More than 300 rice microsatellite markers from the 12 chromosomes (Chrs) of rice [bib_ref] Constructing Genetic Maps with MAPMAKER/EXP 3.0, Ed. 3. Whitehead Institute, Lincoln [/bib_ref] , including all of those used to map the SS18-2 genome [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref] , were screened for polymorphism between EM93-1 and LD. Information on the markers (primer sequences, repeat motifs, and genomic positions) is available in the Gramene database (http://archive.gramene. org/markers/microsat/). DNA extraction, marker amplification by polymerase chain reaction (PCR), and PCR product display by electrophoresis in a 6% nondenatured polyacrylamide gel were performed using the previously described methods [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref]. Marker genotypes were scored using the AlphaEaseFC (Alpha Innotech) gel imaging system. Polymorphic markers with a size difference suitable to score were used to genotype the BC 1 F 1 s to develop a linkage map covering the weedy rice genome. Markers on the Chr or Chr segments heterozygous for the BC 1 F 1 plants #9 and #139 were used to genotype the BC 2 F 1 (9) and BC 2 F 1 (139) populations, respectively, to develop partial linkage maps. Linkage maps were constructed using MAPMAKER/EXP 3.0 [bib_ref] Constructing Genetic Maps with MAPMAKER/EXP 3.0, Ed. 3. Whitehead Institute, Lincoln [/bib_ref]. Map distances in centiMorgan (cM) were converted from recombination fractions using the Kosambi mapping function. Markers were grouped at the LOD score of 3.0 and the maximum distance of 50 cM (equivalent to 0.38 recombination fraction). Linkage groups were assigned to the 12 Chrs based on markers' physical positions [bib_ref] Constructing Genetic Maps with MAPMAKER/EXP 3.0, Ed. 3. Whitehead Institute, Lincoln [/bib_ref]. Orders of closely linked (a few cMs) markers were also checked for physical positions on the Nipponbare genome sequence (International Rice Genome Sequencing Project 2005).
## Data and qtl analysis
Germination data from the BC 1 F 1 population were used to infer correlations for the degree of dormancy between seeds and caryopses and between the AR treatments. Data from the BC 2 F 1 populations were used to estimate correlations of SD with each of the morphological traits. Linear correlation analysis was performed using the SAS CORR program.
For QTL analysis, germination data (x) were transformed by sin 21 (x) 20.5 to improve the normality. The analysis was performed using Windows QTL Cartographer V2.5_011. The interval mapping program was used to scan for QTL at 1 cM walking speed and 1000 permutations at 5% error rate. The composite interval mapping program was used to define QTL peak positions and C.I. (one-LOD support regions), and to estimate the effects of the mapped loci and their contributions to the phenotypic variances (R 2 ).
## Data availability
Data for the origin and differentiation of the parental lines and data for trait correlations in the BC populations are available as Tables S1-S3 in File S1. Phenotypic and genotypic datasets from the mapping populations are available upon request.
# Results
Genetic differentiation and linkage map F 1 plants from the EM93-1/LD cross had $65% seed set, which was $30% lower than the seed set rate for F 1 EM93-1/SS18-2 plants [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref]. Partial sterility is a characteristic of hybrid F 1 s from an intersubspecific cross in rice [bib_ref] Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a..., Oka [/bib_ref]. The common parent EM93-1 in the two crosses is an indica line. The lower seed set rate in the EM93-1/ LD cross supports that LD is japonica-like [bib_ref] Genetic characterization of weedy rices and the inference on their origins, Tang [/bib_ref] and genetic differentiation between the parents is greater than the level for an introsubspecific cross.
DNA polymorphism between EM93-1 and LD was $60%, based on 256 markers amplified for alleles of predicted molecular sizes. Based on the BC 1 F 1 EM93-1//EM93-1/LD (LD-BC 1 F 1 ) population, a linkage map was constructed using 139 markers, with 0.027% missing values. This map covered a total of 1650 cM for the 12 Chrs, with the mean intermarker distance being 12.9 (6 8.9) cM. About 45% of the 139 markers were also located on the linkage map constructed using the BC 1 F 1 EM93-1//EM93-1/SS18-2 (SS18-BC 1 F 1 ) population [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref]. The total genetic distance was 250 cM shorter for the LD than for the SS18-2 genome in the EM93-1 background.
Segregation distortion was observed for some or all of the markers on Chrs 6, 7, and 12 in the LD-BC 1 F 1 population . The segregation ratio was biased against heterozygotes for the markers on Chrs 7 and 12, but toward the heterozygote for RM176 located near the end of the long arm of Chr 6 . Because the BC 1 F 1 population was developed using the F 1 EM93-1/LD plants as the male parent, the distortions must be caused by functionally differentiated genes for male gametophyte development or pollination of the hybrid (sporophyte). Such sporo-gametophytic interaction genes were associated with partial sterility of hybrids from distant crosses in the O. sativa complex [bib_ref] Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a..., Oka [/bib_ref]. In contrast, a segregation distortion was not detected for any of the loci on Chrs 6 and 7, and the markers distal to the major SD QTL qSD12, in the SS18-BC 1 F 1 population [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref].
QTL associated with SD in the BC 1 F 1 population The frequency distribution pattern of percent germination varied with seeds, caryopses, or days of AR (DAR) in the LD-BC 1 F 1 population [fig_ref] Figure 2: Genome-wide scan for seed dormancy QTL in the BC 1 F 1... [/fig_ref]. Correlations (r) of seed germination between any two of the 1, 11, 21, and 31 DAR treatments were significant in File S1), but coefficients of determination were relatively low (R 2 = 0.30-0.77). Similarly, the degree of dormancy between seeds and caryopses was positively correlated, but the R 2 values (0.17-0.21) were even lower than the estimates for seeds at the different DAR in File S1). Therefore, all these measurements were used to detect qSD.
A total of eight qSDs were detected in the population [fig_ref] Figure 2: Genome-wide scan for seed dormancy QTL in the BC 1 F 1... [/fig_ref]. Of them, qSD7-1 was the only one whose effect could be detected by seeds at 1, 11, 21, and 31 DAR. This major QTL contributed more to the variance in germinability for caryopses (R 2 = 0.27) than for seeds (R 2 = 0.07-0.15), and was collocated with Rc. The collocation accounted for the phenotypic correlation between the dormancy and pericarp color traits [bib_ref] Dormancy imposed by covering tissues interrelated with seed shattering and morphological characteristics..., Gu [/bib_ref]. The remaining loci were associated with one to three of the five measurements . LD and EM93-1 contribute n Summary of QTL for seed dormancy (qSD) identified in the BC 1 F 1 (EM93-1//EM93-1/LD) and BC 2 F 1 populations (9) QTL, quantitative trait loci; Chr, chromosome; LR, likelihood ratio; DAR, days after ripening; qSD, QTL for seed dormancy. a Number in the parentheses is the genetic distance of the peak located above (2) or below the marker on the Chr or Chr segment in , , or .
b LR and proportion of the variance explained by the QTL (R 2 ). c Difference between the heterozygous and homozygous genotypes at the locus in arcsine-transformed percent germination for intact seeds at DAR or for caryopsis. the dormancy-enhancing allele to seven and one (qSD1-2) of the eight QTL, respectively.
QTL associated with SD in the BC 2 F 1 (9) population BC 1 F 1 plant #9 is heterozygous for Chr 9 and part of the others except Chrs 5 and 10, while the remainder of the plant genome was synchronized by EM93-1. The total length of the heterozygous regions on the 10 Chrs is $600 cM, as estimated based on the BC 2 F 1 (9) population of 130 plants . The heterozygous regions cover peak-containing (one-LOD support) intervals for qSD7-1, 8, 9, and 12 detected in the BC 1 F 1 population. Phenotypic variation for SD at 7, 14, and 21 DAR , and segregating distortion for the three loci , were observed in the BC 2 F 1 (9) population. A total of five qSDs, including qSD7-1, 8, and 12, but not qSD9, were detected in the advanced BC population . The new locus qSD6-3 was located on the RM528-176 interval near the end of the long arm of Chr 6, and has the dormancy-enhancing allele from LD . The other new locus, qSD7-2, is the second QTL on Chr 7 and has the dormancyenhancing allele from EM93-1.
qSD12 accounted for 67% of the variance in germination percentage at 21 DAR when effects of the other QTL were not significant in the BC 2 F 1 (9) population . However, qSD12's effect was not significant at 7 and 14 DAR when the others were detectable. These results suggest that qSD12 maintained an inhibitory effect on germination longer than the other SD QTL. In addition, the severe segregation distortion for the qSD12-containing region, which greatly reduced the genotypic frequency for heterozygotes (6%) in the BC 2 F 1 population , must also lower the power to evaluate the QTL's effect on germination at an early stage of AR.
QTL associated with SD in the BC 2 F 1 (139) population The BC 1 F 1 plant #139 is heterozygous for Chr 6 and part of the others except Chrs 9 and 12, while the remainder of the plant genome was synchronized by EM93-1. The total length of the heterozygous regions on the 10 Chrs is $550 cM, as estimated based on the BC 2 F 1 (139) population of 151 plants . The heterozygous regions cover peak-containing intervals of qSD4, 6-1, 7-1, 8, and 10 detected in the BC 1 F 1 , and qSD6-3 detected in the BC 2 F 1 (9) population. Phenotypic variation for SD at 7, 14, and 21 DAR , and segregation distortion for RM176 on Chr 6 , were observed in this population. A total of six qSDs, including qSD6-1, 6-3, 7-1, and 10 were detected , but qSD4 and 8 were not significant in the advanced BC population. Two new loci (qSD1-1 and 6-2) were identified in the BC 2 F 1 population and both have the dormancy-enhancing allele from LD . Of the three QTL on Chr 6, qSD6-3 contributed most to the phenotypic variance .
The BC 2 F 1 (139) population segregated on Chr 7 for a segment of $30 cM encompassing qSD7-1 and its flanking markers from RM481 to RM5481 . However, segregation distortion was not detected for these markers, including RM21197 located within the qSD7-1 underlying gene [bib_ref] Association between seed dormancy and pericarp color is controlled by a pleiotropic..., Gu [/bib_ref]. This result indicates that the gene responsible for the segregation distortion in the BC 1 F 1 and BC 2 F 1 (9) populations locates outside the 30 cM segment. Mapping of seed dormancy QTL in the BC 2 F 1 (9) population. (A) A partial linkage map. The map was constructed with markers on 10 Ch or Ch segments segregating in the population. Black bars indicate one-LOD support lengths for qSD, qAn, or qHC. Ovals indicate positions of qSDs previously detected in the BC 1 F 1 EM93-1//EM93-1/SS18-2 population [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref]. (B) Frequency distributions of percent germination. N was the number of BC 2 F 1 plants evaluated at 7, 14, or 21 DAR. (C) Distributions of LRs along the map. qSDs were inferred by peaks of the LR distributions above the threshold. Ch, chromosome; DAR, days of after-ripening; LOD, logarithm of the odds; LR, likelihood ratio; qAn, QTL for awn; qHC, QTL for hull color; qSD, QTL for seed dormancy; QTL, quantitative trait loci.
QTL associated with the seed morphological traits Phenotypic variation for each of the morphological traits and their correlations with SD were observed in the two BC 2 F 1 populations, with the presence of awn, dark pigment on the hull, or red pigment on the pericarp tissue tending to reduce germination percentage in File S1). The phenotypic correlations were similar to those observed in the three BC 1 F 1 populations derived from different lines of weedy rice, including LD and SS18-2 [bib_ref] Dormancy imposed by covering tissues interrelated with seed shattering and morphological characteristics..., Gu [/bib_ref]. Two awn QTL (qAn4-1 and 8) were detected in each of the LD-BC 1 F 1 and two BC 2 F 1 populations . In the BC 2 F 1 populations, the contribution of qAn8 to the phenotypic variance (R 2 ) was three to four times greater for the percentage of awned seeds than for awn length, while qAn4-1 contributed almost equally to the two measurements. qAn4-1 and 8 were linked to but not collocated with qSD4 and 8, respectively .
A major QTL (qHC4) and two modifiers (qHC 2 and 7) were associated with hull color . qHC4 was detected in all of the three populations and contributed most to phenotypic variances in the visual score and component reflection spectra. This major QTL was collocated with qSD4 . The modifiers qHC2 and 7 were detected in the BC 2 F 1 #9 and #139 populations, respectively. qHC7 contributed 8% to the phenotypic variance for the red reflectance only and was collocated with qSD7-1/Rc . It is likely that the modifier qHC7 could be the Rc locus, which was associated with the visual score for the pericarp color in the two BC 2 F 1 populations. This is because the red pigment can be seen on intact straw hull-colored seeds.
# Discussion
Similarity of SD genes between distinct ecotypes of weedy rice A total of 12 SD QTL were identified from the primary and advanced BC populations developed using LD as the nonrecurrent parent. Two-thirds (eight) of these loci were detected from the BC 1 F 1 population, and the remaining 1/3 identified from the BC 2 F 1 populations where $65% of the genome was synchronized by the recurrent parent EM93-1. The temperate ecotype line LD has dormancy-enhancing alleles at 10 (83%) of the 12 loci. The estimate of 83% is close to the previous observation that the tropical ecotype line SS18-2 has dormancy-enhancing alleles at 80% of the 10 QTL detected in the EM93-1 background [bib_ref] New seed dormancy loci detected from weedy rice-derived advanced populations with major..., Ye [/bib_ref].
The SD QTL identified from the populations with LD or SS18-2 as the nonrecurrent parent represent a majority of the reported loci differentiated between wild/weedy and cultivated rice in regard to approximate map positions. For example, qSD6-1, 2, and 3 are similar to those on Chr 6 reported for wild [bib_ref] Genomic regions affecting seed shattering and seed dormancy in rice, Cai [/bib_ref] and weedy [bib_ref] Mapping QTL for seed dormancy in weedy rice, Jing [/bib_ref] rice; qSD4, 7-1, and 7-2 were located on the same marker intervals as the three QTL reported for the three accessions of weedy rice from USA [bib_ref] Genetic architecture of seed dormancy in U.S. weedy rice in different genetics..., Subudhi [/bib_ref] [bib_ref] Quantitative trait locus and haplotype analyses of wild and crop-mimic traits in..., Mispan [/bib_ref] ; and qSD1-1 and sd1 reported for wild rice [bib_ref] Genetic analysis of rice domestication syndrome with the wild annual species, Li [/bib_ref] were both mapped on the top of Chr 1. However, some loci reported by the other groups, such as qSD-2 [bib_ref] Mapping QTL for seed dormancy in weedy rice, Jing [/bib_ref] , qSD3 [bib_ref] Genetic architecture of seed dormancy in U.S. weedy rice in different genetics..., Subudhi [/bib_ref] , and sd12 [bib_ref] Genetic analysis of rice domestication syndrome with the wild annual species, Li [/bib_ref] , were not detected our research. It is possible that some SD genes could have been eliminated from founders of the LD and SS18 populations, or lost during evolution of the weed ecotypes.
The SS18 ($7°N) and LD ($34°N) populations acquired a similar level of SD in geographically isolated ecosystems because they share most genes for the adaptive trait. There are nine common loci (qSD1-1, 1-2, 4, 6-1, 7-1, 7-2, 8, 10, and 12) that are functionally differentiated for SD in both EM93-1/SS18-2 and EM93-1/LD crosses. It is estimated that the tropical and temperate ecotypes are similar in genotype for 75% of the 12 SD loci, if multiple alleles (more than two at a locus) are ignored. The estimated degree of similarity is similar to the report for the dicot model Arabidopsis thaliana [bib_ref] Natural variation for seed dormancy in Arabidopsis is regulated by additive genetic..., Bentsink [/bib_ref]. For example, of a total of 11 SD QTL identified for six ecotypes in the Landsberg erecta background, nine (82%) had an effect on delay of germination in two or n Summary of QTL for the awn and hull color traits identified in the BC 1 F 1 (EM93-1//EM93-1/LD) and BC 2 F 1 populations a Number in the parentheses is the genetic distance of the peak located above (2) or below the marker on the Chr or Chr segment in , , or .
b LR and proportion of the variance explained by the QTL (R 2 ). c Difference between the heterozygous and homozygous genotypes in the trait value. d The trait awn was measured by the percentage of seeds with awn and the mean awn length for seeds from a plant; and the hull color was measured by visual scores (dark vs. straw) for the BC 1 F 1 population and by reflection spectrum readings for darkness (L à ), red redness (a à ), and yellowness (b à ) for the BC 2 F 1 population. more of the Arabidopsis populations [bib_ref] Natural variation for seed dormancy in Arabidopsis is regulated by additive genetic..., Bentsink [/bib_ref]. Thus, the comparative mapping results from the monocot and dicot models strongly suggest that naturally occurring genes controlling SD in a species were highly conserved during evolution.
## Evolutionary mechanisms of sd
The adaptive significance of SD relies on functionally differentiated alleles at multiple loci to regulate the time of germination in local ecosystems. This and the previous research in weedy rice revealed several mechanisms involved in regulating genotypic/allelic frequencies at SD loci in both natural and agricultural ecosystems. The first mechanism is direct selection for the time of germination, which is critical for locally adapted genotypes to complete their life cycle. An extreme example is the domestication of cereal crops by artificial selection for rapid germination mutants [bib_ref] The possible role of weed races in the evolution of cultivated plants, Harlan [/bib_ref]. The second mechanism is indirect selection for wild-type characteristics correlated with SD. A phenotypic selection for the presence of awn, dark hull, or red pericarp tended to enhance SD in File S1). The indirect selections retained the dormancyenhancing alleles at the loci (e.g., qSD4, 7-1, and 8) linked to or collocated with the genes for the interrelated traits ; [bib_ref] Genetic analysis of adaptive syndromes interrelated with seed dormancy in weedy rice..., Gu [/bib_ref] [bib_ref] Quantitative trait locus and haplotype analyses of wild and crop-mimic traits in..., Mispan [/bib_ref]. Some of the "linkage drags" or collocations could be pleiotropic effects of single genes. For example, qSD7-1 and Rc are underlain by the same transcription factor gene (Os07g1120) regulating both abscisic acid (a dormancy-inducing hormone) and flavonoid (red pigments) biosynthesis pathways in early developing seeds [bib_ref] Association between seed dormancy and pericarp color is controlled by a pleiotropic..., Gu [/bib_ref]. The indirect selections explain why SD is generally stronger in black-hulled awned "red" rice populations than in those without the wild-type characteristics. Genome-wide phylogenetic analyses revealed that the hull color, pericarp color, and awn gene-containing regions were intensively selected during domestication and are informative for research on origins of weedy rice [bib_ref] Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a..., Oka [/bib_ref] [bib_ref] Escape to ferality: the endoferal origin of weedy rice from crop rice..., Kanapeckas [/bib_ref] [bib_ref] Signatures of adaptation in the weedy rice genome, Li [/bib_ref].
The third mechanism is indirect selection for crop-mimic traits, such as plant height and flowering time. LD contains dormancy-reducing alleles at qSD1-2 and qSD7-2. Both loci also have an effect on plant height, when the QTL alleles were introduced from SS18-2 into the EM93-1 background [bib_ref] Genetic and physiological characterization of two clusters of quantitative trait loci associated..., Ye [/bib_ref]. qSD1-2 was cloned as semidwarf1 (sd1), a major gene for plant height. The semidwarf line EM93-1 carries a dormancy-enhancing allele, while a vast majority of wild/weedy rice lines (including LD and SS18-2) have a dormancy-reducing allele at qSD1-2/sd1 . A high frequency of the dormancyreducing allele in the nondomesticated germplasm is indicative that the natural selection on such a pleiotropic gene has a greater impact on plant height than on dormancy. Collocation was also reported for the SD/heading date QTL on Chr 3 (Sdr1/Hd8; [bib_ref] Fine linkage mapping enables dissection of closely linked quantitative trait loci for..., Takeuchi [/bib_ref] and 6 (qSD-6-2/qHD-6; [bib_ref] Mapping QTL for seed dormancy in weedy rice, Jing [/bib_ref] , with the dormancy-enhancing alleles delaying flowering. Thus, correlational selections for crop-mimic traits may not favor the retention of a dormancy-enhancing allele, but could contribute to genetic diversity in germination capacity.
The other mechanism was inferred by the three segregation distortion loci (SDL) linked to (qSD7-1) or collocated with (qSD6-3 and 12) an SD locus. The segregation distortion favored a transmission of the dormancy-reducing alleles at qSD7-1 and 12, or the dormancy-enhancing allele at qSD6-3, through gametes produced by heterozygotes from Mapping of qSD in the BC 2 F 1 (139) population. (A) A partial linkage map. The map was constructed with markers on 10 Ch or Ch segments segregating in the population. Black bars indicate one-LOD support intervals for qSD, qAn, or qHC. Ovals indicate positions of qSDs previously detected in the BC 1 F 1 EM93-1//EM93-1/SS18-2 population [bib_ref] Multiple loci and epistasis control genetic variation for seed dormancy in weedy..., Gu [/bib_ref] [bib_ref] Genetic analysis of adaptive syndromes interrelated with seed dormancy in weedy rice..., Gu [/bib_ref] [bib_ref] New seed dormancy loci detected from weedy rice-derived advanced populations with major..., Ye [/bib_ref]. (B) Frequency distributions of percent germination. N was the number of BC 2 F 1 plants evaluated at 7, 14, or 21 DAR. (C) Distributions of LRs along the map. qSDs were inferred by peaks of the LR distributions above the threshold. Ch, chromosome; DAR, days after ripening; LOD, logarithm of the odds; LR, likelihood ratio; qAn, QTL for awn; qHC, QTL for hull color; qSD, QTL for seed dormancy; QTL, quantitative trait loci. the EM93-1/LD cross . A similar pattern of segregation distortion was also observed for a qSD12-containing region, when it was heterozygous for the alleles from SS18-2 and EM93-1; this SDL had a larger effect on eliminating the dormancy-enhancing allele through the male than through the female gametes [bib_ref] Genotyping of endosperms to determine seed dormancy genes regulating germination through embryonic,..., Gu [/bib_ref]. Natural hybridization occurs between weedy and cultivated rice at a low rate [bib_ref] Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a..., Oka [/bib_ref]. Thus, gametophyte development genes that cause segregation distortions in hybrid populations could also influence allelic frequencies for some SD loci, but the influence varies depending on crosses.
## Possible applications of sd genes
In addition to understanding the origins of conspecific/congeneric weeds in agroecosystems [bib_ref] Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a..., Oka [/bib_ref] [bib_ref] Escape to ferality: the endoferal origin of weedy rice from crop rice..., Kanapeckas [/bib_ref] [bib_ref] Signatures of adaptation in the weedy rice genome, Li [/bib_ref] , SD genes conserved across ecotypes could be manipulated to develop a TM strategy. The TM strategy was proposed to complement transgene containment techniques to reduce the risk of gene flow from genetically modified (GM) crops to wild relatives [bib_ref] Tandem constructs: preventing the rise of superweeds, Gressel [/bib_ref]. The basic concept is a built-in linkage between a fitness-enhancing transgene (e.g., herbicide resistance) and a mitigating factor (e.g., reduced SD), which has no negative effect on the GM crop but could reduce the adaptability of weed/crop hybrids to lower the transgene's frequency in a weed population across generations [bib_ref] Tandem constructs: preventing the rise of superweeds, Gressel [/bib_ref]. Silencing SD genes could promote germination uniformity (as for cereal cultivars) and make weeds relatively easy to eliminate by agronomic practices. We are using the qSD7-1 and 12 underlying genes as silencing targets and the RNA interference and genome editing techniques to prove the TM concept in weedy red rice.
[fig] Figure 2: Genome-wide scan for seed dormancy QTL in the BC 1 F 1 EM93-1//EM93-1/LD population. (A) Frequency distributions of percent germination for seeds or caryopses. N was the number of plants evaluated for germination at DAR. (B) Distributions of LR along the 12 Ch. Seed dormancy QTL (qSD) were inferred by peaks of the LR distributions above the threshold. Ch, chromosome; DAR, days after ripening; LR, likelihood ratio; qSD, QTL for seed dormancy; QTL, quantitative trait loci. [/fig]
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Longitudinal report of child with de novo 16p11.2 triplication
Key Clinical Message 16p11.2 deletions and duplications are commonly associated with autism spectrum disorder and linked to mirrored phenotypes of physical characteristics and higher penetrance for deletions. A male with a rare 16p11.2 triplication demonstrated a similar phenotypic presentation to deletion carriers with neurocognitive and adaptive skill deficits and above-average physical growth.
# Introduction
Recurrent deletions and duplications of an approximately 600 kbp region of chromosomal locus 16p11.2 are a frequent genetic etiology of neurodevelopmental disorders such as autism spectrum disorder (ASD), developmental delay, intellectual disability, and schizophrenia [bib_ref] Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded..., Bijlsma [/bib_ref] [bib_ref] Comparative genomics of autism and schizophrenia, Crespi [/bib_ref] [bib_ref] Microduplications of 16p11.2 are associated with schizophrenia, Mccarthy [/bib_ref] [bib_ref] Recurrent reciprocal 16p.11.2 rearrangements associated with global developmental delay, behavioral problems, dysmorphism,..., Shinawi [/bib_ref]. 16p11.2 copy number variations (CNVs) occur with a frequency of one of 1000 in the general population [bib_ref] Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2..., Jacquemont [/bib_ref] and a range of 0.76% to 1.13% in nonsyndromic, idiopathic ASD depending on sample composition [bib_ref] Copy number variation in the dosage-sensitive 16p11.2 interval accounts for only a..., Walsh [/bib_ref] making 16p11.2 one of the most prevalent ASD risk variants. Clinical presentations of 16p11.2 CNVs suggest heterogeneous phenotypes including cognitive, motor, and language delays; behavioral problems; congenital anomalies; and dysmorphic features [bib_ref] Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2..., Jacquemont [/bib_ref] [bib_ref] Copy number variation in the dosage-sensitive 16p11.2 interval accounts for only a..., Walsh [/bib_ref] [bib_ref] Clinical report of microphthalmia and optic nerve coloboma associated with a de..., Bardakjian [/bib_ref] [bib_ref] Phenotypic spectrum associated with de novo and inherited deletions and duplications at..., Fernandez [/bib_ref] [bib_ref] Cognitive and behavioral characterization of 16p11.2 deletion syndrome, Hanson [/bib_ref] [bib_ref] Microdeletion of 16p11.2 associated with endocardial fibroelastosis, Puvabanditsin [/bib_ref] [bib_ref] A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and..., Zufferey [/bib_ref]. 16p11.2 deletions and duplications are also associated with increased incidence of structural brain abnormalities [bib_ref] Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded..., Bijlsma [/bib_ref] [bib_ref] Recurrent reciprocal 16p.11.2 rearrangements associated with global developmental delay, behavioral problems, dysmorphism,..., Shinawi [/bib_ref] [bib_ref] Opposing brain differences in 16p11.2 deletion and duplication carriers, Qureshi [/bib_ref]. It has been suggested the 16p11.2 deletion is more penetrant than the duplication, such that deletion carriers show a higher rate of ASD diagnosis [bib_ref] Phenotypic spectrum associated with de novo and inherited deletions and duplications at..., Fernandez [/bib_ref] and developmental delay [bib_ref] Recurrent reciprocal 16p.11.2 rearrangements associated with global developmental delay, behavioral problems, dysmorphism,..., Shinawi [/bib_ref].
Copy number variation triplications are quite rare, and subsequently, the associated phenotype for 16p11.2 triplication is unknown. Reports of triplication at other loci indicate that triplication cases resemble duplication patients, including behavioral problems associated with 17q21.31 triplication [bib_ref] De novo triplication of the MAPT gene from the recurrent 17q21.31 microdeletion..., Gregor [/bib_ref] and developmental delay associated with 11q12.3 [bib_ref] De novo triplication of 11q12.3 in a patient with developmental delay and..., Yamamoto [/bib_ref]. Although instances of triplication in 16p11.2 have been reported [bib_ref] Discovery of a previously unrecognized microdeletion syndrome of 16p11.2-p12.2, Ballif [/bib_ref] , this is the first study to report a comprehensive phenotype assessment on the developmental phenotype and trajectory of a child with a 16p11.2 triplication over a period of 4 years. 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.
# Methods
The proband was ascertained as part of the longitudinal portion of the Simons Variation in Individuals Project (SVIP) and evaluated during seven study visits between 31 and 72 months old (four clinic visits, three phone calls) with written consent from his parents. This research was approved by the University of Washington Institutional Review Board. Assessments included standardized cognitive, behavioral, adaptive, and ASD evaluations (Appendix S1); neurological examination; and neurologic history and record review by a board-certified pediatric neurologist (KJS). Comparative data for individuals with 16p11.2 deletion or duplication were downloaded from the SFARI Simons VIP cohort. The comparison sample of Simons VIP participants included individuals with the same recurrent 600 kbp BP4-BP5 16p11.2 deletion or duplication without other pathogenic CNVs or known genetic diagnoses identified through clinical evaluations [bib_ref] Developmental trajectories for young children with 16p11.2 copy number variation, Bernier [/bib_ref] [bib_ref] The cognitive and behavioral phenotype of the 16p11.2 deletion in a clinically..., Hanson [/bib_ref] and recruited via referral to the Simons VIP Connect Web site (SimonsVIPConnect.org). See the Simons VIP consortium [bib_ref] Simons Variation in Individuals Project (Simons VIP): a genetics-first approach to studying..., Simons [/bib_ref] for additional information regarding recruitment and eligibility criteria (http://sfari. org/resources/simons-vip).
Three approaches were used to assay chromosome 16p11.2 copy number in the proband and selected relatives: fluorescence in situ hybridization (FISH), wholegenome SNP microarray experiments, and molecular inversion probe (MIP) experiments. Specifically, FISH was performed in a clinical cytogenetics laboratory using a bacterial artificial chromosome (BAC) clone (RP11-74E23) as the probe. Microarray analysis leveraged the Affymetrix Whole Genome-Human SNP Array 6.0, while MIP experiments utilized probes targeting 54 genetic markers (43 informative with high confidence) distinguishing duplicated sequences on each side of the chromosome 16p11.2 critical region (telomeric/BP4, GRCh37/hg19 chr16:29562241-29606852; centromeric/ BP5, GRCh37/hg19 chr16:30302259-30346868) from each other and from all other paralogous sequences [bib_ref] Emergence of a Homo sapiens-specific gene family and chromosome 16p11. 2 CNV..., Nuttle [/bib_ref]. All MIP experiments incorporated a molecular-tagging strategy [bib_ref] Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation, Hiatt [/bib_ref] and a previously described analysis pipeline [bib_ref] Rapid and accurate largescale genotyping of duplicated genes and discovery of interlocus..., Nuttle [/bib_ref].
# Results
# Genetic analysis
FISH experiments performed in a clinical laboratory revealed increased copy number at chromosome 16p11.2 in the proband and in his mother. We verified this diagnosis and assessed inheritance by performing SNP microarray [fig_ref] Figure 1: Genetic Overview [/fig_ref] and/or MIP genotyping [fig_ref] Figure 1: Genetic Overview [/fig_ref] , [bib_ref] Rapid and accurate largescale genotyping of duplicated genes and discovery of interlocus..., Nuttle [/bib_ref] on the proband, his parents, his sister, his maternal aunt (monozygotic twin of his mother), and his maternal grandparents using genomic DNA. These higher-resolution analyses revealed a typical chromosome 16p11.2 duplication in the mother, occurring on the short arm of chromosome [bib_ref] Developmental trajectories for young children with 16p11.2 copy number variation, Bernier [/bib_ref] and spanning approximately 597 kbp (GRCh37/hg19 chr16:29488112-30085308). We identified a triplication of this same region in the proband, likely having originated via nonallelic homologous recombination (NAHR) during meiosis in the mother. No chromosome 16p11.2 rearrangements were observed in the grandparents, and we inferred that the maternal duplication originated in the grandmother [bib_ref] Maternal modifiers and parent-of-origin bias of the autismassociated 16p11. 2 CNV, Duyzend [/bib_ref]. Thus, remarkably, two distinct de novo, likely NAHR-mediated rearrangement events at chromosome 16p11.2, occurred within this family over two generations: an initial de novo duplication followed by the de novo expansion of this duplication to the triplication state (See [fig_ref] Figure 2: Pedigree of 16p11 [/fig_ref] Pedigree of 16p11.2 proband).
## Medical and developmental history
The proband is a Caucasian male with 16p11.2 triplication identified at 18 months. He was born via spontaneous vaginal delivery at 40 weeks gestation to a 31-yearold woman whose pregnancy was complicated only by 3 days of moderate vaginal bleeding of undetermined cause during first trimester. The proband weighed 9lbs 9 oz and measured 21.5 inches long at birth. A medical genetics evaluation at 24 months demonstrated multiple minor dysmorphic features including upslanting palpebral fissures, mild telecanthus, bilateral epicanthal folds, highly arched palate, cupped ears with overfolded helices, a small and broad uvula, a prominent anterior hair whorl, and delays in intramembranous ossification. Echocardiogram identified a likely right-sided aortic arch and two small, nonhemodynamically significant atrial septal defects. Ophthalmologic evaluation revealed mild temporal pallor of the optic nerves in both eyes and mild myopic astigmatism. Other medical issues include a history of chronic constipation and suspected environmental allergies.
## Neuroimaging
An MRI of the brain performed at 14 months demonstrated scattered foci of T 2 hyperintensity in bilateral periventricular and parietal subcortical white matter. These findings were interpreted by the reading radiologist as nonspecific and possibly physiologic for age. Follow-up MRI at 24 months demonstrated unchanged scattered areas of T 2 hyperintensity in the parietal subcortical regions and decreased periventricular T 2 hyperintensities, which were interpreted as remaining nonspecific in etiology, possibly representing in-utero hypoxia/ischemia. from the maternal grandmother (two copies), to the mother with a duplication (three copies), to the proband with triplication (four copies). The logR values increase from generation to generation. In particular, the ratio of the intensity between proband (III-3.) and mother (II-4.) is 1.3 on average, consistent with a ratios of four copies (triplication) to three copies (duplication) (4/3 = 1.3). Green bars indicate the location of segmental duplications associated with breakpoints 4 and 5-collapsed here for ease of display. (B) MIP data (top) and schematics depicting haplotypes inferred from these data (bottom) for the proband (P) carrying a chromosome 16p11.2 triplication, his mother (M) carrying a chromosome 16p11.2 duplication, and his father (F) showing normal copy number status at chromosome 16p11.2. The plots show paralog-specific copy number across a 45-kbp duplication block shared between breakpoint regions flanking the critical region [bib_ref] Emergence of a Homo sapiens-specific gene family and chromosome 16p11. 2 CNV..., Nuttle [/bib_ref]. Points indicate paralog-specific copy number estimates from 43 informative markers targeted in the MIP experiment. Dashed lines signify copy number calls inferred using an automated caller and confirmed by visual inspection.
## Anthropometric and neurological exam findings
Research-based neurological exams starting at 31 months indicated the proband had a single CAL in his left axilla; mild excessive lordosis; and multiple minor dysmorphic features including a broad forehead, broad nasal root, mild hypertelorism, bilateral epicanthal folds, bilateral prominent ears, wide-spaced nipples, small though mildly protuberant ears with thickened superior helices, and a double hair whorl (anterior hairline and cranial vertex). He exhibited facial hypotonia, copious drooling, and hypotonia. Notably, the proband's head circumference was similar to 16p11.2 deletion, and his weight and height were above the mean compared to both deletion and duplication cases [fig_ref] Figure 3: Developmental comparisons to 16p11 [/fig_ref]. By 48 months, dysarthria became notable and persisted, along with significant facial hypotonia and excessive drooling, through the 72-month visit. Lordosis was no longer seen at the 72-month visit, although truncal hypotonia remained notable. At 48 months, the proband's gait was marked by decreased bilateral arm swing and posturing of his arms in front when walking or running. His gait had normalized by 60 months, but he was unable to jump. By 72 months, he had learned to jump, but was still unable to walk on heels, hop on either foot, or balance on either foot for five-seconds.
## Behavioral phenotyping
Experienced, licensed clinicians derived DSM-IV-TRdiagnoses at each on-site visit . The proband demonstrated deficits in adaptive functioning and increasing levels of emotional and behavioral difficulties over time particularly in the areas of pervasive developmental problems, attention, affective problems, and externalizing behaviors (Supporting Information and . To frame behavioral issues in reference to other 16p11.2 CNVs, the proband's individual growth trajectories for the externalizing and internalizing symptoms were compared to the distribution plotted for children with 16p11.2 deletions and duplications [fig_ref] Figure 3: Developmental comparisons to 16p11 [/fig_ref] suggesting the 16p11.2 triplication proband exhibits increasing problems, similar to the 16p11.2 duplication profile.
# Discussion
This is the first in-depth phenotypic description of an individual with triplication within the 16p11.2 locus. The proband's medical history is significant for congenital heart defects, mild visual impairment, dysmorphisms, chronic constipation, and suspected environmental allergies. Additional phenotypic features included long-standing articulation difficulties, hypotonia, copious drooling, and abnormal gait and fine motor coordination. Over the course of the evaluations, the proband met DSM-IV-TR diagnostic criteria for PDD-NOS, mild intellectual disability, ADHD, enuresis, and encopresis. He exhibited developmental delays, impaired adaptive and cognitive functioning, and long-standing pervasive developmental problems in the areas of communication, reciprocal social interaction, repetitive behaviors, and unusual sensory interests and aversions.
CNV triplications are rare chromosomal variants [bib_ref] Intrachromosomal triplications: molecular cytogenetic and clinical studies, Reddy [/bib_ref] , often resulting in a phenotype that is most consistent with duplication of the same locus [bib_ref] A triplication of the Williams-Beuren syndrome region in a patient with mental..., Beunders [/bib_ref] [bib_ref] A girl with inverted triplication of chromosome 3q25.3 -> q29 and multiple..., Ounap [/bib_ref]. The proband exhibited increasing externalizing problems, similar to the Vertical lines indicate scores of borderline concern (dashed, T-scores above 60) and clinical concern (solid, Tscores above 65). The distribution of scores for children with 16p11.2 deletion and duplication are presented for comparison.
duplication carriers. However, other features of the 16p11.2 triplication phenotype more closely resemble the deletion carriers, including patterns of physical growth (e.g., head circumference, height, and weight). In other words, while some features convey a more severe phenotype, phenotypic consequences are not uniform. Recent evidence suggests that 16p11.2 reciprocal CNVs may produce mirrored (i.e., opposing) phenotypes. For instance, deletion carriers tend to exhibit obesity and macrocephaly, while duplication is associated with being underweight and microcephaly [bib_ref] Recurrent reciprocal 16p.11.2 rearrangements associated with global developmental delay, behavioral problems, dysmorphism,..., Shinawi [/bib_ref] [bib_ref] Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2..., Jacquemont [/bib_ref] [bib_ref] KCTD13 is a major driver of mirrored neuroanatomical phenotypes of the 16p11.2..., Golzio [/bib_ref]. These contrasting phenotypes have been proposed to support the presence of dosage sensitive genes within the 16p11.2 locus. This model is also consistent with analyses of gene expression in the context of chromosome 16p11.2 rearrangements, where expression levels of genes within the affected segment have been found to correlate strongly with genomic copy number, with little evidence for dosage compensation [bib_ref] Transcriptional consequences of 16p11. 2 deletion and duplication in mouse cortex and..., Blumenthal [/bib_ref]. Nevertheless, this triplication case conflicts with the notion of a simple graded genetic response between deletion, no-mutation, duplication, and triplication. This may suggest that the genetic micro-overexpression of the 16p11.2 locus (i.e., triplication, four copies instead of two) may be deleterious in a manner similar to a 16p11.2 microdeletion. Perhaps, some genes within the affected interval are sensitive to the precise level of increased dosage, such that triplication (but not duplication) may dysregulate them in a manner functionally equivalent to a deletion. It may also be the case that the haploinsufficient dosage within the triplicated segment is mediated in part by a possible complex chromosomal rearrangement that mimics the effect of a deletion of the same region. It is possible that none of these explanations are fully sufficient to describe the triplication phenotype, but rather together clarify how the perturbation of transcription factors may impact the regulation mechanisms of different genes within the 16p11.2 locus. Currently, the mechanistic bases of chromosome 16p11.2 deletion and duplication phenotypes are not well understood, but cellular and mouse modeling projects promise to provide new insights over the next several years. Further studies of human patients, including the identification and phenotypic characterization of additional triplication cases, will undoubtedly complement these efforts and help resolve basic and clinical questions surrounding one of the most common genetic contributors to abnormal neurodevelopment.
# Acknowledgments
We are sincerely grateful to the proband and his family for their participation in this study and to all the families at the participating Simons Variation in Individuals Project (Simons VIP) sites, as well as the Simons VIP working group Authorship ASW: performed lead role in writing and revision of the manuscript; involved in manuscript concept and design, clinical assessment, data acquisition, and final approval of manuscript for publication. CMH: involved in review of literature, manuscript concept and design, data acquisition, data analysis and interpretation of the results, drafting/revising the manuscript. KJS: completed neurological exams and review of neurological history and medical records for the case subject; involved in manuscript concept and design, data acquisition, drafting/revising the manuscript. JLP: involved in data acquisition, clinical assessment, writing and revision of the manuscript, final approval of the manuscript for publication. TDD: involved in data acquisition, data preparation and interpretation, revising the article for important intellectual content. MHD: involved in writing and revision of the manuscript; performed genetic analysis and interpretation of genetic testing results. XN: involved in writing and revision of the manuscript; performed genetic analysis and interpretation of genetic testing results. EEE: involved in writing and revision of the manuscript; performed genetic analysis and interpretation of genetic testing results. RAB: involved in design and implementation of the research; manuscript concept and design, data acquisition, revising the article for important intellectual content, final approval of the manuscript for publication.
[fig] Figure 1: Genetic Overview. (A) Copy number expansion of the 16p11.2 critical region. LogR (lines) and B-allele frequency (dots) plots show expansion of the 16p11.2 critical region [/fig]
[fig] Figure 2: Pedigree of 16p11.2 proband. De novo 16p11.2 copy number variations noted by shading, including the triplication (proband: III.-3, solid grey) and duplications (mother: II.-4; maternal aunt: II.-5, dashed grey). HTN, hypertension; Hx, history of; Sx, suspected. [/fig]
[fig] Figure 3: Developmental comparisons to 16p11.2 deletions and duplications. Comparison data was obtained from the publically available SFARI Simons VIP cohort. (A) Physical growth trajectories: head circumference, height, and weight physical growth for 16p11.2 triplication case were compared to 16p11.2 deletions (N = 181) and 16p11.2 duplications (N = 95). (B) Growth trajectories for externalizing and internalizing problems are plotted for the 16p11.2 triplication proband across time with age in months indicated at each time point, corresponding to T-scores from the CBCL Externalizing (left panel) and Internalizing (right panel) subscales. [/fig]
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The Evolution of Our Understanding of Immunoproliferative Small Intestinal Disease (IPSID) over Time
Citation: AlYamany, R.; Kharfan-Dabaja, M.A.; Hamadani, M.; Alshaibani, A.; Aljurf, M. The Evolution of Our Understanding of Immunoproliferative Small Intestinal Disease (IPSID) over Time. Curr. Oncol. 2022, 29, 3759-3769. https://
# Introduction
Immunoproliferative small intestinal disease (IPSID) represents a spectrum of indolent lymphoproliferative syndrome that involves different clonal disorder stages. It is considered a type of extra-nodal marginal zone B-cell lymphoma (MZL) of mucosa-associated lymphoid tissue (MALT) [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref].
Marginal zone lymphomas originate from the follicular marginal zone in the lymph nodes and are classified based on their anatomical involvement in three subtypes: splenic MZL, nodal MZL, and extra-nodal MZL [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref].
IPSID was first recognized in the early 1960s when it was termed small intestinal lymphoma; it was first described in the Mediterranean region and was previously known as "Mediterranean lymphoma." [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref]. Later in that decade, patients with Mediterranean lymphoma were noted to have abnormal IgA molecules in their body fluids and serum. These molecules were further studied and found to have a truncated alpha heavy chain; hence, the name was changed to alpha heavy chain disease [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
In 1978, the World Health Organization (WHO) further evaluated the disease and recognized that it was not necessarily a pre-lymphoma stage, but more of a spectrum of diseases, which included alpha heavy chain disease and Mediterranean lymphoma with different stages: benign, intermediate, and malignant. Furthermore, a more descriptive term was given, namely IPSID [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref].
## Epidemiology
IPSID is not common in the Western world. The majority of cases, mostly those described early on, were found in the Mediterranean region with many patients being of Arab and non-European Jewish ancestry. Later, IPSID was identified in other areas, including south and central Africa and Southeast Asia. More recently, sporadic cases have been acknowledged in parts of Europe and the American continent [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Antigen-and/or immune-driven lymphoproliferative disorders, Aljurf [/bib_ref] [bib_ref] Primary Small Intestinal Lymphoma in Adults: A comparative study of IPSID versus..., Salem [/bib_ref]. It is unclear, however, if those cases are the result of migration.
Risk factors that have been commonly associated with the development of IPSID included low socioeconomic status, poor sanitation, and clean water inaccessibility, which might have led to a higher incidence of chronic or recurrent gastrointestinal infections and possibly, at least initially, partially explained the association between IPSID and developing countries [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Antigen-and/or immune-driven lymphoproliferative disorders, Aljurf [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref].
With improved sanitation techniques and the availability of clean water, a decreased incidence of IPSID has been described in those areas considered at a higher risk [bib_ref] Primary Small Intestinal Lymphoma in Adults: A comparative study of IPSID versus..., Salem [/bib_ref] [bib_ref] Changing epidemiology of IPSID in southern Iran, Lankarani [/bib_ref].
An underlying genetic predisposition is suggested, with evidence of human leukocyte antigen (HLA) association, a pattern of elevated alkaline phosphatase noticed in the healthy family members of the affected individuals, and defective cellular and humoral immunity. However, these findings are not considered sufficient evidence of a clear genetic association, highlighting that environmental factors alone are not solely responsible for the occurrence of IPSID [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Antigen-and/or immune-driven lymphoproliferative disorders, Aljurf [/bib_ref]. There have been reports on the association of IPSID with HLA-AW19, HLA-A9, HLA-B12, and the B blood group [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
The majority of patients with IPSID appear to be diagnosed in their second and third decades of life. An estimated ratio of male-to-female incidence of 2.4:1 has been described in some reports [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref].
## Etiology and pathophysiology
Generally, upon exposure to antigens, B lymphocytes bind to TH1 cells and undergo somatic hypermutations to increase antigen-binding specificity and immunoglobulin class-switching from IgM production to the production of IgG, IgA, and IgE subtypes of immunoglobulins [fig_ref] Figure 1: T helper cells bind to B lymphocytes through CD40L and TCR to... [/fig_ref]. IPSID is potentially associated with a chronic inflammatory state, where there is obstinate antigen stimulation, either from persistent infections or other possible inflammatory conditions, such as an autoimmune disease. This persistent stimulation leads to the proliferation of lymphoid cells and the production of increased amounts of IgA immunoglobulins, which typically undergo somatic hypermutations to produce antigen-specific immunoglobulins. However, in IPSID, the persistent antigen presentation with ongoing somatic hypermutations can lead to a clonal production of an abnormal IgA with an abnormal α-heavy chain (α-HC) protein, which lacks the variable heavy chain (V H ), and the first constant domain (C H 1), which is present on the light chain segment of the immunoglobulin [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
The defective α-HC protein loses the ability to bind the light chain, which leads to an abnormal configuration of the produced immunoglobulin . This aberration results from ongoing mutations and alternative splicing along with the abnormally short α-HC mRNA, which results in an abnormal α-HC protein containing various in-frame inserts between the second and third constant regions and the leader peptides, hence producing an unrecognized non-human genomic material [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref].
The hallmark of IPSID is cell clonality, which has been reported at all stages [bib_ref] Antigen-and/or immune-driven lymphoproliferative disorders, Aljurf [/bib_ref]. This clonality suggests that IPSID is lymphomatous at all stages, but it does not always progress to aggressive lymphoma because the cell's normal growth regulators can perhaps balance out the clonality. However, the balance is apparently lost once further genetic modifications occur and anti-apoptotic effects are enhanced. The cells widely proliferate irrepressibly with minimal or no apoptosis, resulting in the accumulation of lymphoid cells and progression to full-blown lymphoma [bib_ref] Antigen-and/or immune-driven lymphoproliferative disorders, Aljurf [/bib_ref] [bib_ref] Immunoglobulin gene rearrangement in immunoproliferative small intestinal disease (IPSID), Smith [/bib_ref].
IPSID shares features with other lymphoproliferative diseases, including MALT lymphoma, lymphoplasmacytic lymphoma, and plasma cell neoplasms [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref]. Both IPSID and MALT lymphoma develop from antigen-driven B lymphocyte activation, which results in clonal disease and later malignant transformation [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref]. Based on the similarities between MALT and IPSID and the association of MALT lymphoma with H. Pylori, some suggested infectious agents may be responsible for the chronic antigen stimulation in IPSID, including small intestinal bacterial overgrowth, giardiasis, and parasitic infections; trichuriasis [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref]. Specific bacterial organisms linked to IPSID on different occasions include Campylobacter jejuni (most common), Vibrio cholera, Vibrio fluvialis, Escherichia coli, and H. pylori, although there is minimal evidence of association with the latter [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Antigen-and/or immune-driven lymphoproliferative disorders, Aljurf [/bib_ref]. Despite the multiple reported findings of C. jejuni-positive cultures in patients with IPSID, the exact mechanism is unknown and definite proof of a seldom association between C. jejuni and IPSID remains unknown. Moreover, it is not clear whether there is a direct association between IPSID and the aforementioned organisms or if it is a mere coincidence due to the poor sanitation setting that IPSID has been associated with [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref]. C. jejuni is hypothesized to be able to generate a mucosal response with the secretion of IgA at high levels, which creates a persistent stimulation and clonal α-heavy chain secretion with no antigen-antibody Fc-dependent suppression [bib_ref] Immunoproliferative small intestinal disease associated with Campylobacter jejuni, Lecuit [/bib_ref].
AlSaleem et al. hypothesized that patients with IPSID, with evidence of C. jejuni infections, might have had a previous infection with V. cholerae, especially since the 1960s cholera epidemic occurred in the same geographic district that IPSID was commonly diagnosed in a couple of years after. The V. cholerae toxin is known to break down the double-stranded DNA, which alters the structure and causes mutations involving the PAX5 gene (encodes typically for the transcription factor B-cell specific activator protein (BSAP)) and other oncogenes in the B lymphocytes, which lead to abnormal lymphopoiesis and the production of the aberrant IgA, and later progress to lymphoma [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref]. The intestinal wall, which creates a barrier between the human body and the external environment, comprises three layers: the outer, middle, and internal layers. The outer layer is composed of the mucous layer, intestinal microbiota, and defense proteins (antimicrobial proteins and IgA). The middle layer is mainly composed of intestinal epithelial cells and, lastly, the inner layer is composed of the innate and adaptive immune cells [bib_ref] Intestinal Barrier in Human Health and Disease, Tommaso [/bib_ref]. It is believed that many intestinal inflammatory conditions are caused by interruption of the intestinal barrier and microbial dysbiosis. This intestinal alteration and overgrowth of specific organisms are thought to contribute to the pathophysiology of IPSID, especially in the early stages, where a complete response is seen with the use of antibiotics alone [bib_ref] An integrative view of microbiome-host interactions in inflammatory bowel diseases, Wlodarska [/bib_ref].
## Clinical features and prognosis
At presentation, common symptoms include non-bloody watery, copious diarrhea, low-grade fevers, night sweats, abdominal pain, weight loss, steatorrhea, nausea, and vomiting, which can lead to malnutrition and electrolytes imbalances manifesting as carpopedal spasms and tetany, among others [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease: A report of 6 cases, Biswas [/bib_ref]. In some patients, fasting appears to decrease symptoms, while in others, consumption of lactose-containing products is associated with worsening symptoms [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref].
Rarely, melena and other forms of gastrointestinal bleeding can occur, with positive occult blood in stool testing. Amenorrhea can be present, possibly as a consequence of severe malnutrition [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease associated with Campylobacter jejuni, Lecuit [/bib_ref].
A physical examination reveals signs of malnutrition and defective absorption, including cachexia, peripheral edema, and finger clubbing, among others. Abdominal distention and tenderness can be present with or without a palpable abdominal mass [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref]. Spreading of the disease beyond the abdomen, including palpable peripheral lymphadenopathy, hepatosplenomegaly, and bone marrow involvement, are not commonly seen in IPSID; however, they can be present (rarely) in advanced stages of the disease [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
If IPSID is left untreated, it can progress and transform into higher grades of B-cell lymphomas and lead to further malabsorption and malnutrition. Other complications seen with the advanced disease include infections, intussusception, intestinal obstruction, gastrointestinal bleeding, and perforation, resulting in severe morbidity and mortality [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease-A Case Report, Afroza [/bib_ref]. Extra-intestinal complications are not common, but reports have described nephropathies, osteoarthropathy, and osteomalacia [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref]. The rate of progression of IPSID to high-grade lymphomas is not well described [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
Most patients are diagnosed when these manifestations develop, and in retrospect, patients frequently report mild symptoms dating back five to ten years preceding the clinical presentation, with an average duration of symptoms lasting between one month to 6 years [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref]. The natural course of the disease is highlighted with episodic manifestations with intervals of improvement in between, likely contributing to a delay in the diagnosis [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref].
## Differential diagnoses
The most important factor in diagnosing IPSID is having a high index of suspicion, especially in patients with unexplained chronic diarrhea from endemic parts of the world [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref]. The presentation of IPSID is very similar to other infectious and inflammatory gastrointestinal conditions, e.g., celiac disease and chronic intestinal infections, tropical sprue, AIDS enteropathy, intestinal tuberculosis, inflammatory bowel disease, and Whipple's disease [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease: A report of 6 cases, Biswas [/bib_ref] [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref]. Hence, it can be challenging to differentiate between these diseases and IPSID; yet some features might favor one diagnosis over the other. For instance, IPSID is usually more intense with involvement of the whole small intestine at times less responsive to a gluten-free diet compared with celiac disease, and stool cultures can be negative, unlike intestinal infections.
Histologically, IPSID and celiac disease both have surface epithelial damage, villous atrophy, and mucosal infiltrates with lympho-plasmacytes; however, IPSID has atrophic crypts, and hyperplastic elongated crypts are seen in celiac disease [bib_ref] Immunoproliferative small intestinal disease: A report of 6 cases, Biswas [/bib_ref] [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref]. Furthermore, serum antibodies to tissue transglutaminase (anti-TTG) positivity help rule in celiac disease while abnormal serum protein electrophoresis would favor IPSID. The development of high-grade lymphomas can complicate both IPSID and celiac disease, yet in the case of celiac disease, if progression to lymphomas ensues, those are T-cell lymphomas and are usually prone to perforation. In the case of IPSID, the disease generally progresses to B-cell lymphomas and it is less likely to present with perforations.
Tropical sprue does not commonly present with abdominal pain and usually has an abnormal D-xylose test [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref]. We acknowledge that differentiating between IPSID and chronic inflammatory gastrointestinal conditions with clinical manifestations and pathology alone might be challenging. Studies to evaluate the heavy chain immunoglobulin (IgH) gene rearrangement and immunohistochemical stains to confirm the expression of heavy chains only from plasma cells can be helpful in such situations.
## Diagnostics and investigations
Reported cases of IPSID were diagnosed between 1 month and 6 years from the initial presentation, with a median time for diagnosis of approximately 10 months [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease: A report of 6 cases, Biswas [/bib_ref]. Most reported cases had similar laboratory [fig_ref] Table 1: Investigational features reported on in the literature in patients diagnosed with IPSID [/fig_ref] , histopathological, and radiological features.
## Radiological findings
Most studies illustrated the findings seen in barium X-ray and CT studies. One report conveyed the findings of an abdominal ultrasound, which showed peripancreatic lymphadenopathy with oval-shaped hypoechoic regions [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref].
Barium X-ray studies were done as part of routine work in the old literature, and the reported transit time was 3-8.5 h with a median of 4.5 h [bib_ref] Immunoproliferative Small Intestinal Disease with Duodenojejunal Lymphoma: Radiologic Changes, Vessal [/bib_ref]. Findings included small intestinal diffuse dilatation, strictures, infiltrative, nodular defects, intussusception, and mucosal fold thickening of various degrees with irregular edges; "Postage stamp" sign, and sprue-like changes [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease with Duodenojejunal Lymphoma: Radiologic Changes, Vessal [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref]. Secretory assessments varied between hypersecretion in most cases, but some had dry malabsorption, similar to findings seen in amyloidosis. An ominous sign that suggests progressive stages of lymphoma is the appearance of widening and displacement of the bowel loops [bib_ref] Immunoproliferative Small Intestinal Disease with Duodenojejunal Lymphoma: Radiologic Changes, Vessal [/bib_ref].
Computed tomography (CT) of the abdomen commonly shows mesenteric and retroperitoneal lymphadenopathy with different degrees of involvement depending on the radiolog-ical stage, as described by Vessal et al. [fig_ref] Table 2: Vessal et al [/fig_ref] [bib_ref] Immunoproliferative Small Intestinal Disease with Duodenojejunal Lymphoma: Radiologic Changes, Vessal [/bib_ref]. Diffuse small bowel mural thickening with the presence of pseudo-polyps with strictures and segmentations, most commonly in the proximal part of the small intestine, are most prevalently in the jejunum [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref] [bib_ref] Immunoproliferative Small Intestine Disease (IPSID): A Case Report, Imanzade [/bib_ref]. The presence of organomegaly has not been commonly documented. Features suggestive of osteomalacia have been noted due to malabsorption.
PET-CT featured hypermetabolic FDG activity involving the small bowel wall and mesenteric lymph nodes [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref]. Hypocholesteremia. Vitamin deficiencies (vitamin B12 and folate). Elevated lactate dehydrogenase (LDH), reported in advanced stages of lymphoma [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref].
Hematology [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref] Complete blood count: mild anemia and leukocytosis. Peripheral blood morphology can demonstrate plasmacytic infiltrates. Bone marrow biopsy rarely is involved and can show plasmacytosis and features of plasma cell leukemia [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref].
Microbiology [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref] [bib_ref] Immunoglobulin gene rearrangement in immunoproliferative small intestinal disease (IPSID), Smith [/bib_ref] [bib_ref] Immunoproliferative Small Intestine Disease (IPSID): A Case Report, Imanzade [/bib_ref] Stool cultures were stated in different reports to be positive for various organisms including Campylobacter jejuni, Vibrio fluvialis, Giardia. Tissue culture was positive for E. coli in one case report.
## Immunology and electrophoresis
Low or normal IgA levels with normal-to-high IgG and IgM levels [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref].
No Bence-Jones proteins in the urine [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref]. Decreased or absent cellular and humoral responses. Immuno-electrophoresis and immunoselection detect α-heavy chain proteins in serum and body fluids, it is considered the most sensitive and specific method and is detected in almost 70% of cases [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref].
Other Positive stool occult [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref]. Sudan III stain of the stool positive, suggestive of malabsorption [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref].
## Radiological stage description
Stage I Focal lymphoma involving the mucosa or submucosa or mesenteric lymph node. Stage II Lymphoma extension to the transmural layer and lymphadenopathy in several regions. Stage III Lymphoma involvement of the bowel and massive mesenteric lymph nodes.
## Stage iv
Lymphoma involving the bowel and extra-mesenteric lymph nodes or parenchymatous organs.
## Esophagogastroduodenoscopy and laparotomy
Endoscopies and laparotomy with full-thickness biopsies at a minimum of three different sites with lymph node biopsies are optimal diagnostic tools, along with immunoselection and electrophoresis to confirm IPSID [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Immunoglobulin gene rearrangement in immunoproliferative small intestinal disease (IPSID), Smith [/bib_ref] [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref]. Patterns often reported include disseminated erythema with atrophic villi and nodular mucosa, ulcerations, small intestinal wall thickening, lymphangiectasis, cobble-stoning appearance, and mesenteric lymphadenopathy [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref] [bib_ref] Immunoproliferative Small Intestine Disease (IPSID): A Case Report, Imanzade [/bib_ref].
Ulcerations can be seen at any stage, but usually indicate a malignant process [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref]. Intestinal masses and dilation were seen on laparotomy in some patients, while others had normal-appearing bowels. The majority of findings were found in the small intestine, mainly in the proximal part; some reviews reported the jejunum to be the most commonly affected site. Gastric and clonal mucosa are usually spared [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref].
## Histopathology and immunohistochemistry stains
Under the microscope, small intestinal villous blunting and shortening with absent crypts and mucosal flattening is seen in patients with IPSID [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestine Disease (IPSID): A Case Report, Imanzade [/bib_ref]. These changes are due to infiltration of the lamina propria with lymphoplasmacytic cells, which secrete the abnormal monoclonal α-HC protein and cause disjunction of the Lieberkühn crypts [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref].
The surface epithelium is usually intact, particularly in the early stages [bib_ref] Treating chronic diarrhea: A systemic review on Immunoproliferative Small Intestinal Disease (IPSID), Evangelista-Leite [/bib_ref]. The earliest pathological feature is the presence of abundant infiltrates of centrocyte-like lymphocytes and plasma cells on the intestinal biopsy; it was previously referred to as stage 0 of IPSID [bib_ref] Mediterranean abdominal lymphoma or immunoproliferative small intestinal disease. Part II: Pathological aspects, Nassar [/bib_ref].
Mucosal infiltrates can be of four different types: diffuse lymphoplasmacytic (DLP), which is commonly benign. Follicular lymphoid (FL) infiltration can be follicular hyperplasia or lymphoma, mixed cellularity of both FL in the superficial mucosa and DLP patterns in the deeper layers; most of these patients were documented as having follicular lymphoma. Lastly, the non-specific type usually causes continuous longitudinal infiltrates with increased cellularity of normal-appearing lymphocytes, plasma cells, and eosinophils [bib_ref] Primary Small Intestinal Lymphoma in Adults: A comparative study of IPSID versus..., Salem [/bib_ref].
In later stages, atypical lymphoid cells with speckled immunoblastic cell aggregates develop and can progress into sheets of dystrophic plasma cells that invade the submucosa and muscularis propria. These cells seem to be interconnected to the development of neoplasm [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Immunoglobulin gene rearrangement in immunoproliferative small intestinal disease (IPSID), Smith [/bib_ref]. Moreover, a predominance of mature plasma cells on the sample could be indicative of a hidden lymphoma in the deeper layers or as a predictive feature of the future development of lymphoma [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref]. Lesions with different stages of the disease can be present within the same part of the intestine [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref].
Based on histological features alone, two variants of IPSID have been described [bib_ref] Clinicopathologic conference case presentation, Ali [/bib_ref].
## 1.
Diffuse infiltration by plasma cells solely or mixed with lymphocytes in the intestine's mucosa at sites other than the neoplastic mass if present. The development of immunoblastic malignant lymphoma with detectable α-HC proteins in body fluids and tissues is commonly seen in association with this variant.
## 2.
Diffuse follicular lymphoid hyperplasia in the mucosa of the small intestine. This variant is commonly associated with diffuse undifferentiated malignant lymphomas and undetectable α-HC proteins.
Galian et al. developed a comprehensive staging system for IPSID relying on the type of cellular infiltrates and mesenteric nodal involvement, classified into stages A, B, and C, in which appropriate treatment is based [fig_ref] Table 3: Galian et al [/fig_ref]. Heavy infiltrations of lamina propria with typical lymphocytes with few dysplastic plasma cells infiltrate with variable atrophic villi in the small intestine.
Few CD20-positive marginal zone B cells with plasmacytic infiltration of mesenteric or other abdominal and retroperitoneal lymph nodes with limited disorganization of histological structure.
## Stage b (intermediate)
Infiltration extending beyond the mucosa with atypical lymphoplasmacytic cells, immunoblastic-like cells with the areas remote from the mucosa, containing many dysplastic cells with total or subtotal villous atrophy.
Atypical plasmacytic and immunoblastic dense infiltrations causing structural changes of the mesenteric and abdominal lymph node construction.
## Stage c (malignant)
Immunoblastic high-grade B-cell lymphomas, some with strong CD20-positivity with plasmacytoid differentiation and proliferative histocytes extending into all layers of the intestinal wall and some forming confined large tumors of malignant formations.
Mesenteric and abdominal lymph nodes with sarcomatous proliferation alter the entire structure.
Immunohistochemistry staining shows monoclonal cytoplasmic defected α-heavy chains that lack light chains and the expression of CD20 positivity on tumor cells, CD138, CD79a, and IgA positivity on plasma cells, and no expressions of CD5 and CD10 on tumor cells.
## Molecular and cytogenetics
Although not frequently documented, some of the genetic abnormalities linked to IPSID include t(9;14), t(2;14), t(5;9), t(21;22) (q22;q11), 14q+ chromosome, abnormal p32 HC locus on chromosome 14 and light chain loci on chromosome 2 and 22. Mutations involving PAX5 genes were also reported [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref]. Although IPSID shares multiple features with MALT lymphoma, the common translocation t(11;18) that is seen frequently in MALT-lymphoma is lacking in IPSID [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
Clonal abnormalities of heavy chains are seen in most cases on molecular testing, even in the earliest phase.
## Atypical ipsid entities
Despite IPSID being an uncommon disease, there are still unique entities less commonly identified in the literature. These infrequent entities include non-secretory IPSID, an under-recognized subtype of IPSID distinguished from the typical secretory IPSID by the histological appearance rather than clinical manifestations, characterized by small centrocyte-like lymphoid cells without extensive plasmacytic differentiation that is usually seen in the secretory form [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
Other rare forms of IPSID include gamma heavy-chain disease and α-HC disease of the colon, stomach, and lungs [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
## Management and outcomes
The management of IPSID depends on the severity, histopathological features, and associated clinical manifestations. Treatment options include supportive measures, pharmacological agents, radiation therapy, and surgery. In the early stages, spontaneous remission has been described [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] ; however, the disease can experience relapse with an advanced stage related to chronic antigenic stimulation; accordingly, early treatment is strongly recommended [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Treatment of alpha chain disease. Result of a prospective study in 21..., Ben Ayed [/bib_ref] [bib_ref] A Rare Presentation of Immunoproliferative Small Intestinal Disease, Nasir [/bib_ref].
## Supportive measurements
Treatment complications of IPSID, including malnutrition, are required to facilitate the response to therapy and recovery of patients. Supportive measures need to consist of correcting unbalanced electrolytes, enhancing nutritional repletion with a high protein diet, and albumin replacement [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref] [bib_ref] Immunoproliferative Small Intestine Disease (IPSID): A Case Report, Imanzade [/bib_ref].
## Pharmacological therapy
Therapeutic options depend on the stage of the disease, whether using antibiotics alone or in combination with chemotherapy; the therapeutic choice is commonly based on the Galian et al. staging system .
In the presence of early-stage disease, stage A, the choice of antibiotics as the only treatment modality is reasonable. Tetracycline alone or combined with metronidazole for 6 months was a popular regimen that resulted in clinical improvement, a reported 71% rate of complete remission, and a 5-year disease-free survival of 43% [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref]. Other antimicrobial options include metronidazole with ampicillin, ciprofloxacin, doxycycline, and a 14-day course of piperacillin-tazobactam have been reportedly used to treat IPSID with observed clinical improvement [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease: A report of 6 cases, Biswas [/bib_ref].
Adjunctive steroids with antibiotics therapy can be of additional benefit in achieving a higher response rate in the early stages [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref]. If no response is seen after completion of an antimicrobial therapy course after 6 months or if patients do not attain a CR in 1 year, repeating a biopsy is recommended, and consideration of starting chemotherapy appears reasonable [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref]. As the disease progresses, the response to antibiotics diminishes, yet it can be used in intermediate stages, stage B, with or without chemotherapy. The suggested approach to stage B is using antibiotics, such as tetracycline with non-intense chemotherapy, for example, CVP (cyclophosphamide, vincristine, and prednisone), which has been reported to prolong remission [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref].
Other reported treatment choices in intermediate stages include anthracycline-based chemotherapy or melphalan [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease and Primary Small Intestinal Lymphoma: Review of Literature, Malik [/bib_ref]. Further options include single chemotherapy (cyclophosphamide) with or without antibiotics and steroids, resulting in clinical, histological, and immunological remission [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref].
In advanced stages, stage C, it is recommended to treat it as aggressive B-cell lymphoma with anthracycline-based chemotherapy and antimicrobial therapy with curative intent, traditionally with six-eight cycles of the CHOP regimen (cyclophosphamide, vincristine, doxorubicin, and prednisone), with a complete remission rate of 50-90% and a 3-year median survival rate of 67% [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative small intestinal disease: A report of 6 cases, Biswas [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease-A Case Report, Afroza [/bib_ref]. Other available chemotherapy options include CHOP-bleomycin, m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone) [bib_ref] Immunoproliferative Small Intestinal Disease: First Case Report from Bangladesh, Gani [/bib_ref].
The use of biological agents, such as rituximab, a monoclonal antibody against CD20, is not ascertained due to the presence of CD20-negative plasma cells but may be considered for CD20-positive lymphocytes. Further studies are needed to better understand the role of rituximab along with anti-myeloma therapeutic regimens [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Report: Immunoproliferative Small Intestinal Disease Associated with Overwhelming Polymicrobial Gastrointestinal Infection with..., Ewers [/bib_ref]. There is no established role for prolonged maintenance with antibiotics [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
## Radiation therapy
Involved site (abdominal) radiation can be used if a fast response is needed, particularly in bulky tumors to relieve symptoms [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
## Surgical intervention
Surgery is usually recommended for selective management of complications, such as obstruction or perforation, and diagnosis and staging with the use of a laparotomy [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref] [bib_ref] Immunoproliferative Small Intestinal Disease in an American Patient with Lymphoma and Macroamylasemia, Blumstein [/bib_ref].
## Stem cell transplantation
The role of an autologous stem cell transplant was recognized in patients with the refractory or relapsed disease [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref]. The efficacy of an allogeneic stem cell transplant is not known, likely due to the low prevalence of the disease.
## Response to therapy and maintenance therapy
Response assessment is based on the improvement of clinical features with the elimination of diarrhea, and weight gain, radiologically by a PET-CT or CT abdomen with the resolution of uptake on PET, and diffuse thickening with regression of lymphadenopathy, along with histological normalization of small intestinal mucosal with the disappearance of monoclonal lymphoplasmacytic infiltrates. Measurement of the α-HC protein in the serum can be used as a marker for a response; however, this method has its limitations in terms of accuracy since (occasionally) the levels of α-HC proteins can regress in the presence of a transformation or relapse [bib_ref] Immunoproliferative small intestinal disease (IPSID): A model for mature B-cell neoplasms, Alsaleem [/bib_ref].
# Conclusions
IPSID is an uncommon disease that is present in certain regions of the world, with the Middle East being one of the earliest (and most prevalent) areas from where cases have been reported. The key tool for diagnosing IPSID is having a high index of suspicion. The optimal treatment choice remains undefined, but the most agreed upon approach depends on the stage of the disease. Therapies include antibiotics alone or in combination with anthracycline-based chemotherapy regimens. Further descriptive studies and clinical trials are needed to establish the most effective therapeutic options. Further research is needed to thoroughly investigate the role of stem cells and targeted therapies in IPSID.
[fig] 3, Figure 1: Curr. Oncol. 2022, 29, FOR PEER REVIEW T helper cells bind to B lymphocytes through CD40L and TCR to the MHC-II and CD40 receptors on the B lymphocytes, promoting different steps responsible for the variable functions of B-cells, including (1) excision of the DNA responsible for IgM transcription and the attachment of the variable regions (VR) to the rest of the DNA leading to the production of different classes of immunoglobulin, a process also known as class-switching. (2) Somatic hypermutation, which is responsible for increasing the antigen-binding specificity. (3) Formation of the germinal center. [/fig]
[fig] Figure 1: T helper cells bind to B lymphocytes through CD40L and TCR to the MHC-II and CD40 receptors on the B lymphocytes, promoting different steps responsible for the variable functions of B-cells, including (1) excision of the DNA responsible for IgM transcription and the attachment of the variable regions (V R ) to the rest of the DNA leading to the production of different classes of immunoglobulin, a process also known as class-switching. (2) Somatic hypermutation, which is responsible for increasing the antigen-binding specificity. (3) Formation of the germinal center. [/fig]
[fig] 1, Figure 2: (A) Normal IgA structure. (B) Aberrant IgA molecule with missing V H and C H 1 regions. [/fig]
[fig] 1, Figure 3: Suggested approach for management of IPSID stages and progression/relapse. [/fig]
[table] Table 1: Investigational features reported on in the literature in patients diagnosed with IPSID. [/table]
[table] Table 2: Vessal et al. radiological staging of IPSID. [/table]
[table] Table 3: Galian et al. histopathological staging of IPSID. [/table]
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Prognostic role and clinicopathological features of SMAD4 gene mutation in colorectal cancer: a systematic review and meta-analysis
Background: Approximately 5.0-24.2% of colorectal cancers (CRCs) have inactivating mutations in SMAD4, making it one of the frequently mutated genes in CRC. We thus carried out a comprehensive system review and meta-analysis investigating the prognostic significance and clinicopathological features of SMAD4 gene mutation in CRC patients.Methods:A detailed literature search was conducted in PubMed, Web of Science and Embase databases to study the relationship between SMAD4 mutations and the demographic and clinicopathological characteristics in CRC patients. The hazard ratios (HRs) with 95% confidence intervals (CI) were used to evaluate the effect of SMAD4 mutations on overall survival (OS) and progression-free survival (PFS)/recurrence-free survival (RFS).Results: Ten studies enrolling 4394 patients were eligible for inclusion. Data on OS were available from 5 studies and data on PFS/RFS were available from 3 studies. Comparing SMAD4-mutated CRC patients with SMAD4 wild-type CRC patients, the summary HR for OS was 1.46 (95% CI 1.28-1.67, P = 0.001), the summary HR for PFS/RFS was 1.59 (95% CI 1.14-2.22, P = 0.006). In terms of clinicopathology parameters, 9 studies have data that can be extracted, SMAD4 mutations were associated with tumor location (odds ratio [OR] = 1.15, colon/rectum, 95% CI 1.01-1.31, P = 0.042), TNM stage (OR = 1.28, stage IV/I-III, 95% CI 1.03-1.58, P = 0.025), lymph node metastasis (OR = 1.42, N1 + N2/N0, 95% CI 1.20-1.67, P < 0.001), mucinous differentiation (OR = 2.23, 95% CI 1.85-2.70, P < 0.001) and rat sarcoma viral oncogene homolog (RAS) mutation status (OR = 2.13, 95% CI 1.37-3.34, P = 0.001). No connection was found with age, gender, tumor grade, microsatellite instability status and b-viral oncogene homolog B1 mutation status. Besides, publication bias was not observed in any study.Conclusions:This meta-analysis suggests that SMAD4 mutation was associated with OS, PFS/RFS, and clinicopathological parameters, including tumor site, disease stage, RAS status, lymph node metastasis and mucinous differentiation. Our meta-analysis indicated that SMAD4 mutations could predict the poor prognosis and aggressive clinicopathological characteristics of CRC. More large-sample cohort studies are needed to confirm this conclusion. Since SMAD4 mutations are closely related to RAS mutations, their relationship warrants further investigation. metastasis are still the main causes of death in newly diagnosed CRC patients, and the overall survival (OS) rate of advanced CRC is still unsatisfactory.
The Cancer Genome Atlas database revealed that the mutation frequency of SMAD4 is 10%, which is one of the most common mutated genes in CRC. SMAD4 is an established tumor suppressor gene located in chromosome band 18q21, and one of the most commonly destroyed gene in cancer among SMAD family genes [bib_ref] Transforming growth factor-beta signaling pathway in colorectal cancer and its tumor microenvironment, Itatani [/bib_ref]. This gene encodes a member of the Smad family of signal transduction proteins, that is phosphorylated and activated by transmembrane serine-threonine receptor kinases in response to transforming growth factor beta (TGF-β) signal transduction. The product of this gene forms homomeric complexes and heteromeric complexes with other activated Smad proteins in the context of activating by TGF-β receptors, then accumulate in the nucleus and regulate the transcription of target genes [bib_ref] Smad4/DPC4 silencing and hyperactive Ras jointly disrupt transforming growth factor-beta antiproliferative responses..., Calonge [/bib_ref]. Mutations or deletions in the SMAD4 gene have been shown to result in pancreatic cancer, juvenile polyposis syndrome [bib_ref] Juvenile polyposis syndrome, Brosens [/bib_ref] , and hereditary hemorrhagic telangiectasia [bib_ref] Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia, Shovlin [/bib_ref]. In the past 2 decades, many studies had shown that SMAD4 mutation can not cause tumorigenesis by itself, but it can promote tumor progression caused by other genes [bib_ref] The role of TGF-beta/SMAD4 signaling in cancer, Zhao [/bib_ref]. The role of SMAD4 in CRC is similar to that in pancreatic cancer. The prevalence of SMAD4 mutations have recently been reported in 5.0-24.2% of several retrospective studies of sporadic CRC from 1999 to 2020 [bib_ref] SMAD3 and SMAD4 mutations in colorectal cancer, Fleming [/bib_ref] [bib_ref] Association of SMAD4 mutation with patient demographics, tumor characteristics, and clinical outcomes..., Sarshekeh [/bib_ref] [bib_ref] SMAD4 gene mutation predicts poor prognosis in patients undergoing resection for colorectal..., Mizuno [/bib_ref] [bib_ref] Higher frequency of Smad4 gene mutation in human colorectal cancer with distant..., Miyaki [/bib_ref] [bib_ref] Mutation status of RAS, TP53, and SMAD4 is superior to mutation status..., Kawaguchi [/bib_ref] [bib_ref] Mutations of key driver genes in colorectal cancer progression and metastasis, Huang [/bib_ref]. However, whether pathogenic mutation of SMAD4 reduces the OS in all CRC patients remains unclear. Therefore, we conducted a meta-analysis to assess the association of SMAD4 mutations with OS and PFS/RFS, as well as the relationship between SMAD4 mutations and clinicopathological characteristics of early and advanced CRC.
# Methods
## Search strategy
We conducted this study based on the preferred reporting items for Systematic Reviews and Meta-Analyses 2009 guidelines and registered with the International Prospective Register of Systematic Reviews, PROSPERO (identification code CRD42021244570). Systematic review of several databases was conducted in December 2020 with no lower limit set for date of publication. Search for related articles published in English or Chinese in the following electronic databases: PubMed, Web of Science, and Embase. The keywords "SMAD4" or "DPC4" and "colorectal cancer" or "colon cancer" or "rectum cancer" were used for relative articles searching.
## Study selection and inclusion criteria
All articles are limited to human studies published in English or Chinese that based on the following selection criteria: (1) Researches involved the prognostic of SMAD4 mutations in CRC patients, and provided sufficient information to obtain the Hazard ratios (HRs) and 95% confidence interval (CI) of OS or progression-free survival (PFS)/recurrence-free survival (RFS) directly or indirectly from the Kaplan-Meier curve. (2) Studies using surgical resection specimen of tumor to detect SMAD4 mutation in CRC. (3) The odds ratio (OR) associated with clinicopathologic features is given directly or can be obtained from computable data. (4) The study does not include CRC patients who received preoperative chemotherapy or radiotherapy. (5) Duplicate report results are unified by the latest or largest version.
## Data extraction and quality assessment
The two authors (F.T. and L.T) extracted all data sets from the selected studies independently, if there are any objections, we resolved through consensus or consultation with the corresponding author. The following information was collected from each study: first author, year of publication, country, time of diagnosis, sample size, CRC cases with SMAD4 gene mutations, sequencing methods of SMAD4 gene, mean follow-up periods and participants' characteristics, including median age, gender, lymph node metastasis status, rat sarcoma viral oncogene homolog (RAS), b-viral oncogene homolog B1 (BRAF), microsatellite instability (MSI) status and mucinous differentiation as well as tumor stage. HRs and 95% CI of OS and PFS/RFS were extracted directly from papers, if not, we choose to extract from Kaplan-Meier Curve via Engauge Digitizer Version 4.1 (http:// marku mmitc hell. github. io/ engau ge-digit izer/). We used the Newcastle-Ottawa Scale to assess the methods and report quality of the included studies, and ranked them by score (8-9 points for high quality; 5-7 points for medium quality; less than 5 points for low quality) [bib_ref] Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality..., Stang [/bib_ref].
## Statistical analyses
HRs and ORs with their 95% CI were calculated. P value less than 0.05 was considered statistically significant. The Q statistic and I 2 tests was used to estimate Heterogeneity among studies. The I 2 statistic was ranged from 0 to 1. A random effect model was used for I 2 > 0.5, which represented strong heterogeneity. Otherwise, fixed-effect model would be applied. The analysis was performed to evaluate the impact of SMAD4 gene mutation on the prognosis of CRC. In addition, we evaluated the correlation between SMAD4 mutation status and different tumor grades, tumor differentiation, lymph node metastasis, and MSI/BRAF/RAS status. Sensitivity analysis was used to check data stability. The Egger's test and Begg's test were used for detection of publication bias, and P < 0.05 indicated significant bias. All analysis was performed with STATA 16.0 (Stata Corporation, College Station, TX, USA).
# Results
## Selection of studies
The flowchart of the study selection is shown in [fig_ref] Figure 1: Schematic flow diagram for selection of included studies [/fig_ref]. There were 465 articles identified from PubMed, 686 articles from Web of Science, 895 articles from Embase database. A total of 2045 articles were initially identified by the search strategy, and 657 full-text articles were retrieved after screening. Each selected article is tracked forward and backward, in case they contain another research of interest that has not yet been identified. Second, 1280 unrelated titles and abstracts were excluded from the study, and 108 full-text articles were evaluated for applicability, 7 articles were found to have no available outcome indicators or clinicopathologic features, 63 articles relate to SMAD4 protein expression and survival data, 12 articles were non-human trials and 15 articles were reviews, letters or case reports. A total of 4394 patients were included in the final ten studies [bib_ref] SMAD3 and SMAD4 mutations in colorectal cancer, Fleming [/bib_ref] [bib_ref] Association of SMAD4 mutation with patient demographics, tumor characteristics, and clinical outcomes..., Sarshekeh [/bib_ref] [bib_ref] SMAD4 gene mutation predicts poor prognosis in patients undergoing resection for colorectal..., Mizuno [/bib_ref] [bib_ref] Higher frequency of Smad4 gene mutation in human colorectal cancer with distant..., Miyaki [/bib_ref] [bib_ref] SMAD4 alteration associates with invasive-front pathological markers and poor prognosis in colorectal..., Oyanagi [/bib_ref] [bib_ref] Clinicopathological characterization of SMAD4-mutated intestinal adenocarcinomas: a case-control study, Liao [/bib_ref] [bib_ref] Single-nucleotide variants, tumour mutational burden and microsatellite instability in patients with metastatic..., Stahler [/bib_ref] [bib_ref] Prognostic value of loss of heterozygosity and sub-cellular localization of SMAD4 varies..., Jia [/bib_ref] [bib_ref] Analysis of SMAD4/DPC4 gene alterations in multiploid colorectal carcinomas, Ando [/bib_ref] [bib_ref] Prognostic implications of mucinous differentiation in metastatic colorectal carcinoma can be explained..., Khan [/bib_ref]. The detailed features of these [fig_ref] Table 3: Quality assessment according to the Newcastle-Ottawa scale of the included studies [/fig_ref].
## Relationship between smad4 mutations and crc prognosis
A total of 5 articles provided OS related data. Due to the moderate heterogeneity (I 2 = 41.6%, P heterogeneity = 0.144), we use the fixed-effect model to pool HR. Comparing SMAD4 mutant patients with SMAD4 wild-type patients in CRC, the summary HR for OS was 1.46 (95% CI 1.28-1.67, P = 0.001) [fig_ref] Figure 2 a: Forest plot for meta-analysis of the association of SMAD4 mutations with OS... [/fig_ref]. A total of 3 articles provided PFS/RFS related data. Comparing SMAD4 mutant patients with SMAD4 wild-type patients in CRC, the summary HR for PFS/ RFS was 1.59 (95% CI 1.14-2.22, P = 0.006) [fig_ref] Figure 2 a: Forest plot for meta-analysis of the association of SMAD4 mutations with OS... [/fig_ref] and there was moderate heterogeneity between the studies (I 2 = 48.2%, P heterogeneity = 0.145), so we use the fixed-effect model to pool HR.
## Relationship between smad4 mutations and clinicopathologic features of crc
A total of 9 studies have data that can be extracted from clinicopathologic results, the specific characteristics of which are detailed in
## Sensitivity analysis and publication bias
Our analysis of publication bias using correlation test revealed that there is no obvious publication bias for OS (P = 0.277 for Begg's test and 0.221 for Egger's test) and PFS/RFS (P = 0.235 for Begg's test and 1.000 for Egger's test) . In addition, the sensitivity analysis confirmed that the results were reliable for OS and PFS/RFS .
# Discussion
The role of Smad4 mutations of the prognosis and clinicopathological parameters in CRC has been investigated in several studies, but the results are inconsistent. In addition, no meta-analysis has been conducted to evaluate the impact of SMAD4 gene on the prognosis of CRC. Therefore, we conducted a meta-analysis and suggested that SMAD4 pathogenic mutations were associated with poor prognosis in CRC. Compared with the SMAD4 wild-type controls, SMAD4 mutations are associated with worse OS (pooled HR = 1.46, 95% CI 1.28-1.67, P < 0.001) and worse PFS/RFS (HR = 1.59, 95% CI 1.14-2.22, P = 0.006). In order to further investigate the role of SMAD4 gene in CRC, we also analyzed the relationship between SMAD4 status with clinical pathological parameters of CRC, the results show that patients with SMAD4 mutations have higher pathological TNM stages (stage IV/I-III; pooled OR = 1.28; 95% CI 1.03-1.58), that is, distant metastasis is more likely to occur in patients with SMAD4 mutations. And SMAD4 mutant patients were more likely to feature mucinous differentiation (pooled OR = 2.23; 95%CI 1.85-2.70, P = 0.000), tumors are more likely to occur in the colon (pooled OR = 1.15; 95% CI 1.01-1.31; P = 0.042), more prone to lymph node metastasis (N1 + N2/N0; pooled OR = 1.42; 95% CI 1.20-1.67; P = 0.000), and to harbor concurrent RAS mutations (pooled OR = 2.13; 95% CI 1.37-3.34; P = 0.001). Importantly, all of these parameters generally indicate a poor prognosis. Combined OR suggested that SMAD4 gene mutation has nothing to do with age, gender, tumor grade, MSI or BRAF status. The effect of SMAD4 gene on MSI or BRAF status remains to be elucidated. Another meta-analysis [bib_ref] Mutations of key driver genes in colorectal cancer progression and metastasis, Huang [/bib_ref] showed that SMAD4-mutated patients were at a higher risk of distant metastasis (combined OR 2.04, 95% CI 1.41-2.95), which is consistent with our results.
Over the past 2 decades, many studies have shown that SMAD4 mutation does not cause tumorigenesis by itself, but it can promote tumor progression caused by other genes [bib_ref] The role of TGF-beta/SMAD4 signaling in cancer, Zhao [/bib_ref]. Ohtaki et al [bib_ref] Somatic alterations of the DPC4 and Madr2 genes in colorectal cancers and..., Ohtaki [/bib_ref]. reported that the frequency of SMAD4 mutations were significantly higher in tumors with liver metastasis than in those without such metastasis. Inamoto et al. [bib_ref] Loss of SMAD4 promotes colorectal cancer progression by accumulation of myeloid-derived suppressor..., Inamoto [/bib_ref] reported that SMAD4-deficient colorectal tumor cells secreted more CCL9 and CCL15, these two chemokines recruit CCR1 + myeloid cells through CCL9-CCR1 and CCL15-CCR1 axis, resulting in metastasis. Vauthey et al. [bib_ref] Innovation and future perspectives in the treatment of colorectal liver metastases, Vauthey [/bib_ref] concluded that patients with SMAD4 mutations are less likely to undergo repeated hepatectomy due to recurrent disease after the initial tumor resection. Alhopuro et al. [bib_ref] SMAD4 levels and response to 5-fluorouracil in colorectal cancer, Alhopuro [/bib_ref] showed that SMAD4 is a predictive biomarker for 5-fluorouracil (5-Fu) based chemotherapy in CRC patients. Zhang et al. [bib_ref] Smad4 sensitizes colorectal cancer to 5-fluorouracil through cell cycle arrest by inhibiting..., Zhang [/bib_ref] discovered a novel mechanism mediated by SMAD4 to trigger 5-Fu chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade. Mei et al. [bib_ref] SMAD4 and NF1 mutations as potential biomarkers for poor prognosis to cetuximab-based..., Mei [/bib_ref] suggested that SMAD4 mutations could be potential biomarkers for poor prognosis of cetuximab-based therapy, which needs to be further validated in a larger patient cohort. Lin et al. [bib_ref] Silencing Smad4 attenuates sensitivity of colorectal cancer cells to cetuximab by promoting..., Lin [/bib_ref] found that silencing SMAD4 reduces the sensitivity of CRC cells to cetuximab by promoting epithelial-mesenchymal transition (EMT), while the high expression of Smad4 may be clinically beneficial to cetuximab-based therapy. Mizuno et al. [bib_ref] SMAD4 gene mutation predicts poor prognosis in patients undergoing resection for colorectal..., Mizuno [/bib_ref] found that SMAD4 mutation was significantly associated with poor OS following hepatic resection, which was independent of RAS mutation status. These findings indicate that SMAD4 pathogenic variants play a key role in tumor progression and the efficacy of target therapy in CRC patients.
In the current analysis, researchers found that SMAD4 gene alteration was significantly associated with loss of SMAD4 expression in CRC, and loss of SMAD4 disrupts canonical TGF-β signaling [bib_ref] Novel targeting approaches and signaling pathways of colorectal cancer: an insight, Tiwari [/bib_ref] , because it is a signaling transcription factor. In addition, it is reported that the loss of SMAD4 function is independently associated with the reduction of RFS and OS in CRC patients, especially patients with advanced disease [bib_ref] SMAD4 loss in colorectal cancer patients correlates with recurrence, loss of immune..., Wasserman [/bib_ref]. In contrast, CRC patients with high Smad4 expression had a much longer median OS than those with low Smad4 expression [bib_ref] High SMAD4 levels appear in microsatellite instability and hypermethylated colon cancers, and..., Isaksson-Mettavainio [/bib_ref]. Germline mutations of TGF-β family signaling pathway genes significantly increase the risk of having colonic neoplasia [bib_ref] Tgf-beta signaling alterations and colon cancer, Bellam [/bib_ref]. The canonical TGF-β/Smad4 signaling pathway acts as a tumor suppressor in early stages, which is characterized by its anti-proliferative activity, ability to induce apoptosis and promote genome stability, while TGF-β acts as a metastasis promoter to stimulate the development of advanced tumors [bib_ref] Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream..., Javelaud [/bib_ref].
EMT is a well-coordinated process in which epithelial cells lose cell connectivity and polarity and transform into mesenchymal cells with migration and invasion capabilities. Studies have suggested that EMT is a key step in tumor progression and metastasis, and the TGF-β1 signaling plays a key role in EMT [bib_ref] Ginsenoside Rb2 inhibits epithelial-mesenchymal transition of colorectal cancer cells by suppressing TGF-beta/Smad..., Dai [/bib_ref]. Functional study results indicate that TGF-β-induced Smad4-dependent EMT followed by apoptosis in CRC cells [bib_ref] Overview of the oncogenic signaling pathways in colorectal cancer: mechanistic insights, Farooqi [/bib_ref] [bib_ref] TGFbeta-induced SMAD4-dependent apoptosis proceeded by EMT in CRC, Siraj [/bib_ref]. Siraj et al. [bib_ref] Ras alters epithelial-mesenchymal transition in response to TGFbeta by reducing actin fibers..., Safina [/bib_ref] identified TGF-β-induced EMT was insufficient to obtain invasive potential, while the activated RAS would alter the reaction, imparting tumorigenic and invasive potential. Therefore, the synergistic effect between Ras-Raf-MAPK and TGF-β/Smad cascades is a necessary condition for the acquisition of aggressive phenotype in cancer. At present, RAS has been recognized as tumor driver gene, predictive biomarker and therapeutic target in CRC. The expression of RAS up-regulates the expression of phosphotyrosine kinase receptors ERBB1 (EGFR) and ERBB2 (HER2) and induces an aggressive phenotype. Smad4-dependent signal transduction negatively regulates the expression of these receptors and inhibits Ras-induced upregulation of EGFR and ERBB2, thus exerting an antiproliferative effect. The loss of oncogenic RAS and SMAD4 signals synergistically upregulate the abnormal expression of EGFR and ERBB2, leading to the development of neoplasm and the metastasis and spread of the primary tumor [bib_ref] Expression of oncogenic K-ras and loss of Smad4 cooperate to induce the..., Zhao [/bib_ref] [bib_ref] The crosstalk of RAS with the TGF-beta family during carcinoma progression and..., Grusch [/bib_ref]. TGF-β can quickly activated RAS and ERK pathway [bib_ref] Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming..., Yue [/bib_ref] , in contrast, the ERK pathway inhibits the TGF-β/Smad4 pathway by phosphorylating Smad2 and Smad3 at serine or threonine residues in the linker region, so epithelial cells with oncogenic RAS mutations usually exhibit loss of TGF-β antiproliferative response [bib_ref] The role of TGF-beta/SMAD4 signaling in cancer, Zhao [/bib_ref]. Patients with RAS wild-type tumors and retained SMAD4 wild-type had longer OS than patients with both mutations [bib_ref] Long-term survivors of pancreatic adenocarcinoma show low rates of genetic alterations in..., Masetti [/bib_ref]. However, SMAD4 mutations were significantly associated with poorer OS regardless of RAS mutation status or other clinicopathological factors. The precise cooperative mechanisms of SMAD4 with other genes of influence also requires further examination.
Given the relative frequency of SMAD4 mutations in CRC patients, routine SMAD4 testing may be appropriate. For individualized treatment of CRC, SMAD4, as a driver mutation, will become a novel target for precision medical treatment of CRC, and further research should be done for guiding clinical decision-making.
No heterogeneity or publication bias was found in this meta-analysis, and sensitivity analysis shows that our results are reliable. However, this analysis has several limitations. First, our meta-analysis included studies of qualified articles published in English or Chinese, and did not include relevant articles written in other languages or unpublished papers, which is likely to result in selection bias. Second, the use of specific therapies and tumor stage differed among the included articles. Third, the HR calculated from the data or extracted from the survival curve may not be as reliable as the HR calculated directly using the analysis of variance. Therefore, the results should be carefully interpreted. However, as far as we know, this is the first meta-analysis to demonstrate SMAD4 mutation by evaluating the pathological features and prognostication in CRC.
# Conclusion
In conclusion, we found that SMAD4 mutation was associated with poor prognosis in CRC, but has nothing to do with MSI status, BRAF status or tumor grade. Further studies are needed to evaluate these findings and the clinical significance of SMAD4 status in CRC.
[fig] Figure 1: Schematic flow diagram for selection of included studies [/fig]
[fig] Figure 2 a: Forest plot for meta-analysis of the association of SMAD4 mutations with OS of patients with CRC. b Forest plot for meta-analysis of the association of SMAD4 mutations with PFS/RFS of patients with CRC [/fig]
[fig] Figure 3 a 297, Figure 4 a: Forest plot of Egger's test for publication bias of OS. b Forest plot of Egger's test for publication bias of PFS/RFS Fang et al. BMC Gastroenterol (2021) 21:Sensitivity analysis of meta-analysis of the association of SMAD4 mutations with OS in CRC patients. b Sensitivity analysis of meta-analysis of the association of SMAD4 mutations with PFS/RFS in CRC patients [/fig]
[table] Table 1: Main characteristics of studies includedNR not report, MA mean age, MF median follow-up, Mut mutation, WT wild type, HR hazard ratio, e estimate, NGS next generation sequencing, NOS Newcastle-Ottawa scale, OS overall survival, PFS progression-free survival, RFS recurrence-free survival [/table]
[table] Table 2: Data extracted from studies included Mut mutated, WT wild type, WMD well to moderately differentiated, PD poorly differentiated, LN lymph node metastases, NR not reported Female Male Colon Rectum I-III IV WMD PD Stable Unstable WT Mut WT Mut Yes No Yes No [/table]
[table] Table 3: Quality assessment according to the Newcastle-Ottawa scale of the included studies [/table]
[table] Table 4: Relationship of SMAD4 gene and clinicopathologic characteristics of colorectal cancer MSI microsatellite instability, RAS rat sarcoma viral oncogene homolog, BRAF b-viral oncogene homolog B1 [/table]
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Caregiver Perception of Weight Status in 5-Year-Old Children From a Community of High Socioeconomic Deprivation in New Zealand
Background: Early childhood obesity is highly prevalent in Aotearoa New Zealand (NZ). Little is known about caregiver perception of children's weight status among those living in areas of high socioeconomic deprivation, particularly Māori and Pacific children.Aims: To explore caregiver perception of weight status among children starting school in areas of high socioeconomic deprivation and examine potential associations between the child's body mass index (BMI) z-score and their caregiver's perception of their child's body size or health.Methods: Participants were 5-year-old children living in a community of high socioeconomic deprivation and their caregivers. Children had their weight and height measured. BMI z-scores were calculated according to World Health Organization standards. Caregivers were asked to assess their child's BMI and health status, and choose a silhouette that best represented their child's body size.Results: One hundred and six children (>75% Māori or Pacific) were included. Over half (58%) had overweight or obesity, with only 16% correctly perceived by their caregiver as overweight. These children tended to have higher BMI z-scores than those not correctly perceived as overweight. Caregivers chose larger silhouettes to represent children's body sizes as children's BMI z-scores increased. There was no discernible association between children's BMI z-scores and caregiver perception of children's health.Conclusions: Caregivers appeared to judge their child's body size in comparison to other children. The normalization of childhood obesity and infrequent caregiver recognition of this condition in children in communities with a high prevalence may impact the uptake and efficacy of intervention initiatives.
# Introduction
Overweight in early childhood is estimated to affect 38 million children worldwide. In Aotearoa New Zealand (NZ), despite some recent evidence for a slight decline, the prevalence of overweight/obesity in early childhood remains high (>30%) [bib_ref] Improving rates of overweight, obesity and extreme obesity in New Zealand 4-year-old..., Shackleton [/bib_ref]. Childhood obesity tracks into later life [bib_ref] The use of measures of obesity in childhood for predicting obesity and..., Simmonds [/bib_ref] , and is associated with increased risk of comorbidities both in childhood and adolescence [bib_ref] A systematic review and meta-analysis estimating the population prevalence of comorbidities in..., Sharma [/bib_ref] , and adulthood [bib_ref] Childhood obesity as a predictor of morbidity in adulthood: a systematic review..., Llewellyn [/bib_ref]. Thus, the early prevention and treatment of childhood obesity remain important public health concerns in NZ and elsewhere.
Much interest has been placed on parental perception of their child's weight status, based on the belief that accurate parental perception of childhood overweight or obesity will facilitate better weight outcomes for children [bib_ref] Asian parents' perception of child weight status: a systematic review, Park [/bib_ref] [bib_ref] Parental perception of child's body weight: a systematic review, Tompkins [/bib_ref] [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref] [bib_ref] Parental underestimates of child weight: a meta-analysis, Lundahl [/bib_ref] [bib_ref] Overweight but unseen: a review of the underestimation of weight status and..., Robinson [/bib_ref]. However, internationally, the majority of studies have reported parents do not accurately identify overweight or obesity in children, and frequently under-estimate their weight status [bib_ref] Asian parents' perception of child weight status: a systematic review, Park [/bib_ref] [bib_ref] Parental perception of child's body weight: a systematic review, Tompkins [/bib_ref] [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref] [bib_ref] Parental underestimates of child weight: a meta-analysis, Lundahl [/bib_ref]. Common factors influencing parental perception of children's weight status include: child's age and sex, parental education, and socioeconomic status [bib_ref] Asian parents' perception of child weight status: a systematic review, Park [/bib_ref] [bib_ref] Parental perception of child's body weight: a systematic review, Tompkins [/bib_ref] [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref] [bib_ref] Parental underestimates of child weight: a meta-analysis, Lundahl [/bib_ref]. Parental perception of their child's overall health and lifestyle may be important too, with qualitative research suggesting parents of young children do not feel concerned by their child's weight status if they perceive them to be in good health and/or engaged in regular physical activity [bib_ref] A little on the heavy side": a qualitative analysis of parents' and..., Eli [/bib_ref] [bib_ref] Health and happiness is more important than weight': a qualitative investigation of..., Syrad [/bib_ref] [bib_ref] Why don't low-income mothers worry about their preschoolers being overweight?, Jain [/bib_ref] [bib_ref] Parental perceptions of overweight during early childhood, Goodell [/bib_ref]. Additionally, systematic reviews have reported improved parental accuracy in studies which use visual assessments such as silhouettes, rather than verbal assessments requiring parents to select the words "overweight" or "obese", which are potentially more subject to stigma [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref].
Three previous studies have examined parental perceptions of their children's weight status in NZ [bib_ref] Agreement between parental perception of child weight status and actual weight status..., Williams [/bib_ref] [bib_ref] DXA measurements confirm that parental perceptions of elevated adiposity in young children..., Miller [/bib_ref] , with all reporting high rates of under-perception of excess weight status among parents of children with overweight or obesity [bib_ref] Agreement between parental perception of child weight status and actual weight status..., Williams [/bib_ref] [bib_ref] DXA measurements confirm that parental perceptions of elevated adiposity in young children..., Miller [/bib_ref]. Of these studies, two did not break down parental perception by ethnicity [bib_ref] DXA measurements confirm that parental perceptions of elevated adiposity in young children..., Miller [/bib_ref] , while the other reported that misperception did not differ by ethnicity [bib_ref] Agreement between parental perception of child weight status and actual weight status..., Williams [/bib_ref]. However, children included in the latter study were predominantly of NZ Europeans, while only 1 in 5 lived in areas of high socioeconomic deprivation [bib_ref] Agreement between parental perception of child weight status and actual weight status..., Williams [/bib_ref]. A greater proportion of children of Māori (indigenous people of NZ) and Pacific ethnicity have early childhood obesity (20.0 and 30.2%, respectively) compared to European or Asian children (12.7 and 8.1%, respectively) (2). Furthermore, obesity affects <10% of 5-year-olds living in the least deprived areas of NZ, while it affects just over 20% of those living in the most deprived areas [bib_ref] Improving rates of overweight, obesity and extreme obesity in New Zealand 4-year-old..., Shackleton [/bib_ref]. Therefore, the aim of this study was to explore the accuracy of caregiver perception of weight status at school entry among children living in an Auckland community of high socioeconomic deprivation (predominantly Māori and Pacific). We also aimed to examine potential associations between children's body mass index (BMI) z-scores and caregiver perception of children's body size when assessed with silhouettes or verbally, and their perception of their child's health.
# Methods
Participants were drawn from the Welcome-to-School Study, which has been described previously [bib_ref] The prevalence of refractive error and visual impairment among New Zealand children..., Findlay [/bib_ref]. Briefly, the study recruited 5-year-old children (and their caregivers) at school entry living in a central Auckland community of high socioeconomic deprivation [bib_ref] The prevalence of refractive error and visual impairment among New Zealand children..., Findlay [/bib_ref]. Children underwent comprehensive health, developmental, educational, and social assessments with appropriate referrals and linkages made if any issues were identified [bib_ref] The prevalence of refractive error and visual impairment among New Zealand children..., Findlay [/bib_ref]. All children had their heights and weights measured while barefoot and wearing light clothing by a research nurse, following a standardized protocol. Height was measured using a SECA 217 portable stadiometer (SECA, Hamburg, Germany) to the nearest mm, while weight was measured with a SECA 874 digital scale to the nearest 0.01 kg. Assessments occurred at either the child's home or school, according to caregiver preference.
Caregivers were interviewed face-to-face about their child's health and development by the research nurse using a standardized questionnaire [bib_ref] The prevalence of refractive error and visual impairment among New Zealand children..., Findlay [/bib_ref]. The specific questions included in this study were those asking caregivers about their perceptions of their child's weight (underweight, normal weight, overweight, or don't know), body size (visual assessment), and current health (poor, fair, good, very good, excellent, or don't know) (Supplementary Material). The tool used for visual assessment of child's body size was sourced from a previous study about overweight in Latino preschoolers [bib_ref] Overweight in Latino preschoolers: do parental health beliefs matter?, Kersey [/bib_ref] , and was composed of 12 line drawn silhouettes of girls and boys (six each) ranked from "C" (representing relatively thin) to "H" (representing severe obesity) (Supplementary Material). Caregivers were not informed of weight status attributed to the silhouettes.
Demographic information was collected, including caregiver education level, and child's age, sex, and ethnicity. Caregiver education was stratified as high school or less, certificate/diploma, or University degree. Children's ethnicity was classified using the Statistics NZ order of prioritization, with the exception that all children not classified as either Māori or Pacific were classified as "Other" due to their small number. The participant's home address was used to derive their level of socioeconomic deprivation (NZDep2013 score), ranging from 1 (least deprived) to 10 (most deprived), which were stratified into quintiles.
One hundred and twenty-one children were enrolled and examined for the Welcome-to-School Study over the 12-month study period, which equated to approximately: 75% of the eligible children starting school in the area. Only those with available data on weight, height, and caregiver perception of weight status were included in the present study (n = 106, 88% of study participants or 67% of the total cohort of children starting school).
## Statistical analyses
BMI was calculated and transformed into age-and sex-adjusted z-scores as per World Health Organization (WHO) standards [bib_ref] Development of a WHO growth reference for school-aged children and adolescents, De Onis [/bib_ref]. The child's BMI status was subsequently defined as: normal weight or underweight [BMI z-score < 1.036 (i.e., <85th percentile)], overweight [≥1.036 but <1.645 (i.e., <95th percentile)], or obesity (z-score ≥ 1.645) [bib_ref] Expert committee recommendations regarding the prevention, assessment, and treatment of child and..., Barlow [/bib_ref].
The BMI of children with overweight or obesity correctly perceived as such by their caregiver were compared to those who were not with a two-sample t-test. BMI z-scores among children identified as Māori, Pacific, or "Other" were compared using a general linear model. The associations between the child's BMI z-score and the caregiver's choice in the picture scale or their perception of the child's health were examined using non-parametric Spearman's rank correlations, with results reported as the Spearman's rho coefficient (ρ) and the respective 95% confidence interval (CI) and p-value. Differences in the caregiver ranking of children along the picture scale according to ethnicity and gender were compared using non-parametric Kruskal-Wallis tests.
Group summary data are provided in the text as means ± standard deviations, except for picture scale data reported as medians, quartile 1 (i.e., 25th percentile), and quartile 3 (i.e., 75th percentile). Data were analyzed using SPSS v25 (IBM Corp, Armonk, NY, USA) and Minitab v21 (Pennsylvania State University, State College, Pennsylvania, USA). All statistical tests were two-sided with significance set at p < 0.05. Figures were created using GraphPad Prism v8.2 (GraphPad Software Inc., San Diego, CA, USA).
# Results
## Participant characteristics
There were 117 children with BMI data in the Welcome-to-School Study. However, caregiver perception of their child's weight status was not available for nine participants, and two caregivers responded "don't know"; leaving 106 children in this study with valid data [fig_ref] TABLE 1 |: Sociodemographic characteristics of participants [/fig_ref].
Just under half were female (46%). More than half (n = 61; 58%) had overweight or obesity [fig_ref] TABLE 1 |: Sociodemographic characteristics of participants [/fig_ref]. Few caregivers (16%) had a University education [fig_ref] TABLE 1 |: Sociodemographic characteristics of participants [/fig_ref]. Over 80% lived in areas with the highest quintile of socioeconomic deprivation [fig_ref] TABLE 1 |: Sociodemographic characteristics of participants [/fig_ref]. Almost all questionnaires were completed by a female primary caregiver (mostly the child's mother). [fig_ref] TABLE 2 |: Caregiver perceived vs [/fig_ref] summarizes caregiver perceived vs. actual weight status of children. There were no children of underweight, normal weight, or overweight status perceived as overweight by their caregiver [fig_ref] TABLE 2 |: Caregiver perceived vs [/fig_ref]. Only 10 out of 39 (26%) children with obesity were perceived as having overweight, while three children with obesity were perceived as underweight [fig_ref] TABLE 2 |: Caregiver perceived vs [/fig_ref]. Supplementary [fig_ref] TABLE 1 |: Sociodemographic characteristics of participants [/fig_ref] shows that the proportions of girls (23.1%) and boys (26.9%) with obesity considered overweight by their caregiver were similar.
## Caregiver perception of child's weight status
Further highlighting the association between caregiver perception of child's weight status and BMI z-score, children with overweight or obesity correctly recognized as such by their caregiver tended to have higher BMI z-scores than those who were not (4.66 ± 1.92 vs. 2.00 ± 1.32, respectively; p < 0.0001) . One notable exception was a boy with a confirmed BMI z-score of 10.06 perceived as underweight by his mother. BMI, body mass index. Data are n (%) or mean ± SD. a n = 106, except for "Education" (n = 101) and "Socioeconomic deprivation" (n = 94). b Socioeconomic deprivation was measured using the NZDep2013 (23).
Child's weight status was defined based on body mass index (BMI) z-scores standardized for age and sex as per World Health Organization standards (24), as follows: overweight, BMI ≥ 85th to <95th percentile (z-score ≥ 1.036 and <1.645); and obesity, BMI ≥ 95th percentile (z-score ≥ 1.645) [bib_ref] Expert committee recommendations regarding the prevention, assessment, and treatment of child and..., Barlow [/bib_ref]. shows caregiver choice of a silhouette in relation to children's BMI z-scores. Not surprisingly, caregivers tended to choose larger silhouettes as their children's BMI z-scores increased (ρ = 0.61; p < 0.0001) . However, many caregivers selected a slim silhouette for their children, despite them having overweight or obesity . These findings were largely unchanged regardless of whether children were identified as Māori, Pacific, or Other .
The only observable ethnic differences were lower BMI zscores among children identified as "Other" ethnicities (0.46 ± 0.88) compared to children identified as Māori (1.37 ± FIGURE 1 | Caregiver recognition of overweight in children with overweight or obesity and the children's respective body mass index (BMI) z-scores (n = 61). BMI z-scores were standardized for age and sex as per World Health Organization standards [bib_ref] Development of a WHO growth reference for school-aged children and adolescents, De Onis [/bib_ref]. Horizontal black bars represent the median and the interquartile range, while the p-value for the comparison between groups was derived from a t-test.
## Caregiver perception of child's weight vs. child's health status
There was no correlation between caregiver perception of their child's health status and the child's BMI z-score [ρ = 0.02 (95% CI −0.18, 0.21); p = 0.85], with many children across the BMI range considered to be healthy . In fact, very few children were deemed to have "poor" or "fair" health, and several children with very severe obesity (BMI z-score 4.20-10.06) were considered to have "very good" or "excellent" health . Of the 10 children with obesity who were correctly recognized as having "overweight", only one was considered to have "fair" health, while the rest were considered to have "good" (n = 3), "very good" (n = 3), or "excellent" (n = 3) health.
# Discussion
In a population of 5-year-old children in NZ living in an Auckland suburb of high socioeconomic deprivation, over half had overweight or obesity [compared to one in three overall in the country [bib_ref] Improving rates of overweight, obesity and extreme obesity in New Zealand 4-year-old..., Shackleton [/bib_ref] ], but few were perceived as overweight by their caregivers. It was uncommon for caregivers of children FIGURE 2 | Caregiver choice of silhouette and child body mass index (BMI) z-score (n = 103). Children's BMI z-scores were standardized for age and sex as per World Health Organization standards [bib_ref] Development of a WHO growth reference for school-aged children and adolescents, De Onis [/bib_ref]. The two red lines parallel to the x-axis represent the thresholds for overweight and obesity, defined as: overweight, BMI ≥ 85th to <95th percentile (z-score ≥1.036 and <1.645); and obesity, BMI ≥ 95th percentile (z-score ≥ 1.645) [bib_ref] Expert committee recommendations regarding the prevention, assessment, and treatment of child and..., Barlow [/bib_ref]. Figure of silhouettes reproduced with permission from Kersey et al. [bib_ref] Overweight in Latino preschoolers: do parental health beliefs matter?, Kersey [/bib_ref]. The association between the two parameters is reported as the Spearman's rank correlation coefficient (ρ) and the respective 95% confidence interval and p-value.
with overweight or obesity to choose larger silhouettes as representative of their child's body size, even though children correctly perceived as overweight tended to have higher BMI zscores than those not perceived as such. In addition, we observed that caregiver perception of children's health status was not associated with children's BMI z-scores.
Similar to previous research in NZ [bib_ref] Agreement between parental perception of child weight status and actual weight status..., Williams [/bib_ref] [bib_ref] DXA measurements confirm that parental perceptions of elevated adiposity in young children..., Miller [/bib_ref] and elsewhere [bib_ref] Asian parents' perception of child weight status: a systematic review, Park [/bib_ref] [bib_ref] Parental perception of child's body weight: a systematic review, Tompkins [/bib_ref] [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref] [bib_ref] Parental underestimates of child weight: a meta-analysis, Lundahl [/bib_ref] , caregiver recognition of overweight in children with overweight or obesity was infrequent in our study, even though Children perceived as overweight tended to be on the higher end of the BMI z-score distribution. It may be that the high prevalence of obesity in children and adults in communities represented by our study population [i.e. large proportions of Māori or Pacific people and those living in areas of high socioeconomic disadvantage], has created a "normalizing" effect [bib_ref] Overweight but unseen: a review of the underestimation of weight status and..., Robinson [/bib_ref] , whereby overweight or obesity is so common it is considered the norm, so that only children with exceptionally high BMI z-scores may be perceived to carry excessive weight. Indeed, previous longitudinal studies have reported increased frequency of parental underestimation of weight status in children with overweight co-occurring with an increased prevalence of childhood obesity in a given population [bib_ref] Overweight but unseen: a review of the underestimation of weight status and..., Robinson [/bib_ref]. Our findings from the silhouette assessment provide further evidence to support the normalization theory. There was a clear pattern of caregivers choosing increasingly larger silhouettes to represent their child's body size as children's BMI z-scores increased. Of note, many caregivers of children with a BMI zscore on the lower end of the obesity range chose slim silhouettes for their children. Therefore, it seems likely that caregiver judgment was influenced by their perception of their child's body size compared to other children in their community, rather than their child's BMI status per se. This phenomenon has been reported in different populations [bib_ref] Overweight but unseen: a review of the underestimation of weight status and..., Robinson [/bib_ref] and was consistent across the ethnicities in our study.
There was no apparent link between caregiver perception of children's health status and their BMI z-scores, with children across the BMI z-score range considered to be in "very good" or "excellent" health. This disconnect between weight status and health has been reported previously, particularly among caregivers of young children who point to other indicators of their child's health, including their happiness [bib_ref] Health and happiness is more important than weight': a qualitative investigation of..., Syrad [/bib_ref] [bib_ref] The complexity of food provisioning decisions by Māori caregivers to ensure the..., Glover [/bib_ref] , dietary intake [bib_ref] Health and happiness is more important than weight': a qualitative investigation of..., Syrad [/bib_ref] [bib_ref] Why don't low-income mothers worry about their preschoolers being overweight?, Jain [/bib_ref] , or levels of physical activity [bib_ref] Health and happiness is more important than weight': a qualitative investigation of..., Syrad [/bib_ref] [bib_ref] Why don't low-income mothers worry about their preschoolers being overweight?, Jain [/bib_ref] [bib_ref] Parental perceptions of overweight during early childhood, Goodell [/bib_ref] [bib_ref] The complexity of food provisioning decisions by Māori caregivers to ensure the..., Glover [/bib_ref]. In NZ, Māori caregivers of children aged 5 years and younger believed that once children started school, they would burn off any excess fat through increased physical activity [bib_ref] The complexity of food provisioning decisions by Māori caregivers to ensure the..., Glover [/bib_ref]. However, elsewhere, parents of 7-year-old children were more accurate in recognizing their child's overweight or obesity when they perceived their health as poor [bib_ref] Are parental perceptions of child activity levels and overall health more important..., Vangeepuram [/bib_ref]. Indeed, parental recognition of overweight or obesity is more accurate in older than younger children [bib_ref] Parental perception of child's body weight: a systematic review, Tompkins [/bib_ref] [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref] [bib_ref] Parental underestimates of child weight: a meta-analysis, Lundahl [/bib_ref]. Therefore, a link between caregiver perception of health and weight may not be seen among caregivers of children soon after starting school, such as those in our study, as weight-related comorbidities are less of immediate concern for them.
## Implications for public health
Our study has shown that in this community with a high prevalence of overweight or obesity, only a few children with very high BMI z-scores were perceived as overweight. Therefore, if caregiver recognition of excessive weight in their child is deemed important for early intervention, this may be challenging in communities with a high prevalence of childhood obesity where normalization and infrequent caregiver recognition of the condition may impact uptake and efficacy of interventions.
Theoretically, it would make sense that caregiver recognition of a weight issue in children would be followed by intervention. To this end, public health interventions in the UK and US have focused on providing caregivers of school children with a BMI report card so that they are aware of their child's weight status [bib_ref] UK children's body mass index report cards: public benefit or poor policy?, Hardy [/bib_ref]. In NZ, caregivers of children identified as "extremely overweight" at the B4School Check (a comprehensive check of the development and growth of children aged 4-5 years) are informed of their child's weight status, and their child is referred for weight management. However, longitudinal studies not involving any form of intervention have reported increased weight gain among children who have been recognized as having overweight or obesity by their parents [bib_ref] Parental perception of weight status and weight gain across childhood, Robinson [/bib_ref] [bib_ref] Parental perception of child's weight status and subsequent BMIz change: the KOALA..., Gerards [/bib_ref] [bib_ref] Parents' perceptions of their children as overweight and children's weight concerns and..., Robinson [/bib_ref]. Furthermore, BMI report cards have not been as effective as hoped; [bib_ref] UK children's body mass index report cards: public benefit or poor policy?, Hardy [/bib_ref] in the UK, for example, there were minimal parental-reported changes in health behaviors among parents who were informed that their child was above a healthy weight [bib_ref] The benefits and harms of providing parents with weight feedback as part..., Falconer [/bib_ref]. Therefore, as we previously concluded [bib_ref] Parental perceptions of obesity in school children and subsequent action, Butler [/bib_ref] , it seems unlikely that caregiver recognition of childhood obesity alone is sufficient for behavioral change. Supportive holistic interventions are needed to assist families in achieving a healthy weight for their children. Future research should also consider the longitudinal impact of accurate caregiver perception of children's weight status on childhood weight gain in the NZ context.
# Limitations and strengths
Our study is limited by the relatively small number of participants, restricting our ability to generalize our findings to a wider population. However, our focus on children living in a community of high socioeconomic deprivation, in particular of Māori or Pacific ethnicity, and our sample comprising more than two thirds of the cohort of children starting school in a community of interest are key strengths. This is important given the higher prevalence of early childhood obesity among children in these groups in NZ [bib_ref] Improving rates of overweight, obesity and extreme obesity in New Zealand 4-year-old..., Shackleton [/bib_ref]. In addition, the images used for caregiver visual assessment of child body size were not culturally adapted to caregivers of Māori or Pacific preschool children. Still, their use meant that our study did not solely rely on a scale using words, which are more likely to be subject to misreporting due to stigma [bib_ref] Difference between parental perception and actual weight status of children: a systematic..., Rietmeijer-Mentink [/bib_ref].
# Conclusions
In this sample of predominantly Māori or Pacific children living in an urban NZ community of high socioeconomic deprivation, few (16%) were correctly perceived as overweight by their caregivers. Caregivers appeared to judge their child's body size in comparison to other children in their community rather than by their actual weight status, potentially contributing to the infrequent recognition of overweight. There did not appear to be a link between children's BMI z-scores and caregiver perception of children's health for those included in our study. The normalization of childhood obesity in communities with a high prevalence may impact the uptake and efficacy of intervention initiatives.
# Data availability statement
The datasets presented in this article are not readily available because of the strict conditions of the ethics approval. However, the anonymised data on which this article was based could be made available to other investigators upon bona fide request, and following all the necessary approvals (including ethics approval) of the detailed study proposal and statistical analyses plan. Requests to access the datasets should be directed to AL, [email protected].
# Ethics statement
Welcome-to-School Study was approved by the Central Disability Ethics Committee of the NZ Ministry of Health (15/CEN/224/AM04). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
# Author contributions
Welcome-to-School Study: AL was the principal investigator and obtained funding. AL and AB contributed to study design. AB enrolled participants, performed assessments, and collected and curated data. Current study: WC and JD obtained funding. ÉB, JD, WC, and AL contributed to study design. JD and ÉB were responsible for data curation and analyses. ÉB wrote the manuscript with assistance from JD and critical input from WC, AL, and AB. All authors have approved the submission of the manuscript in its final form.
# Funding
ÉB was supported by a scholarship from A Better Start -National Science Challenge, which was funded by the NZ Ministry of Business, Innovation and Employment. The Welcome-to-School Study was supported by funding from Cure Kids (#4001), A+ Charitable Trust (6995), and Joyce Fisher Charitable Trust.
[table] TABLE 1 |: Sociodemographic characteristics of participants. [/table]
[table] TABLE 2 |: Caregiver perceived vs. actual weight status of the child (n = 106). [/table]
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Relation of Leptin, Ghrelin and Inflammatory Cytokines with Body Mass Index in Pulmonary Tuberculosis Patients with and without Type 2 Diabetes Mellitus
Background: Pulmonary tuberculosis (TB) patients often suffer from anorexia and poor nutrition, causing weight loss. The peptide hormones leptin and its counterpart ghrelin, acting in the regulation of food intake and fat utilization, play an important role in nutritional balance. This study aimed to investigate the association of blood concentrations of leptin, ghrelin and inflammatory cytokines with body mass index (BMI) in TB patients with and without type 2 diabetes mellitus (T2DM).Methods: BMI, biochemical parameters and plasma levels of leptin, ghrelin and inflammatory cytokines were measured before the start of treatment in 27 incident TB patients with T2DM, 21 TB patients and 23 healthy subjects enrolled in this study.Results:The levels of leptin were significantly higher in TB patients (35.2±19.1 ng/ml) than TB+T2DM (12.6±6.1 ng/ml) and control (16.1±11.1 ng/ml) groups. The level of ghrelin was significantly lower in TB (119.9±46.1 pg/ml) and non-significantly lower in TB+T2DM (127.7±38.6 pg/ml) groups than control (191.6±86.5 pg/ml) group. The levels of TNF-α were higher, while IFN-γ and IL-6 levels were lower in patients than in the control group. Leptin showed a negative correlation with BMI in TB (r=-0.622, p<0.05) and TB+T2DM (r= -0.654, p<0.05) groups, but a positive correlation with BMI in the control group (r=0.521, p<0.05). Contrary ghrelin showed a positive correlation with BMI in TB (r=0.695, p<0.05) and TB+T2DM (r= 0.199, p>0.05) groups, but negative correlation with BMI in the control (r=-0.693, p<0.05) group. Inflammatory cytokines were poorly correlated with BMI in this study. Only IFN-γ showed a significant negative correlation with BMI in the control group (r=-0.545, p<0.05).Conclusions: This study may suggest that possible abnormalities in ghrelin and leptin regulation (high levels of leptin and low levels of ghrelin) may be associated with low BMI and may account for the poor nutrition associated with TB and TB+T2DM.
# Introduction
Pulmonary tuberculosis (TB) is a major cause of mortality around the world, nearly one-third of the world's population is infected, and 8~12 million people become newly infected each year. TB incidence is influenced by several social and economic factors, such as poverty or poor nutrition [bib_ref] Drivers of tuberculosis epidemics: the role of risk factors and social determinants, Lönnroth [/bib_ref] , as well as by other diseases, such as diabetes mellitus (DM). Many studies now show that DM may be associated with an increased risk (almost triple) of developing active TB [bib_ref] Diabetes mellitus increases the risk of active tuberculosis: a systematic review of..., Jeon [/bib_ref] [bib_ref] Diabetes and the risk of tuberculosis: a neglected threat to public health?, Stevenson [/bib_ref] [bib_ref] Cross-sectional assessment reveals high diabetes prevalence among newly-diagnosed tuberculosis cases, Restrepo [/bib_ref] , and TB patients who also have diabetes may have higher rates of treatment failure and death [bib_ref] Impact of diabetes mellitus on treatment outcomes of patients with active tuberculosis, Dooley [/bib_ref] [bib_ref] The effect of type 2 diabetes mellitus on the presentation and treatment..., Alisjahbana [/bib_ref]. China has the second highest rate of TB morbidity in the world and DM rates are reaching epidemic proportions [bib_ref] Asia-Pacific faces diabetes challenge, Cheng [/bib_ref]. This harmful synergy of TB and DM has led public health systems in China to attempt to tackle the two diseases concurrently. A study was initiated in which active TB patients in poverty zones are screened for DM and the effect of a diet and lifestyle intervention is evaluated [bib_ref] Screening and intervention of diabetes mellitus in patients with pulmonary tuberculosis in..., Wang [/bib_ref].
Poor nutrition represented by wasting and anorexia, is a prominent feature of both TB and DM, being a hypercatabolic state, characterised by accelerated protein degradation and muscle wasting, resulting in weight loss, and deteriorating clinical functions with resultant poor prognosis [bib_ref] Diabetes and the risk of tuberculosis: a neglected threat to public health?, Stevenson [/bib_ref]. The appetite-related hormones, leptin and ghrelin, may be new candidate causes of TB-associated malnutrition [bib_ref] Leptin and energy metabolism in pulmonary tuberculosis, Schwenk [/bib_ref] [bib_ref] Decreased plasma leptin concentrations in tuberculosis patients are associated with wasting and..., Van Crevel [/bib_ref] [bib_ref] The relation between serum leptin levels and body fat mass in patients..., Yüksel [/bib_ref]. Leptin is a protein hormone of 167 amino acids. Its main effect relates to energy exhaustion and control of food intake, implicated as an anorexigenic factor that reduces appetite [bib_ref] Weight-reducing effects of the plasma protein encoded by the obese gene, Halaas [/bib_ref].The connection of leptin to adipose tissue emphasizes the endocrine function of that compartment. Conversely, ghrelin is a 28-amino-acid peptide, which is produced by the stomach and has recently attracted interest as a novel anti-catabolic and orexigenic factor, that is increased in anorexic conditions and stimulates appetite [bib_ref] Physiological, pathological and potential therapeutic roles of ghrelin, Leite-Moreira [/bib_ref]. Many studies have also revealed that both leptin and ghrelin are immune system regulators in addition to their effect on food intake [bib_ref] Physiological, pathological and potential therapeutic roles of ghrelin, Leite-Moreira [/bib_ref] [bib_ref] Leptin modulates the T-cell immune response and reverses starvationinduced immunosuppression, Lord [/bib_ref]. Plasma levels of leptin and ghrelin can be altered in disease states associated with anorexia [bib_ref] Leptin and energy metabolism in pulmonary tuberculosis, Schwenk [/bib_ref] [bib_ref] Decreased plasma leptin concentrations in tuberculosis patients are associated with wasting and..., Van Crevel [/bib_ref] [bib_ref] The relation between serum leptin levels and body fat mass in patients..., Yüksel [/bib_ref]. However, previous data regarding leptin levels in TB patients are conflicting. One study has shown that pretreatment plasma leptin levels were lower in TB patients than in healthy controls and there was a strong positive correlation between leptin concentration and body mass index (BMI) in both the control and patient group [bib_ref] Decreased plasma leptin concentrations in tuberculosis patients are associated with wasting and..., Van Crevel [/bib_ref]. Serum leptin level was found to be higher in TB patients than controls in other studies [bib_ref] The relation between serum leptin levels and body fat mass in patients..., Yüksel [/bib_ref] [bib_ref] Relation of leptin and tumor necrosis factor alpha to body weight changes..., Cakir [/bib_ref] in which leptin levels were positively related to BMI only in the control group. Ghrelin has not been widely studied in patients with TB [bib_ref] The role of feed regulating peptides on weight loss in patients with..., Yurt [/bib_ref] [bib_ref] Relation of ghrelin, leptin and inflammatory markers to nutritional status in active..., Kim [/bib_ref] [bib_ref] Gut hormones, appetite suppression and cachexia in patients with pulmonary TB, Chang [/bib_ref] , let alone patients with TB+DM. There may be increased activation of the inflammatory system and alterations of the immune system in TB and TB+T2DM [bib_ref] Tuberculosis, malnutrition and wasting, Schwenk [/bib_ref] [bib_ref] Cytokines related to nutritional status in patients with untreated pulmonary tuberculosis in..., Karyadi [/bib_ref] [bib_ref] Potential role of interleukin 6 in reactive thrombocytosis and acute phase response..., Unsal [/bib_ref]. Leptin and ghrelin secretion and their circulating levels are effected by diet, adiposity, energy balance, and hormonal factors together with many intrinsic adiposity factors and cytokines [bib_ref] The adipocyte specific transcription factor C-EBPalpha modulates human ob gene expression, Miller [/bib_ref]. Tumor necrosis factoralpha (TNF-α) shows antimycobacterial activity and promotes granuloma formation in TB patients [bib_ref] The immunoendocrine component in the pathogenesis of tuberculosis, Bottasso [/bib_ref]. The increase of TNF-α· may cause anorexia and consequent weight loss in TB patients [bib_ref] Cachectin: More than a tumor necrosis factor, Beutler [/bib_ref]. Interferon-gamma (IFN-γ), a Th1-type cytokine, is known to be a key cytokine in the host immune response to tuberculosis infection. If IFN-γ cannot be produced or cannot exert its effects, TB infection is more severe and often fatal [bib_ref] The immunoendocrine component in the pathogenesis of tuberculosis, Bottasso [/bib_ref].Some studies showed there were negative correlations between inflammatory mediators like CRP, IL-1 and TNF-α with BMI in patients with active lung tuberculosis [bib_ref] Decreased plasma leptin concentrations in tuberculosis patients are associated with wasting and..., Van Crevel [/bib_ref] [bib_ref] Relation of leptin and tumor necrosis factor alpha to body weight changes..., Cakir [/bib_ref] [bib_ref] Increased interleukin-1 production and monocyte suppressor cell activity associated with human tuberculosis, Fujiwara [/bib_ref]. However, evidence for a link between the inflammatory response and malnutrition is still equivocal and Incomplete [bib_ref] Cytokines related to nutritional status in patients with untreated pulmonary tuberculosis in..., Karyadi [/bib_ref] [bib_ref] Anorexia of infection: current prospects, Langhans [/bib_ref]. Whether weight loss in tuberculosis is probably due to over release of cytokines remains unknown.
This study was undertaken to investigate whether the plasma levels of leptin, ghrelin and inflammatory cytokines are associated with BMI (reflecting nutritional status) in TB patients with and without type 2 diabetes (T2DM).
# Materials and methods
# Ethics statement
Permission from the ethics committee of the affiliated hospital of Medical School of Qingdao University was obtained before the study. And study was conducted according to the principles outlined in the Declaration of Helsinki. Study subjects were informed, each submitted written informed consent before the study.
## Subjects
27 patients with TB +T2DM from the Chest Hospital of Qingdao were enrolled prospectively in this cross sectional study. TB+T2DM patients had positive sputum culture of mycobacterium tuberculosis and positive chest X ray, also had a fasting plasma glucose ≥7.0 mmol/l (mM) or a random blood sugar >11.1 mM 21 TB patients (also from chest hospital of Qingdao) and 23 healthy subjects from medical center of the affiliated hospital of Qingdao medical college both with similar age and gender distribution were enrolled. Patients with type 1 diabetes, miliary TB, non-tuberculous mycobacteria (NTM), or human immunodeficiency virus co-infection were excluded. Patients and control subjects who had any other serious concomitant diseases or had been previously treated with anti-TB drugs were also excluded. Informed consent was obtained from all subjects.
## Measurements
A blood sample was collected before any treatment had been given via a venous catheter into a Heparin Sodium tube and non-anticoagulation tube (5ml respectively), between 7 and 8 AM after overnight fasting. After centrifugation of the heparin sodium tube, plasma was stored at -80°C. Samples with non-anticoagulation tubes, were water bathed for 20~30min at 37°C, and then centrifuged at 1580g for 5 min. All biochemical analyses (including fasting plasma glucose, hemoglobin, lipids, hepatic function parameter) in nonanticoagulation tubes were performed on 7600-210 automatic biochemistry analyzer (HITACHI, Inc, Japan), using Synchron reagents provided by leadmanbio, Beijing. Blood samples collected in the Heparin Sodium tubes for the following assesments: plasma levels of leptin (eBioscience; BMS2039INST, USA), total ghrelin (Millipore, EZGRA-88K, USA), TNF-α (Peprotech; 900-M25, USA), interleukin-6 (IL-6) (Peprotech; 900-M16, USA) and IFN-γ (Peprotech; 900-M27, USA) were determined using enzyme-linked immuno sorbent assay. Subjects were weighed barefoot with minimum clothing using an electronic weighing scale, body weight was recorded to the nearest 0.1 kg. Height was measured to the nearest 0.1 cm using stadiometer, the BMI was calculated as Weight (kg)/ height 2 (m).
# Statistical analysis
The sample size was estimated with a two-sided alpha of 5% and a power of 80%. Data were tested for normal distribution using the Kolmogorov-Smirnov test. If the distribution appeared nonnormal, the continuous variables were transformed by calculating the log 10 prior to using parametric test statistics. Results are expressed as mean±SD for normally distributed data and geometric means for log 10 transformed data. Statistical testing for transformed variables were carried out by ANOVA if the data were normally distributed, and by non-parametric tests if the data were non-normally distributed. Correlations between transformed variables were estimated using pearson correlation coefficient. Multivariate linear regression analysis was carried out to evaluate the relationship between the parameters. SPSS 11.5 software was used for all statistical analyses and a p-value <0.05 was deemed statistically significant.
# Results
The anthropometric and biochemical characteristics of the 3 groups are shown in . TB (23.0±4.3 kg/m 2 ) and particularly TB+T2DM (22.2±3.5 kg/m 2 ) groups had lower levels of BMI than the control group (24.8±3.6 kg/m 2 ). TB+T2DM showed a higher level of serum glucose than TB and control groups (10.6±4.4 versus 5.1±0.5 mM, 10.6±4.4 versus 5.2±0.5 mM, respectively, p<0.05). The control group had higher level of HDL-cholesterol, proteins and uric acid than patients groups (p<0.05).
The levels of peptide hormones and inflammatory cytokines are shown in [fig_ref] Table 2: Circulating levels of appetite-related hormones and inflammatory cytokines in patients and healthy... [/fig_ref] [fig_ref] Table 3: Correlation of characteristics with BMI among different groups [/fig_ref] shows the correlation of appetite-related hormones and inflammatory cytokines with BMI among the 3 groups. Leptin showed a negative correlation with BMI in TB (r=-0.622, p<0.05) and TB+T2DM (r= -0.654, p<0.05) groups, but positive correlation with BMI in control group (r=0.521, p<0.05). Ghrelin showed positive correlation with BMI in the TB (r=0.695, p<0.05) and TB+T2DM (r= 0.199, p>0.05) groups, but negative correlation with BMI in control group (r=-0.693, p<0.05). There were positive nonsignificant correlations for all 3 cytokines in the TB group, negative nonsignificant correlations for TNF-α and IFN-γ and a positive nonsignificant correlation for IL-6 in the TB+T2DM group. In the control group, all correlations were negative and nonsignificant, except for IFN-γ (r=-0.545, p<0.05).
Multivariate linear regression of BMI as dependent variable with age, sex, appetite-related hormones and inflammatory cytokines in the three groups are showen in [fig_ref] Table 4: Multivariate linear regression of BMI with age, sex, appetite-related hormones and inflammatory... [/fig_ref]. Leptin was significantly inversely associated with BMI in the TB (p=0.009) and TB+T2DM (p=0.012) groups, but positively in the control group (p=0.436). Ghrelin had a significant positive association with BMI in the TB (p=0.026) and TB+T2DM (p=0.049) groups, but an inverse association in the control group (p=0.233). There were no important associations between BMI and age, sex, inflammatory cytokines in 3 groups respectively. Leptin was the major factor affecting BMI in the TB group, while ghrelin was the major factor in the TB+T2DM group.
# Discussion
In our study, we found TB patients with or without T2DM had a worse nutritional status (lower BMI and plasma protein levels) than controls. Our results demonstrate that plasma leptin levels were significantly higher in the TB group compared to the TB +T2DM and control groups, whereas levels of plasma ghrelin were lower in patients than in the control group. The association with BMI was negative for leptin and positive for ghrelin in the patients whereas these associations were opposite in control group. Levels of TNF-α were higher and IFN-γ were lower in the patients compared with the controls. However, in mulivariate analysis, the association of leptin and ghrelin with BMI was not explained by the cytokines.
Our study examined not only TB and healthy subjects, but TB+T2DM patients as well, a group which has rarely received attention until now. This relates to the special situation in China where TB and DM constitute a double-burden. Patients in our study were not yet treated by anti-tuberculosis drugs. Limitations were the relatively small sample size, and the small number females within group, so the sex effect on related hormones and inflammatory cytokines could not be investigated. We have measured total ghrelin levels in the plasma, which contains both "active" and "inactive" forms. However, it has been reported that total ghrelin levels can reflect the level of the active form in the plasma [bib_ref] The relationship between active ghrelin levels and human obesity involves alterations in..., Marzullo [/bib_ref] [bib_ref] Assessment of ghrelin, Espelund [/bib_ref]. In this study, we chose BMI as a correlate of body fat. The hospital in our area could not afford equipment to measure the percent body fat. Otherwise, body fat estimated from skinfold thickness is dependent on accurate measurements at the right sites (biceps, triceps, subscapular, and suprailiac regions), this requires skillful staff to carry out the work. However, patients in our study were mainly from counties and villages in poor rural areas, and although skinfold thickness was measured, it was difficult to train local staffs to perform standardized measurements for percent body fat measurement. According to many reports, BMI could effectively predict the percentage of body fat. Suchanek P et al [bib_ref] Which index best correlates with body fat mass: BAI, BMI, waist or..., Suchanek [/bib_ref] aimed to compare estimates of body fat content, like BMI, body adiposity index (BAI), waist-hip ratio (WHR), with respect to their ability to predict the percentage of body fat. They found BMI index was the better universally valid index to predict the percentage of body fat than BAI index and WHR index. Also others reported a good correlation of BMI and percentage of body fat especially in Asian. The correlation coefficient (R) could go up to 0.90 [bib_ref] Healthy percentage body fat ranges: an approach for developing guidelines based on..., Gallagher [/bib_ref]. Researchers in China also made many investigations to evaluate the possibilities to use BMI as predictor of body fat status and diseases caused by obesity [bib_ref] Verification of BMI classification reference for overweight and obesity in Chinese children..., Ma [/bib_ref] [bib_ref] Relationship of body mass index, waist circumference and cardiovascular risk factors in..., Du [/bib_ref] [bib_ref] Comparison of various anthropometric and body fat indices in identifying cardiometabolic disturbances..., Zhang [/bib_ref]. With respect to difference according to race, Asian people have less muscle and skeleton tissue than westerns with the same BMI, and the percentage of body fat is higher. So given the quality of the skinfold thickness measurements in our study and based on the reports of correlation between BMI and body fat in Asians, we prefer to use BMI as a correlate of body fat applicable to individuals in China.
TNF-α induces fever and weight loss, which are prominent symptoms of TB. It has been shown to be associated with both protection and pathogenesis in mycobacterial infections [bib_ref] Effects of tumornecrosis factor alpha on host immune response in chronic persistent..., Mohan [/bib_ref]. Raised concentrations of TNF-α may underly much of the metabolic clustering due to diabetes mellitus [bib_ref] Evaluation of TNF-α and IL-6 Levels in Obese and Non-obese Diabetics: Pre-and..., Goyal [/bib_ref]. In our study, the level of TNF-α was higher in TB patients, especially those with T2DM. This finding may suggest that mycobacterium tuberculosis (MTB), mainly multiplying in macrophages ， stimulates TNF-α release, especially in the presence of high glucose concentrations. IFN-γ as one of the typical Th1 cytokines is a key cytokine in the host immune response to tuberculosis infection. We found that TB patients with or without T2DM had lower levels of IFN-γ than controls like others reported [bib_ref] Gamma interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances..., Douvas [/bib_ref] , which may indicate that they have a weaker defense against tuberculosis infection.
Leptin is mainly synthesized by adipose tissue. But under inflammatory conditions, one study suggested a ''cytokineleptin hypothesis'' implying that higher multiple cytokine levels are correlated with increased leptin levels [bib_ref] Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in..., Sarraf [/bib_ref]. In the TB patient group, we found higher levels of TNF-α and leptin compared to the control group. However, the TB+T2DM group had higher levels of TNF-α in combination with lower leptin levels compared to the control group in our study. Experimental studies showed that the leptin levels in patients with type 2 diabetes are diverse and seem to be related to the duration of the disease. In subjects with poorly controlled type 2 diabetes who lost weight rapidly, with lower levels of BMI and leptin, levels of TNF-α were reported to be higher than in controls [bib_ref] Leptin deficiency causes insulin resistance induced by uncontrolled diabetes, German [/bib_ref] [bib_ref] Central insulin and leptin-mediated autonomic control of glucose homeostasis, Marino [/bib_ref]. So possibly in patients with type 2 diabetes, leptin and TNF-α status can not be explained by the ''cytokine-leptin hypothesis''. Therefore in TB patients with T2DM, the pathogenesis of wasting may be different. To our knowledge there have been no reports in the literature on this. Maybe there is a more complicated mechanism at work when TB and T2DM are combined. Leptin may be an important factor involved in the mechanism behind wasting, because both in TB patients with and without T2DM, leptin showed a negative correlation with BMI. Two articles [bib_ref] The role of feed regulating peptides on weight loss in patients with..., Yurt [/bib_ref] [bib_ref] Gut hormones, appetite suppression and cachexia in patients with pulmonary TB, Chang [/bib_ref] found that ghrelin in TB patients is elevated compared to controls, and correlates negatively with BMI [bib_ref] Gut hormones, appetite suppression and cachexia in patients with pulmonary TB, Chang [/bib_ref]. But Kim et al [bib_ref] Relation of ghrelin, leptin and inflammatory markers to nutritional status in active..., Kim [/bib_ref] found that plasma ghrelin levels were significantly lower in malnourished patients than in well nourished patients with TB before treatment. In correlation analysis, ghrelin levels were negatively correlated to the malnutrition score. Our results were similar showing a positive correlation of ghrelin with BMI in TB and TB+T2DM groups as Kim et al [bib_ref] Relation of ghrelin, leptin and inflammatory markers to nutritional status in active..., Kim [/bib_ref]. Interestingly, the correlation of leptin and ghrelin with BMI in the patient groups is contrary to what is observed in the healthy control group where it is correlated with the amount of fat tissue.as reported in the literature [bib_ref] The relation between serum leptin levels and body fat mass in patients..., Yüksel [/bib_ref] [bib_ref] Relation of leptin and tumor necrosis factor alpha to body weight changes..., Cakir [/bib_ref]. In TB patients high leptin levels may be a cause of the low BMI instead of being a consequence of the amount of fat tissue. Inflammatory cytokines were poorly correlated with BMI in our patients, although there was a negative association of leptin and TNF-α with BMI. Cakir B et al [bib_ref] Relation of leptin and tumor necrosis factor alpha to body weight changes..., Cakir [/bib_ref] have found increased and correlated levels of TNF-α and leptin in tuberculosis patients, and his interpretation was that the elevated leptin level leads to weight loss, and through that may contribute to the inflammatory process. However, Kim JH et al [bib_ref] Relation of ghrelin, leptin and inflammatory markers to nutritional status in active..., Kim [/bib_ref] found higher levels of TNF-α in the TB group, that were poorly correlated with weight loss, whereas leptin levels were positively correlated with TNF-α, although not statistically significant. Combined with our findings, higher levels of leptin and TNF-α may indicate that although TNF-α may correlate with leptin, it is not directly responsible for the lower BMI. Although some reports have shown that plasma TNF-α levels are associated with weight loss in TB, we were unable to demonstrate this. Although high inflammatory status maybe primarily responsible for low BMI in patients with TB, low BMI in TB cannot be explained by the investigated inflammatory cytokines only [bib_ref] Tuberculosis, malnutrition and wasting, Schwenk [/bib_ref] [bib_ref] Interleukin-6 and human immunodeficiency virus load, but not plasma leptin concentration, predict..., Van Lettow [/bib_ref]. This may suggest that externalization of leptin and ghrelin in these patients is not determined by nutritional status (amount of fat tissue), but in fact is a causal factor in the development of low BMI. The effect of leptin and ghrelin on BMI seems to be largely independent of the investigated cytokines. Further studies are needed to investigate the complex mechanisms.
# Conclusions
This study provides novel evidence of status and relationship between appetite-related hormones, inflammatory cytokines and BMI in TB patients with or without T2DM. We conclude that possible abnormalities in leptin and ghrelin regulation may be associated with the development of poor nutrition (low BMI) during the inflammatory response in TB patients with or without T2DM. TB patients with T2DM may have more complex and different pathogenesis compared to TB patients only. Given the complexity of the interaction of appetite-related hormones and cytokines, further studies are needed to clarify this issue and underlying mechanisms.
[table] Table 2: Circulating levels of appetite-related hormones and inflammatory cytokines in patients and healthy subjects (N= 71). [/table]
[table] Table 3: Correlation of characteristics with BMI among different groups. [/table]
[table] Table 4: Multivariate linear regression of BMI with age, sex, appetite-related hormones and inflammatory cytokines in different groups. Dependent Variable: BMI. Abbreviations: IL-6, interleukin-6; TNF-α, denotes tissue necrosis factor-α; IFN-γ,interferon-γ. doi: 10.1371/journal.pone.0080122.t004 [/table]
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On the Versatile Role of Electrospun Polymer Nanofibers as Photocatalytic Hybrid Materials Applied to Contaminated Water Remediation: A Brief Review
Citation: Cordoba, A.; Saldias, C.; Urzúa, M.; Montalti, M.; Guernelli, M.; Focarete, M.L.; Leiva, A. On the
# Introduction
Nowadays, one of the most critical problems facing humanity is the increasing water contamination, mainly attributed to the negative impact of anthropogenic activities on varied aquatic environments. It is well-documented that skillful and environmentally friendly polluted water and wastewater management has a pivotal role in effectively mitigating adverse environmental impacts and the availability of, e.g., freshwater sources, especially in less developed countries. In recent years, an increasing number of pollutants, such as dyes, pharmaceuticals, pesticides, and other organic molecules, have been detected in water sources. The wastewater treatments available to date, such as coagulation, flocculation, or adsorption, merely concentrate or transfer the contaminants from one phase to another without their complete elimination [bib_ref] Recent developments in photocatalytic water treatment technology: A review, Chong [/bib_ref].
In this context, advanced oxidation processes emerge as an innovative green chemical strategy to degrade the main organic pollutants present in wastewater. Interestingly, this type of process could involve photocatalytic reactions that generate highly reactive oxygen species (e.g., OH, O 2 , O 3 , and H 2 O 2 ) [bib_ref] Evaluation of advanced oxidation processes for water and wastewater treatment-A critical review, Miklos [/bib_ref]. The photogenerated reactive oxygen species can completely degrade most organic pollutants, bacteria, and viruses. The used photocatalysts are commonly based on nanosized semiconductor compounds (e.g., ZnO, CuO, TiO 2 , Fe 2 O 3 , CdS, PdS, AgBr, and ZnS) [bib_ref] Remediation of wastewater using various nanomaterials, Anjum [/bib_ref]. Using these nanomaterials makes it feasible to simultaneously take advantage of the semiconductor photo-properties and their high active surface area. These attributes strongly contribute to improve the efficiency of light-driven processes [bib_ref] Polymer-supported titanium dioxide photocatalysts for environmental remediation: A review, Singh [/bib_ref]. However, a decrease in the photocatalytic activity of nanoparticles (NPs) has been observed after a certain number of cycles of use, an effect which is essentially ascribed to their tendency to agglomerate, reducing the effective surface area spontaneously. Moreover, the efficient recovery of NPs from the reaction medium for subsequent reuse has also been a challenging task to date [bib_ref] Polymer-supported titanium dioxide photocatalysts for environmental remediation: A review, Singh [/bib_ref].
In order to solve these drawbacks, different types of structural supports for nanoparticles have been proposed. Glass mats, inorganic-carbon fibers, polymers, among others, are some materials typically used [bib_ref] Evaluation of advanced oxidation processes for water and wastewater treatment-A critical review, Miklos [/bib_ref]. Electrospun polymer nanofibers (NFs) have emerged as a promising alternative to support and stabilize NPs in many chemical environments, preserving their highly valuable properties over time and after repeated use cycles [bib_ref] nanofibers: A highly stable catalyst for Heck reaction, Du [/bib_ref]. Materials composed by NFs display unique functional properties, such as nanosized diameter, large specific surface area, high aspect ratio, high degree of porosity, and pore interconnectivity. These materials can be obtained as macroscopic mat-like structures, characterized by flexibility and elasticity, which can be advantageously used as a platform for numerous emerging environmental applications, e.g., the filtration of liquids, separation of fine particles for water treatment and technology environmental remediation processes, as well as, as photocatalyst support [bib_ref] Nanofiber technology: Current status and emerging developments, Lim [/bib_ref]. The high surface-to-volume ratio and continuous matrix structure, characterized by small pores, confer to these materials several advantages in the separation process of contaminants, especially when the surface/interface interactions play an essential role [bib_ref] Nanofiber technology: Current status and emerging developments, Lim [/bib_ref]. Additionally, another significant advantage is the enormous surface modification possibilities that polymer nanofibers offer [bib_ref] Fabrication and Plasma Modification of Nanofibrous Tissue Engineering Scaffolds, Asadian [/bib_ref].
Considering all these aspects, electrospun nanofiber mats are excellent candidates to act as a support matrix for various metal or semiconducting NPs, giving stable hybrid materials. Specifically, efficient hybrid systems consisting of photocatalytic nanoparticles supported on polymeric nanofibers can be obtained simply, incorporating semiconducting NPs onto the surface of preformed polymeric NFs or introducing the NPs in the polymer solution before the electrospinning process. Although several works have been conducted on the application of hybrid electrospun nanofiber systems to degrade toxic organic compounds in wastewater via photocatalysis, to the best of our knowledge, there is no comprehensive review that summarizes the relevant literature and provides critical insights, especially from a perspective of the role developed by the polymeric nanofibers. Consequently, with the rapid increase in this research topic, in this brief review, advances in the use of electrospun NFs based materials with photocatalytic activity in light-driven wastewater remediation processes are presented. The role played by various electrospun polymer nanofibers in the preparation of these systems and their application in the photodegradation of different types of organic pollutants is described. Finally, concluding remarks and future perspectives of this research area of technological interest are also outlined.
## Electrospun polymer nanofibers
Electrospinning is a versatile technology to produce polymer nanofibers, from solutions or melts, using electrostatic forces. Typically, an electrospinning setup is composed of a syringe, a collector, and a high voltage power supply (See [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref]. The syringe acts as a polymer solution reservoir and has a metal capillary at the tip connected to the positive terminal of a high voltage source. The negative terminal is connected to a metal collector that, in most cases, is simply grounded. The flow of the electrospinning process is controlled by a pump that generates adequate drops at the needle tip. When the charges accumulate on the drop's surface (due to the high voltage), mutual charge repulsion and the contraction of the surface charges to the counter electrode cause a force directly opposite to the surface tension [bib_ref] Polymer nanofibers assembled by electrospinning, Frenot [/bib_ref]. When the intensity of the electric field increases, the surface The tunable process parameters governing electrospinning are the applied voltage, the distance between the tip and the collector, the feed rates of solutions, the rotational speed of the collector, and the type of collector. At the same time, some intrinsic parameters of the polymeric solutions that affect the characteristics of the final material are surface tension, viscosity, concentration, conductivity, dielectric constants of the solvent and polymer, polymer molecular weight and polydispersity, polymer chemical structure, and presence of interactions, and electrostatic forces. Moreover, some environmental parameters, such as humidity and temperature, affect the electrospinning process [bib_ref] Solution Selection Model for Coaxial Electrospinning and Its Application to Nanostructured Hydrogen..., Kurban [/bib_ref] [bib_ref] Controlling the Fiber Diameter during Electrospinning, Fridrikh [/bib_ref] [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref] [bib_ref] Advances in drug delivery via electrospun and electrosprayed nanomaterials, Ramakrishna [/bib_ref] [bib_ref] Electrospun polymer biomaterials, Ding [/bib_ref] [bib_ref] Polymer Nanofibre Adsorbent Applications for Metal Ion Removal, Pereao [/bib_ref]. Several reviews have addressed the effect of these parameters on the properties of the obtained electrospun NFs [bib_ref] Controlling the Fiber Diameter during Electrospinning, Fridrikh [/bib_ref] [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref] [bib_ref] Advances in drug delivery via electrospun and electrosprayed nanomaterials, Ramakrishna [/bib_ref]. It is worth noting that the parameters governing conventional electrospinning to obtain single nanofibers are shared with coaxial electrospinning, a process aimed at obtaining core-shell type nanofibers with various morphologies from two or more different polymer solutions.
However, it should be noted that, among others, the two main parameters that strongly affect the electrospinning process are polymer concentration and electrical potential [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref]. In particular, the concentration of the solution strongly affects the size of the fibers, e.g., the diameter of the electrospun NFs increases with the concentration of the solution according to a ratio of the power-law [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref]. At the same time, electrospinning voltage strongly correlates with the formation of defects in the fibers [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref].
In general, many highly soluble polymers, which give rise to sufficiently concentrated solutions, can be processed through the electrospinning process to obtain fibers. It is estimated that more than 200 different polymers and copolymers, both synthetics, and naturals, have been electrospun to obtain NFs, including the following examples, poly The tunable process parameters governing electrospinning are the applied voltage, the distance between the tip and the collector, the feed rates of solutions, the rotational speed of the collector, and the type of collector. At the same time, some intrinsic parameters of the polymeric solutions that affect the characteristics of the final material are surface tension, viscosity, concentration, conductivity, dielectric constants of the solvent and polymer, polymer molecular weight and polydispersity, polymer chemical structure, and presence of interactions, and electrostatic forces. Moreover, some environmental parameters, such as humidity and temperature, affect the electrospinning process [bib_ref] Solution Selection Model for Coaxial Electrospinning and Its Application to Nanostructured Hydrogen..., Kurban [/bib_ref] [bib_ref] Controlling the Fiber Diameter during Electrospinning, Fridrikh [/bib_ref] [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref] [bib_ref] Advances in drug delivery via electrospun and electrosprayed nanomaterials, Ramakrishna [/bib_ref] [bib_ref] Electrospun polymer biomaterials, Ding [/bib_ref] [bib_ref] Polymer Nanofibre Adsorbent Applications for Metal Ion Removal, Pereao [/bib_ref]. Several reviews have addressed the effect of these parameters on the properties of the obtained electrospun NFs [bib_ref] Controlling the Fiber Diameter during Electrospinning, Fridrikh [/bib_ref] [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref] [bib_ref] Advances in drug delivery via electrospun and electrosprayed nanomaterials, Ramakrishna [/bib_ref]. It is worth noting that the parameters governing conventional electrospinning to obtain single nanofibers are shared with coaxial electrospinning, a process aimed at obtaining core-shell type nanofibers with various morphologies from two or more different polymer solutions.
However, it should be noted that, among others, the two main parameters that strongly affect the electrospinning process are polymer concentration and electrical potential [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref]. In particular, the concentration of the solution strongly affects the size of the fibers, e.g., the diameter of the electrospun NFs increases with the concentration of the solution according to a ratio of the power-law [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref]. At the same time, electrospinning voltage strongly correlates with the formation of defects in the fibers [bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref].
In general, many highly soluble polymers, which give rise to sufficiently concentrated solutions, can be processed through the electrospinning process to obtain fibers. It is estimated that more than 200 different polymers and copolymers, both synthetics, and naturals, have been electrospun to obtain NFs, including the following examples, poly (lactic acid), poly (lactic-co-glycolic acid), poly (caprolactone), poly (ethylene oxide), poly (vinyl alcohol), and cellulose [bib_ref] Plasma Modification of Poly Lactic Acid Solutions to Generate High Quality Electrospun..., Rezaei [/bib_ref] [bib_ref] Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering, Kumbar [/bib_ref] [bib_ref] Drug delivery system based on cyclodextrin-naproxen inclusion complex incorporated in electrospun polycaprolactone..., Canbolat [/bib_ref] [bib_ref] Preparation of antibacterial PVA and PEO nanofibers containing Lawsonia Inermis (henna) leaf..., Avci [/bib_ref] [bib_ref] Obtaining cellulose nanofibers with a uniform width of 15 nm from wood, Abe [/bib_ref]. In , some examples of synthetic and natural polymers that have been electrospun, including the solvent used, are summarized [bib_ref] Electrospinning and Electrospun Nanofibers: Methods, Materials, and Applications, Xue [/bib_ref].
Interestingly, some inorganic solids, such as metals, metal oxides/carbides/nitrides, as well as carbon and doped carbon, which have been processed as fibers using polymers as carriers, are also included in .
## Hybrid photocatalysts based on polymer nanofibers in the degradation of water pollutants
The organic contaminants in water originally come from domestic, municipal, and industrial wastewater, and therefore there are various organic and inorganic pollutants. Organic contaminants, such as dyes, antibiotics, analgesics, herbicides, pesticides, and stimulants, have become the main sources of water contamination. Therefore, research for developing new approaches for the effective removal of these pollutants, such as photocatalytic processes, has received increasing attention. Generally, the photocatalytic capacity of a material in the degradation of organic pollutants in water is determined first using model molecules of organic pollutants. Organic dyes are the most studied molecules to test the degradation activity of many materials. Dyes are commonly used in industrial and laboratory activities, although it is recognized that they have a substantial negative impact on the environment.
Interestingly, dye degradation (usually denoted by changes of color or discoloration phenomena) can be observed even by the naked eye and easily monitored by UV-Vis absorption measurements. Some of the most widely used organic dyes are azo dyes, such as Reactive Black 5 (RB5), Reactive Red 120 (RR120), Reactive Orange 16 (RO16), among others [bib_ref] TiO2-Doped Electrospun Nanofibrous Membrane for Photocatalytic Water Treatment, Blanco [/bib_ref] [bib_ref] Novel CdPdS/PVAc core-shell nanofibers as an effective photocatalyst for organic pollutants degradation, Unnithan [/bib_ref]. Additionally, it is nowadays known that pharmaceutical molecules are not only beneficial drugs for the cure of diseases, but also are possible water contaminants. Recently, about 3000 different biologically active compounds have been reported in the water cycle, including surface water, wastewater, groundwater, and, on a smaller scale, drinking water [bib_ref] Photocatalytic degradation of Ibuprofen, Naproxen, and Cetirizine using PAN-MWCNT nanofibers crosslinked TiO..., Uheida [/bib_ref].
As mentioned, polymer nanofibers have proven to be a good alternative to support photocatalyst particles due to their advantageous characteristics, such as high porosity, high specific surface area, flexibility, and the chemical functionalization possibilities that these materials present. Thus, hybrid polymeric NFs, in the form of permeable free-standing or supported layer on porous substrates, have been demonstrated to act not only as good support and stabilizer for photocatalytic NPs, but also as an active phase aiding in the degradation of pollutants, e.g., mitigating the inhibition process of oxidative capacity that the co-occurring organics commonly produce by the scavenging of ROS and electron holes during photocatalysis [bib_ref] Porous Electrospun Fibers Embedding TiO 2 for Adsorption and Photocatalytic Degradation of..., Lee [/bib_ref]. Indeed, NFs can improve the effectiveness of the photocatalytic water remediation process by attracting the contaminants towards the NPs or favoring the predominant transport phenomena (e.g., diffusion) of water in the photoactive material.
The following sections summarize the research found in the literature on the application of photocatalytic hybrid nanofibers in water and wastewater remediation to date. The research cases reviewed are presented grouped into four categories according to the polymeric material used to obtain the nanofibers, i.e., Section 3.1 synthetic polymers, Section 3.2 natural polymers, Section 3.3 copolymers and polymeric blends, and Section 3.4 polymers blended with carbon nanostructures.
## Electrospun nanofibers of synthetic polymers decorated with photocatalytic nanoparticles
Most of the electrospun nanofibers that have been reported as supports of photocatalysts for the degradation of organic water pollutants are formed by synthetic polymers. To obtain the hybrid material, photocatalytic nanoparticles can be either mixed with the polymer before electrospinning or deposited on the polymer nanofibers after electrospinning. In this section, the use of different synthetic polymers to obtain hybrid polymer nanofibers/photocatalytic nanoparticles is featured, especially highlighting the contribution made by the polymer in the photocatalytic performance of the material.
A representative work was developed by Blanco and coworkers, who prepared electrospun polyamide 6 (PA6, a thermoplastic polymer with good mechanical properties) nanofibrous membranes containing TiO 2 nanoparticles [bib_ref] TiO2-Doped Electrospun Nanofibrous Membrane for Photocatalytic Water Treatment, Blanco [/bib_ref]. The authors incorporated the nanoparticles into the polymer solution before the electrospinning process. Thus, to prepare the hybrids NFs, 25 wt.% TiO 2 NPs were added to a PA6 solution (12 w/w in a mixture 2:1 of acetic acid: formic acid) using a conventional ultrasonic bath and sodium dodecyl sulfate to stabilize the NPs dispersion. The electrospun nanofibrous membranes were obtained at 23 ± 2 - C and 50 ± 5% of relative humidity; the collector distance and voltage were set at 170 mm and 75 kV, respectively. The authors estimated that most of the NPs were inside the PA6 NFs, which is based on the significant increase in the diameters of the NFs ranging from 60-100 nm for bare PA6 NFs to 110-260 nm for PA6/25 wt.% TiO 2 NFs. The FTIR characterization corroborated the presence of the TiO 2 NPs, showing an increase in the band centered at 550 cm −1 , which was attributed to Ti-O bending. However, the FTIR spectrum of NFs modified with TiO 2 NPs did not show significant changes that could reflect interactions between PA6 and TiO 2 compared to the spectrum of non-modified nanofibers. Therefore, if there are interactions, these would not be strong enough to be detected by FTIR spectroscopy. The absence of strong interactions between TiO 2 NPs and PA6 was also demonstrated by the TiO 2 NPs agglomeration in the PA6/25% TiO 2 NFs observed by SEM images in [fig_ref] Figure 2: SEM images of [/fig_ref]. In the case of photodegradation study, 80% degradation of Remazol Black B, a synthetic diazo reactive dye used as a model of organic water pollutants, was observed in a solution with the hybrid nanofibrous membranes immersed after 240 min of irradiation with 365 nm UV light. This photocatalytic process was monitored following the decrease in the dye absorbance using UV-Vis spectroscopy. Notably, the photocatalytic hybrid nanofibers maintain their effectiveness when reused for three cycles. Additionally, the authors also studied the antibacterial activity of PA6/TiO 2 electrospun NFs against E. coli, which was eliminated after 24 h of contact with the material upon UV light irradiation. Well-documented homopolymer Nylon 6,6, a polyamide commonly obtained the by polycondensation of hexamethylenediamine and adipic acid, has also been used to prepare polymer-inorganic core-shell NFs, with a polymeric core and an inorganic shell of zinc oxide, combining electrospinning and atomic layer deposition (ALD) techniques [bib_ref] Polymer-Inorganic Core-Shell Nanofibers by Electrospinning and Atomic Layer Deposition: Flexible Nylon-ZnO Core-Shell..., Kayaci [/bib_ref]. Nanofibers of Nylon 6,6 were obtained by electrospinning using 5 wt.% and 8 wt.% polymer concentrations and two different solvents, formic acid (FA) and hexafluoro-2-propanol (HFIP). The electrospinning was carried out at 23 °C and 36% relative humidity, and syringes with metallic needles of 0.8 mm of inner diameter were used, while a feed rate of 1 mL/h, 15kV of applied voltage, and a tip-to-collector distance of 10 cm were set up. Later, ZnO with precise thickness control was deposited onto NFs using the ALD technique. The average diameter of the Nylon 6,6 NFs showed dependence on both the concentration of the electrospun solution and the solvent used. However, the TEM images revealed that the thickness of the ZnO shell layer was about 90 nm for each Nylon 6,6-ZnO nanofiber sample, independently of the average fiber diameters of the bare polymer previous to performing the ALD technique [fig_ref] Figure 3: Representative TEM images of [/fig_ref]. Remarkably, the fibrous structure of Nylon 6,6 was not disturbed during the ALD of ZnO shell layer, a process developed at 200 °C. The core−shell nylon 6,6-ZnO nanofibers showed unique properties, such as structural flexibility, provided by the polymeric core and good photocatalytic activity due to the ZnO shell layer. The photocatalytic activity of the nanofibers obtained was tested by monitoring the photocatalytic decomposition of Rhodamine-B, an organic dye molecule used as a model of organic waste compound. The Nylon 6,6-ZnO nanofiber mat of thinner average fiber diameter (~80 nm) showed better photocatalytic efficiency (93% RhB degradation after 16 h of 365 nm UV irradiation) when compared to the nanofiber mat having AFD of ~650 nm, likely due to the higher surface area of this sample. The structural and chemical stabilities of the Nylon 6,6-ZnO nanofiber mats were tested by TEM imaging and XPS measurement after the UV irradiation experiment. TEM images showed that the samples maintained their nanofibrous structure without deformation, only a few spots due to the destruction of ZnO layer were detected, and the XPS study of the samples corroborated these observations. Well-documented homopolymer Nylon 6,6, a polyamide commonly obtained the by polycondensation of hexamethylenediamine and adipic acid, has also been used to prepare polymer-inorganic core-shell NFs, with a polymeric core and an inorganic shell of zinc oxide, combining electrospinning and atomic layer deposition (ALD) techniques [bib_ref] Polymer-Inorganic Core-Shell Nanofibers by Electrospinning and Atomic Layer Deposition: Flexible Nylon-ZnO Core-Shell..., Kayaci [/bib_ref]. Nanofibers of Nylon 6,6 were obtained by electrospinning using 5 wt.% and 8 wt.% polymer concentrations and two different solvents, formic acid (FA) and hexafluoro-2-propanol (HFIP). The electrospinning was carried out at 23 - C and 36% relative humidity, and syringes with metallic needles of 0.8 mm of inner diameter were used, while a feed rate of 1 mL/h, 15 kV of applied voltage, and a tip-to-collector distance of 10 cm were set up. Later, ZnO with precise thickness control was deposited onto NFs using the ALD technique. The average diameter of the Nylon 6,6 NFs showed dependence on both the concentration of the electrospun solution and the solvent used. However, the TEM images revealed that the thickness of the ZnO shell layer was about 90 nm for each Nylon 6,6-ZnO nanofiber sample, independently of the average fiber diameters of the bare polymer previous to performing the ALD technique [fig_ref] Figure 3: Representative TEM images of [/fig_ref]. Remarkably, the fibrous structure of Nylon 6,6 was not disturbed during the ALD of ZnO shell layer, a process developed at 200 - C. The core−shell nylon 6,6-ZnO nanofibers showed unique properties, such as structural flexibility, provided by the polymeric core and good photocatalytic activity due to the ZnO shell layer. The photocatalytic activity of the nanofibers obtained was tested by monitoring the photocatalytic decomposition of Rhodamine-B, an organic dye molecule used as a model of organic waste compound. The Nylon 6,6-ZnO nanofiber mat of thinner average fiber diameter (~80 nm) showed better photocatalytic efficiency (93% RhB degradation after 16 h of 365 nm UV irradiation) when compared to the nanofiber mat having AFD of 650 nm, likely due to the higher surface area of this sample. The structural and chemical stabilities of the Nylon 6,6-ZnO nanofiber mats were tested by TEM imaging and XPS measurement after the UV irradiation experiment. TEM images showed that the samples maintained their nanofibrous structure without deformation, only a few spots due to the destruction of ZnO layer were detected, and the XPS study of the samples corroborated these observations. Unnithan and coworkers incorporated photocatalytic nanoparticles ex situ to the electrospinning process to produce hybrid NFs. They report the preparation of cadmium palladium sulfide (CdS/PdS) alloy NPs embedded in the NFs of thermoplastic poly(vinyl acetate) (PVAc), known for its adhesion properties [bib_ref] Novel CdPdS/PVAc core-shell nanofibers as an effective photocatalyst for organic pollutants degradation, Unnithan [/bib_ref]. In this case, the synthesis of the semiconducting photocatalytic nanoparticles was developed in situ in the polymer solution. The in situ synthesis of CdS/PdS alloy NPs was carried out by mixing ammonium sulfide with the respective salt precursors of Cd and Pd in a PVAc DMF solution [bib_ref] Novel CdPdS/PVAc core-shell nanofibers as an effective photocatalyst for organic pollutants degradation, Unnithan [/bib_ref]. The electrospinning of the colloidal solution thus obtained produced nanofibers of core-shell structure. NFs with uniform diameters in the range of 200-250 nm were obtained. The core-shell configuration of NFs was evidenced by TEM and HRTEM imaging [fig_ref] Figure 4: Illustration showing the mechanism of forming the core-shell structure [/fig_ref]. To explain the results, the authors suggested that the water molecules from the reagents promoted the assembly of the NPs to form initially nanoparticle clusters and, subsequently, the NPs clusters would coalesce and form the core section of the NFs, facilitated by the hydrophobic nature of the polymer. The core-shell NFs were tested against two different azo dyes, RB5 and RO16, showing almost 100% of dye degradation after 180 and 120 min under sunlight irradiation, respectively. Notably, the efficiency was not affected after three consecutive cycles. The PVAc shell did not influence the photocatalytic activity of the PdS/CdS NPs. The authors attribute this to the suitable electric conductivity of the PVAc. This polymer would form a conductive network on the surface of NPs, which can promote the photogeneration of electron (e−) and holes (h+) and subsequently prevent their recombination. Unnithan and coworkers incorporated photocatalytic nanoparticles ex situ to the electrospinning process to produce hybrid NFs. They report the preparation of cadmium palladium sulfide (CdS/PdS) alloy NPs embedded in the NFs of thermoplastic poly(vinyl acetate) (PVAc), known for its adhesion properties [bib_ref] Novel CdPdS/PVAc core-shell nanofibers as an effective photocatalyst for organic pollutants degradation, Unnithan [/bib_ref]. In this case, the synthesis of the semiconducting photocatalytic nanoparticles was developed in situ in the polymer solution. The in situ synthesis of CdS/PdS alloy NPs was carried out by mixing ammonium sulfide with the respective salt precursors of Cd and Pd in a PVAc DMF solution [bib_ref] Novel CdPdS/PVAc core-shell nanofibers as an effective photocatalyst for organic pollutants degradation, Unnithan [/bib_ref]. The electrospinning of the colloidal solution thus obtained produced nanofibers of core-shell structure. NFs with uniform diameters in the range of 200-250 nm were obtained. The core-shell configuration of NFs was evidenced by TEM and HRTEM imaging [fig_ref] Figure 4: Illustration showing the mechanism of forming the core-shell structure [/fig_ref]. To explain the results, the authors suggested that the water molecules from the reagents promoted the assembly of the NPs to form initially nanoparticle clusters and, subsequently, the NPs clusters would coalesce and form the core section of the NFs, facilitated by the hydrophobic nature of the polymer. The core-shell NFs were tested against two different azo dyes, RB5 and RO16, showing almost 100% of dye degradation after 180 and 120 min under sunlight irradiation, respectively. Notably, the efficiency was not affected after three consecutive cycles. The PVAc shell did not influence the photocatalytic activity of the PdS/CdS NPs. The authors attribute this to the suitable electric conductivity of the PVAc. This polymer would form a conductive network on the surface of NPs, which can promote the photogeneration of electron (e−) and holes (h+) and subsequently prevent their recombination. Panthi and coworkers reported a similar photocatalytic system; they prepared hybrid electrospun NFs of PVAc containing PdS/ZnS NPs [bib_ref] Fabrication of PdS/ZnS NPs doped PVAc hybrid electrospun nanofibers: Effective and reusable..., Panthi [/bib_ref]. The NPs were synthesized in situ by the following summarized procedure. ZnAc and PdAc with 5:1 weight ratio were dissolved in DMF. The prepared solution was mixed with 18% (w/w) PVAc solution in DMF. The resulting solution was kept under stirring for 2 h to ensure proper mixing, and then ammonium sulfide was added dropwise with vigorous stirring for 5 h to promote the fine dispersion of PdS-doped ZnS NPs. Finally, the polymer and PdS/ZnS NPs solution was electrospun. Hybrid NFs with smooth surfaces and uniform diameters in the range of 200-300 nm were obtained. The absence of PdS and ZnS NPs on the surface of the hybrid nanofibers was confirmed by FESEM images, which indicated that the PdS and ZnS NPs were incorporated into the polymer nanofibers. In [fig_ref] Figure 5: TEM images of [/fig_ref] , TEM and HRTEM images of pristine PVAc and hybrid ZnS-PVAc and PdSZnS-PVAc electrospun nanofibers are shown. The images of hybrid electrospun NFs show well-doped nanoparticles inside the fiber matrix. In both cases, the size of the nematic phase constituted of NPs was in the range of 5-8 nm. The authors propose that the position of metal ions (M 2+ ) on electrospun nanofibers should be adjusted by controlling the interaction between the polymer and metal ions. During the electrospinning process, Coulomb repulsions between the charged metal nanoparticles (M 2+ ) could be the main factor responsible for the NPs homogeneously dispersed, favoring polymer/metal ion interactions. Remarkably, the photocatalytic activity of PdSZnS-PVAc hybrid nanofibers was higher than that of ZnS-PVAc hybrid nanofibers. The complete degradation of the MB dye was achieved with PdS/ZnS-PVAc in 100 min, while only 75% of the dye was degraded simultaneously with ZnS-PVAc. Panthi and coworkers reported a similar photocatalytic system; they prepared hybrid electrospun NFs of PVAc containing PdS/ZnS NPs [bib_ref] Fabrication of PdS/ZnS NPs doped PVAc hybrid electrospun nanofibers: Effective and reusable..., Panthi [/bib_ref]. The NPs were synthesized in situ by the following summarized procedure. ZnAc and PdAc with 5:1 weight ratio were dissolved in DMF. The prepared solution was mixed with 18% (w/w) PVAc solution in DMF. The resulting solution was kept under stirring for 2 h to ensure proper mixing, and then ammonium sulfide was added dropwise with vigorous stirring for 5 h to promote the fine dispersion of PdS-doped ZnS NPs. Finally, the polymer and PdS/ZnS NPs solution was electrospun. Hybrid NFs with smooth surfaces and uniform diameters in the range of 200-300 nm were obtained. The absence of PdS and ZnS NPs on the surface of the hybrid nanofibers was confirmed by FESEM images, which indicated that the PdS and ZnS NPs were incorporated into the polymer nanofibers. In [fig_ref] Figure 5: TEM images of [/fig_ref] , TEM and HRTEM images of pristine PVAc and hybrid ZnS-PVAc and PdSZnS-PVAc electrospun nanofibers are shown. The images of hybrid electrospun NFs show well-doped nanoparticles inside the fiber matrix. In both cases, the size of the nematic phase constituted of NPs was in the range of 5-8 nm. The authors propose that the position of metal ions (M 2+ ) on electrospun nanofibers should be adjusted by controlling the interaction between the polymer and metal ions. During the electrospinning process, Coulomb repulsions between the charged metal nanoparticles (M 2+ ) could be the main factor responsible for the NPs homogeneously dispersed, favoring polymer/metal ion interactions. Remarkably, the photocatalytic activity of PdSZnS-PVAc hybrid nanofibers was higher than that of ZnS-PVAc hybrid nanofibers. The complete degradation of the MB dye was achieved with PdS/ZnS-PVAc in 100 min, while only 75% of the dye was degraded simultaneously with ZnS-PVAc.
Rosman and coworkers used the highly chemically inert thermoplastic fluoropolymer polyvinylidene fluoride PVDF to obtain a series of electrospun NFs with embedded NPS of the heterojunction photocatalysts of ZnO with different Ag-based compounds. To prepare the NFs, first, a polymer solution of PVDF in DMAc/acetone blend, was doped with different photocatalysts (ZnO, ZnO/Ag 2 CO 3 , ZnO/Ag 2 CO 3 /Ag 2 O, and ZnO/Ag 2 O) by stirring, and then the resulting doped solution was used to fabricate electrospun NFs via electrospinning. The set parameters were: flux 1 mL/h, a voltage of 8 kV, and a distance of 150 mm between the spinneret and the collector. The NFs showed a rough surface, and the ZnO/Ag 2 CO 3 /Ag 2 O nanoparticles were homogeneously distributed in PVDF NFs [fig_ref] Figure 6: TEM images of PVDF/ZnO/Ag 2 CO 3 /Ag 2 O electrospun nanofiber [/fig_ref]. The FTIR spectra of the nanofibers with the embedded heterojunction photocatalysts ZnO/Ag 2 CO 3 , ZnO/Ag 2 O, or ZnO/Ag 2 CO 3 /Ag 2 O demonstrated the structural integrity of the photocatalysts. Significantly, the main signals of ZnO, Ag 2 CO 3 , and Ag 2 O were not overlapped after being incorporated in the PVDF nanofiber. The NFs with the ZnO/Ag 2 CO 3 /Ag 2 O photocatalyst showed faster degradation of Reactive Red 120 (RR120) dye as a model pollutant under 312 nm UV light irradiation. In fact, 99.62% dye degradation when 300 min of light exposure was reached, maintaining a relatively high photocatalytic activity after five cycles. This good activity was attributed to a synergistic effect of adsorption and enhanced photocatalytic degradation. According to contact angle and AFM measurements, the system catalyst embedded into the PVDF NFs would exhibit lower hydrophobization and higher surface roughness, which would facilitate the dye molecule adsorption adjacent to their photocatalytic surface sites. On the other hand, the heterojunction between the ZnO and Ag 2 CO 3 /Ag 2 O mixed phase would narrow the bandgap. The authors highlight the affinity of PVDF and RR120, indicating that bare PVDF nanofibers showed adsorption of approximately 10% of dye after 300 min of UV light irradiation. Rosman and coworkers used the highly chemically inert thermoplastic fluoropolymer polyvinylidene fluoride PVDF to obtain a series of electrospun NFs with embedded NPS of the heterojunction photocatalysts of ZnO with different Ag-based compounds. To prepare the NFs, first, a polymer solution of PVDF in DMAc/acetone blend, was doped with different photocatalysts (ZnO, ZnO/Ag2CO3, ZnO/Ag2CO3/Ag2O, and ZnO/Ag2O) by stirring, and then the resulting doped solution was used to fabricate electrospun NFs via electrospinning. The set parameters were: flux 1 mL/h, a voltage of 8 kV, and a distance of 150 mm between the spinneret and the collector. The NFs showed a rough surface, and the ZnO/Ag2CO3/Ag2O nanoparticles were homogeneously distributed in PVDF NFs [fig_ref] Figure 6: TEM images of PVDF/ZnO/Ag 2 CO 3 /Ag 2 O electrospun nanofiber [/fig_ref]. The FTIR spectra of the nanofibers with the embedded heterojunction photocatalysts ZnO/Ag2CO3, ZnO/Ag2O, or ZnO/Ag2CO3/Ag2O demonstrated the structural integrity of the photocatalysts. Significantly, the main signals of ZnO, Ag2CO3, and Ag2O were not overlapped after being incorporated in the PVDF nanofiber. The NFs with the ZnO/Ag2CO3/Ag2O photocatalyst showed faster degradation of Reactive Red 120 (RR120) dye as a model pollutant under 312 nm UV light irradiation. In fact, 99.62% dye degradation when 300 min of light exposure was reached, maintaining a relatively high photocatalytic activity after five cycles. This good activity was attributed to a synergistic effect of adsorption and enhanced photocatalytic degradation. According to contact angle and AFM measurements, the system catalyst embedded into the PVDF NFs would exhibit lower hydrophobization and higher surface roughness, which would facilitate the dye molecule adsorption adjacent to their photocatalytic surface sites. On the other hand, the heterojunction between the ZnO and Ag2CO3/Ag2O mixed phase would narrow the bandgap. The authors highlight the affinity of PVDF and RR120, indicating that bare Ramasundaram and coworkers reported the fabrication of hybrid NFs PVDF-photocatalytic nanoparticles, using different photocatalytic NPs [bib_ref] Photocatalytic applications of paper-like poly(vinylidene fluoride)-titanium dioxide hybrids fabricated using a combination..., Ramasundaram [/bib_ref]. In their work, TiO2 NPs were incorporated by electrospray on electrospun NFs of PVDF, i.e., the NP incorporation was made after the electrospinning process. First, the PVDF NFs mat was prepared by the electrospinning process using a PVDF solution in a DMF/acetone mixture. The electrospinning setup used comprised a needle with an inner diameter of 330 μm, a spinning speed of 50 μL/min, 10 cm distance between the tip and the collector, and 15 kV of applied Ramasundaram and coworkers reported the fabrication of hybrid NFs PVDFphotocatalytic nanoparticles, using different photocatalytic NPs [bib_ref] Photocatalytic applications of paper-like poly(vinylidene fluoride)-titanium dioxide hybrids fabricated using a combination..., Ramasundaram [/bib_ref]. In their work, TiO 2 NPs were incorporated by electrospray on electrospun NFs of PVDF, i.e., the NP incorpora-tion was made after the electrospinning process. First, the PVDF NFs mat was prepared by the electrospinning process using a PVDF solution in a DMF/acetone mixture. The electrospinning setup used comprised a needle with an inner diameter of 330 µm, a spinning speed of 50 µL/min, 10 cm distance between the tip and the collector, and 15 kV of applied voltage. Then, a TiO 2 dispersion in DMF was electrosprayed on both sides of the PVDF NFs mats using the same process parameters used for PVDF electrospinning. According to the authors, DMF is one of the best solvents for PVDF based on solubility parameter theory. Thus, at room temperatures, DMF could penetrate the amorphous regions of PVDF, while the crystalline regions remain mostly unaffected, producing minimal swelling. This effect of the solvent on the polymer would allow to immobilize electrosprayed nanomaterials efficiently on the surface of the PVDF matrix. The resultant PVDF-TiO 2 hybrid NFs presented TiO 2 NPs mainly immobilized on the outer surface of the mat. Notably, the hybrid PVDF-TiO 2 NFs completely degraded cimetidine, bisphenol A (BPA), and 4-chlorophenol upon 40, 80, and 100 min of UV irradiation, respectively. The photocatalytic activity of the PVDF-TiO 2 was observed during 10 cycles of catalysis; prior to each cycle, the photocatalyst was rinsed with deionized water and immersed in a freshly prepared target solution before new UV irradiation. Interestingly, the authors report no noticeable reduction in photocatalytic activity after the 10 cycles, indicating the high reusability of the hybrid system. Additionally, no trace of Ti into the treated aqueous solutions was detected, confirming the robustness of the PVDF-TiO 2 hybrids NFs, a product of the above-mentioned DMF effect on the polymer and the interactions between PVDF and TiO 2 nanoparticles.
Another synthetic polymer that has been frequently used in the production of synthetic nanofibers to immobilize photocatalytic nanostructures is polyacrylonitrile (PAN), a vinyl polymer derived from the acrylate family. For example, Guo and coworkers reported the use of PAN to immobilize nanostructures of 2,9,16,23-tetranitro phthalocyanine copper (II) (CuTNPc) and bismuth oxychloride nanosheets (BiOCl) [bib_ref] Bismuth oxychloride (BiOCl)/copper phthalocyanine (CuTNPc) heterostructures immobilized on electrospun polyacrylonitrile nanofibers with..., Guo [/bib_ref]. PAN NFs were obtained by simple electrospinning of a solution in DMF, using 12 kV of operating voltage and 15 cm of distance between the syringe tip and the collector. Scanning electron microscopy (SEM) images of the obtained PAN NFs showed ultra-long one-dimensional nanostructures, with a high aspect ratio and a relatively smooth surface, their diameter distribution ranging from 200 to 300 nm. Two consecutive solvothermal treatments modified PAN NFs. Firstly, the NFs were treated at 160 - C for 12 h with a 4-nitrophthalonitrile, Cu(Ac) 2 H 2 O, and ammonium molybdate in ethylene glycol solution to form CuTNPc/PAN NFs. Secondly, CuTNPc/PAN NFs were reacted with Bi(NO 3 ) 3 5H 2 O and NaCl dissolved in ethylene glycol at the same temperature for 24 h to obtain BiOCl/CuTNPc/PAN NFs. Note that both processes were carried out in a 25 mL Teflon-lined stainless-steel autoclave. After the first solvothermal treatment, the PAN NFs surface was covered with a secondary nanostructure of CuTNPc, maintaining the non-woven nanofiber structure. In this case, a noticeable color change from white to black was observed. After secondary solvothermal reaction, the BiOCl nanosheets were uniformly grown on the surface of CuTNPc/PAN nanofibers without aggregation. Thus, the three-dimensional macroporous structure was well maintained. The BiOCl/PAN nanofibers were also prepared similarly. [fig_ref] Figure 7: SEM images and EDX spectra of samples [/fig_ref] shows typical SEM images of PAN, CuTNPc/PAN, BiOCl/CuTNPc/PAN, and BiOCl/PAN nanofibers mats, as well as their respective energy-dispersive X-ray (EDX) spectra.
The analysis of the thermogravimetric profiles of pristine PAN NFs and modified NFs (CuTNPc/PAN, BiOCl/CuTNPc/PAN, and BiOCl/PAN) revealed a decrease in the thermal stability of the modified NFs compared to those of bare PAN. This allowed inferring the existence of interactions between PAN and metal atoms, explaining the uniform distribution of heterostructures in NFs and the absence of aggregation observed. Concerning their photocatalytic activities, BiOCl/CuTNPc/PAN NFs showed 75% RhB degradation after 180 min under UV irradiation, while for CuTNPc/PAN nanofibers and BiOCl/PAN nanofibers, the values were only 24% and 20%, respectively. Interestingly, the authors reported an adsorption of approximately 5% of RhB by PAN NFs, allowing to infer that an additional effect produced by PAN could exist, e.g., favoring the diffusion of the dye toward the photoactive sites on the NFs. Note that the flexible nanofibrous mat easily floated on the surface of the liquid, optimizing the absorption of sunlight during the photocatalysis step.
NPc/PAN NFs. Note that both processes were carried out in a 25 mL Teflon-lined stainless-steel autoclave. After the first solvothermal treatment, the PAN NFs surface was covered with a secondary nanostructure of CuTNPc, maintaining the non-woven nanofiber structure. In this case, a noticeable color change from white to black was observed. After secondary solvothermal reaction, the BiOCl nanosheets were uniformly grown on the surface of CuTNPc/PAN nanofibers without aggregation. Thus, the three-dimensional macroporous structure was well maintained. The BiOCl/PAN nanofibers were also prepared similarly. [fig_ref] Figure 7: SEM images and EDX spectra of samples [/fig_ref] shows typical SEM images of PAN, CuTNPc/PAN, BiOCl/CuT-NPc/PAN, and BiOCl/PAN nanofibers mats, as well as their respective energy-dispersive X-ray (EDX) spectra. On the other hand, Qayum and coworkers proposed an attractive strategy to promote the photocatalytic degradation of the widely used salicylic acid (SA), using PAN nanofibers as support for photocatalytic nanoparticles [bib_ref] Efficient decontamination of multi-component wastewater by hydrophilic electrospun P.A.N./AgBr/Ag fibrous membrane, Qayum [/bib_ref]. In this study, a combination of electrospinning, wet-chemical methods, and thermal treatment to obtain hydrophilic PAN/AgBr/Ag fibrous membranes was employed. The fabrication procedure involved four steps: (i) firstly, a PAN-AgNO 3 NFs mat was prepared by electrospinning a sol of AgNO 3 and PAN into DMF. The set electrospinning parameters were a needle with a diameter of 0.7 mm, distance of 20.0 cm between the needle and collector, 18 kV of voltage, and feeding rate of 0.1 mL/min; (ii) secondly, thermal treatment of the mat at 140 - C, to promote the transformation from AgNO 3 to Ag nanoparticles, was performed; (iii) the treated membrane was immersed in FeBr 3 solution for 40 min for the dissolution of AgNO 3 and formation of AgBr nanoparticles; (iv), and finally, UV irradiation was applied to generate the AgBr/Ag Schottky junction. The procedure is summarized in the scheme of. Notably, according to the described procedure, the precursor PAN/AgNO 3 nanofibers were subjected to three considerable modifications to generate the final PAN-AgBr/Ag nanofibrous material.
secondly, thermal treatment of the mat at 140 °C, to promote the transformation from AgNO3 to Ag nanoparticles, was performed; (iii) the treated membrane was immersed in FeBr3 solution for 40 min for the dissolution of AgNO3 and formation of AgBr nanoparticles; (iv), and finally, UV irradiation was applied to generate the AgBr/Ag Schottky junction. The procedure is summarized in the scheme of. Notably, according to the described procedure, the precursor PAN/AgNO3 nanofibers were subjected to three considerable modifications to generate the final PAN-AgBr/Ag nanofibrous material. SEM images and energy disperse X-ray spectrometry (EDX) measurements of PAN/AgBr/Ag fibrous membranes are depicted in. The images show that C and SEM images and energy disperse X-ray spectrometry (EDX) measurements of PAN/ AgBr/Ag fibrous membranes are depicted in. The images show that C and N were uniformly distributed on the surface of the fibers, while Ag and Br were preferentially concentrated on the surface of NPs, demonstrating the deposition of the AgBr and Ag NPs in the outer layer of the fiber. PAN/AgBr/Ag fibrous membranes showed good stability of the porous structures along with adequate mechanical strength, even allowing their use in filtration processes. This material could degrade 96% of SA upon 5 h of UV-visible irradiation, even after five cycles of use. Interestingly, the antibacterial activity of the nanocomposite fibers was also demonstrated; the performance of the material was tested using a wastewater sample containing SA, E. coli, and dispersed red 1 (an azo dye).
## Electrospun nanofibers of natural, biobased and/or biodegradable polymers decorated with photocatalytic nanoparticles
The use of natural and biodegradable polymers for various purposes, including the production of hybrid nanofibers containing photocatalytic NPs, has inherent environmental advantages over synthetic polymers, the latter being obtained from fossil-based raw materials and contributing to the increasingly dramatic and unpopular plastic pollution. Additionally, natural, biobased and/or biodegradable polymers might have chemical functionalities that favor the generation of favorable interactions with photocatalytic nanoparticles and organic pollutants. Thus, the polymer-NPs interactions contribute to the stability of the hybrid NFs, while the polymer-organic pollutant interactions facilitate the contact of the organic molecules with the active sites of photocatalysis, favoring their photodegradation. However, the studies reported in the literature using these types of polymers are still scarcely addressed. This section summarizes some reports on the use of natural, biobased and/or biodegradable polymers, such as chitosan, cellulose, polycaprolactone, and polylactide, to prepare hybrid photocatalytic nanofibers.
Park and coworkers successfully prepared electrospun nanofibrous mats of biodegradable, biocompatible, and hydrophobic polyester polycaprolactone (PCL) containing TiO 2 NPs. In their study, they prepared PCL NFs with TiO 2 NPs at various concentrations (1, 3, 5, and 7 wt.%). The electrospinning solutions were prepared from a pre-dispersion of the TiO 2 powders in a methylene chloride/DMF polymer solution that was stirred for 5 h and then blended with a 12.5 wt.% PCL solution for 6 h. The set electrospinning parameters were: tip-to-collector distance 20 cm, capillary tip with 0.51 mm of inner diameter, and a voltage of 18-21 kV. Subsequently, the NFs were treated by atmospheric pressure plasma with oxygen gas to decompose the polymer at the surface and expose TiO 2 at the exterior of nanofibers. The treatment with high-energy plasma etched the polymer, and as a consequence, the exposing of TiO 2 clusters at the surface of PCL NFs increased the number of active sites of TiO 2 . The results showed that a concentration of TiO 2 up to 3 wt.% had a reinforcing effect on the polymeric membranes. Conversely, above 5 wt.% TiO 2 , the tensile strength decreased probably because of the severe agglomeration of the TiO 2 nanopowder that was observed in SEM images. The material showed 70-90% RB5 degradation (3 mg/mL solution) after 120 min of 254 nm of UV irradiation, the degradation increasing with the increase in the amount of TiO 2 NPs. The material with 3 wt.% TiO 2 NPs showed the best compromise between mechanical strength and improved photocatalytic capacity. The photocatalytic activity of these systems was explained by two concomitant effects resulting from the plasma treatment: the exposure of the NPs at the fiber surface due to polymer etching and a high increase in fiber hydrophilicity evidenced by contact angle measurements. The authors suggested that, even if the TiO 2 NPs played a role in increasing in part the hydrophilicity of the nanofibers, the most important factor would be the generation of polar OH groups at the polymer surface by atmospheric pressure plasma treatment, corroborated by FTIR. This would allow the water to spread completely over the surface and increase the effective number of photocatalytic active sites on the large surface. Thus, the authors demonstrated that a plasma treatment can efficiently modify PCL/TiO 2 hybrid nanofibers from a hydrophobic to a hydrophilic state.
Another biodegradable and biobased thermoplastic polyester, Poly-L-Lactide (PLLA), was used by Sugunan and coworkers [bib_ref] Radially Oriented ZnO Nanowires on Flexible Poly-l-Lactide Nanofibers for Continuous-Flow Photocatalytic Water..., Sugunan [/bib_ref] to prepare a highly flexible hybrid material consisting of PLLA NFs with radially oriented ZnO nanoneedles. To obtain the NFs, the electrospinning of a 7 wt.% PLLA/chloroform solution was carried out. In the process, a 0.8 mm diameter needle, a voltage of 10 kV, 10 cm distance from the tip of the needle to the collector and a flow rate of 1 mL/h were used. Later, PLLA NFs were immersed for 30 min into a colloidal ZnO suspension and then allowed to dry. Then, the "seeded" NFs were immersed into a mixed aqueous solution of Zn(NO 3 ) 2 and hexamine and heated to 75 - C for 6 h. The NFs obtained in this way showed a hierarchical three-dimensional nanostructure consisting of polymeric NFs (~200 nm before deposition) that were densely covered by radially oriented ZnO nanoneedles (~50 nm), as [fig_ref] Figure 9: Hierarchical fibrous polymer/ZnO nanostructures [/fig_ref] shows. The authors determined that a typical 10 × 10 cm mat had a surface area equal to a square of Si/glass substrate of 75 cm side length through BET surface area measurements.
Interestingly, the hierarchical PLLA-ZnO nanostructure retains the high flexibility of the original PLLA nanofibrous mat. This last allowed the authors to design a water treatment by setting up a "continuous flow" through fixed-bed photocatalytic material placed inside a glass tube and using a 100 W mercury vapor lamp as a UV light source. A 90% of methylene blue decomposition was obtained after 80 min of circulation of a 5 ppm solution in the system, while at least a 50% of degradation of two hazardous compounds, MCP an organophosphate plaguicide and diphenylamine were achieved after 100 min of irradiation. Additionally, the material exhibited the high structural and chemical stability of the hierarchical structures in the continuous flow regime. min into a colloidal ZnO suspension and then allowed to dry. Then, the "seeded" NFs were immersed into a mixed aqueous solution of Zn(NO3)2 and hexamine and heated to 75 °C for 6 h. The NFs obtained in this way showed a hierarchical three-dimensional nanostructure consisting of polymeric NFs (~200 nm before deposition) that were densely covered by radially oriented ZnO nanoneedles (~50 nm), as [fig_ref] Figure 9: Hierarchical fibrous polymer/ZnO nanostructures [/fig_ref] shows. The authors determined that a typical 10 × 10 cm mat had a surface area equal to a square of Si/glass substrate of 75 cm side length through BET surface area measurements. Interestingly, the hierarchical PLLA-ZnO nanostructure retains the high flexibility of the original PLLA nanofibrous mat. This last allowed the authors to design a water treatment by setting up a "continuous flow" through fixed-bed photocatalytic material placed inside a glass tube and using a 100 W mercury vapor lamp as a UV light source. A 90% of methylene blue decomposition was obtained after 80 min of circulation of a 5 ppm solution in the system, while at least a 50% of degradation of two hazardous compounds, MCP an organophosphate plaguicide and diphenylamine were achieved after 100 min of irradiation. Additionally, the material exhibited the high structural and chemical stability of the hierarchical structures in the continuous flow regime.
Chitosan, a non-toxic, biodegradable, and environmentally friendly biopolymer, is considered a promising material for several applications, e.g., as a carrier due to its hydroxyl and amino groups, which allow it to interact with various molecules and nanoparticles. Accordingly, chitosan nanofibers are highly interesting for the generation of hybrid materials; however, the electrospinning of chitosan presents considerable difficulty due to its polyelectrolyte nature and problems generated by their charges in the electrospinning process, Chitosan, a non-toxic, biodegradable, and environmentally friendly biopolymer, is considered a promising material for several applications, e.g., as a carrier due to its hydroxyl and amino groups, which allow it to interact with various molecules and nanoparticles. Accordingly, chitosan nanofibers are highly interesting for the generation of hybrid materials; however, the electrospinning of chitosan presents considerable difficulty due to its polyelectrolyte nature and problems generated by their charges in the electrospinning process, such as instabilities in the electrospinning jet, high electrical conductivity, and viscosity of polyelectrolyte solutions (at the concentrations suitable for electrospinning) [bib_ref] Chitosan-functionalized nanofibers: A comprehensive review on challenges and prospects for food applications, De Farias [/bib_ref].
Consequently, the electrospinning of chitosan remains a challenge not completely solved at present. Fakhri and coworkers prepared chitosan and polycaprolactone biodegradable polymer nanofibers decorated with Tungsten disulfide (WS 2 ) and studied these hybrid nanocomposites in the photocatalytic degradation of Neomycin (NEO), an aminoglycoside antibiotic [bib_ref] Preparation and characterization of WS 2 decorated and immobilized on chitosan and..., Fakhri [/bib_ref]. The chitosan NFs were obtained by electrospinning of low molecular weight chitosan in trifluoroacetic acid solution; the nanofiber neutralization was performed in an aqueous solution of K 2 CO 3 and a final drying at 60 - C. The electrospinning setting was a flow rate of 1 mL/h, voltage of 13-15 kV, needle diameter of approximately 0.1 mm, 25 - C, and 50-60% relative humidity. A similar procedure was used to obtain PCL (80,000 g/mol) NFs. The WS 2 nanoparticles immobilized on CS and PCL nanofibers were synthesized in situ by the following procedure. First, the CS and PCL nanofibers were immersed in a suspension of 0.005 M (NH 4 ) 6 W 7 O 24 ·4H 2 O, 0.02 g PVP, and C 2 H 5 NS (0.04 M). Secondly, an aqueous solution of acetic acid (40 mL, 5 % vol) was added under vigorous stirring. Finally, the mixture was placed in a Teflon autoclave and sealed at 140-190 - C for 8 h. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] shows SEM images and the energy dispersive spectroscopy (EDS) analysis of WS 2 NPs and SEM images of chitosan and PCL NFs, WS 2 /chitosan and WS 2 /PCL nanocomposites. The degradation efficiencies of NEO by the photocatalytic activity of WS 2 /CS nanofibers and WS 2 /PCL nanofibers were determined following the change in characteristic absorbance of NEO solutions by UV-Vis spectroscopy. The optimal time of UV-light irradiation and pH values for high efficiency of NEO decomposition (between 80-90%) were pH 7, and 40 min. Interestingly the WS 2 /chitosan and WS 2 /PCL NFs showed higher NEO photodegradation than the WS 2 NPs, indicating an enhancement by including polymers. WS2/chitosan and WS2/PCL nanocomposites. The degradation efficiencies of NEO by the photocatalytic activity of WS2/CS nanofibers and WS2/PCL nanofibers were determined following the change in characteristic absorbance of NEO solutions by UV-Vis spectroscopy. The optimal time of UV-light irradiation and pH values for high efficiency of NEO decomposition (between 80-90%) were pH 7, and 40 min. Interestingly the WS2/chitosan and WS2/PCL NFs showed higher NEO photodegradation than the WS2 NPs, indicating an enhancement by including polymers. The preparation of a hybrid photocatalyst based on cellulose acetate nanofibers, the most important synthetic cellulose ester, using TiO 2 -doped chitosan microspheres and the study of its performance in the photocatalytic degradation of methyl orange (MO) in water solution was reported by Xuejuan Shi and al. [bib_ref] TiO 2 -Doped Chitosan Microspheres Supported on Cellulose Acetate Fibers for Adsorption..., Shi [/bib_ref]. In a first step, the authors obtained cellulose acetate NFs by electrospinning of a 10 wt.% CA (w/w) in an acetone/deionized water (85/15, w/w) solution. The electrospinning conditions included an applied voltage of 20 kV, 15 cm of distance between the tip of the needle and the collector, and 1.0 mL/h flow rate. Later, chitosan microspheres doped with TiO 2 nanoparticles were embedded onto cellulose acetate nanofibers by electrospraying. A 2 wt.% chitosan (w/w) dissolved in 90% acetic acid (v/v) solution and containing different amounts of TiO 2 nanoparticles (from 1%, to 5%, w/w) was used to electrospray. The results showed that the diameter of TiO 2 -doped chitosan microspheres lightly increased with the increase in TiO 2 content from 1 wt.% to 3 wt.%, while that at higher content of TiO 2 (4 and 5%), the NPs experimented agglomeration. The BET method was employed to estimate the specific surface area of nanofibrous material. For bare chitosan and chitosan-TiO 2 spheres embedded on CA NFs, the values corresponded to 301.30 and 342.10 m 2 /g, respectively. These results reflected an increase due to TiO 2 NPs inclusion. The removal of MO in the presence of chitosan-TiO 2 spheres embedded in CA NFs catalyzed by visible light showed an increase up to 3 wt.% of TiO 2 NPs on chitosan spheres and decreased when the content of TiO 2 increased to 4 and 5 wt.%. The authors attributed this behavior to the reduction in photocatalytic reaction sites due to TiO 2 agglomeration. It is necessary to emphasize that the MO removal capacity was not only due to photocatalytic reactions, but also due to the adsorption of the dye mainly propitiated by chitosan, which was inferred from the FTIR and XPS results. Furthermore, recycling experiments showed that fiber membranes had excellent chemical stability and reusability.
Cellulose acetate (CA) has also been used to obtain electrospun fibers decorated with photocatalytic NPs. High surface area photocatalytic cellulosic fibers containing TiO 2 NPs were prepared by Bedford and coworkers [bib_ref] Photocatalytic cellulosic electrospun fibers for the degradation of potent cyanobacteria toxin microcystin-LR, Bedford [/bib_ref] by first electrospinning solutions of CA into non-woven fiber meshes. The electrospinning experiments were performed under ambient conditions with a voltage 25 kV, flow rate of 0.5-0.6 mL/h, and 11 cm distance between spinneret and collector. Interestingly, the obtained CA nanofibers were converted to succinylated cellulose fibers through a deacetylation using KOH in ethanol and the corresponding neutralization followed by a succinylation of the hydroxyl groups using a succinic anhydride in DMF solution. Finally, the nanofibers were loaded with dispersions of 1% titania nanoparticles, previously homogenized by ultrasound, at different pH for one hour at 75 - C, and then rinsed with distilled water under ultrasound to remove unbound titania. The electrospun fiber diameter and BET surface area were varied by controlling the concentration of acetic acid in the electrospinning solution. A higher fiber diameter at a higher concentration of acetic acid was obtained. Consequently, the BET surface area showed an inverse variation, i.e., a higher surface area at minor fiber diameter, therefore at a lower acetic acid concentration. The conversion of the CA fibers to cellulose fibers and then to succinylated cellulose fibers produced a slight change in average diameter and BET surface area of the fibers. Carboxylic acid functionality was generated because these groups are known to bind titania better than acetyl and hydroxyl groups found in the CA and cellulose fibers. When identical concentrations of P25 TiO 2 NPs were loaded onto cellulose fibers and succinylated cellulose fibers, a clear difference in the amount of titania loaded onto the carboxylic acid functionalized fibers respect FE-SEM images observed the hydroxyl containing cellulosic fibers. The TiO 2 doped nanofibers were tested to degrade toxin microcystin-LR (MC-LR), one of the most common toxins generated by cyanobacterial algal blooms that frequently occur in various water sources around the world. The photocatalytic degradation of MC-LR by photocatalytic cellulosic fibers loaded with titania under visible and solar light was reached. The results indicated that a large surface coverage of titania and an active surface area of fiber mat are highly desirable conditions for effective MC-LR degradation.
The use of natural cotton cellulose (CC) to prepare nanofibers decorated with CdS NPs and the study of their photocatalytic activity for the photodegradation of rhodamine B (RhB) was reported by Qiuyan Liu et al. [bib_ref] CdS nanoparticle-functionalized natural cotton cellulose electrospun nanofibers for visible light photocatalysis, Liu [/bib_ref]. The methodology to obtain the hybrid NFs combines the electrospinning technique and the chemical bath deposition. To prepare the cotton cellulose nanofibers, 1.15 wt.% cotton was added to 8.5 wt.% LiCl/DMAc solution under vigorously stirring at 80 - C for 4 h. The parameters used in the electrospinning process were: applied voltage of 18 kV, the syringe to collector distance 15 cm, humidity under 40%, flow rate of 0.8 mL/h, and room temperature. The CdS nanoparticles were formed in situ at the surface of cotton cellulose nanofibers. For this, CC nanofibers were immersed in 0.1 M Cd(NO 3 ) 2 ethanol solution for 3 min and were rinsed with deionized water twice. Then, the Cd 2+ ion-loaded nanofibers were immersed into 0.05 M Na 2 S solution for 3 min, followed by a second rinsing. The cycle was repeated by 5, 10, 20, and 30 times. In between cycles, the sheet was kept drying at 60 - C for 5 min. Correspondingly, the obtained CC/CdS nanocomposites were labeled as CC/CdS-5, CC/CdS-10, CC/CdS-20 and CC/CdS-30. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] shows the SEM images of bare CC NFs and CC/CdS NFs.
In general, more agglomerated and larger CdS nanoparticles were deposited on the uniform and smooth surfaces of CC nanofibers as the number of cycles of CdS NPs synthesis increased. The obtained CC/CdS nanocomposites showed excellent photocatalytic efficiency to RhB dye degradation. After 40 min of visible light irradiation, the CC/CdS-20, CC/CdS-10, CC/CdS-30, CC/CdS-5, and CdS nanoparticles were able to degrade ca. 99%, 93%, 83%, 78% and 51% of the RhB dye, respectively. Additionally, the NFs decorated with CdS NPs exhibited remarkable photostability. Importantly, a slight loss of photocatalytic activity after three cycles of use was observed.
formed in situ at the surface of cotton cellulose nanofibers. For this, CC nanofibers were immersed in 0.1 M Cd(NO3)2 ethanol solution for 3 min and were rinsed with deionized water twice. Then, the Cd 2+ ion-loaded nanofibers were immersed into 0.05 M Na2S solution for 3 min, followed by a second rinsing. The cycle was repeated by 5, 10, 20, and 30 times. In between cycles, the sheet was kept drying at 60 °C for 5 min. Correspondingly, the obtained CC/CdS nanocomposites were labeled as CC/CdS-5, CC/CdS-10, CC/CdS-20 and CC/CdS-30. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] shows the SEM images of bare CC NFs and CC/CdS NFs. In general, more agglomerated and larger CdS nanoparticles were deposited on the uniform and smooth surfaces of CC nanofibers as the number of cycles of CdS NPs synthesis increased. The obtained CC/CdS nanocomposites showed excellent photocatalytic efficiency to RhB dye degradation. After 40 min of visible light irradiation, the CC/CdS-20, CC/CdS-10, CC/CdS-30, CC/CdS-5, and CdS nanoparticles were able to degrade ca. 99%, 93%, 83%, 78% and 51% of the RhB dye, respectively. Additionally, the NFs decorated with CdS NPs exhibited remarkable photostability. Importantly, a slight loss of photocatalytic activity after three cycles of use was observed.
## Electrospun nanofibers of copolymers and polymer blends decorated with photocatalytic nanoparticles
Despite the use of copolymers and polymer blends to produce electrospun hybrid nanofibers could present several advantages, such as facilitating the electrospinning process and the combination of polymers or polymer building blocks with different structure and functional groups, which could confer different properties to the NFs; the studies reported to date are scarce.
The preparation of hybrid nanofibers of poly (vinylidene difluoride-co-trifluoroethylene) (P(VDF-TrFE)) copolymer, containing TiO2 nanoparticles (P(VDF-TrFE)/TiO2) and TiO2/Graphene oxide nanocomposite (P(VDF-TrFE)/TiO2/GO), was reported by Almeida
## Electrospun nanofibers of copolymers and polymer blends decorated with photocatalytic nanoparticles
Despite the use of copolymers and polymer blends to produce electrospun hybrid nanofibers could present several advantages, such as facilitating the electrospinning process and the combination of polymers or polymer building blocks with different structure and functional groups, which could confer different properties to the NFs; the studies reported to date are scarce.
The preparation of hybrid nanofibers of poly (vinylidene difluoride-co-trifluoroethylene) (P(VDF-TrFE)) copolymer, containing TiO 2 nanoparticles (P(VDF-TrFE)/TiO 2 ) and TiO 2 / Graphene oxide nanocomposite (P(VDF-TrFE)/TiO 2 /GO), was reported by Almeida and coworkers [bib_ref] TiO 2 /graphene oxide immobilized in P(VDF-TrFE) electrospun membranes with enhanced visible-light-induced..., Almeida [/bib_ref]. To obtain these photocatalytic systems, in both cases (TiO 2 and TiO 2 /GO NPs), different amounts of NPs were sonicated for 2 h with a P(VDF-TrFE) solution in N,Ndimethyl formamide/methyl ethyl ketone (85/15, v/v) to achieve a good dispersion. Later, the solution was kept under vigorously stirring for 2 h. Finally, the electrospinning process was carried out at 1.5 kV, flux of 0.5 mL/h, and the electrospun fibers were collected at 15 cm from the needle tip. The electrospun mats showed smooth fibers with the presence of beads. The authors attributed this to the low viscosity of the solution and a low concentration that does not allow the adequate entanglement of the polymer chains. Furthermore, the addition of TiO 2 /GO NPs would increase the solution electrical conductivity and, consequently, smaller fiber diameter due to the extra mechanical stretch promoted by the applied electrical field. This could explain that the number of beads present in the NFs mat increases with NPs addition, giving rise to a necklace-like fiber structure. SEM studies proved that bare fibers have an average fiber diameter of 518 ± 119 nm, while nanocomposite mats showed a decrease in the average fiber diameter varying from 318 ± 126 down to 226 ± 93 nm, for the samples from 3 to 8 wt.% of TiO 2 /GO, respectively.
The results obtained showed that GO incorporation substantially improves MB adsorption, reaction rate, and removal efficiency under UV and Visible radiation. Additionally, all the samples containing GO could remove 100% of MB in less than 90 min, under visible light irradiation. The authors attribute this improved photocatalytic activity to the high surface area and porosity of the electrospun samples and the advantageous electrical and structural properties of graphene oxide. It is likely that bead-shaped fiber mats could also enhance the photocatalytic activity for the degradation of organic pollutants under visible light irradiation due to the increase in light scattering and photoactive surface area.
Random block copolymers, such as poly(dimethylsiloxane-block-etherimide) (PSEI), have also been used. Lee and coworkers [bib_ref] Enhanced Photocatalytic Activity using Layer-by-Layer Electrospun Constructs for Water Remediation, Lee [/bib_ref] prepared nanofibers of this random block copolymer containing 35-40 wt.% dimethyl siloxane copolymerized with polyetherimide (PEI) units and modified their surface with TiO 2 NPs by the layer by layer (LBL) technique. The copolymer nanofibers were obtained by the electrospinning of a 22 wt.% PSEI solution in a mixture of DMF and pyridine, the electrospinning parameters, voltage, solution flow rate, and the collector distance were set up to 30 kV, 0.01 mL min −1 , and 35 cm, respectively. The authors indicate that the siloxane units provide NFs flexibility and resistance against ultraviolet-light-aging effect and variations in temperature. The average diameter of the PSEI fibers prior to coating, determined from SEM images [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] , was 650 ± 180 nm and the surface area determined by BET analysis was 12 m 2 g −1 . The LBL deposition of TiO 2 NPs on electrospun fibers was carried out after the plasma treatment of PSEI.
NFs. An electrostatically bonded coating on fibers was prepared by alternating dipping in (octa(3-ammoniumpropyl) octa-silsesquioxane octachloride) POSS-8NH 3 + 8Cl − and TiO 2 dispersion. Ten bilayers of negatively charged colloidal TiO 2 NPs and positively charged polyhedral oligomeric silsesquioxanes were LBL-assembled on the plasma-treated PSEI NFs mesh (TiO 2 LBL-NFs). The amount of TiO 2 on the PSEI fibers was determined using inductively coupled plasma-atomic emission spectroscopy; 16.9 µg mg −1 or 1.4 mg m −2 of the nanofibers were obtained. Transmission electron micrographs showed that POSS/TiO 2 layers were uniformly deposited on the PSEI fibers [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref]. Finally, hybrid NFs were tested in the photocatalytic degradation of Bisphenol A BPA, an additive used in the plastics industry, acting as an endocrine disruptor in living organisms. The decomposition of BPA with TiO 2 LBL-NFs reached 89.3%, resulting in an 18.4% higher degradation compared to colloidal TiO 2 NPs. The multilayer-assembled TiO 2 nanofibers TiO 2 LBL-NFs, in addition to exhibiting a higher degree of BPA decomposition, maintained first-order kinetics for over 12 h, while the photocatalytic activities of colloidal TiO 2 NPs were initially higher; however, this behavior exhibited an abrupt decrease due to nanoparticles agglomeration. The authors determined that BPA adsorption on TiO 2 LBL-NF was significantly higher than TiO 2 NPs. The above was attributed to the increase in the binding affinity due to the additional adsorption sites presented by cationic species of polyhedral oligomeric silsesquioxanes POSS. Poly (vinylpyrrolidone) (PVP), a synthetic polymer with a unique combination of relevant physical and chemical properties, soluble in water and many organic solvents, has also been used to obtain hybrid nanofibers with photocatalytic NPs. For example, the preparation of PVDF/PVP blend NFs containing TiO2 NPs has been reported [bib_ref] Porous Electrospun Fibers Embedding TiO 2 for Adsorption and Photocatalytic Degradation of..., Lee [/bib_ref]. To obtain the photocatalytic NFs, PVDF and PVP were dissolved at various ratios (PVDF(wt.%):PVP(wt.%) = 18:0, 12:6, 9:9, and 6:12) in DMAc/acetone (1:1 v:v along with Poly (vinylpyrrolidone) (PVP), a synthetic polymer with a unique combination of relevant physical and chemical properties, soluble in water and many organic solvents, has also been used to obtain hybrid nanofibers with photocatalytic NPs. For example, the preparation of PVDF/PVP blend NFs containing TiO 2 NPs has been reported [bib_ref] Porous Electrospun Fibers Embedding TiO 2 for Adsorption and Photocatalytic Degradation of..., Lee [/bib_ref]. To obtain the photocatalytic NFs, PVDF and PVP were dissolved at various ratios (PVDF(wt.%):PVP(wt.%) = 18:0, 12:6, 9:9, and 6:12) in DMAc/acetone (1:1 v:v along with TiO 2 (4 wt.%). The solutions were vigorously stirred at 60 - C for 1 h and cooled to room temperature before the electrospinning process. The hybrid nanomaterial PVP acts as a sacrificial polymer to increase the surface area and improve light access to TiO 2 . To wash out the PVP and generate electrospun porous fibers, the electrospun mat was immersed in water, sonicated, and placed at 60 - C for 24 h. The blend composition showed directly affects the surface morphology and hydrophobicity of the electrospun porous fiber mat. The SEM images of the NFs with TiO 2 adding and subsequent washing of the sacrificial PVP show NFs of rougher surface [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref]. The authors determined that PVP removal by washing to generate porous structure significantly increased the BET surface area, e.g., from 30.6 to 117.2 m 2 /g for PVDF(12%)/PVP(6%)-TiO 2 . Interestingly, TEM analysis showed that TiO 2 was present in the fiber's interior throughout the cross-section. For MB removal, the authors exploited the "bait-hook and destroy" strategy, wherein dye adsorption (bait-hook) and photocatalytic dye degradation (destroy) are carried out simultaneously. The strategy is based on using an appropriate support material that offers an active surface to adsorb and bring priority pollutants near the photocatalytic sites. Thus, short-lived ROS would be more efficiently exploited. As a result, the excellent adsorption of MB on the porous fiber mats was obtained, and then, the almost complete dye degradation was achieved using UV light irradiation for 120 min. Additionally, over 10 reusing cycles, there was no change in MB adsorption capacity and the photocatalytic degradation, reflecting high stability and reusability. The authors concluded that effective immobilization of TiO 2 on polymers with an affinity for specific priority pollutants helps to increase the efficiency and reduce the energy requirements of photocatalytic water treatment. For example, the TiO 2 -embedded PVDF fiber mats' hydrophobic surface would facilitate the adsorption and concentration of nonpolar organic contaminants near photocatalytic sites. Zheng and coworkers reported the same blend of PVDF/PVP to prepare a novel composite material with high photocatalytic activity starting with the nanofibers of the blend with the immobilized nanoparticles of the core-shell structure of the photocatalyst TiO2@g-C3N4 (TCN) [bib_ref] A novel PVDF-TiO2@g-C 3 N 4 composite electrospun fiber for efficient photocatalytic..., Zheng [/bib_ref]. For the preparation of the nanofibers, PVDF and PVP were dissolved in a DMAC/acetone solvent mixture (v:v = 1:1) in the ratio PVDF (wt.%): PVP (wt.%) = 8%:2%. The core-shell nanoparticles of TiO2 and the metal-free polymer n-type semiconductor graphite-like carbon nitride (g-C3N4) were added. The resulting solution was stirred at 60 °C for 2 h and subjected to ultrasound for 4 h to achieve good dispersion. The electrospinning conditions were: an applied voltage of 20 kV, a flow rate of 2 mL h −1 , 15 cm between the needle and the collector, and a 1.07 mm blunt needle (G17) was used. Next, the electrospun nanofibers were immersed in deionized water and subjected to son- Zheng and coworkers reported the same blend of PVDF/PVP to prepare a novel composite material with high photocatalytic activity starting with the nanofibers of the blend with the immobilized nanoparticles of the core-shell structure of the photocatalyst TiO 2 @g-C 3 N 4 (TCN) [bib_ref] A novel PVDF-TiO2@g-C 3 N 4 composite electrospun fiber for efficient photocatalytic..., Zheng [/bib_ref]. For the preparation of the nanofibers, PVDF and PVP were dissolved in a DMAC/acetone solvent mixture (v:v = 1:1) in the ratio PVDF (wt.%):PVP (wt.%) = 8%:2%. The core-shell nanoparticles of TiO 2 and the metal-free polymer ntype semiconductor graphite-like carbon nitride (g-C 3 N 4 ) were added. The resulting solution was stirred at 60 - C for 2 h and subjected to ultrasound for 4 h to achieve good dispersion. The electrospinning conditions were: an applied voltage of 20 kV, a flow rate of 2 mL h −1 , 15 cm between the needle and the collector, and a 1.07 mm blunt needle (G17) was used. Next, the electrospun nanofibers were immersed in deionized water and subjected to sonication to remove PVP. Finally, the NFs were dried under vacuum at 90 - C for 24 h. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] shows SEM images of PVDF and PVDF-TCN-2 g nanofibers. The PVDF nanofibers obtained showed uniform distribution and sizes, with diameters between 300-400 nm. Removing the sacrificial PVP produced a rougher surface with pore formation, increasing the specific area and facilitating interaction with the catalyst nanoparticles. For fibers prepared with 0.2 g of catalyst PVDF-TCN-2 g, SEM images show that the catalyst has a small effect on the nanofiber structure, with the nanofibers showing a slight increase in surface roughness. According to SEM elemental mapping scanning spectra for the fiber surface of carbon, fluorine, oxygen, and titanium, catalyst nanoparticles were located on the surface of nanofibers and in the pores. Atomic force microscopy measurements corroborated the higher roughness of the catalyst-containing nanofibers. Additionally, the authors established by water contact angle measurements on the nanofiber mats that the inclusion of the catalyst nanoparticles conferred high hydrophilicity to the surface. The degradation of tetracycline, an oral antibiotic, was evaluated using PVDF and PVDF-TCN electrospun nanofibers under visible light irradiation. PVDF nanofibers showed no photocatalytic activity to degrade tetracycline. At the same time, for PVDF-TCN fibers, the degradation capacity increased up to 0.2 g catalyst content, reaching a degradation efficiency of 81.9%, 92.3%, and 97.0% after 300 min of irradiation for PVDF-TCN 0.1 g, PVDF-TCN 0.15 g, PVDF-TCN 0.2 g, respectively. For a TCN content of 0.3 g, the catalytic activity decreased. The adsorption of tetracycline on these substrates was also evaluated under dark conditions for 30 min. The adsorption capacity of PVDF fibers, PVDF-TCN 0.1 g, PVDF-TCN 0.15 g, PVDF-TCN 0.2 g and pure TCN were 3.97%, 4.95%, 9.17%, 16.42% and 12.9%, respectively. The above would indicate that the adsorption of tetracycline by both the polymer and the catalyst contribute to its degradation, facilitating the encounter of the drug molecules with the catalytic sites.
## Electrospun nanofibers of polymers/carbon nanostructures blends containing photocatalytic nps
According to the reviewed literature, another strategy that has proven to be effective in preparing hybrid nanofibers with photocatalytic properties for the degradation of wa- The degradation of tetracycline, an oral antibiotic, was evaluated using PVDF and PVDF-TCN electrospun nanofibers under visible light irradiation. PVDF nanofibers showed no photocatalytic activity to degrade tetracycline. At the same time, for PVDF-TCN fibers, the degradation capacity increased up to 0.2 g catalyst content, reaching a degradation efficiency of 81.9%, 92.3%, and 97.0% after 300 min of irradiation for PVDF-TCN 0.1 g, PVDF-TCN 0.15 g, PVDF-TCN 0.2 g, respectively. For a TCN content of 0.3 g, the catalytic activity decreased. The adsorption of tetracycline on these substrates was also evaluated under dark conditions for 30 min. The adsorption capacity of PVDF fibers, PVDF-TCN 0.1 g, PVDF-TCN 0.15 g, PVDF-TCN 0.2 g and pure TCN were 3.97%, 4.95%, 9.17%, 16.42% and 12.9%, respectively. The above would indicate that the adsorption of tetracycline by both the polymer and the catalyst contribute to its degradation, facilitating the encounter of the drug molecules with the catalytic sites.
## Electrospun nanofibers of polymers/carbon nanostructures blends containing photocatalytic nps
According to the reviewed literature, another strategy that has proven to be effective in preparing hybrid nanofibers with photocatalytic properties for the degradation of water pollutants is incorporating carbon nanostructures in these materials. For example, Uheida and coworkers prepared polyacrylonitrile/multiwall carbon nanotubes composite nanofibers containing surface-modified TiO 2 nanoparticles PAN-CNT/TiO 2 -NH 2 [bib_ref] Photocatalytic degradation of Ibuprofen, Naproxen, and Cetirizine using PAN-MWCNT nanofibers crosslinked TiO..., Uheida [/bib_ref]. Firstly, the solution to be electrospun was prepared by the addition of PAN/DMF solution to 3 wt.% of surface-activated CNTs in a DMF dispersion. The electrospinning process was carried out at room temperature with a voltage of 25 kV, the flow rate was set 0.5 mL/h, and the distance from the needle tip to the collector was 15 cm. The electrospun PAN/CNT mat was dried in a vacuum oven to remove the remaining amount of solvent and later was immersed into the crosslinking medium containing 2.5 wt.% glutaraldehyde (GA), kept shaking for 24 h at room temperature. After the activation reaction was completed, PAN/CNT composite nanofibers mat was removed and then immersed into 2 mL of an aqueous dispersion of TiO 2 -NH 2 NPs and kept shaking for 24 h. Finally, the crosslinked composite nanofibers were washed with ethanol, deionized water, and dried in air at room temperature. The SEM and TEM images of the fabricated PAN-CNT/TiO 2 -NH 2 composite nanofibers [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] show smooth nanofibers with an average diameter of 126 ± 4 nm and show that amino-functionalized TiO 2 NPs are attached to the surface of the PAN/CNT nanofibers as a result of the crosslinking process. The PAN-CNT/TiO 2 -NH 2 composite nanofibers were evaluated in the photocatalytic degradation of pharmaceuticals ibuprofen, cetirizine, and naproxen. Interestingly, the incorporation of CNTs enhanced the mechanical strength of the composite NFs and contributed as a photoactive phase (acting as an effective electron donor) in the photodegradation process of the studied drugs. Remarkably, the complete photodegradation of ibuprofen, naproxen, and cetirizine was achieved at 210, 90, and 50 min, respectively, under visible light irradiation. The photodegradation efficiency of PAN-CNT/TiO 2 -NH 2 composite nanofibers remained stable during five sequential cycles, indicating good stability and reusability of the catalyst.
The same research group also reported the preparation of electrospun composites nanofibers of PAN and CNT and incorporating TiO 2 NPs surface-modified TiO 2 -NH 2 to generate PAN-CNT/TiO 2 -NH 2 NFs. However, in this report, the authors studied and established the physicochemical parameters involved in phenol photodegradation in an aqueous solution [bib_ref] Photodegradation of phenol using composite nanofibers under visible light irradiation, Mohamed [/bib_ref]. The same methodologies were used for NFs preparation and modification. TiO 2 NPs (diameters ranging from 5 to 30 nm) were strongly attached to the PAN-CNT NFs. At optimum conditions, a 99.8% phenol degradation in the presence of PAN-CNT/TiO 2 -NH 2 and irradiation with a 100 W halogen lamp as the visible light source was achieved in a time lesser than 20 min.
Sharma and coworkers used graphene oxide (GO) to prepare GO/PAN composite nanofibers by electrospinning. The GO/PAN NFs were later modified with TiO 2 NPs, and finally, their performance in the photodegradation of Rhodamine 6G (Rh6G) dye was studied [bib_ref] Reusable graphene oxide nanofibers for enhanced photocatalytic activity: A detailed mechanistic study, Sharma [/bib_ref]. The solution for electrospinning was prepared by stirring a mixture of PAN (1.75 g), GO (20 mg), and DMF (20 mL) for 24 h. A syringe with a 0.5 mm inner diameter capillary, a high potential of 13 kV, a distance of 15 cm between the syringe and the collector, and a flow rate of 0.3 mL/h were used for electrospinning. The incorporation of TiO 2 nanoparticles into PAN/GO nanofibers was achieved by immersion in a TiO 2 sol-gel solution for 15 min. Finally, the nanofibers were dried at 65 - C for 1 h in air. FTIR and UV-Vis spectroscopy confirmed the strong interaction between the nanofibers and rhodamine. PAN/GO composite nanofibers showed a good photocatalytic effect on rhodamine degradation; 65% of dye was degraded after 12 h under natural sunlight with only 6 mg addition of PAN/GO nanofibers in 20 mL of dye solution, while with TiO 2loaded PAN/GO nanofibers, the photocatalytic degradation of the dye was enhanced with UV irradiation compared to visible light. Additionally, the TiO 2 loading on the nanofiber shows a faster degradation of the dye with UV light compared to visible light.
hanced the mechanical strength of the composite NFs and contributed as a photoactive phase (acting as an effective electron donor) in the photodegradation process of the studied drugs. Remarkably, the complete photodegradation of ibuprofen, naproxen, and cetirizine was achieved at 210, 90, and 50 min, respectively, under visible light irradiation. The photodegradation efficiency of PAN-CNT/TiO2-NH2 composite nanofibers remained stable during five sequential cycles, indicating good stability and reusability of the catalyst. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref]. SEM images of (a) PAN-CNT (b) and PAN-CNT/TiO2-NH2, and TEM Images of (c) PAN-CNT/TiO2-NH2 nanofibers composite. (Reprinted from the work of Uheida et al. [bib_ref] Photocatalytic degradation of Ibuprofen, Naproxen, and Cetirizine using PAN-MWCNT nanofibers crosslinked TiO..., Uheida [/bib_ref]. Copyright 2019, with permission from Elsevier).
The same research group also reported the preparation of electrospun composites nanofibers of PAN and CNT and incorporating TiO2 NPs surface-modified TiO2-NH2 to generate PAN-CNT/TiO2-NH2 NFs. However, in this report, the authors studied and established the physicochemical parameters involved in phenol photodegradation in an aqueous solution [bib_ref] Photodegradation of phenol using composite nanofibers under visible light irradiation, Mohamed [/bib_ref]. The same methodologies were used for NFs preparation and modification. TiO2 NPs (diameters ranging from 5 to 30 nm) were strongly attached to the PAN-CNT NFs. At optimum conditions, a 99.8% phenol degradation in the presence of PAN-CNT/TiO2-NH2 and irradiation with a 100 W halogen lamp as the visible light source was achieved in a time lesser than 20 min.
Sharma and coworkers used graphene oxide (GO) to prepare GO/PAN composite nanofibers by electrospinning. The GO/PAN NFs were later modified with TiO2 NPs, and finally, their performance in the photodegradation of Rhodamine 6G (Rh6G) dye was studied [bib_ref] Reusable graphene oxide nanofibers for enhanced photocatalytic activity: A detailed mechanistic study, Sharma [/bib_ref]. The solution for electrospinning was prepared by stirring a mixture of PAN (1.75 g), GO (20 mg), and DMF (20 mL) for 24 h. A syringe with a 0.5 mm inner diameter On the other hand, an interesting way to obtain carbon nanofibers containing photocatalytic nanoparticles starting from polymer nanofibers has been used. In this case, the polymer NFs, after being obtained, were subjected to thermal treatments to produce the calcination of the polymer and carbon generation. A fascinating example is the work of Zhang and coworkers, in which they report the obtaining of peapod-like electrospun nanofiber membranes, consisting of TiO 2 NPs, graphene oxide and carbon, (TiO 2 @GO@C NFs), which showed excellent efficiency in the photocatalytic degradation of methylene blue [bib_ref] Controllable synthesis of peapod-like TiO 2 @GO@C electrospun nanofiber membranes with enhanced..., Zhang [/bib_ref]. To fabricate the TiO 2 @GO@C electrospun nanofiber membranes, a solution of the TiO 2 precursor titanium isopropylate TTIP, graphene oxide and PAN was prepared in DMF using magnetic stirring and ultrasound for homogenization. The solution was then electrospun using a syringe with a 0.6 mm diameter needle, 13-15 kV and 13.5 cm tip-collector distance. After 12 h of ripening, the membranes were preoxidized at 280 - C for 4 h. Finally, the membranes were carbonized at 600-900 - C under an Ar atmosphere with a flow rate of 200 mL min −1 , using a heating rate of 5 - C min −1 . Using this process, TiO 2 -precursor@GO@PAN nanofiber membranes form TiO 2 @GO@C peapod-like nanofiber membranes. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] shows TEM images of (A) TiO 2 @GO and (B)TiO 2 @GO@C nanofiber membranes. It is observed that the TiO 2 NPs are almost wrapped by GO forming a peashaped TiO 2 @GO structure. Moreover, in [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] , it can be observed that TiO 2 @GO is wrapped with the carbon nanofiber, forming peapod-shaped TiO 2 @GO@C nanofiber membranes. The excellent photocatalytic degradation efficiency of MB, 98.5% over 3 h was obtained using peapod-like TiO 2 @GO@C nanofiber membranes with 0.3 wt.% GO. The authors ascribed these promising results to the large surface area, along with abundantly generated functional groups and excellent thermal conductivity of GO that improve the transport of carriers generated by TiO 2 NPs, in photocatalysis. Additionally, good mechanical properties are provided by the inclusion of a pea-like TiO 2 @GO structure, which shows a strengthening effect in the carbon nanofibers, improving their resistance to fracture. authors ascribed these promising results to the large surface area, along with abundantly generated functional groups and excellent thermal conductivity of GO that improve the transport of carriers generated by TiO2 NPs, in photocatalysis. Additionally, good mechanical properties are provided by the inclusion of a pea-like TiO2@GO structure, which shows a strengthening effect in the carbon nanofibers, improving their resistance to fracture. Another interesting report describes obtaining process of a porous nanofibrous membrane consisting of PAN, β-cyclodextrin, TiO 2 NPs and graphene oxide, PAN/b-CD/TiO 2 / GO, and the evaluation of this material in the photocatalytic degradation of methyl orange and methylene blue under natural sunlight [bib_ref] Enhanced photocatalytic degradation of organic dyes by ultrasonic-assisted electrospray TiO 2 /graphene..., Zhang [/bib_ref]. In this work, the solution for electrospinning was prepared by mixing TiO 2 NPs and graphene oxide in different proportions in DMF; then, the mixture was subjected to an ultrasonic bath; subsequently, PAN and β-cyclodextrin were added, and the mixture was stirred up to complete dissolution. To obtain electrospun NFs, a syringe with a 0.7 mm needle, voltage set at 18 kV, flow rate at 0.4 mL/h and 18-20 cm between the needle and the manifold were used. Apparently, PAN nanofibrous membranes trended to plasticize and swell in aqueous environments, limiting their ability to adsorb contaminants. For this reason, PAN is often combined with other materials for applications in aqueous media in order to moderate hydrophilic/hydrophobic balance. Specifically, the incorporation of β-cyclodextrin (β-CD) would include a hydrophobic cavity and a hydrophilic outer wall. [fig_ref] Figure 1: Scheme of the electrospinning setup and process [/fig_ref] shows that the nanofiber membrane has darkened areas due to the presence of GO. SEM images show rough nanofibers due to the presence of TiO 2 NPs and GO clusters. As the amount of GO incorporated increases, there is a decrease in the average diameter of the NFs, an effect that was attributed to the increase in the conductivity of the electrospun solution. TiO 2 NPs and GO were uniformly dispersed in the PAN/β-CD nanofibrous membranes without showing aggregation. The PAN/β-CD/TiO 2 /GO nanofibers showed good stability and reusability in MB and MO adsorption and photodegradation tests. During the first three cycles of use, the degradation efficiency of MB and MO by the nanofibrous membranes remained at 80%, then decreased by approximately 10% in the fifth cycle. The authors attribute the good efficiency obtained to the low density of PAN and the porosity of the PAN/β-CD/TiO 2 /GO nanofibrous membranes, which allow their flotation in the dye solution, increasing the contact surface and facilitating light penetration.
showing aggregation. The PAN/β-CD/TiO2/GO nanofibers showed good stability and reusability in MB and MO adsorption and photodegradation tests. During the first three cycles of use, the degradation efficiency of MB and MO by the nanofibrous membranes remained at 80%, then decreased by approximately 10% in the fifth cycle. The authors attribute the good efficiency obtained to the low density of PAN and the porosity of the PAN/β-CD/TiO2/GO nanofibrous membranes, which allow their flotation in the dye solution, increasing the contact surface and facilitating light penetration.
## Concluding remarks
A wide range of polymers have been proposed to obtain hybrid materials of polymeric nanofibers/photocatalytic nanoparticles with potential applications in the lightdriven removal of organic pollutants from water. Most of the studies carried out to date exploited synthetic polymers, and only in a few cases natural polymers, biopolymers, or
## Concluding remarks
A wide range of polymers have been proposed to obtain hybrid materials of polymeric nanofibers/photocatalytic nanoparticles with potential applications in the light-driven removal of organic pollutants from water. Most of the studies carried out to date exploited synthetic polymers, and only in a few cases natural polymers, biopolymers, or biodegradable polymers were considered. This is probably due to the difficulty of finding an adequate compromise between polymer degradation and the durability of the photocatalytic material during use. In our opinion, future designs should consider the increased use of naturally occurring polymers. In this perspective, blending the materials with other environmentally friendly polymers (such as PCL, PLA PVP, and PEO, among others) may be helpful for the processing of the natural polymers. The blending strategy can be also exploited to modify and improve the properties of natural polymers (such as mechanical properties, conductivity, hydrophobicity, etc.). Among the different polymers that have been used in hybrid photocatalytic materials, we believe that optically active polymers (conductive, donor-acceptor, -conjugated) could also be considered as interesting options since these types of polymers are expected to contribute actively to the photodegradation of pollutants and/or enhance the effect of the light.
A relevant aspect that should be considered, in our view, is the role played by the polymeric electrospun nanofibers, which extends much further than simply being a support for the photocatalytic material. In fact, the polymer can actively contribute to enhancing the adsorption of pollutants, facilitating their approach to the photocatalytic sites or it can interact with the surface of the catalytic hybrid material to improve its performance. An outstanding example of the possible "active" role of polymers is represented by cellulose acetate NFs, which, thanks to the presence of carboxylic acid groups, bind very efficiently to photocatalytic titania nanoparticles. Hence, using this polymeric precursor, it was possible to obtain a photocatalytic hybrid material based on polymeric nanofibers with excellent performance and durability. Another relevant strategy for designing photocatalytic hybrid materials exploits a matrix composed of nanofibers of a polymer blend. In this case, high porosity in the nanofibers can be achieved by removing one of the polymers, for example, with a specific solvent. As an advantage, the enhanced porosity significantly increases the contact area and the availability of photocatalytic sites, leading to a considerable improvement of the photocatalytic performance of the material. An alternative approach to optimize photocatalytic activity is the calcination of hybrid polymer nanofibers to obtain carbon nanofibers or to produce a one-dimensional structure of semiconductor crystals. The interaction of the catalytic nanoparticles with the included carbon nanostructures, such as graphene derivatives and carbon nanotubes, yields highly performing materials.
Regarding the final application of the hybrid electrospun systems reviewed in this review, we would like to point out that important challenges still remain, and more issues should therefore be fixed, such as the control of the fouling and the increase in the permeate fluxes that are still relatively low. In addition, more investigations are needed to demonstrate the durability of the membranes in terms of photocatalytic activity, as well as the cost-effective synthesis of these hybrid nanomaterials for large scale applications.
[fig] Figure 1: Scheme of the electrospinning setup and process. [/fig]
[fig] Figure 2: SEM images of (a) PA6 NFs and (b) NFs of PA6 with 25 wt.% TiO2 nanoparticles. (Reprinted from the work of Blanco et al.[25]). [/fig]
[fig] Figure 3: Representative TEM images of (a) 8%-nylon 6,6/FA-ZnO, (b) 5%-nylon 6,6/HFIP-ZnO, (c) 8%-nylon 6,6/HFIP-ZnO core−shell nanofibers; (d) representative SAED pattern of the core−shell nylon 6,6-ZnO nanofibers (8%-nylon 6,6/HFIP-ZnO NF). (Reprinted from the work of Kayaci et al.[31]. Copyright 2012, with permission from the American Chemical Society). [/fig]
[fig] Figure 4: Illustration showing the mechanism of forming the core-shell structure. (Reprinted from the work of Unnithan et al.[27]; Copyright 2012, with permission from Elsevier). [/fig]
[fig] Figure 5: TEM images of (a) pristine PVAc electrospun nanofibers, (b) ZnS-PVAc hybrid electrospun nanofibers, (c) PdSZnS-PVAc hybrid electrospun nanofibers, and (b1,c1) HRTEM for the marked area in (b,c) hybrid electrospun nanofiber mats, respectively. (Reprinted from the work of Panthi et al.[32], Copyright 2015, with permission from Elsevier). [/fig]
[fig] Figure 6: TEM images of PVDF/ZnO/Ag 2 CO 3 /Ag 2 O electrospun nanofiber; inset SAED pattern. (Reprinted from the work of Rosman et al.[28]). [/fig]
[fig] Figure 7: SEM images and EDX spectra of samples: (a,b) PAN, (c,d) CuTNPc/PAN, (e,f) BiOCl/CuT-NPc/PAN, and (g,h) BiOCl/PAN. (Reprinted from the work of Guo et al. [34]. Copyright 2018, with permission from Elsevier).The analysis of the thermogravimetric profiles of pristine PAN NFs and modified NFs (CuTNPc/PAN, BiOCl/CuTNPc/PAN, and BiOCl/PAN) revealed a decrease in the thermal stability of the modified NFs compared to those of bare PAN. This allowed inferring the existence of interactions between PAN and metal atoms, explaining the uniform distribution of heterostructures in NFs and the absence of aggregation observed. Concerning their photocatalytic activities, BiOCl/CuTNPc/PAN NFs showed 75% RhB degradation after 180 min under UV irradiation, while for CuTNPc/PAN nanofibers and Bi-OCl/PAN nanofibers, the values were only 24% and 20%, respectively. Interestingly, the authors reported an adsorption of approximately 5% of RhB by PAN NFs, allowing to [/fig]
[fig] Figure 8: (a) Schematic diagram for the fabrication procedure of PAN/AgBr/Ag fibrous membrane and (b) STEM image and corresponding EDX elemental mapping of PAN/AgBr/Ag nanofibrous membrane. (Adapted from the work of Qayum et al. [35], Copyright 2019, with permission from Elsevier). [/fig]
[fig] Figure 9: Hierarchical fibrous polymer/ZnO nanostructures. Low-magnification SEM image of the hierarchical nanostructure consisting of radially aligned ZnO nanowires grown on electrospun poly-L-lactide nanofibers. Inset: schematic representation of the hierarchical nanostructure. (Reprinted from the work of Sugunan et al. [36]. Copyright 2021, with permission from John Wiley and Sons). [/fig]
[fig] Author: Contributions: M.L.F. and A.L. contributed equally to this work. M.L.F., A.L. and A.C. conceived the idea, subject matter and structure of the manuscript, and drafted the manuscript with the help of, C.S., M.U., M.M. and M.G. All authors have read and agreed to the published version of the manuscript. Funding: A.C. would like to thank to Conicyt for the PhD scholarship 21191257. A. Leiva thanks the FONDECYT project 1211124 and FONDAP 15110019. [/fig]
[table] Table 1 Table 1: Electrospun polymers, solvents used and carrier if corresponding. Adapted with permission from Xue et al.[23]. Copyright © 2019, American Chemical Society. Cont. [/table]
[bib_ref] The effect of processing variables on the morphology of electrospun nanofibers and..., Deitzel [/bib_ref] |
RNA in Cancer Immunotherapy: Unlocking the Potential of the Immune System
◥Recent advances in the manufacturing, modification, purification, and cellular delivery of ribonucleic acid (RNA) have enabled the development of RNA-based therapeutics for a broad array of applications. The approval of two SARS-CoV-2-targeting mRNAbased vaccines has highlighted the advances of this technology. Offering rapid and straightforward manufacturing, clinical safety, and versatility, this paves the way for RNA therapeutics to expand into cancer immunotherapy. Together with ongoing trials on RNA cancer vaccination and cellular therapy, RNA therapeutics could be introduced into clinical practice, possibly stewarding future personalized approaches. In the present review, we discuss recent advances in RNA-based immuno-oncology together with an update on ongoing clinical applications and their current challenges.
# Introduction
RNA has become a widely popular tool for vaccination against infectious diseases following the SARS-CoV2 pandemic [bib_ref] The tangled history of mRNA vaccines, Dolgin [/bib_ref]. Potentially, this molecule can be exploited for many more applications, especially in cancer immunotherapy, offering a broad range of RNAbased therapeutic strategies [bib_ref] mRNA in cancer immunotherapy: beyond a source of antigen, Van Hoecke [/bib_ref]. Since its discovery in 1961 by Brenner and colleagues, RNA has evolved from what was initially considered a mere intermediary between DNA and protein to a versatile molecule operating at multiple cellular levels, exploitable for immunotherapeutic applications [bib_ref] The infinite possibilities of RNA therapeutics, Mollocana-Lara [/bib_ref]. In vitro transcribed messenger RNA (iVT-mRNA), self-amplifying RNA (SAM), antisense oligonucleotides (ASO), aptamers, small interfering RNA (siRNA), and microRNA (miRNA) are the most studied RNA formats at present, which can be categorized into two main groups: (i) coding RNA (cRNA) translating into protein, including mRNA and SAM, and (ii) noncoding RNA (ncRNA) that does not translate into proteins but rather regulates cell physiology and functions, including among others ASOs, aptamers, siRNA, and miRNA. Based on their role related to cancer biology, ncRNA can function as oncogenes or tumor suppressor genes [fig_ref] Figure 1: Overview of coding and noncoding RNA structures [/fig_ref] ; ref. 5) and therefore be implemented for therapeutic use. Initially, RNA was not considered a suitable therapeutic tool due to its unstable nature and susceptibility to rapid degradation by ubiquitous ribonucleases (RNases). Other concerns regarding potential toxicity, unspecific immune activation, and unknown effectiveness also needed further investigation [bib_ref] Paving the road for RNA therapeutics, Dammes [/bib_ref]. At present, many limitations have been overtaken, including chemical modification of the RNA structure, paralleled with the development of novel technologies for RNA delivery and protection. This allows fast, cost-effective, and versatile generation of mRNA suitable for clinical application in the context of cancer and infectious diseases [bib_ref] Recent advances in mRNA vaccine technology, Pardi [/bib_ref] , introducing RNA as a promising pharmaceutical product ; refs. [bib_ref] Turning the corner on therapeutic cancer vaccines, Hollingsworth [/bib_ref] [bib_ref] Beyond just peptide antigens: the complex world of peptide-based cancer vaccines, Stephens [/bib_ref] [bib_ref] Fotin-Mleczek M. mRNA: a novel avenue to antibody therapy?, Schlake [/bib_ref] [bib_ref] A comparison of plasmid DNA and mRNA as vaccine technologies, Liu [/bib_ref] [bib_ref] mRNA: a versatile molecule for cancer vaccines, Diken [/bib_ref].
## In vitro transcribed mrna and self-amplifying rna
Mature eukaryotic mRNA consists of highly conserved molecular features, including a cap structure at the 5 0 terminus, two extended untranslated regions (UTR) at the 5 0 and 3 0 end of the open reading frame (ORF), and a poly-A tail at the 3 0 terminus (13), all influencing mRNA degradation, stability, immunogenicity, and translatability [bib_ref] Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity..., Holtkamp [/bib_ref]. For the generation of iVT-mRNA, a DNA template is required, commonly derived from a linearized plasmid [bib_ref] mRNA vaccines -a new era in vaccinology, Pardi [/bib_ref] , from which mRNA is transcribed using RNA polymerase enzymes such as T7, SP6, or T3 [bib_ref] In vitro transcription of long RNA containing modified nucleosides, Pardi [/bib_ref]. Other main components of the iVT reaction mix comprise a RNase inhibitor, pyrophosphatases, and a reaction buffer including the four ribonucleoside triphosphates (rNTP) and a capping reagent (ARCA, Clean Cap TM ). The final concentration of each component, the reaction temperature, and time will determine the final mRNA yield obtained [fig_ref] Figure 1: Overview of coding and noncoding RNA structures [/fig_ref] refs. [bib_ref] In vitro transcription of long RNA containing modified nucleosides, Pardi [/bib_ref] [bib_ref] Karik o K. HPLC purification of in vitro transcribed long RNA, Weissman [/bib_ref].
After the iVT reaction, the DNA template, all enzymes used, organic and inorganic contaminants and secondary transcription products, such as incomplete/truncated RNA molecules and double-stranded RNA (dsRNA) have to be removed [bib_ref] A facile method for the removal of dsRNA contaminant from in vitro-transcribed..., Baiersd€ Orfer [/bib_ref]. A DNase digestion is performed to eliminate the DNA template, followed by a salt and ethanol precipitation or more accurately by high-pressure/performance liquid chromatography (HPLC) to obtain pure mRNA [bib_ref] Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and..., Karik O [/bib_ref]. The purification process reduces immune activation triggered by contaminants and therefore can improve mRNA translation upon delivery into the cell [bib_ref] Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and..., Karik O [/bib_ref]. In addition to manufacturing and purification, thorough quality controls are performed to assess the final RNA integrity, purity, concentration, capping efficiency, sequence, and poly-A tail length [bib_ref] Analytical techniques currently used in the pharmaceutical industry for the quality control..., Demelenne [/bib_ref].
Poor mRNA uptake and in vivo expression are often observed due to extracellular RNase activity [bib_ref] mRNA vaccines -a new era in vaccinology, Pardi [/bib_ref] and intrinsic mRNA immunogenicity [bib_ref] Three decades of messenger RNA vaccine development, Verbeke [/bib_ref] , triggering interferon (IFN) pathway activation [bib_ref] Recent advances in mRNA vaccine technology, Pardi [/bib_ref]. Packaging mRNA into lipid nanoparticles (LNP) has proven an effective solution to ensure protection and successful RNA delivery into the cell [bib_ref] Delivering the messenger: advances in technologies for therapeutic mRNA delivery, Kowalski [/bib_ref]. A multitude of nanoparticles have been tested and extensively reviewed by others [bib_ref] Lipid nanoparticles for mRNA delivery, Hou [/bib_ref] [bib_ref] Formulation and delivery technologies for mRNA vaccines, Zeng [/bib_ref] [bib_ref] Nanomedicine-based approaches for mRNA delivery, Uchida [/bib_ref]. At present, the LNP-mRNA COVID-19 vaccines Tozinameran and Elasomeran, with LNPs composed of ionizable lipids, phospholipids, cholesterol, and PEGylated lipids, have been clinically approved [bib_ref] Difference in the lipid nanoparticle technology employed in three approved siRNA (Patisiran)..., Suzuki [/bib_ref]. Moreover, RNA chemical modifications such as N1-methyladenosine, 5-methylcytidine, pseudouridine, N6-methyladenosine, 5-methyl deoxycytidine, and inosine pioneered by Kariko and colleagues [bib_ref] Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification..., Karik O [/bib_ref] [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref] [bib_ref] Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational..., Karik O [/bib_ref] [bib_ref] Chemical and structural effects of base modifications in messenger RNA, Harcourt [/bib_ref] have been exploited to generate stable, nonimmunogenic, and high-translatable RNA molecules, including also modified base analogs as 5`cap structures [bib_ref] Cap 1 messenger RNA synthesis with co-transcriptional CleanCap, Henderson [/bib_ref]. Other work on the optimization of the poly-A tail length showed enhanced mRNA stability and lowered immunogenic profile [bib_ref] Short poly(A) tails are a conserved feature of highly expressed genes, Lima [/bib_ref]. Additional optimization of UTRs and codon optimization paired with sequence design of the encoding sequence (34) aimed to improve RNA lifetime stability and expression [bib_ref] Improving mRNA-based therapeutic gene delivery by expression-augmenting 3' UTRs identified by cellular..., Orlandini Von Niessen [/bib_ref] [bib_ref] Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA, Asrani [/bib_ref] [bib_ref] Human 5' UTR design and variant effect prediction from a massively parallel..., Sample [/bib_ref].
## Noncoding and antisense oligonucleotides
Solid-phase synthesis is mostly used for manufacturing (antisense) oligonucleotides. The first nucleotide of the desired molecule is attached to a solid phase and elongated using a repetition of a fourstep cycle, adding one nucleotide per cycle until the oligonucleotide is fully synthesized. The four-step cycle includes detritylation/deblocking, coupling, capping, and oxidation or thiolation. The synthesis is performed in a column reactor under programmed delivery of all reagents [fig_ref] Figure 1: Overview of coding and noncoding RNA structures [/fig_ref]. A 70% to 80% success synthesis rate is achieved of the desired oligonucleotide length, requiring a final HPLC purification step, removing all polymers showing incorrect length (n AE x; ref. 4). Chemical modifications were implemented to this process, aiming for improved pharmacokinetics [bib_ref] Therapeutic oligonucleotides: state of the art, Smith [/bib_ref]. The most used modifications include a substitution of the phosphodiester bond with a phosphorothioate, proving to increase resistance toward nuclease degradation . Advantages and disadvantages of RNA-compared with DNA-and protein-based therapeutics (data from references 8-12).
## Rna dna protein
[formula] Synthesis þ þ À Manufacture time a þ/À þ /À À Genome integration À þ /À À Self-adjuvancy þ/À þ /À À Storage b þ/À þ /À þ Administration þ þ À a [/formula]
RNA manufacture time is fast but depends on a DNA template. b Storage requirements depend on the formulation used for the therapeutic product.
and reduce binding to plasma proteins, and decreasing renal clearance [bib_ref] RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform, Bennett [/bib_ref]. However, reduced target affinity is also observed, and therefore 2 0 -methoxyethyl, 2 0 -deoxy-2 0 -fluoro, and 2 0 -O-methyl modifications were introduced on the ribose of the RNA to improve specificity [bib_ref] Analytical techniques currently used in the pharmaceutical industry for the quality control..., Demelenne [/bib_ref]. A similar manufacturing approach is exploited for the synthesis of RNA aptamers. A widely used method to design aptamers is by Systematic Evolution of Ligands by Exponential enrichment (SELEX). In short, a high diversity library of single-stranded RNA (ssRNA) is synthetized by in vitro transcription [bib_ref] SELEX tool: a novel and convenient gel-based diffusion method for monitoring of..., Liu [/bib_ref]. From the RNA library, a selective target binding ssRNA is isolated through repeated rounds of exposure, binding, selection, and amplification. Once the desired sequence/design of the RNA aptamers is obtained, the aptamers are manufactured using solid-phase synthesis [bib_ref] Aptamers and the RNA world, past and present, Gold [/bib_ref].
## Rna as a vaccination strategy
Therapeutic cancer vaccines aim to generate antigen-specific T-cell responses targeting tumor cells and potentially achieve long-term clinical benefits [bib_ref] Therapeutic cancer vaccines revamping: technology advancements and pitfalls, Antonarelli [/bib_ref] [bib_ref] Therapeutic cancer vaccines, Saxena [/bib_ref]. The approval of two mRNA vaccines for COVID-19 prevention has highlighted the potential of mRNA technology [bib_ref] From COVID-19 to cancer mRNA vaccines: moving from bench to clinic in..., Chakraborty [/bib_ref]. Two main RNA-based approaches have been extensively explored for cancer vaccination: ex vivo mRNA-loaded dendritic cell (DC) vaccines [bib_ref] Recent advances in experimental dendritic cell vaccines for cancer, Filin [/bib_ref] [bib_ref] Therapeutic cancer vaccination with ex vivo RNA-transfected dendritic cells-an update, D€ Orrie [/bib_ref] and mRNA-LNP vaccines (ref. [bib_ref] mRNA vaccine for cancer immunotherapy, Miao [/bib_ref] ; [fig_ref] Figure 2: Figure 2 [/fig_ref]. In both strategies, mRNA is used to deliver the tumor-associated antigen (TAA) or tumor-specific antigen to elicit an antitumor immune response. Upon cellular entry followed by translation of the mRNA, the proteasome processes the mRNA-encoded protein into peptides that ultimately will be processed and presented by human leucocyte antigen (HLA) class I molecules to CD8 þ T cells [bib_ref] Learning from the proteasome how to fine-tune cancer immunotherapy, Vigneron [/bib_ref]. CD4 þ T-cell stimulation is also recommended to support the CD8 þ T-cell response [bib_ref] Adot evi O. Interest of tumor-specific CD4 T helper 1 cells for..., Galaine [/bib_ref]. In this regard, coupling an HLA class II sorting signal, such as the signal sequence of the invariant chain, lysosomalassociated membrane protein (LAMP), or DC-LAMP, to the antigen sequence is required to ensure that antigen-derived peptides enter the HLA class II presentation pathway, despite the protein being synthesized in the cytosol of the cell [bib_ref] Messenger RNA-electroporated dendritic cells presenting MAGE-A3 simultaneously in HLA class I and..., Bonehill [/bib_ref] [bib_ref] Efficient presentation of known HLA class II-restricted MAGE-A3 epitopes by dendritic cells..., Bonehill [/bib_ref].
Cancer vaccines, mostly targeting cancer-testis and differentiation antigens, as monotherapy, have not shown significant activity thus far. Most clinical trials have been ineffective, and the induced immune reactivity was insufficient, limited in time, and narrow. The lack of clinical efficacy from vaccine treatment alone can be attributed to weak antigen delivery modalities that induced low T-cell titers as well as immune checkpoints remaining intact, which ultimately prevented tumor cell killing. It is now thought that neoantigens generated by somatic alterations could be differentially recognized by the immune system as these proteins/peptides would be unique to the tumor and the T-cell repertoire recognizing such neoantigens would not have been subjected to central tolerance mechanisms, as is the case for cancer-testis-and differentiation antigen-specific T cells [bib_ref] The changing landscape of therapeutic cancer vaccines: novel platforms and neoantigen identification, Jou [/bib_ref]. Overview of active mRNA-based immunotherapeutic strategies. Left, LNP-antigen mRNA vaccine delivered systemic or locally, followed by antigen expression resulting in T-cell priming and eventually cancer cell killing. Right, monocytes or hematopoietic progenitor cells are isolated from blood, further cultured, and differentiated into DCs. mRNA is then used to load the DCs ex vivo with tumor antigens. The modified DCs are administered to patients, where they will prime T cells, eventually resulting in the killing of cancer cells. Adapted from an image created with BioRender.com.
## Mrna-based dendritic cell vaccines
mRNA-based DC cancer vaccination was introduced more than two decades ago [bib_ref] Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells, Hsu [/bib_ref]. At present, more than 30 clinical trials have been published, recently reviewed by Dorrie and colleagues [bib_ref] Therapeutic cancer vaccination with ex vivo RNA-transfected dendritic cells-an update, D€ Orrie [/bib_ref]. For DC generation, monocytes or hematopoietic progenitor cells are isolated from blood and further cultured and differentiated into DCs [bib_ref] Engineering dendritic cell vaccines to improve cancer immunotherapy, Perez [/bib_ref]. mRNA is then used to load the DCs ex vivo with tumor antigens. The modified DCs are administered to patients [fig_ref] Figure 2: Figure 2 [/fig_ref] via intravenous, intradermal, subcutaneous, or intranodal injections [bib_ref] mRNA-based dendritic cell vaccines, Benteyn [/bib_ref]. DC maturation is most often induced using a cytokine cocktail (56), feasible with both protein and mRNA delivery [bib_ref] Enhancing the T-cell stimulatory capacity of human dendritic cells by coelectroporation with..., Bonehill [/bib_ref]. Examples of mRNAinduced functional manipulation of DCs include TriMix mRNA, a mixture consisting of CD40 Ligand (CD40L), CD70, and constitutively active Toll-like receptor 4 (TLR4) encoding mRNA. Several studies showed that TriMix-DC vaccination induces robust, TAA-specific T-cell responses in the majority of analyzed patients [bib_ref] Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients..., Wilgenhof [/bib_ref] [bib_ref] Dendritic cells loaded with mRNA encoding full-length tumor antigens prime CD4þ and..., Van Nuffel [/bib_ref] [bib_ref] A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy..., Wilgenhof [/bib_ref]. Other strategies to improve the activation of T cells by mRNA-modified DC have been studied and show promising results [bib_ref] CD83 expression on dendritic cells and T cells: correlation with effective immune..., Aerts-Toegaert [/bib_ref] [bib_ref] Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function..., Tuyaerts [/bib_ref] [bib_ref] Interference with PD-L1/PD-1 co-stimulation during antigen presentation enhances the multifunctionality of antigen-specific..., Pen [/bib_ref] [bib_ref] Engineering monocyte-derived dendritic cells to secrete interferon-a enhances their ability to promote..., Willemen [/bib_ref] [bib_ref] Characterization of interleukin-15-transpresenting dendritic cells for clinical use, Van Den Bergh [/bib_ref].
DC-based vaccines have shown to induce adaptive immune responses, and RNA transfection is emerging as an ideal method for antigen-loading and functional manipulation of the applied cells. DC vaccination rarely produces adverse events and has a highly safe profile [bib_ref] Therapeutic cancer vaccination with ex vivo RNA-transfected dendritic cells-an update, D€ Orrie [/bib_ref]. However, challenges that call for imperative improvements such as the manufacturing process, the optimal choice of DC subset or their in vitro generation, the antigen choice, the route of administration, and vaccination schedule still need to be addressed [bib_ref] Ex vivo pulsed dendritic cell vaccination against cancer, Gu [/bib_ref].
## Mrna-based vaccines
First clinical results were observed with intradermal or intranodal naked mRNA administration, resulting in mRNA uptake by antigen-presenting cells in the dermis or lymph nodes followed by antigen presentation and T-cell stimulation [bib_ref] Recent developments of RNA-based vaccines in cancer immunotherapy, Faghfuri [/bib_ref]. Even though it has a positive clinical outcome and favorable safety profile, the many limitations, such as limited mRNA uptake, resulting in low bioavailability of antigens and lowered immune responses, hampered its implementation [bib_ref] mRNA vaccines -a new era in vaccinology, Pardi [/bib_ref].
Recent attempts opted for intramuscular or intravenous administration of mRNA encapsulated by delivery carriers [bib_ref] Formulation and delivery technologies for mRNA vaccines, Zeng [/bib_ref]. Four recent clinical trials, using mRNA packaged in LNPs, have shown promising clinical and immunologic results in patients with solid tumors. In the Lipo-Merit trial (NCT02410733; [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] , a vaccine consisting of nonmodified lipoplexed mRNA targeting a variety of TAAs (NY-ESO-1, MAGE-A3, tyrosinase, and TPTE) was administered intravenously to advanced melanoma patients. The adverse events (pyrexia, chills, and flu-like symptoms) were mild to moderate and transient. Expansion and activation of antigen-specific T cells with cytolytic activity against tumor cells could be documented. Continuous vaccination resulted in the persistence of antigen-specific memory T cells. Encouraging clinical responses have been reported [bib_ref] An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma, Sahin [/bib_ref].
In the R07198457-trial (NCT03815058 [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] , the administration of nonmodified mRNA encoding up to 20 patient-specific neoantigens has been studied as monotherapy and in combination with a PD-L1 inhibitor, in patients with advanced solid tumors (breast cancer, prostate cancer, ovarian cancer, melanoma, non-small cell lung cancer, bladder cancer, and colorectal cancer; refs. [bib_ref] Abstract CT169: a phase Ia study to evaluate RO7198457, an individualized neoantigen..., Braiteh [/bib_ref] [bib_ref] Abstract CT301: a phase Ib study to evaluate RO7198457, an individualized neoantigen..., Lopez [/bib_ref]. Also, in this trial, the adverse events were mostly of grade 1-2 and transient. Neoantigen-specific T-cell responses were observed in most of the patients. Promising clinical results in these often heavily pretreated patients were noted.
In both the KEYNOTE-603 trial (NCT03313778; [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] and the KEYNOTE-942 trial (NCT03897881; [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] ; Moderna and Merck), the safety and immunogenicity of intramuscularly administered lipid-protected modified mRNA encoding neoantigens were evaluated, either as monotherapy or in combination with anti-PD-1 monoclonal antibodies (mAb, pembrolizumab) in patients with solid tumors [bib_ref] A phase I multicenter study to assess the safety, tolerability, and immunogenicity..., Burris [/bib_ref] [bib_ref] 798 Safety, tolerability, and immunogenicity of mRNA-4157 in combination with pembrolizumab in..., Bauman [/bib_ref]. This mRNA-based personalized cancer vaccine has an acceptable safety profile along with observed clinical responses in combination with pembrolizumab. Preliminary efficacy analysis from checkpoint inhibition-na€ ve relapsed/refractory human papillomavirus (HPV) negative head and neck squamous cell carcinoma (cohort suggests activity of this combination [bib_ref] 798 Safety, tolerability, and immunogenicity of mRNA-4157 in combination with pembrolizumab in..., Bauman [/bib_ref].
An alternative mRNA-based approach uses SAM, originating from positive ssRNA alphaviruses, consisting of the RNA replication machinery of the alphavirus (self-assembly genes; [fig_ref] Figure 1: Overview of coding and noncoding RNA structures [/fig_ref] and replacing other genetic regions with the gene sequence encoding the antigen(s) of interest. SAM amplifies over time (up to 2 months) and consequently induces more potent and persistent immune responses [bib_ref] Self-amplifying RNA viruses as RNA vaccines, Lundstrom [/bib_ref] [bib_ref] mRNA vaccine: a potential therapeutic strategy, Wang [/bib_ref]. Clinical applications using SAM have been promising in preventing infectious diseases [bib_ref] Self-amplifying RNA vaccines for infectious diseases, Bloom [/bib_ref] and are transitioning into the cancer immunotherapy field. Gritstone, a Californiabased company, is performing clinical studies, where a viral prime and a SAM boost are used to induce immune responses against private or shared neoantigens.
## Rna in passive immunotherapy
Passive immunotherapy is used as an umbrella term to describe any strategy designed to help a patient to fight disease by administration of immune system components that have been generated in the laboratory, including delivery of proinflammatory cytokines, immune-modulatory mAbs, or ex vivo manipulated autologous effector immune cells [bib_ref] Revisiting immunotherapy: a focus on prostate cancer, Cha [/bib_ref] [bib_ref] mRNA as novel technology for passive immunotherapy, Schlake [/bib_ref]. As with active immunotherapies, passive strategies can also benefit from the implementation of not only iVT-mRNA but also ncRNA, as passive immunotherapy covers a broader range of strategies [fig_ref] Figure 3: Overview of passive immunotherapeutic strategies [/fig_ref].
## Mrna for protein therapy in vivo
In vivo delivery of antibody encoding mRNA
Since the development of the hybridoma technique in 1975 by Milstein and K€ ohler, therapeutic mAbs have been introduced for numerous indications. The mAb-mediated blockade of immune checkpoints, such as programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), has revolutionized cancer treatment [bib_ref] Combination of CTLA-4 and PD-1 blockers for treatment of cancer, Rotte [/bib_ref] [bib_ref] The yin and yang of co-inhibitory receptors: toward anti-tumor immunity without autoimmunity, Schnell [/bib_ref] [bib_ref] Manipulation of the immune system for cancer defeat: a focus on the..., D'arrigo [/bib_ref]. Besides full-sized mAb, antibody fragments, such as single-chain variable fragments (scFv) and heavy-chain V H domains, have been heavily studied. In parallel, a variety of tumorassociated targets, such as TAAs, vascular, and stromal cells, have been explored as targets [bib_ref] Fotin-Mleczek M. mRNA: a novel avenue to antibody therapy?, Schlake [/bib_ref] [bib_ref] mRNA as novel technology for passive immunotherapy, Schlake [/bib_ref] [bib_ref] Theranostics in immuno-oncology using nanobody derivatives, Lecocq [/bib_ref] [bib_ref] Antibody and antibody fragments for cancer immunotherapy, Chen [/bib_ref].
Although mRNA-based antibody therapies have yet to face technical and clinical challenges (e.g., frequency and route of administration; ref. [bib_ref] Advancements in mRNA encoded antibodies for passive immunotherapy, Deal [/bib_ref] , the use of mRNA for in vivo production of therapeutic antibodies remains a promising approach (10).
In 2019, Rybakova and colleagues demonstrated the delivery of mRNA encoding the humanized anti-human epidermal growth factor receptor 2 (HER2) antibody, trastuzumab, via LNP in tumor-bearing mice. The reported serum-antibody concentrations were detectable up to 14 days after LNP injection, demonstrating more favorable pharmacodynamics compared with the recombinant mAbs. In general, the mRNA transcribed antibody retained its cell toxicity properties in vivo, which contributed to a significant delay in HER2-positive tumor growth when administered weekly [bib_ref] mRNA delivery for therapeutic anti-HER2 antibody expression in vivo, Rybakova [/bib_ref].
The potential of bispecific T-cell-engaging antibodies is high, but their manufacturing is often challenging. Stadler and colleagues tested the in vivo production of bispecific antibodies by treating mice with pharmacologically optimized, nucleoside-modified iVT-mRNA encoding the bispecific antibody. Sustained endogenous synthesis of the bispecific antibody was achieved, eliminating advanced tumors as effectively as the corresponding purified bispecific antibody was achieved. This approach could accelerate the clinical development of novel bispecific antibodies [bib_ref] Elimination of large tumors in mice by mRNA-encoded bispecific antibodies, Stadler [/bib_ref]. A clinical trial by BioNTech is investigating the safety and pharmacokinetics of BNT141 (NCT04683939; [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] and BNT142, both mRNA encoding antibodies targeting CLDN18.2 and CD3 À CLDN6, respectively, in unresectable or metastatic claudin 18.2-positive solid tumors for which no available standard therapy is likely to confer clinical benefits.
## Mrna-engineered adoptive cell therapies
Although immune-checkpoint blocking and mAb-based methods have shown promising results in breast cancer [bib_ref] PD-L1 status in breast cancer: current view and perspectives, Vranic [/bib_ref] , melanoma [bib_ref] Targeted agents and immunotherapies: optimizing outcomes in melanoma, Luke [/bib_ref] , and non-small cell lung carcinoma [bib_ref] Immunotherapy for non-small cell lung cancer: current concepts and clinical trials, Mayor [/bib_ref] [bib_ref] Cancer immunotherapy using checkpoint blockade, Ribas [/bib_ref] , among others, many patients still develop disease progression after these therapies, requiring additional treatment options [bib_ref] mRNA as novel technology for passive immunotherapy, Schlake [/bib_ref] [bib_ref] How mRNA therapeutics are entering the monoclonal antibody field, Van Hoecke [/bib_ref] [bib_ref] Adoptive cellular therapies: the current landscape, Rohaan [/bib_ref]. This demand was met with the introduction of adoptive cell therapy (ACT), pioneered by Steven A. Rosenberg, who demonstrated the in vivo antitumor activity of tumor-infiltrating lymphocytes (TIL; refs. [bib_ref] mRNA as novel technology for passive immunotherapy, Schlake [/bib_ref] [bib_ref] Elimination of large tumors in mice by mRNA-encoded bispecific antibodies, Stadler [/bib_ref] [bib_ref] Adoptive cellular therapies: the current landscape, Rohaan [/bib_ref]. Over the years, ACT moved beyond the use of TILs. New strategies include ex vivo expansion and modification of tumor-residing or peripheral T cells with TCRs or CARs that convey specificity for the cancer cells [bib_ref] Adoptive cellular therapies: the current landscape, Rohaan [/bib_ref] [bib_ref] The antigen-binding moiety in the driver's seat of CARs, Hanssens [/bib_ref]. Although ACT mainly focuses on the introduction of T cells expressing a TCR or CAR, Exteberria and colleagues showcased the therapeutic effect of the intratumoral administration of T cells transiently expressing IL12 in combination with transient CD137 ligand expression resulting in antitumor toxicity [bib_ref] Intratumor adoptive transfer of IL-12 mRNA transiently engineered antitumor CD8, Etxeberria [/bib_ref].
Successful clinical outcome has been reported with CAR-T therapies in hematologic malignancies. For instance, CAR-T therapy targeting CD19 in chronic lymphocytic and acute lymphoblastic leukemia (97), CD33 and CD123 CAR targeting in acute myeloid leukemia (AML; refs. [bib_ref] In vitro-transcribed mRNA chimeric antigen receptor T cell (IVT mRNA CAR T)..., Soundara Rajan [/bib_ref] [bib_ref] Karik o K. The emerging role of in vitro-transcribed mRNA in adoptive..., Foster [/bib_ref] , and anti-BCMA CAR-T cell therapy in multiple myeloma (MM; ref. [bib_ref] Overview of anti-BCMA CAR-T immunotherapy for multiple myeloma and relapsed/refractory multiple myeloma, Feng [/bib_ref]. This success was unmet when translated to solid malignancies as it is among others more challenging to identify the right target molecules.
At present, the clinical implementation of engineered T cells still raises many questions, including cross reactivity, controllability of permanently modified cellular products, and safety assessments [bib_ref] Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy, Mensali [/bib_ref]. Because of these safety concerns, there has been an increasing interest for mRNA-based T-cell manipulation as transient expression is ensured in both TCR and CAR approaches [bib_ref] In vitro-transcribed mRNA chimeric antigen receptor T cell (IVT mRNA CAR T)..., Soundara Rajan [/bib_ref] [bib_ref] Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy, Mensali [/bib_ref]. However, despite this major advantage, other disadvantages such as insufficient longevity of mRNA-encoded CAR or TCR expression need to be addressed. This drawback also results in higher T-cell demands as repeated administration would be necessary to compensate the reduced half-life of CAR or TCR expression [bib_ref] In vitro-transcribed mRNA chimeric antigen receptor T cell (IVT mRNA CAR T)..., Soundara Rajan [/bib_ref].
In 2020, Parayath and colleagues reported on the use of an injectable nanocarrier to deliver CAR or TCR encoding mRNA directly to circulating T cells, eliminating the need for ex vivo T-cell expansion [bib_ref] In vitrotranscribed antigen receptor mRNA nanocarriers for transient expression in circulating T..., Parayath [/bib_ref]. In this study, using leukemia and prostate cancer mouse models, the nanoparticles were manufactured using poly b-amino ester, which self-assembles into nanocomplexes when interacting with anionic nucleic acids. These nanoparticles specifically targeted CD8 þ T cells by incorporation of an anti-CD8-linked polyglutamic acid. For CAR therapy, the nanoparticles were administered weekly, as the CAR expression lasted up to 8 days. A similar duration of TCR expression was achieved. The authors demonstrated that the use of injectable nanocarrier for mRNA delivery was sufficient to bring disease regression [bib_ref] In vitrotranscribed antigen receptor mRNA nanocarriers for transient expression in circulating T..., Parayath [/bib_ref]. More recently, the successful in vivo generation of CAR-T cells by delivery of modified mRNA packaged into T-cell targeted LNPs was reported. Transient expression and functionality of the CAR were observed [bib_ref] CAR T cells produced in vivo to treat cardiac injury, Rurik [/bib_ref]. Phase I trials in mesothelioma (NCT01355965) and pancreatic cancer (NCT01897415) have been initiated using autologous T cells transfected with mRNA encoding a mesothelin targeting CAR. The use of such mRNA-engineered T cells appeared to be feasible and safe. Early signs of antitumor activity and absence of overt off-tumor ontarget toxicity were observed [bib_ref] Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid..., Beatty [/bib_ref]. A phase I clinical trial using autologous cMet-redirected T cells administered intratumorally in patients with breast cancer (NCT01837602) has shown that cMet-CAR-T cell injections were well tolerated, as no patients experienced above grade 1 adverse events, whereas tumor necrosis, and a consequential inflammatory response, was present when IHC was performed on tumor resections [bib_ref] Safety and efficacy of intratumoral injections of chimeric antigen receptor (CAR) T..., Tchou [/bib_ref].
## Mrna-based modulation of the tumor microenvironment
The intratumoral delivery of therapeutic compounds is an attractive option to increase the in situ bioavailability and, thus, the efficacy of immunotherapies. This applies to compounds targeted to tumor tissue as well as for compounds targeting immune cells that play an important role in immune evasion, such as regulatory T cells, tumorassociated macrophages (TAM), neutrophils (TAN), and immature DCs [bib_ref] Intratumoral immunotherapy: from trial design to clinical practice, Champiat [/bib_ref] [bib_ref] Intratumoural administration and tumour tissue targeting of cancer immunotherapies, Melero [/bib_ref] [bib_ref] Targeting the tumor microenvironment to enhance antitumor immune responses, Van Der Jeught [/bib_ref] [bib_ref] Intratumoral delivery of mRNA: overcoming obstacles for effective immunotherapy, Van Der Jeught [/bib_ref] [bib_ref] Current strategies for intratumoural immunotherapy: beyond immune checkpoint inhibition, Yuan [/bib_ref]. Therefore, delivery of mRNA encoding such compounds can contribute to antitumor immunity, as shown before by delivery of mRNA encoding a fusokine consisting of IFNb and the ectodomain of the TGFb type III receptor [bib_ref] Intratumoral administration of mRNA encoding a fusokine consisting of IFN-b and the..., Van Der Jeught [/bib_ref]. In a study reported by Haabeth and colleagues, charge-altering releasable transporters were used for the intratumoral delivery of mRNA encoding immune modulators [bib_ref] Local delivery of Ox40l, Cd80, and Cd86 mRNA kindles global anticancer immunity, Haabeth [/bib_ref]. In this study, a monotherapy with mRNA encoding for IFNg, IL12, CD70, CD80, CD86, and CD40L was investigated, and in particular a significant tumor growth delay was observed for CD40L, CD80, and CD86, as confirmed also by Van Lint and colleagues through intratumoral delivery of TriMix mRNA [bib_ref] Intratumoral delivery of TriMix mRNA results in T-cell activation by crosspresenting dendritic..., Van Lint [/bib_ref]. More recent studies have published similar results with mRNA formulated in saline solution [bib_ref] Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across..., Hotz [/bib_ref]. mRNA encoding IFNa, IL12 single chain, granulocytemonocyte colony stimulation factor (GM-CSF), and IL15 sushi was administered intratumorally, resulting in an increase of immune cell populations accompanied by intratumoral IFNg induction, systemic antigen-specific T-cell expansion, increased granzyme B þ T-cell infiltration, and formation of immune memory [bib_ref] Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across..., Hotz [/bib_ref]. In another preclinical study, iVT-mRNA encoding IL15 was administered in vivo. This mRNA was complexed using a protamine/liposome system. In both local and systemic administration, the CLLP/IL15 mRNA resulted in significant tumor-inhibitory effects in subcutaneous, abdominal cavity, and pulmonary metastasis models [bib_ref] Efficient colorectal cancer gene therapy with IL-15 mRNA nanoformulation, Lei [/bib_ref].
Several clinical trials using intratumoral delivery of mRNA are ongoing. A phase I study (NCT03739931; [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] is evaluating the intratumoral delivery of LNP-encapsulated mRNA encoding human OX40L, IL23, and IL36g, either as monotherapy or in combination with immune-checkpoint blockade in patients with advanced malignancies. BioNTech is testing the intratumoral delivery of BNT131 or a mRNA mixture encoding IL12 single chain, IFN-alpha2b, GM-CSF, and IL15 sushi as monotherapy and in combination with PD-1 targeting cemiplimab in advanced solid tumors. Another phase I study by eTheRNA in collaboration with VUB-UZ aims to deliver synthetic naked mRNA encoding TriMix intratumorally in early-stage breast cancer (NCT03788083; [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref].
ncRNA for the reduction of expression RNA interference-based therapeutic interventions
All the above-mentioned applications involve the use of mRNA to mediate the expression of immune-boosting proteins. Notably, progressively more studies have focused on RNA as an intermediary, not only for expression but also for the regulation of expression, since RNA interference (RNAi) has been discovered in 1998 [bib_ref] Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Fire [/bib_ref]. The starting feature of RNAi is siRNA, short hairpin RNA (shRNA), or miRNA [bib_ref] Emerging role of RNA interference in immune cells engineering and its therapeutic..., Monty [/bib_ref] [bib_ref] Gene editing and RNAi approaches for COVID-19 diagnostics and therapeutics, Berber [/bib_ref] , which after cleavage by the Dicer enzyme and after association with the RISC/ago2 enzyme complex has the capacity to hybridize with a complementary mRNA strand and results in the cleaving of that strand [bib_ref] Revealing the world of RNA interference, Mello [/bib_ref] [bib_ref] The current state and future directions of RNAibased therapeutics, Setten [/bib_ref]. This was successfully applied in immuno-oncology by Li and colleagues, where siRNA-PD-L1 (siPD-L1) was codelivered with imatinib in liposomal nanoparticles, resulting in the reduced expression of PD-L1, synergistically causing a tumor delay more significant than the monotherapies [bib_ref] Melanoma cancer immunotherapy using PD-L1 siRNA and imatinib promotes cancer-immunity cycle, Li [/bib_ref]. Similar results were achieved in a mouse melanoma model by Wang and colleagues [bib_ref] Polymer-lipid hybrid nanovesicle-enabled combination of immunogenic chemotherapy and RNAimediated PD-L1 knockdown elicits..., Wang [/bib_ref] , indicating that, in combination with chemotherapy, or extracellular targeting, RNAi-mediated cell disruption can significantly promote the antitumor effects of already clinically available cancer treatment [bib_ref] Emerging role of RNA interference in immune cells engineering and its therapeutic..., Monty [/bib_ref]. Moreover, RNAi can as well be exploited beyond the alleviation of inhibitory pathways, but also for TME remodeling on TAMs, TANs, and immature DCs [bib_ref] Folate receptor-targeted RNAi nanoparticles for silencing STAT3 in tumor-associated macrophages and tumor..., Chen [/bib_ref] [bib_ref] Cancer immunotherapy: targeting tumor-associated macrophages by gene silencing, Zins [/bib_ref] and in conjugation with mRNA for DC vaccination [bib_ref] CD83 expression on dendritic cells and T cells: correlation with effective immune..., Aerts-Toegaert [/bib_ref] [bib_ref] Attenuated expression of A20 markedly increases the efficacy of double-stranded RNA-activated dendritic..., Breckpot [/bib_ref]. Although RNAi holds promise, important improvements regarding clinical applications, such as pharmacodynamics and pharmacokinetics, as well as toxicity need to be addressed [bib_ref] The current state and future directions of RNAibased therapeutics, Setten [/bib_ref]. However, improvements regarding toxicity have already been booked with LNP formulations, as toxicity is often due to unintended and on-target off-tissue RNAi activity.
## Beyond rnai
Besides their role in RNAi, small ncRNA, such as miRNA, could act not only as tumor suppressor miRNA (TS-miR), but also as oncogenes (oncomiR), depending on the target [bib_ref] Targeting miR-155-5p and miR-221-3p by peptide nucleic acids induces caspase-3 activation and..., Milani [/bib_ref]. miRNA mimics, classifiable as ASOs can be implemented for anticancer therapy. Around 20-25 bases long, ASOs bind to their miRNA targets, preventing interaction of that miRNA with its target mRNA, and resulting in RNase Hmediated degradation [bib_ref] RNA-based therapeutics: from antisense oligonucleotides to miRNAs, Bajan [/bib_ref]. The use of ASOs has already been demonstrated in a preclinical setting for glioma using anti-miR-21 with miR-21 being on oncomiR, suppressing IL12 (126) for anticancer therapy. Around 20-25 bases long, ASOs bind to their miRNA targets, preventing the interaction of that miRNA with its target mRNA and resulting in RNase H mediated degradation [bib_ref] RNA-based therapeutics: from antisense oligonucleotides to miRNAs, Bajan [/bib_ref]. The use of ASOs has already been demonstrated in a preclinical setting for glioma using anti-miR-21 with miR-21 being on oncomiR, suppressing IL12 (126). Next to ASOs, aptamers have also entered the scope of RNA-mediated immunotherapy. Aptamers possess a small molecular weight, making them suitable for TME entry. Moreover, their longer shelf-life and low immunogenicity, combined with their possibility of cell-free manufacturing, give them advantageous features for clinical applicability [bib_ref] Immune modulatory short noncoding RNAs targeting the glioblastoma microenvironment, Wei [/bib_ref]. In a study by Gao and colleagues, aptamers targeting PD-L1 were developed and validated [bib_ref] Anti-PD-L1 DNA aptamer antagonizes the interaction of PD-1/PD-L1 with antitumor effect, Gao [/bib_ref]. Besides this, aptamers targeting CXCL12 (NOX-A12) and CCL-2 (NOX-E36) have been tested in clinical trials [bib_ref] Aptamers: cutting edge of cancer therapies, Shigdar [/bib_ref] , from which monotherapy of NOX-A12 showed induction of T helper 1 cytokines and resulted in prolonged time on treatment versus prior therapy in 35% of patients with metastatic microsatellite stable colorectal or pancreatic cancer in combination with pembrolizumab [bib_ref] Abstract CT117: phase 1/2 study with CXCL12 inhibitor NOX-A12 and pembrolizumab in..., Halama [/bib_ref]. These studies concluded that aptamers can be considered a valid alternative compared with mAbs, as the production costs are significantly lower and similar tumor inhibition and binding affinity as for mAbs was obtained [bib_ref] Immune modulatory short noncoding RNAs targeting the glioblastoma microenvironment, Wei [/bib_ref].
# Conclusion and perspectives
The SARS-CoV-2 pandemic has unlocked the great potential of mRNA as a therapeutic agent, due to the extreme need for a prompt development of an effective COVID-19 vaccine. This rapid progress was possible only because of the preexisting long-term experience and already developed mRNA technology of the past three decades. Next, the mRNA format's high versatility could push further the implementation of mRNA-based personalized cancer therapies into the clinic, relying on an easily convertible manufacturing process [bib_ref] Neo-antigen mRNA vaccines, Esprit [/bib_ref]. Nevertheless, personalized therapies still require the identification of novel, cancer-specific targets (including neoantigens) for which abundance and immunogenicity studies remain the main challenge. However, improvements in in silico prediction algorithms and nextgeneration sequencing (are expected to address this implementation; refs. [bib_ref] Neo-antigen mRNA vaccines, Esprit [/bib_ref] [bib_ref] Identification of neoantigens for individualized therapeutic cancer vaccines, Lang [/bib_ref].
Despite high versatility, reduced costs, and quick manufacturing of mRNA vaccines, further insights are still required, especially regarding the mechanisms of action and therefore understanding the contribution of the innate immunogenicity of mRNA [bib_ref] mRNA as a transformative technology for vaccine development to control infectious diseases, Maruggi [/bib_ref]. The two SARS-CoV-2 mRNA-based vaccines, BNT162b2 (Tozinameran) and mRNA-1273 (Elasomeran), showed that mRNA chemical modifications and purity play an important role in reducing intrinsic immunogenicity, and this is key for intramuscular injected prophylactic vaccines [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref]. Low intrinsic immunogenicity is also necessary for other RNA therapeutics, where the protein level needs to be as high as possible including antibody encoding mRNA and mRNA-based modulation of the TME [bib_ref] Impact of mRNA chemistry and manufacturing process on innate immune activation, Nelson [/bib_ref]. Furthermore, the use of adjuvants and even the mRNA self-adjuvancy level has not yet been extensively evaluated in terms of potential benefits or adverse effects in mRNA cancer vaccine studies [bib_ref] Three decades of messenger RNA vaccine development, Verbeke [/bib_ref] [bib_ref] Tailoring mRNA vaccine to balance innate/adaptive immune response, Linares-Fern Andez [/bib_ref].
The prompt optimization of LNP for clinical formulations contributed to the success of RNA as a therapeutic agent. However, many parameters need further investigation, such as biodegradability, tissue and cell tropism, long-term side effects, route of administration and delivery, all having a major effect on the overall cost, efficacy, and safety profile of LNP [bib_ref] Lipid nanoparticles for mRNA delivery, Hou [/bib_ref] [bib_ref] COVID-19 vaccines: the status and perspectives in delivery points of view, Chung [/bib_ref].
Regarding RNAi, hereditary transthyretin amyloidosis and acute hepatic porphyria can already benefit from treatment options [bib_ref] Cancer biology functional genomics: from small RNAs to big dreams, Sundara Rajan [/bib_ref]. Nevertheless, for the treatment of cancer, RNAi and ncRNA formulations have not yet been approved. The main challenge here is the scarce delivery of effector molecules in tumor cells to induce a clinically significant response. Two clinical trials (NCT01676259 and NCT01591356) are ongoing, using RNAi targeting KRAS and EPHA2, respectively. Positive results from this work could further accelerate the implementation of RNAi into the clinic [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref].
The use of RNA in passive immunotherapeutic approaches is catalyzed by current results from ongoing clinical trials [fig_ref] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics [/fig_ref] ref. 2). More preclinical studies are necessary to thoroughly investigate RNA kinetics and dosage [bib_ref] mRNA as novel technology for passive immunotherapy, Schlake [/bib_ref].
If, on the one hand, RNA therapeutics boast of a high safety profile (138) due to a transient dwelling time, on the other hand, more frequent administrations are required, which in terms of ACT might hamper the manufacturing process, as T cells are limited. In addition to this, injected T cells might also fail to induce a potent response as expression could be lost before reaching the tumor site. Local administrations could work as an efficient alternative, but to date have been unsuccessful in human clinical trials [bib_ref] Karik o K. The emerging role of in vitro-transcribed mRNA in adoptive..., Foster [/bib_ref].
Altogether, the mentioned developments in the RNA field indicate its potential as an ideal candidate anticancer therapeutic agent, expanding on its current use for antiviral vaccination. Because of its manufacturing benefits, RNA therapeutics could establish a general presence in the drug development industry, going even beyond implementation for cancer immunotherapy.
# Authors' disclosures
K. Thielemans reports a patent for WO2009/034172 issued and licensed to eTheRNA and a patent for WO2021/185833 pending. K. Breckpot reports grants from Research Council VUB, Stichting tegen Kanker, and VLAIO and other support from Oncology Research Center and Scientific Fund Willy Gepts during the conduct of the study. No disclosures were reported by the other authors.
[fig] Figure 1: Overview of coding and noncoding RNA structures. Left, in vitro transcription (iVT) of messenger RNA (mRNA). mRNA has several conserved features, including a 5 0 cap structure, two extended untranslated regions (UTR) at the 5 0 and 3 0 end of the ORF, and a 3 0 poly-A tail. The final iVT product can be mRNA or self-amplifying mRNA. Right, solid-phase synthesis of noncoding and antisense oligonucleotides. Abbreviations: ASO, antisense oligonucleotides; mRNA, messenger RNA; UTR, untranslated region; rNTP, ribonucleoside triphosphates; miRNA, microRNA; siRNA, small interfering RNA. Adapted from an image created with BioRender.com. [/fig]
[fig] Figure 2: Figure 2. Overview of active mRNA-based immunotherapeutic strategies. Left, LNP-antigen mRNA vaccine delivered systemic or locally, followed by antigen expression resulting in T-cell priming and eventually cancer cell killing. Right, monocytes or hematopoietic progenitor cells are isolated from blood, further cultured, and differentiated into DCs. mRNA is then used to load the DCs ex vivo with tumor antigens. The modified DCs are administered to patients, where they will prime T cells, eventually resulting in the killing of cancer cells. Adapted from an image created with BioRender.com. [/fig]
[fig] Figure 3: Overview of passive immunotherapeutic strategies. Left, systemic administration of TCR and CAR LNP-mRNA causes specific uptake by CD8 þ T cells, followed by expression and antigen recognition, resulting in cancer cell death. Top right, ex vivo TCR/CAR LNP-mRNA manipulation of T cells, followed by systemic administration. Bottom right, intratumoral or systemic delivery of LNP-mRNA encoding monoclonal antibodies (mAbs), immune-inducing cytokines, or stimulatory receptors. Abbreviations: TCR, T-cell receptor; CAR, chimeric antigen receptor. Adapted from an image created with BioRender.com. [/fig]
[table] Table 2: Overview of active and recruiting clinical trials using mRNA-based therapeutics. [/table]
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The Role of Autophagy and Related MicroRNAs in Inflammatory Bowel Disease
Accumulating evidence demonstrates that microRNA-(miR-) mediated posttranscriptional regulation plays an important role in autophagy in inflammatory bowel disease (IBD), a disease that is difficult to manage clinically because of the associated chronic recurrent nonspecific inflammation. Research indicates that microRNAs regulate autophagy via different pathways, playing an important role in the IBD process and providing a new perspective for IBD research. Related studies have shown that miR-142-3p, miR-320, miR-192, and miR-122 target NOD2, an IBD-relevant autophagy gene, to modulate autophagy in IBD. miR-142-3p, miR-93, miR-106B, miR-30C, miR-130a, miR-346, and miR-20a regulate autophagy by targeting ATG16L1 through several different pathways. miR-196 can downregulate IRGM and suppress autophagy by inhibiting the accumulation of LC3II. During the endoplasmic reticulum stress response, miR-665, miR-375, and miR-150 modulate autophagy by regulating the unfolded protein response, which may play an important role in IBD intestinal fibrosis. Regarding autophagy-related pathways, miR-146b, miR-221-5p, miR-132, miR-223, miR-155, and miR-21 regulate NF-κB or mTOR signaling to induce or inhibit autophagy in intestinal cells by releasing anti-or proinflammatory factors, respectively.
# Introduction
Inflammatory bowel disease (IBD) is divided into Crohn's disease (CD) and ulcerative colitis (UC). As a chronic nonspecific disease, the recurrent and persistent features of IBD are difficult to comprehensively cure, and the disease has a serious impact on patient quality of life. With the acceleration of urbanization and modernization, the incidence of IBD has increased to more than 0.5% of the total population of developed countries, such as in Europe and the United States [bib_ref] Increasing incidence and prevalence of the inflammatory bowel diseases with time, based..., Molodecky [/bib_ref]. The prevalence of IBD has also risen significantly in Asia, with rates in East Asia more than doubling in the past few decades [bib_ref] Changing epidemiological trends of inflammatory bowel disease in Asia, Ng [/bib_ref]. IBD is generally accepted to be caused by interactions among immune deficiencies, genetic factors, and environmental factors in susceptible populations, though the specific pathogenesis remains unclear [bib_ref] Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in..., Packey [/bib_ref]. Based on recent molecular studies, researchers are recognizing that genetic factors involved in the inflammatory response and immune function and related pathways (including micro-RNAs and autophagy) play important roles in the pathophysiology of IBD.
MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, single-stranded, noncoding RNAs that bind to the 3 ′ untranslated region (UTR) and 5 ′ UTR or partially translated region of a target mRNA [bib_ref] Recent insights and novel bioinformatics tools to understand the role of microRNAs..., Sacco [/bib_ref] , inhibiting transcription of the mRNA by blocking its translation [bib_ref] MicroRNAs: target recognition and regulatory functions, Bartel [/bib_ref]. Wu et al. [bib_ref] MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage..., Wu [/bib_ref] first evaluated abnormal expression of miRNAs in the intestinal tissue of UC patients in 2008, finding a specific miRNA expression pattern: three miRNAs (miR192, miR375, and miR422b) were markedly downregulated and eight (miR16, miR21, miR23a, miR24, miR29a, miR126, miR195, and let7f) notably upregulated in active UC tissues [bib_ref] miRNAs as new molecular insights into inflammatory bowel disease: crucial regulators in..., Xu [/bib_ref]. Since then, further studies have confirmed that miRNAs participate in the occurrence and development of IBD (including inactive UC) through the immune system, related inflammatory pathways, and other pathways. In addition to being related to colon cancer and inflammatory-associated cell senescence, miR-21 expression is significantly increased in intestinal fibroblasts and may participate in the IBD process through NOS2 and CD68 [bib_ref] Macrophages, nitric oxide and microRNAs are associated with DNA damage response pathway..., Sohn [/bib_ref].
Autophagy is a eukaryotic cellular response to starvation, hypoxia, toxicity, other external pressures, or other stimuli. The process involves the formation of a membrane surrounding internal structures, such as organelles and cytoplasmic macromolecules, phagocytosis of intracellular components, and their presentation to the lysosome for degradation and recycling. Although the main function of autophagy is to maintain cellular homeostasis [bib_ref] Eaten alive: a history of macroautophagy, Yang [/bib_ref] [bib_ref] Autophagy and the immune system, Kuballa [/bib_ref] , it also plays an important role in host defense, especially in regulating inflammation [bib_ref] Modulation of inflammation by autophagy: consequences for human disease, Netea-Maier [/bib_ref] [bib_ref] Inflammatory bowel disease, past, present and future: lessons from animal models, Mizoguchi [/bib_ref]. Autophagy defects can lead to a series of problems, including intestinal epithelial dysfunction, innate immune dysfunction, and intracellular pathogen clearance. For example, when autophagy is defective, adherentinvasive Escherichia coli (AIEC) is not addressed by the cell, and proinflammatory cytokine secretion (such as TNF-α and IL-6) increases in the intestinal tract [bib_ref] Defects in autophagy favour adherent-invasive Escherichia coli persistence within macrophages leading to..., Lapaquette [/bib_ref] [bib_ref] Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to..., Lapaquette [/bib_ref].
There is also recent evidence showing that miRNAs regulate autophagy via different molecular pathways and have key roles in IBD [bib_ref] Role of MiRNAs in inflammatory bowel disease, Cao [/bib_ref] [bib_ref] MicroRNAs at the epicenter of intestinal homeostasis, Belcheva [/bib_ref] [bib_ref] The emerging role of miRNAs in inflammatory bowel disease: a review, Chapman [/bib_ref] [bib_ref] Novel players in inflammatory bowel disease pathogenesis, Murphy [/bib_ref]. miRNAs can regulate intestinal autophagy by targeting IBD-relevant autophagy genes such as NOD2, ATG16L1, and IRGM, thus modulating innate intestinal immunity and intestinal epithelial function. These miRNAs are also involved in autophagy by regulating the unfolded protein response (UPR) during endoplasmic reticulum stress, which contributes to IBD intestinal fibrosis. Studies of cellular pathways have found that miRNAs can induce or inhibit intestinal autophagy by regulating NF-κB and mTOR signaling, thereby affecting inflammatory factors and anti-inflammatory or proinflammatory effects [fig_ref] Table 1: Summary of miRNAs that regulate autophagy in IBD [/fig_ref] and [fig_ref] Figure 1: miRNAs regulate cell autophagy via different molecular pathways in the process of... [/fig_ref].
Several review articles to date have discussed the role of miRNAs in IBD [bib_ref] miRNAs as new molecular insights into inflammatory bowel disease: crucial regulators in..., Xu [/bib_ref] [bib_ref] Role of MiRNAs in inflammatory bowel disease, Cao [/bib_ref] [bib_ref] The emerging role of miRNAs in inflammatory bowel disease: a review, Chapman [/bib_ref] [bib_ref] MicroRNAs: how many in inflammatory bowel disease?, Schaefer [/bib_ref] [bib_ref] MicroRNAs in inflammatory bowel disease, Pekow [/bib_ref] [bib_ref] The role of microRNA in inflammatory bowel disease, Dalal [/bib_ref] [bib_ref] Functional role and therapeutic targeting of microRNAs in inflammatory bowel disease, Soroosh [/bib_ref] , However, there are no reviews that focus on the IBD field and on miRNAs that regulate autophagy and associated pathways. Elucidation of the interaction between miRNAs and autophagy in IBDspecific mechanisms will help to explain the occurrence, development, and future molecular targets of IBD therapy. This article aims at summarizing the mechanism of miRNAs related to IBD that regulate autophagy via different pathways and to provide a theoretical reference for further research.
## Ibd-relevant autophagy genes
Widely accepted IBD-relevant autophagy genes, including NOD2, ATG16L1, and IRGM, were identified through genome-wide association studies (GWASs) and subsequent meta-analyses of GWAS and immune chip data [bib_ref] Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease, Jostins [/bib_ref]. These studies have been crucial to elucidating the mechanisms underlying IBD, particularly autophagy and innate immunity in CD and intestinal epithelial barrier dysfunction in UC. They have also provided clues for novel IBD therapeutic strategies.
2.1. NOD2. NOD2 is the first gene thought to predict increased susceptibility to CD. The NOD2 protein is a cytoplasmic receptor that senses bacterial wall peptides and promotes clearance by initiating proinflammatory transcription [bib_ref] Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease, Hugot [/bib_ref]. Because NOD2 signaling can activate autophagy, it has recently been shown that the NOD2 pathway and autophagy are cross-regulated, and this relationship plays a positive role in intracellular bacterial clearance [bib_ref] ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's..., Homer [/bib_ref] [bib_ref] NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen..., Cooney [/bib_ref].
It has also been reported that miR-142-3p effectively inhibits autophagy in a NOD2-dependent manner and downregulates IL-8 mRNA expression [bib_ref] Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p, Zhai [/bib_ref]. There is a negative correlation between NOD2 and miR-320 expression: miR-320 downregulates NOD2 mRNA and protein expression. In IBD, expression of miR-320 in intestinal mucosa is significantly decreased, which may explain the observed rise in NOD2 expression [bib_ref] NOD2 is regulated by Mir-320 in physiological conditions but this control is..., Pierdomenico [/bib_ref] [bib_ref] Expression of NOD2 is increased in inflamed human dental pulps and lipoteichoic..., Keller [/bib_ref]. Researchers have also identified the interaction between miR-192 and NOD2 as possibly being related to the pathogenesis of IBD. miR-192, the most highly expressed UC-associated miRNA, appears to be dysfunctional in the intestinal epithelial cells of patients with IBD. Additionally, miR-192 significantly alters expression of NOD2 mRNA and protein and significantly reduces phosphorylation of NF-κB and expression of IL-8 and CXCL3 [bib_ref] MicroRNAs at the epicenter of intestinal homeostasis, Belcheva [/bib_ref] [bib_ref] NOD2 expression is regulated by microRNAs in colonic epithelial HCT116 cells, Chuang [/bib_ref].
As NF-κB has an inhibitory effect on autophagy (see Section 3.1) [bib_ref] Autophagy and NF-κB: fight for fate, Xiao [/bib_ref] , it may have a key function in the pathogenesis of IBD and the innate immune response of intestinal epithelial cells [bib_ref] NOD2 expression is regulated by microRNAs in colonic epithelial HCT116 cells, Chuang [/bib_ref]. miR-122 regulates the PI3K/Akt/mTOR/ p70S6K pathway in breast cancer and downregulates NOD2 expression [bib_ref] Inhibition of breast cancer cell proliferation and tumorigenesis by long non-coding RNA..., Zhang [/bib_ref]. In CD, miR-122 targets NOD2 and reduces lipopolysaccharide-(LPS-) induced apoptosis by inhibiting NOD2, a process that activates the NF-κB pathway. Because activation of NF-κB has an inhibitory effect on autophagy, miR-122 can play an autophagy-related role in IBD [bib_ref] miR-122 targets NOD2 to decrease intestinal epithelial cell injury in Crohn's disease, Chen [/bib_ref]. In addition, by regulating NOD2 and proinflammatory cytokines, miR-146a may be important in IBD; however, it still needs to be verified whether miR-146a affects autophagy via NOD2 [bib_ref] NOD2-nitric oxide-responsive microRNA-146a activates sonic hedgehog signaling to orchestrate inflammatory responses in..., Ghorpade [/bib_ref].
## Atg16l1. atg16l1
is an important adapter protein in the autophagosome formation that occurs during autophagy [bib_ref] ATG16L1: a multifunctional susceptibility factor in Crohn disease, Salem [/bib_ref]. Some human and animal studies have shown that ATG16L1 dysfunction is closely related to intestinal inflammation in CD [bib_ref] Role of MiRNAs in inflammatory bowel disease, Cao [/bib_ref] [bib_ref] Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine, Cadwell [/bib_ref]. miR-142-3p was recently shown to negatively regulate ATG16L1 in CD colon epithelial cells. Upregulation of miR-142-3p decreases expression of ATG16L1 mRNA and protein, thereby reducing the autophagic activity of related cells [bib_ref] Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p, Zhai [/bib_ref] [bib_ref] miR-142-3p regulates autophagy by targeting ATG16L1 in thymic-derived regulatory T cell (tTreg), Lu [/bib_ref]. AIEC infection can lead to overexpression of miR-93 and miR-106B, inhibition of ATG16L1, and downregulation of ATG5, causing a reduction in autophagosome formation and thus interfering with the autophagy pathway and bacterial clearance [bib_ref] MIR106B and MIR93 prevent removal of bacteria from epithelial cells by disrupting..., Lu [/bib_ref]. In intestinal epithelial HCT116 cells, miR-106b targets ATG16L1 and modulates autophagy, partially through a binding site at the 3′ end of the ATG16L1 3′ UTR. In addition, miR-106aregulated autophagy may also occur in an ATG16L1-independent manner [bib_ref] miR-106b fine tunes ATG16L1 expression and autophagic activity in intestinal epithelial HCT116..., Zhai [/bib_ref]. In ovarian cancer cells, NF-κB transcriptionally upregulates miR-130a expression in response to inflammatory stimuli, and miR-130a can increase levels [bib_ref] A lentiviral sponge for miRNA-21 diminishes aerobic glycolysis in bladder cancer T24..., Yang [/bib_ref] 2014 [bib_ref] Upregulation of miR-21 in cisplatin resistant ovarian cancer via JNK-1/c-Jun pathway, Echevarria-Vargas [/bib_ref] 2018 [bib_ref] MiR-21 promotes ECM degradation through inhibiting autophagy via the PTEN/akt/mTOR signaling pathway..., Wang [/bib_ref] ※ refers to more closely related to IBD; ※※ refers to the most studied in IBD [bib_ref] miRNAs as new molecular insights into inflammatory bowel disease: crucial regulators in..., Xu [/bib_ref] [bib_ref] Role of MiRNAs in inflammatory bowel disease, Cao [/bib_ref] [bib_ref] Functional role and therapeutic targeting of microRNAs in inflammatory bowel disease, Soroosh [/bib_ref]. of p-mTOR and impair LC3-II accumulation, leading to blockade of rapamycin-or starvation-induced autophagy [bib_ref] miR-130a upregulates mTOR pathway by targeting TSC1 and is transactivated by NF-κB..., Wang [/bib_ref]. AIEC also enhances expression of miR-30C and miR-130a by activating NF-κB, resulting in the downregulation of ATG5 and ATG16L1 and inhibition of autophagy. This study also revealed that the signaling pathway through which this occurs may be effective in inducing the proinflammatory cytokine IL-8, leading to defects in autophagy-mediated clearance of AIEC [bib_ref] Crohn's diseaseassociated adherent invasive Escherichia coli modulate levels of microRNAs in intestinal..., Nguyen [/bib_ref].
Vitamin D receptor (VDR) activation downregulates expression of ATG16L1 and its related lysozyme, impairing the antibacterial effect of Paneth cells and resulting in defective autophagic function in intestinal inflammatory cells [bib_ref] Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis, Wu [/bib_ref]. VDR is the target of miR-346, and TNF-α induces miR-346 expression during intestinal mucosal inflammation, which downregulates VDR in the intestinal epithelium and affects autophagy [bib_ref] MicroRNA-346 mediates tumor necrosis factor α-induced downregulation of gut epithelial vitamin D..., Chen [/bib_ref]. Gene set enrichment analysis (GSEA) has demonstrated that miR-20a expression is negatively correlated with the autophagy-lysosome pathway. Indeed, miR-20a regulates several genes related to autophagy and inhibits ATG16L1, BECN1, and SQSTM1 protein expression [bib_ref] MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage..., Liu [/bib_ref]. One study found miR-20a to be significantly increased in the intestinal mucosa of pediatric CD patients [bib_ref] Altered mucosal expression of microRNAs in pediatric patients with inflammatory bowel disease, Béres [/bib_ref].
## Irgm.
Immunity-related GTPase family M protein (IRGM) has been considered to be associated to autophagy since 2006, though its specific molecular association with autophagy remains unclear [bib_ref] Mechanism of action of the tuberculosis and Crohn disease risk factor IRGM..., Chauhan [/bib_ref]. More recent studies have shown that IRGM proteins play important roles in innate immunity against intracellular pathogens (such as CD-associated AIECs) [bib_ref] Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to..., Lapaquette [/bib_ref] [bib_ref] Human IRGM induces autophagy to eliminate intracellular mycobacteria, Singh [/bib_ref]. IRGM-dependent autophagy plays an important role in combating pathogenic AIEC, and the abundance of pathogenic AIEC has been demonstrated to be higher in the intestinal mucosa of CD patients than in that of healthy controls. Furthermore, studies have shown that IRGM regulates autophagy by affecting mitochondrial division [bib_ref] Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria, Singh [/bib_ref]. By regulating IRGM expression, miR-196 may participate in the endogenous fine-tuning of autophagic pathway initiation and in the control of intracellular pathogen degradation in human cells. miR-196 has also been shown to be overexpressed in inflammatory intestinal epithelial cells in CD. This expression downregulates the IRGM protective variant (c.313C) but not the riskassociated allele (c.313T), suggesting that CD-associated risk (T allele) and protective (C allele) haplotypes confer differences in IRGM expression under the control of miR-196. Notably, this regulatory mechanism does not appear to occur in all cell types. Additionally, overexpression of miR-196 can also induce downregulation of LC3II transformation, inhibiting LC3II accumulation. In other words, miR-196 inhibits autophagy from its initiation step. Downregulation or loss of IRGM expression may also impair the control of autophagy (xenophagy) of CD-associated AIEC in cellular replication [bib_ref] A synonymous variant in IRGM alters a binding site for miR-196 and..., Brest [/bib_ref]. increased in inflammatory intestinal epithelial cells [bib_ref] Genetic association and functional role of Crohn disease risk alleles involved in..., Hoefkens [/bib_ref]. ER stress is a cellular process that is triggered by the folding of multiple interfering proteins in the endoplasmic reticulum. This process allows cells to cope with an excess of misfolded or expanded proteins through a complex signaling network known as the UPR [bib_ref] ER stress-induced cell death mechanisms, Sano [/bib_ref] , which can rebalance the ER through an adaptive mechanism involving autophagy. Defects in autophagy invoke the ER stress response [bib_ref] Crohn's disease: NOD2, autophagy and ER stress converge, Fritz [/bib_ref] , promoting autophagy [bib_ref] ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy, Qin [/bib_ref]. XBP1 and ORMDL3 are two genes involved in UPR that are thought to be associated with IBD, and lack of their expression can promote intestinal inflammation [bib_ref] Functional analysis of the impact of ORMDL3 expression on inflammation and activation..., Hsu [/bib_ref] [bib_ref] XBP1 links ER stress to intestinal inflammation and confers genetic risk for..., Kaser [/bib_ref]. Deletion of XBP1 enhances activation of IRE1 and sensitizes intestinal epithelial cells to inflammatory cytokines [bib_ref] XBP1 links ER stress to intestinal inflammation and confers genetic risk for..., Kaser [/bib_ref]. Directly upstream of XBP1, IRE1 is a kinase that can induce activation of JNK signals under ER stress through interaction with TRAF2 [bib_ref] Coupling of stress in the ER to activation of JNK protein kinases..., Urano [/bib_ref]. JNK directly induces formation of autophagosomes by LC3 [fig_ref] Figure 2: Endoplasmic reticulum stress and autophagy [/fig_ref] [bib_ref] Linking of autophagy to ubiquitin-proteasome system is important for the regulation of..., Ding [/bib_ref]. ORMDL3 regulates Ca 2+ uptake in the ER, and ORMDL3 dysfunction results in protein misfolding and increased sensitivity to ER stress. The underlying mechanism of these molecular interactions with regard to intestinal inflammation remains unclear [bib_ref] Autophagy, microbial sensing, endoplasmic reticulum stress, and epithelial function in inflammatory bowel..., Kaser [/bib_ref] [bib_ref] The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular..., Cantero-Recasens [/bib_ref].
## Endoplasmic reticulum stress pathway
## Regulation of mirnas via the er stress pathway.
Studies have shown that miRNAs can regulate ER stress and induce autophagy [bib_ref] Gene expression profiling indicate role of ER stress in miR-23a~27a~24-2 cluster induced..., Chhabra [/bib_ref]. miR-665 has been shown to be upregulated in the intestinal mucosa of patients with IBD and can downregulate expression of XBP1 and ORMDL3 during ER stress, increasing JNK activity and leading to an increase in inflammatory factor-induced apoptosis and autophagy sensitivity.
Additionally, miR-665 may be important in the positive feedback regulation of the miR-665/ER/NF-κB loop, resulting in chronic inflammation of the gastrointestinal tract, but the mechanism requires further validation [bib_ref] Upregulation of miR-665 promotes apoptosis and colitis in inflammatory bowel disease by..., Li [/bib_ref]. Expression of miR-375, which regulates ER stress, is elevated in normal colon tissue and significantly reduced in intestinal inflammation [bib_ref] The role of microRNAs in mitochondria: small players acting wide, Duarte [/bib_ref] [bib_ref] Sa1870 MiR-375 is a key regulator of intestinal homeostasis in response to..., Ambrose [/bib_ref]. However, because its specific mechanism is not clear, involvement of miR-375 in regulating autophagy in IBD through ER stress modulation requires further study. In HeLa cells, miR-346 was found to be induced under ER stress and to modulate autophagic flux. GSK3B has been shown to be the target of miR-346 and to participate in ER stress-related autophagy. miR-346 activates autophagy by interrupting the association between BCL2 and BECN1 in a GSK3B-dependent manner. However, whether this mechanism plays a role in IBD remains to be studied [bib_ref] miR-346 functions as a pro-survival factor under ER stress by activating mitophagy, Guo [/bib_ref]. Additionally, miR-150 may regulate fibroblast autophagy via ER stress and function in IBD intestinal fibrosis. Studies have shown that IRE1a has a splicing effect on XBP1. The IRE1α-XBP1 axis leads to the expansion of the ER, enhancing its ability to secrete extracellular matrix proteins and activate myofibroblasts. An increase in αSMA expression is a characteristic of fibroblast transformation into myofibroblasts. miR-150 inhibits expression of the transcription factor c-Myb (the primary target of miR-150, which can increase expression of αSMA), thereby reducing expression of αSMA to exert an antifibrosis effect. Although IRE1α can directly regulate degradation of miR-150, the loss of IRE1α activity may lead to an increase in miR-150 levels [bib_ref] Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1..., Heindryckx [/bib_ref]. Because the absence of XBP1 enhances activation of IRE1 during endoplasmic reticulum stress [bib_ref] XBP1 links ER stress to intestinal inflammation and confers genetic risk for..., Kaser [/bib_ref] , expression of miR-150 is speculated to be reduced, and any antifibrosis effect is expected to be weakened. miR-150 is expressed in the serum of IBD (UC) tissuesand may thus regulate fibroblast autophagy via ER stress and play a role in IBD intestinal fibrosis [fig_ref] Figure 2: Endoplasmic reticulum stress and autophagy [/fig_ref]. IRE1 interacts with TRAF2 to activate JNK signaling, and JNK directly induces formation of autophagosomes via LC3, leading to enhanced autophagy. The IRE1α-XBP1 axis leads to the expansion of the endoplasmic reticulum, enhancing its ability to secrete extracellular matrix proteins and activate myofibroblasts. XBP1 deficiency in endoplasmic reticulum stress leads to enhanced IRE1 activation, decreased miR-150 expression, and reduced c-Myb inhibition, leading to greater expression of αSMA, expansion of the endoplasmic reticulum, enhancement of extracellular matrix protein secretion capacity, and activation of fibroblast transformation into myofibroblasts, ultimately leading to fibrosis.
## Signaling pathway
4.1. NF-κB, MAPK (JNK), and FOXO3a. NF-κB activates a variety of signaling molecules, including Toll-like receptors and cytokine receptors, which play an important role in inflammation, immune responses, cell proliferation, cell differentiation, and apoptosis [bib_ref] NF-κB: a key role in inflammatory diseases, Tak [/bib_ref]. Autophagy inhibits NF-κB activation, and NF-κB suppresses autophagy [bib_ref] Autophagy and NF-κB: fight for fate, Xiao [/bib_ref]. Autophagy attenuates NF-κB in inflammation, whereas inhibition of autophagy activates NF-κB by blocking the formation of autophagosomes and promoting autophagosome accumulation. Activation of NF-κB also induces the upregulation of p62 and JNK to form a positive feedback loop that inhibits autophagy [bib_ref] Autophagy joins the game to regulate NF-κB signaling pathways, Djavaheri-Mergny [/bib_ref] [bib_ref] NF-κB signaling activation induced by chloroquine requires autophagosome, p62 protein, and c-Jun..., Yang [/bib_ref]. c-Jun N-terminal protein kinase (JNK) is a serine/threonine kinase and mitogen-activated protein kinase (MAPK) in mammals that is a core component of the autophagic signaling pathway. Phosphorylated JNK promotes autophagy through upstream Bcl2 family protein kinases [bib_ref] Colistin-induced autophagy and apoptosis involves the JNK-Bcl2-Bax signaling pathway and JNK-p53-ROS positive..., Lu [/bib_ref]. FoxO3a enhances autophagy in response to environmental stress and activates autophagy by binding directly to the promoters of autophagy-related genes such as LC3, Beclin1, and Atg12. FoxO3a can also be phosphorylated and inactivated by PI3K/Akt. Akt phosphorylation reduces FoxO3a transcriptional activation by inhibiting FoxO3a translocation to the nucleus. Autophagy might be induced by the Akt/FoxO3a pathway to protect cells in the early stage of environmental stress. Autophagy is decreased in the later stage, but FoxO3a expression continues to increase, subsequently upregulating p-Akt expression [bib_ref] FoxO3a regulates inflammation-induced autophagy in odontoblasts, Li [/bib_ref]. Additionally, mTOR enhances the NF-κB signaling pathway by downregulating expression of FOXO3, suggesting an ability to inhibit autophagy [bib_ref] miR-155 controls lymphoproliferation in LAT mutant mice by restraining T-cell apoptosis via..., Rouquette-Jazdanian [/bib_ref]. Studies have shown that miR-146b overexpression activates and upregulates the NF-κB pathway, thereby inhibiting autophagy, improving intestinal epithelial function, reducing intestinal inflammation in dextran sulfate sodium-(DSS-) induced colitis mice, and increasing the survival rate of fatal colitis [bib_ref] MicroRNA-146b improves intestinal injury in mouse colitis by activating nuclear factor-κB and..., Nata [/bib_ref]. Substance P (SP) also increases NF-κB pathway activation in colonic epithelial cells [bib_ref] Substance P enhances NF-κB transactivation and chemokine response in murine macrophages via..., Sun [/bib_ref] [bib_ref] Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via the..., Ramnath [/bib_ref] by regulating expression of miR-221-5p via the MAPK and NF-κB pathways, and miR-221-5p negatively regulates expression of proinflammatory cytokines in colonic epithelial cells by responding to SP. The increased expression of miR-221-5p in human and mouse colitis tissues, which elevates SP expression, suggests a role in IBD autophagy [bib_ref] 196 identification of a novel Substance P (SP)-Neurokinin-1 Receptor (NK-1R) microRNA-221 inflammatory..., Fang [/bib_ref]. Moreover, because they are upregulated in IBD patients and in mouse inflammatory bowel tissues, miR-132 and miR-223 have been reported to be important mediators in IBD pathogenesis. They can inhibit expression of IκBα by negatively regulating FOXO3a, leading to the enhancement of NF-κB signaling and increasing the production of proinflammatory cytokines [bib_ref] MicroRNA-132 and microRNA-223 control positive feedback circuit by regulating FOXO3a in inflammatory..., Kim [/bib_ref]. In another study [bib_ref] Loss of miR-223 and JNK signaling contribute to elevated stathmin in malignant..., Birnie [/bib_ref] , JNK signaling was shown to be upstream of miR-223, and activation of JNK induced miR-223 expression. Therefore, we speculate that JNK signaling is activated in IBD and that it upregulates miR-223 expression, leading to the enhancement of NF-κB signaling and downregulation of FOXO3a. This process plays a role in inhibiting autophagy. In addition, miR-21 is overexpressed in intestinal inflammation and tissue injury, which can suppress autophagy (see Section 3.2). Upregulation of miR-21 has been found in ovarian cancer cells in association with activation of the JNK-1/c-Jun pathway [bib_ref] Upregulation of miR-21 in cisplatin resistant ovarian cancer via JNK-1/c-Jun pathway, Echevarria-Vargas [/bib_ref]. Nonetheless, it remains unclear whether these mechanisms regulate (inhibit) autophagy in IBD.
4.2. mTOR, Akt, PI3K, and PTEN. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase. Inhibition of mTORC1-induced autophagy using genetic or pharmacological methods was first achieved in yeast studies [bib_ref] Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast, Noda [/bib_ref]. In mammals, mTORC1 inhibits autophagy initiation through phosphorylation [bib_ref] mTOR: a pharmacologic target for autophagy regulation, Kim [/bib_ref] [bib_ref] mTOR and autophagy: a dynamic relationship governed by nutrients and energy, Dunlop [/bib_ref]. Akt is an intracellular serine/threonine kinase that is downstream of phosphatidylinositol-3 kinase (PI3K). Phosphorylated Akt (p-Akt) activates mTOR, as reflected by an increase in p-mTOR expression, leading to autophagy inhibition [bib_ref] Selenizing astragalus polysaccharide attenuates PCV2 replication promotion caused by oxidative stress through..., Liu [/bib_ref]. Related studies have shown that miR-155 can increase Akt activation by decreasing expression of SHIP-1, resulting in enhanced mTORC1 expression, and inhibition of miR-155 can reduce the inflammatory reaction in experimental colitis. The PI3K-AKT signaling pathway was strongly activated by sustained overexpression of miR-155 in DHL16 cells [bib_ref] miR-155 controls lymphoproliferation in LAT mutant mice by restraining T-cell apoptosis via..., Rouquette-Jazdanian [/bib_ref] [bib_ref] MicroRNA-155 promotes the pathogenesis of experimental colitis by repressing SHIP-1 expression, Lu [/bib_ref] [bib_ref] miR-155 deficiency protects mice from experimental colitis by reducing T helper type..., Singh [/bib_ref] [bib_ref] Quantitative proteomics reveals that miR-155 regulates the PI3K-AKT pathway in diffuse large..., Huang [/bib_ref]. However, whether miR-155 plays a role in autophagy in IBD remains to be studied. Knockdown of mouse miR-21 reduces the effect of inflammation on tissue damage and increases survival rates in DSS-induced lethal colitis [bib_ref] MicroRNA-21 knockout improve the survival rate in DSS induced fatal colitis through..., Shi [/bib_ref]. miR-21 has also been shown to specifically inhibit autophagy in a variety of diseases. For example, overexpression of Rab11a increases expression of LC3II and beclin-1 in renal ischemia. miR-21 directly inhibits Rab11a expression, thereby inhibiting autophagy, and participates in the Akt/PTEN pathway to inhibit autophagy in primary liver cancer [bib_ref] MicroRNA-21 knockout improve the survival rate in DSS induced fatal colitis through..., Shi [/bib_ref] [bib_ref] MiR-21 inhibits autophagy by targeting Rab11a in renal ischemia/ reperfusion, Liu [/bib_ref] [bib_ref] MiR-21 mediates sorafenib resistance of hepatocellular carcinoma cells by inhibiting autophagy via..., He [/bib_ref] [bib_ref] Targeting miR-21 induces autophagy and chemosensitivity of leukemia cells, Seca [/bib_ref] [bib_ref] Silencing of microRNA-21 confers radio-sensitivity through inhibition of the PI3K/AKT pathway and..., Gwak [/bib_ref]. In contrast, some studies have shown that miR-21 inhibits p-Akt and deactivates mTOR to induce autophagy [bib_ref] A lentiviral sponge for miRNA-21 diminishes aerobic glycolysis in bladder cancer T24..., Yang [/bib_ref] [bib_ref] MiR-21 regulates biological behavior through the PTEN/PI-3 K/Akt signaling pathway in human..., Xiong [/bib_ref]. PTEN, a potential target gene of miR-21, acts as a lipid phosphatase that antagonizes PI3K signaling. miR-21 inhibition increases PTEN protein levels and suppresses AKT phosphorylation [bib_ref] A lentiviral sponge for miRNA-21 diminishes aerobic glycolysis in bladder cancer T24..., Yang [/bib_ref] [bib_ref] MiR-21 promotes ECM degradation through inhibiting autophagy via the PTEN/akt/mTOR signaling pathway..., Wang [/bib_ref]. More research is needed to determine whether miR-21 inhibits or induces autophagy in IBD via these pathways. One study has also demonstrated that miR-106b and miR-93 regulate cell progression by suppressing PTEN and enhancing activity of the PI3K/Akt pathway. Regardless, it remains to be verified whether these miRNAs play the same role in IBD [bib_ref] MiR-106b and miR-93 regulate cell progression by suppression of PTEN via PI3K/Akt..., Li [/bib_ref].
# Conclusion
The important role of miRNA in IBD pathophysiology has been clarified in recent years with increasing molecular evidence on IBD occurrence and development. Current studies on miRNA-related mechanisms in IBD mainly focus on three components: immune homeostasis disorder, intestinal epithelial barrier dysregulation, and autophagy regulation [bib_ref] Role of MiRNAs in inflammatory bowel disease, Cao [/bib_ref]. However, few studies have attempted to identify which miRNA may be more linked to autophagy in IBD, and the functions of miRNAs in the autophagy pathway in IBD have only been addressed with cellular experiments but few in vivo experiments. In addition, the mechanisms of the NOD2 and NF-κB pathways appear to have garnered the most attention. It is likely that other signal pathways will be linked to IBD, including the classic autophagy pathway LKB1/AMPK-PI3K/AKT and beclin-1/bcl-2. Therefore, more studies must be performed in the future to expand these fields. Additionally, although many studies have demonstrated that miRNAs regulate related proteins in the autophagy pathway, only a few studies have investigated the effect of miRNA on autophagy flux. As autophagy is a dynamic biological process, it would be interesting to elucidate the role of miRNAs in autophagic flux.
Study of the molecular expression mechanisms of miR-NAs in IBD may lead to IBD molecular targeted therapy. Given the function of miRNAs in regulating autophagy in IBD pathogenesis, exploring the potential of miRNAautophagy-based therapeutics is also needed. As it has been identified as a new diagnostic/prognostic tool, miR-21 may serve as a therapeutic target in the future. Nonetheless, before application of this basic research to the clinic can occur, many technical issues need to be overcome. For example, a single miRNA can interact with multiple genes or tissues in vivo, and it may be involved in multiple signaling pathways. Using inhibitors may lead to suboptimal side effects by affecting many pathways in different type of cells. Therefore, identification of IBD-associated miRNA autophagic networks and exploration of specific miRNAs as targets for the treatment of IBD is very important. There are two completed phase I trials (NCT00688012 for a single ascending dose and NCT00979927 for multiple ascending doses; drug: SPC3649) funded by Santaris Pharma related to a locked nucleic acid-(LNA-) modified oligonucleotide that specifically inhibits endogenous miR-122, resulting in prolonged dose-dependent reductions in hepatitis C virus (HCV) RNA levels without evidence of viral resistance [bib_ref] Functional role and therapeutic targeting of microRNAs in inflammatory bowel disease, Soroosh [/bib_ref] [bib_ref] MicroRNA-targeting therapeutics for hepatitis C, Baek [/bib_ref].
In summary, both miRNAs and autophagy play an important role in IBD. The effect of miRNA on autophagy is a promising field of study for exploring IBD treatment. A deeper understanding of the complex dialog between miR-NAs and autophagy and their exact roles in IBD may lead to a great success in the future.
## Conflicts of interest
The authors declare that there is no conflict of interest regarding the publication of this article.
# Authors' contributions
[fig] Figure 1: miRNAs regulate cell autophagy via different molecular pathways in the process of IBD. [/fig]
[fig] Figure 2: Endoplasmic reticulum stress and autophagy. [/fig]
[fig] Shiyuan: Wang, Yan Huang, and Cili Zhou have the same rights; Wang, Yan Huang, and Cili Zhou compiled the complete draft and modification of the manuscript; Jimeng Zhao, Luyi Wu, Min Zhao, and Fang Zhang reviewed the literature; Huirong Liu and Huangan Wu conceptualized the study; and all authors approved the final version of the manuscript. [/fig]
[table] Table 1: Summary of miRNAs that regulate autophagy in IBD.SP regulates miR-221-5p expression through the MAPK and NF-κB pathways; miR-221-5p negatively regulates expression of proinflammatory cytokines in colonic epithelial cells in response to SP.Unclear/decreases phosphorylated AKT and deactivates the mTOR NK-1/c-Jun pathway; promotes miR-21 upregulation. miR-21 inhibition enhances PTEN protein levels and inhibits AKT phosphorylation. Inhibits autophagy via the PTEN/Akt/mTOR pathway. [/table]
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Multiple regions of the genome have been associated with the risk of prostate cancer in Caucasians, particularly including several polymorphisms located at 8q24. Region 2 of 8q24 has been repeatedly found to be associated with the risk of prostate cancer among men of African descent, although one study performed in the Caribbean island of Jamaica did not report this finding. In this study, the single nucleotide polymorphism rs16901979, located in region 2 of 8q24, was genotyped in 498 cases of histologically confirmed prostate cancer and 541 controls from the French Caribbean islands of Guadeloupe, where the population is largely of African descent. The AA genotype and the A allele at rs16901979 were associated with elevated risks of prostate cancer (odds ratios [ORs] = 1.84, 95% confidence interval [95% CI] = 1.26-2.69, P = 0.002 and OR = 1.36, 95% CI = 1.13-1.64, P = 0.001, respectively). Following stratification of the patients by disease aggressiveness, as defined by the Gleason score, the pooled genotypes AC + AA were associated with a higher risk of a Gleason score ≥7 at diagnosis (OR = 1.79, 95% CI = 1.17-2.73, P = 0.007). In summary, the A allele at rs16901979 was associated with the risk of prostate cancer in the Caribbean population of Guadeloupe, confirming its involvement in populations of African descent. Moreover, our study provides the first evidence of an association between this variant and the risk of aggressive prostate cancer.
# Introduction
Over the last few years, several genome-wide association studies have been performed on patients with prostate cancer and controls of Caucasian origin. These studies identified more than 70 loci or regions associated with the risk of prostate cancer. [bib_ref] Identification of 23 new prostate cancer susceptibility loci using the iCOGS custom..., Eeles [/bib_ref] Notably, at least five different regions on the long arm of chromosome 8 at 8q24 have been implicated. [bib_ref] Multiple loci on 8q24 associated with prostate cancer susceptibility, Olama [/bib_ref] These 8q24 loci were also studied in various populations of African descent, which have a higher incidence of prostate cancer and a higher associated mortality rate than Caucasian men. Among these loci, markers in region 2 of 8q24 were consistently found to be associated with prostate cancer susceptibility in men of African ancestry; [bib_ref] Multiple independent genetic variants in the 8q24 region are associated with prostate..., Salinas [/bib_ref] [bib_ref] Replication of prostate cancer risk loci on 8q24, 11q13, 17q12, 19q33, and..., Hooker [/bib_ref] in particular, the polymorphism rs16901979 is associated with a higher risk of prostate cancer in African-American men, [bib_ref] Confirmation study of prostate cancer risk variants at 8q24 in African Americans..., Robbins [/bib_ref] [bib_ref] Prostate cancer risk associated loci in African Americans, Xu [/bib_ref] [bib_ref] Multiple regions within 8q24 independently affect risk for prostate cancer, Haiman [/bib_ref] [bib_ref] 8q24 sequence variants in relation to prostate cancer risk among men of..., Benford [/bib_ref] [bib_ref] Evidence for an association between prostate cancer and chromosome 8q24 and 10q11..., Wang [/bib_ref] [bib_ref] Validation of genome-wide prostate cancer associations in men of African descent, Chang [/bib_ref] Afro-Caribbean men (on Tobago island), [bib_ref] Chromosome 8q24 variants are associated with prostate cancer risk in a high..., Okobia [/bib_ref] and West African men (Nigerians). [bib_ref] 8q24 risk alleles in West African and Caribbean men, Murphy [/bib_ref] However, no association was reported between this single nucleotide polymorphism (SNP) and the risk of prostate cancer in men in Cameroon (Africa) or Jamaica (an Afro-Caribbean population). [bib_ref] 8q24 risk alleles in West African and Caribbean men, Murphy [/bib_ref] This discrepancy may be attributable to heterogeneity in African ancestry and/or the relatively small samples ORIGINAL ARTICLE Region 2 of 8q24 is associated with the risk of aggressive prostate cancer in Caribbean men of African descent from Guadeloupe (French West Indies) distribution of the general population and invited to participate. The rate of participation exceeded 95% for both cases and controls. For both groups, inclusion criteria were as follows: no history of (or current) hormone treatment, including treatment with 5-α reductase inhibitors, and at least one parent born on a Caribbean island with a population recognized as being predominantly of African descent. Additional inclusion criteria for controls were normal findings upon digital rectal examination and a total plasma prostate-specific antigen concentration no higher than the 75 th percentile of that of the appropriate age group of African-American men without clinical evidence of prostate cancer. [bib_ref] Age-specific reference ranges for prostate-specific antigen in black men, Morgan [/bib_ref] Information was collected from both patients and controls concerning demographic characteristics, place of birth, parents' place of birth, use of medication, and family history of prostate cancer. Participants were also asked to provide a blood sample. DNA was extracted from leucocytes using a standardized protocol. Eligible cases (n = 498) and controls (n = 537) had a DNA sample available at the time of genotyping. Their ages ranged from 40 to 94 years [fig_ref] Table 1: Characteristics of the cases and controls [/fig_ref]. The clinical characteristics of the prostate cancer cases are also described in [fig_ref] Table 1: Characteristics of the cases and controls [/fig_ref]. The study was approved by the Guadeloupean Ethics Committee for studies involving human subjects and written informed consent was obtained from each participant.
## Single nucleotide polymorphism genotyping
Genotyping was performed by the 5′ nuclease PCR method, using commercially available TaqMan® assays (Applied Biosystems, Foster City, CA, USA). Briefly, the final volume for PCR was 10 µl, which contained 10 ng DNA, 0.25 µl 40× Assay Mix, and 5 µl TaqMan® Universal PCR master mix (Applied Biosystems, Foster City, CA, USA). A first step at 92°C for 10 min was followed by 90 cycles of 92°C for 15 s and 60°C for 1 min. After PCR, end-point fluorescence was measured, and allelic discrimination was carried out using the ABI 7000 Sequence Detector (Applied Biosystems).
## Statistical analyses
Genotypes were tested for consistency with the expected genotype frequencies under Hardy-Weinberg equilibrium in the control population. For the analysis of aggressiveness, the prostate cancer patients were stratified by the aggressiveness of their cancers, as defined by the Gleason score. Specifically, low aggressiveness was defined as a Gleason score <7 and high aggressiveness was defined as a Gleason score ≥7. A case-only analysis was performed. Odds ratios (ORs), as a measure of risk, and 95% confidence intervals (95% CIs) were estimated from logistic regression models with adjustment for age and Caribbean origin. P < 0.05 were considered as statistically significant. All statistical tests were two-tailed and were carried out using StatView version 5.0 (SAS Institute Inc., Cary, NC, USA).
# Results and discussion
We successfully determined the SNP rs16901979 genotype of 489 prostate cancer cases and 534 controls [fig_ref] Table 1: Characteristics of the cases and controls [/fig_ref]. The genotype frequencies for healthy controls were in Hardy-Weinberg equilibrium. The frequency of the A allele in the control population (40%) was not different from the frequencies reported for African-Americans (42.5% 5 and 42% 6,10 ) and was statistically lower than the frequencies reported for West Africans (57% among Cameroonian controls and 50% among Nigerian controls). [bib_ref] 8q24 risk alleles in West African and Caribbean men, Murphy [/bib_ref] The AA genotype and the A allele at rs16901979 were associated with elevated risks of prostate cancer (OR = 1.84, 95% CI = 1.26-2.69, P = 0.002 and OR = 1.36, 95% CI = 1.13-1.64, P = 0.001, respectively) [fig_ref] Table 2: Associations between rs16901979 variants and the risk of prostate cancer [/fig_ref]. This finding is in agreement with the study by Okobia et al. [bib_ref] Chromosome 8q24 variants are associated with prostate cancer risk in a high..., Okobia [/bib_ref] of African-Caribbean men from Tobago, which found a significant association between this AA genotype and the risk of prostate cancer (OR = 1.57, 95% CI = 1.06-2.32, P = 0.02). In contrast, Murphy et al. [bib_ref] 8q24 risk alleles in West African and Caribbean men, Murphy [/bib_ref] reported no association between this variant and the risk of prostate cancer in the population of the Caribbean island of Jamaica. The discrepancies between these three studies cannot be explained by the differences between the frequencies of the A allele in the three Caribbean populations: 42% among the controls from Guadeloupe (this study), 46% among those from Tobago, [bib_ref] Chromosome 8q24 variants are associated with prostate cancer risk in a high..., Okobia [/bib_ref] and 50% among the Jamaican controls. [bib_ref] 8q24 risk alleles in West African and Caribbean men, Murphy [/bib_ref] Thus, the A allele was statistically less frequent in controls from Guadeloupe than in controls from Tobago and Jamaica. By contrast, no difference was found between the controls from Tobago and Jamaica. Yet, despite the similar allele frequencies in Tobago and Jamaica, the significant association with prostate cancer risk in Tobago was not observed in Jamaica. The lack of an observed association in the population from Jamaica may be a consequence of the small numbers of cases (n = 96) and controls (n = 118) included the noted study.
Even if the frequency of the A allele at rs16901979 differs greatly according to the origin of the population, it appears that this variant is largely associated with the risk of prostate cancer. Indeed, it has now been implicated in populations of Caucasian, 5,16-18 African, [bib_ref] Confirmation study of prostate cancer risk variants at 8q24 in African Americans..., Robbins [/bib_ref] [bib_ref] Prostate cancer risk associated loci in African Americans, Xu [/bib_ref] [bib_ref] Multiple regions within 8q24 independently affect risk for prostate cancer, Haiman [/bib_ref] [bib_ref] 8q24 sequence variants in relation to prostate cancer risk among men of..., Benford [/bib_ref] [bib_ref] Evidence for an association between prostate cancer and chromosome 8q24 and 10q11..., Wang [/bib_ref] [bib_ref] Validation of genome-wide prostate cancer associations in men of African descent, Chang [/bib_ref] [bib_ref] Chromosome 8q24 variants are associated with prostate cancer risk in a high..., Okobia [/bib_ref] and even Asian 19,20 origin.
In this study, our comparison of prostate cancer cases with low and high aggressiveness (Gleason score <7 vs ≥7) revealed that the pooled genotypes AC + AA were associated with a higher risk of a Gleason score ≥7 at diagnosis (OR = 1.79, 95% CI = 1.17-2.73, [fig_ref] Table 3: Association between rs16901979 variants and risk of prostate cancer stratified by disease... [/fig_ref]. This is the first report of an association between the SNP rs16901979 and the risk of more aggressive prostate cancer in a population of African ancestry. However, another polymorphism in region 2 of 8q24, broad11934905, has similarly been shown to be associated with an increase in nonorgan confined disease and early biochemical recurrence after radical prostatectomy in African-American men. [bib_ref] Prostate cancer risk allele specific for African descent associates with pathologic stage..., Whitman [/bib_ref] In addition, rs16901979 is associated with the risk of advanced disease in men of European [bib_ref] 8q24 and prostate cancer: association with advanced disease and meta-analysis, Cheng [/bib_ref] and Asian 22 origin. However, other studies did not find any statistically significant difference between aggressive and nonaggressive cases in men of either European or African ancestry. [bib_ref] Multiple independent genetic variants in the 8q24 region are associated with prostate..., Salinas [/bib_ref] [bib_ref] Prostate cancer risk associated loci in African Americans, Xu [/bib_ref] [bib_ref] Common variants in 8q24 are associated with risk for prostate cancer and..., Pal [/bib_ref] Bensen et al. [bib_ref] Genetic polymorphism and prostate cancer aggressiveness: a case-only study of 1,536 GWAS..., Bensen [/bib_ref] recently performed a large case-only study of 1536 candidate SNPs in 1066 African-American men and 1087 European men. They showed that the polymorphisms rs13254738 and rs1456305 in region 2 of 8q24 were associated with prostate cancer aggressiveness in African-American men and that rs6993569 in the same region was associated with prostate cancer aggressiveness in European men. They suggested that the apparent inconstancies between the two populations might reflect ethnic differences in the linkage disequilibrium structure of this region. Nevertheless, this study of a large series of African-American and Caucasian patients provided further evidence that region 2 of 8q24 is important in prostate cancer aggressiveness. In agreement with these results, linkage analyses performed by the International Consortium of Prostate Cancer Genetics found LOD scores (log 10 of the ORs) over 3.0 at 8q24 in families with multiple cases and more aggressive disease. [bib_ref] Inherited susceptibility for aggressive prostate cancer, Isaacs [/bib_ref]
[formula] P = 0.007) ( [/formula]
# Conclusions
We found that the A allele at rs16901979 was associated with the risk of prostate cancer in the population of the Caribbean archipelago of Guadeloupe. Our findings confirm the involvement of the A allele at rs16901979 in populations of African descent, irrespective of their locations or their migration stories (the United States, the Caribbean Islands, and West Africa). Furthermore, we report a positive association between this variant and the risk of aggressive prostate cancer.
# Author contributions
GCT, LM, and OC conceived, designed, and supervised the study. GCT and LM performed the statistical analyses and drafted the manuscript. GCT, LM, MR, CG, and PB participated in data acquisition and management. All authors read and approved the manuscript.
[table] Table 1: Characteristics of the cases and controls [/table]
[table] Table 2: Associations between rs16901979 variants and the risk of prostate cancer [/table]
[table] Table 3: Association between rs16901979 variants and risk of prostate cancer stratified by disease aggressiveness (as defined by the Gleason score)Number of patients with low or high disease aggressiveness as defined by the Gleason score (low: Gleason score<7 and high: Gleason score≥7); b OR adjusted for age and Caribbean origin; c 95% CI. CI: confidence interval; OR: odds ratio [/table]
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Cardiovascular risk factors among Chamorros
Background: Little is known regarding the cardiovascular disease risk factors among Chamorros residing in the United States.Methods:The Chamorro Directory International and the CDC's Behavioral Risk Factor Surveillance System Questionnaire (BRFSS) were used to assess the health related practices and needs of a random sample of 228 Chamorros.Results: Inactivity, hypertension, elevated cholesterol and diabetes mellitus were more prevalent in this Chamorro sample compared to the US average. Participants who were 50-and-older or unemployed were more likely to report hypertension, diabetes and inactivity, but they were also more likely to consume more fruits and vegetables than their younger and employed counterparts. Women were more likely to report hypertension and diabetes, whereas men were more likely to have elevated BMI and to have never had their blood cholesterol checked.
# Background
Cardiovascular disease, which encompasses heart disease, stroke and hypertension, was responsible for 16.7 million deaths in 2003. This number represented one-third of total deaths worldwide. In the United States, cardiovascular disease alone is responsible for approximately 1 in every 5 American deaths. It is currently the number one cause of death in developed countries, and the World Health Organization (WHO) projects that by 2010, cardiovascular disease will also be the major cause of morbidity and mortality in developing countries. Cardiovascular disease is the leading cause of death across all ethnic groups, including Caucasians, African-Americans, Hispanics, American Indians/Alaskan Natives. Among Asian Americans and Pacific Islanders (AAPI) [bib_ref] Deaths: Leading causes for, Anderson [/bib_ref] it is virtually tied with cancer as the leading causes of death. Compared to other ethnic groups, the frequency of cardiovascular disease is relatively low in the AAPI community [bib_ref] Summary health statistics for U.S. adults: National Health Interview Survey, Lucas [/bib_ref]. This has resulted in a relative lack of research of the AAPI populations as a focus group among public health efforts to prevent or delay cardiovascular disease [bib_ref] White paper on the health status of Asian Americans and Pacific Islanders..., Louie [/bib_ref]. The groups within the AAPI community, such as the Chamorro community which is discussed in this paper, have received even less attention. Cardiovascular disease is a major cause of morbidity and mortality within the AAPI community, accounting for over 25% of all AAPI deaths in 2001 [bib_ref] Deaths: Leading causes for, Anderson [/bib_ref]. Complicating the assessment of cardiovascular disease and health promotion interventions in the AAPI community is the way that Census data were collected for the Asian and Pacific Islander community. Prior to the 1990 Census, Asians and Pacific Islanders were aggregated into a single group. While the 1990 Census allowed Asians and Pacific Islanders to select a subgroup for membership, it did not allow respondents to select multiple sub-groups, a particular problem for a community where a high proportion of people are multiracial. For the 2000 Census, it was possible for respondents to select multiple racial categories and thereby more accurately depict their racial heritage . Certain subgroups within the AAPI conglomerate suffer more from cardiovascular disease than others. For example, data collected by the Missouri State Health Department showed that overall, AAPI mortality from heart disease is half the rate of Caucasians, and one-third the rate of African-American. However, closer scrutiny of the AAPI data, reveals that the Hawaiian subgroup, for example, has mortality rates 2.4 times that of Caucasians and 1.6 times that of African-American [bib_ref] Health status of Asian Pacific Americans in Missouri, Kwon [/bib_ref]. The AAPI population is highly heterogeneous. Understanding the specific variations that exist in the backgrounds, cultural practices, and morbidity and mortality rates of AAPI subgroups will facilitate the creation of optimal health interventions that can be "narrow casted" to those AAPI sub-groups known to have the greatest risk of developing premature cardiovascular disease [bib_ref] A debunking of the myth of healthy Asian Americans and Pacific Islanders, Chen [/bib_ref]. Narrowcasting of health promotion messages involves the development of a social marketing campaign that is to be focused on a narrowly defined audience.
Thus to develop a niche social marketing campaign, it is first necessary to understand the audience, the outcomes to be accomplished, and the messages to be delivered toward this end. In this study, Chamorros and cardiovascular disease were the focus of the research team. While Pacific Islanders comprise only 0.3% of the total U.S. population, they are the second fastest growing ethnic group after Asians at-large. The Census Bureau projections indicate that by 2050, the number of Pacific Islanders living in the United States will have tripled. Thus it is imperative that adequate public health information be gathered about this group so that optimal health promotion intervention can be provided to this growing community.
The Pacific Islands are divided into three large geographical regions: Micronesia, Polynesia, and Melanesia.
Chamorros are indigenous people of the Mariana Islands, a sub-division of Micronesia. The vast majority of this ethnic group is found in Guam, an island of the Marianas archipelago, and the continental United States. According to the 2000 Census, of those Chamorros living in the continental United States, more than one-third resided in California, with San Diego County home to the largest Chamorro community outside of Guam. Thus, San Diego is an ideal site for a study of the continental U.S. Chamorro population. The health needs of the Chamorros have often been overshadowed by the larger, more visible AAPI ethnicities. An exhaustive search of the scientific literature yielded little information regarding the health status of the Chamorro people residing on the continental United States, especially in relation to cardiovascular disease [bib_ref] Cancer risk factor assessment among Chamorro women, Nguyen [/bib_ref] [bib_ref] Cancer risk factor assessment among Chamorro men in San Diego, Wu [/bib_ref] [bib_ref] Diabetes management in San Diego's Chamorro community, Wu [/bib_ref] [bib_ref] Health effects of westernization and migration among Chamorros, Reed [/bib_ref] [bib_ref] Guam: Health Needs of Chamorros, Rodriguez [/bib_ref] [bib_ref] Food Sources of Macronutrients in the Diets of Fifth Grade Children on..., Pobocik [/bib_ref] [bib_ref] The Influence of Obesity on the Self-Reported Health Status of Chamorros and..., Pinhey [/bib_ref]. Even in the 2001 Center for Disease Control and Prevention, National Center for Health Statistics (CDC/NCHS) publication, no reliable data were reported for the incidence of cardiovascular disease, even in the larger category of Pacific Islanders [bib_ref] Summary health statistics for U.S. adults: National Health Interview Survey, Lucas [/bib_ref].
In this paper, the authors report on the risk factors for cardiovascular disease that were identified in San Diego's Chamorro community. By documenting such baseline data in this rapidly growing minority group, health care providers and community leaders can identify and create culturally competent, specific interventions for Chamorros.
# Methods
## Subject recruitment
The Center for Disease Control and Prevention's 2001 core Behavioral Risk Factor Surveillance System (BRFSS)and five of 14 optional modules were used to survey participants. The national data gathered using the 2001 BRFSS were used as the reference point to which the data collected from the Chamorro community was compared.
The Chamorro Directory International (a telephone directory composed exclusively of people who self-identified as members of the Chamorro community) was used to reach members of the local Chamorro community. Leaders of the Chamorro Community have maintained the Directory since 1995, updating and reprinting it every 5 years. Data is gathered by word-of-mouth requests for entry information and more recently, by Internet solicitations. As identifiable information is received, every potential entry is contacted to confirm the accuracy of the information and to secure written approval to publish the information. Using a University of California San Diego IRB-approved protocol, all 658 names in the Chamorro Directory Internationalfor the local region were entered into a computerized database. Then a computer-generated call list was created to determine who would be called and in what order they would be called. Calls were made until at least 100 men and 100 women from the Chamorro community had been recruited to the study. The sample size was limited by the modest size of the planning grant ($10,000) that was received to help the community conduct a health assessment. The first adult to answer the phone was invited to complete the survey. The invitation to participate included the IRB-approved verbal informed consent script which included a strong statement of confidentiality. If he/she refused to participate, other household members were invited and likewise consented. Of those households contacted, the individual response rate was 62.8%. To obtain a sample diverse in age and socioeconomic status, calls were made seven days a week, from early morning until late evening. The survey workers were bilingual members of the Chamorro community. The community newspaper and a word-of-mouth campaign by the Pacific Islander Cancer Control Network (PICCN) leaders helped encourage participation in the study.
A midpoint review of the sample's socio-demographic data indicated an under-representation of men and male and female Chamorros less than 50 years of age. To address this problem, the recruitment strategy was modified such that surveyors randomly asked to speak with the oldest adult, youngest adult, or any adult male in the family throughout the remainder of the data collection process. Completed surveys were individually reviewed for specific health problems and risk factors. Based on their perceived needs, participants were mailed a packet of personalized health promotion information such as preventive care and self-help resources. At the end of the project, a second packet was mailed containing a letter describing the project's findings and information regarding the community's most pressing overall health risks and needs. All transactions were mailed first-class, so that undelivered mail could be tracked. Only one packet of the 250 mailed was returned as undeliverable. Three of the 228 participants requested that no packet be mailed. All were given this opportunity.
The collected data were entered into an ACCESS 2000 database and analyzed with SPSS for Windows 10.0. Chi-squared tests were used to establish relationships between socioeconomic characteristics and cardiovascular disease risk factors. A p-value of < 0.05 was considered statistically significant. Risk factors were analyzed by gender, age (< 50 versus ≥ 50), and employment status to determine whether differences existed within those sample subsets.
Frequencies of the eight known risk factors for cardiovascular disease (BMI, physical activity, diet, hypertension, blood cholesterol, diabetes, smoking, and alcohol consumption) were also contrasted with the American Heart Association data.
# Results
## Sample description
This study sample consisted of 100 male and 128 female Chamorros from San Diego County. The male participants ranged in age from 18 to 87 years. Average age for the men was 48.7 years (SD = 17.7); the median, 47.0. The female participants ranged in age from 19 to 88 years. Average age for the women was 54.7 years (SD = 15.2); the median, 57.5. The median education category for this sample group was "1 to 3 years of college," with a range from grade school to at least four years of college. More than half of the study participants withheld information regarding household income, so all results related to income should be interpreted with caution. Of the 49.6% who reported an income, the median household income was "$35,000 -$50,000." Women were much more willing than men to volunteer their income data: 63% of female versus only 33% of male participants reported their income. The vast majority (93%) of participants had healthcare coverage, while a large portion (47%) reported excellent/very good health. Most of the participants were middle-aged (47%), married (61%), and had no children living in the household (62%). Participants' sociodemographic data are presented in [fig_ref] Table 1: Participants' Sociodemographic Data [/fig_ref].
## Eight risk factors of cardiovascular disease 1. body mass index (bmi)
According to the National Heart, Lung and Blood Institute (NHLBI), an ideal BMI ranges from 18.5 to 24.9. Any person with a BMI of 25 to 29.9 is considered overweight, while a BMI of 30 and greater is considered obese . In this study sample, the BMI was not available for 21% of the participants as 28 males and 20 females declined to provide either their height or weight. Of those who reported their height and weight, the average BMI was 27.4 (male 28.0, female 27.0) with a range of 17.2 to 47.7 (male 18.3 -44.1, female 17.2 -47.7) and an overall standard deviation of 5.6 (male 4.4, female 6.3). The majority of study participants were either overweight or obese: 31.1% were overweight while an additional 21.1% were obese [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. Men were significantly more likely than women to be overweight or obese (p < .05). Participants who were employed were much more likely than the unemployed to be overweight or obese: (p < .05). Higher income also correlated with an elevated BMI (0r = .250, p < .05).
## Physical activity
Physical activity was calculated as number of days/week multiplied by amount of time/day a person spent doing either moderate or strenuous physical exercise. The CDC recommends a minimum of 150 minutes of moderate exercise or 60 minutes of vigorous exercise per weekand the American Heart Association's (AHA) recommended 90 minutes of moderate physical activity per week. Among the participants, 66.7% did less than 150 minutes of moderate exercise per week, 67.4% did less than 60 minutes of vigorous exercise per week, and 34% did not exercise at all [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. Participants who were 50 and older or those not employed were at significantly higher risk of failing the AHA guideline (p < .05).
## Diet
Daily consumption of fruits and vegetables was calculated for each participant. Total fruit included fruit and fruit juices, whereas total vegetables included green salad, nonfried potatoes, carrots, and others. The AHA's Food Pyramid recommends eating at least 3 servings of vegetable and 2 servings of fruit per day. Sixty percent of the study participants failed to consume the minimum of 5 servings of vegetable and fruit per day [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. Participants who were younger than 50 or employed outside of the home tended to eat less fruit or vegetable than their older and unemployed counterparts (p < .05).
## Hypertension
Of the 228 study participants, 42.5% reported they had been diagnosed with hypertension, but only 34.2% reported taking medication for their hypertension [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. Females, those aged 50 and over, and those unemployed were diagnosed with hypertension at significantly greater frequencies than their male, younger, and employed counterparts (p < .05). Among the female participants hypertension was associated with less education, but this trend was not seen in the male participants. Less educated women reported hypertension at a much greater frequency than their more educated counterparts (p < .05).
## Blood cholesterol
The AHA recommends that all adults starting at age 20 should obtain a full lipid profile once every five years. Seventy percent of study participants reported compliance, but 27% never had blood drawn for a lipid panel [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. This is especially true among the male participants. Significantly more men than women had never had their blood cholesterol checked (p < .05). Those participants who were male, aged less than 50, and employed were significantly more likely than their counterparts to have never checked their blood cholesterol (p < .05).
## Diabetes
Of those Chamorros surveyed, 16.2% were diabetic, and an additional 2.6% reported gestational diabetes [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. The mean age of diagnosis for men was 58.0 years (median age 60, SD = 11.3) and 46.1 years for women (median 50, SD = 9.1). Participants who were female, *Not married -divorced, widowed, separated, never married, member of an unmarried couple **Unemployed -out of work, homemaker, student, retired, unable to work ***Health coverage -health insurance, prepaid plans, or government plans such as Medicare aged 50 and above, and not employed were significantly more likely to have diabetes than those who were male, younger than 50, and employed (p < .05).
## Tobacco use
Fifteen percent of study participants were smokers. Half (51%) of those who smoked had tried to quit within the last year [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref]. The average age for first time smoking cigarettes was 14.9 years (± 4.2 SD) for men and 19.1 years (± 7.8 SD) for women. The average age when they first started smoking regularly was 16.8 years (± 4.2 SD) for men and 20.9 years (± 5.9 SD) for women. Forty-one participants (21 men and 20 women) reported smoking at least 100 cigarettes in their lifetime but were current nonsmokers. The survey also queried about smoking restrictions encountered at home and in the workplace. Within the home, 83% of the 35 smokers indicated they could not smoke inside the house at all, 3% could smoke in certain designated areas or at certain times, 6% could smoke anywhere, and 8% had no rules about smoking inside the home. Of those same 35 smokers, 57% worked indoors, and 80% of these indoor workers were prohibited from smoking in public and work areas.
## Alcohol consumption
The daily consumption of alcoholic beverages was calculated for each participant as the number of days per month in which one had at least one drink, multiplied by the average number of drinks consumed on each of those days, then divided by 30 to obtain a daily average. The AHA recommends drinking should be limited to an average of one to two drinks per day for men, and one drink per day for women. Out of the 228 participants, only four averaged more than one drink per day, and most (63.6%) had not drunk at all in the month preceding the survey [fig_ref] Table 2: Cardiovascular Disease Risk Factors [/fig_ref].
## Cumulative risk factors
The mean number of cumulative cardiovascular disease risk factors (CRF) for this study population was 2.01 per person. As seen in [fig_ref] Table 3: Chamorro Cumulative Risk Factors [/fig_ref] , 35% had at least three CRF. Participants who were 50 years and older, unemployed, *Body Mass Index (BMI) = weight (lbs)/height (in) 2 * 703 **Smoker -one who has smoked at least 100 cigarettes (5 packs) in his/her lifetime and who is smoking some days or everyday. ***Non-smoker -one who has not smoked at least 100 cigarettes (5 packs) in his/her lifetime and is not currently smoking some days or everyday.
and less educated reported substantially higher CRF than their younger, employed, and more educated counterparts (p < .05). [fig_ref] Table 4: Weight Control [/fig_ref] shows that more than half of the study participants were actively managing their weight: 25% were trying to lose weight, 25% were trying to maintain current weight, and 5% responded "yes" to both options. Women were more likely than men to report that they were trying to lose or maintain their weight (p < .05
## Weight management
# Discussion
Cardiovascular disease is the number one cause of death in the U.S., making it a major national health threat with far-reaching consequences. Since individual health behaviors are highly predictive of disease development and mortality, an effective disease prevention campaign begins with an assessment of modifiable disease behav- iors. This study provides an assessment of the cardiovascular disease-related health behaviors of San Diego's Chamorro population. The data can be used by health care providers, public health advocates, and indigenous community leaders to develop cardiovascular disease prevention and intervention strategies that are culturally specific, sensitive, and appropriate to this AAPI ethnic subgroup.
While the data in this study has not been age-adjusted, compared to state and national datathe Chamorros in this study appeared more susceptible to four of the eight risk factors: low physical activity, hypertension, high blood cholesterol, and diabetes. Chamorros (66.7%) appear to be less likely to adhere to the CDC's weekly recommendation for moderate exercise compared to state (53.3%) and national (52.8%) averages. Hypertension and diabetes were present among the Chamorros at an alarming rate. Chamorros in this study reported hypertension (42.5%) at a much greater frequency than other Californians (23.4%) and nation-wide numbers (24.8%).
The prevalence of diabetes among Chamorros (16.2%) was more than twice the state (7.2%) and national (7.1%) figures. Gestational diabetes among Chamorros (2.6%) reflected similarly disproportionate figures compared to the state (1.1%) and national (0.7%) rates. In addition, Chamorro women reported high blood cholesterol (40.6%) more frequently than the average Californian woman (30.5%) and national average (32.1%). In contrast, the prevalence of high blood cholesterol was slightly lower in Chamorro men (31.0%) than their state (35.1%) and national (33.8%) counterparts.
As expected, the sociodemographic characteristics of age, gender, and employment status were often aligned with the behavioral risk factors. Participants who were 50 years and older and unemployed tended to report hypertension, diabetes, and low physical activity, but they also consumed more fruits and vegetables than their younger and employed counterparts. Women were more likely than men to be hypertensive and diabetic; but men were more likely than women to have an elevated BMI and to have never had their blood cholesterol checked. Men were more likely than women to have set a optimal weight goal that would result in an unhealthy BMI. These are clearly opportunities for interventions by primary care health providers, public health policy makers, the media, and community leaders. Women started smoking later than men, at an age (19.1 years ± 7.8 SD) which coincides with the beginning of gynecology care. This may indicate a role for gynecologists in the primary prevention of smoking in Chamorro women. Conversely, the fact that male participants started smoking at a much younger age (14.9 years ± 4.2 SD) indicates the need for earlier intervention in the male Chamorro population.
Among the eight behavioral risk factors examined, BMI is considered to be of great importance due to its relationship to the other risk factors. There has been discussion within the scientific community as to whether the BMI used in the U.S. is applicable to other ethnic minority groups. Some research suggests that there are bone size differences among various ethnic groups that make the simple calculation using height and weight recordings less accurate than a calculation that includes bone size considerations. Further, while these studies suggest that ethnic specific cut-off points for BMI would be more appropriate, there are no widely accepted standards for Pacific Islanders. Therefore, for this community health assessment, the standard BMI cut-off point was necessarily used.
Reducing BMI has also been shown to reduce one's risk of developing diabetes, hypertension, and elevated blood cholesterol. Given the immense health benefits of BMI reduction to patients, it is important to ensure patients' related health knowledge and access to supportive services. If health professionals can broach and discuss the topic in a culturally sensitive manner, patients will be more receptive to recommendations and also more likely to cooperate. When helping patients plan a weight loss program, it is important to set attainable goals. The concept of an ideal weight is inherently related to ideal body image, which in turn is determined partly by culture. It is a well-known fact that body image varies widely among cultures. Fat and obesity carry a powerful stigma in modern-day Western society, but are held in high regard among many of the Pacific Islander traditional cultures [bib_ref] The relationship between ethnicity and obesity in Asian and Pacific Islander populations:..., Davis [/bib_ref]. Among Hawaiians and Samoans, for example, corpulence was dignifying and indicative of wealth [bib_ref] Do Polynesians still believe that big is beautiful? Comparison of body size..., Craig [/bib_ref] [bib_ref] Development of a community-based diabetes management program for Pacific Islanders, Wang [/bib_ref]. A cross-cultural comparison between Australian and Samoan women indicated overweight Samoan women felt healthier and more attractive than their Australian equivalents. One study comparing body size perceptions between different ethnic groups living in New Zealand revealed that Pacific Islanders most frequently underestimated their body weight [bib_ref] Ethnic differences in perceptions of body size in middle-aged European, Maori and..., Metcalf [/bib_ref]. These findings may indicate that among Pacific Islanders, being overweight or obese is more socially acceptable. Since continental US Chamorros are descendants of the Pacific Island heritage, they may also possess this cultural propensity for obesity. To be effective counselors, health professionals should first determine how the patient perceives his or her own body weight currently, and then ascertain that an "ideal weight" in the clinical sense matches the patient's expectations.
Once a patient's weight and fitness goals have been established, the next step is to devise a weight loss and exercise program that will fit into the community's cultural structure. An earlier paper that assessed this community's risk for diabetes began to address the cultural challenges health care providers will need to be sensitive to, as they strive to help the community improve its over all health status [bib_ref] Diabetes management in San Diego's Chamorro community, Wu [/bib_ref]. Diet has an important cultural context among Chamorros. Researchers have cited Western influence on the native Chamorro's diet as a cause of adverse health effects. A study of children's diet in Guam documented a drastic change from the traditional vegetarian and fish diet to a Westernized, meat-based diet consisting of fatty and processed food [bib_ref] Food Sources of Macronutrients in the Diets of Fifth Grade Children on..., Pobocik [/bib_ref]. Chamorro populations with increasing Western influence were found to eat a higherfat diet and suffer higher coronary heart disease mortality [bib_ref] Health effects of westernization and migration among Chamorros, Reed [/bib_ref]. Chamorros living in the continental US also experience this "Westernization." Health educators can take advantage of cultural pride by encouraging Chamorro patients to modify their diet toward the more traditional ethnic diet of plants and fish [bib_ref] Diabetes management in San Diego's Chamorro community, Wu [/bib_ref]. It has been postulated that Westernization has made a greater impact on Pacific Islanders' diet and lifestyle than it has on their cultural perspectives on body image [bib_ref] The relationship between ethnicity and obesity in Asian and Pacific Islander populations:..., Davis [/bib_ref]. The basis for this statement arises from a study of Samoans' body size perception where, although the Samoan defined thinner bodies as ideal, which reflected a definite Western influence, they were not overtly concerned with striving for this ideal [bib_ref] Perceptions of body size in Pacific Islanders, Brewis [/bib_ref]. Overweight individuals were quite satisfied with their large body size, and obese Samoan women were no more likely to try and lose weight than their thinner counterparts. The same may also hold true for continental US Chamorros, and the resulting health effects can be detrimental.
The Chamorro leaders who helped to initiate this community health assessment have demonstrated their ability to undertake a campaign to improve the health of their community. Through focus groups and community town hall meetings, as well as the exchange of information with other Chamorro communities, these same leaders can next begin to evaluate the universality of these findings. Where appropriate, they can also begin to develop strategies to address identified problems or develop plans for further investigation. The interventions they develop are likely to be closely aligned with the values and social fabric of their community. To be optimally resourceful in their program development, Chamorro leaders will need to call upon available public health educators and clinicians to help them interpret the latest medical literature to assure that the health promotion goals they set are grounded on evidence-based research.
# Limitations
This study is only the first step in the process of understanding how to optimize the health and well being of the Chamorro community. This study has definite limitations as regards the generalizability of the findings, including the potential for self-selection bias (62.8% response rate), the use of self-reported information that could not be ver-ified in person, the small sample size, the unwillingness of those contacted to report about household income and body mass index and the narrow geographic eligibility for the sample. However, as an exploratory study, the information gathered and analyzed can lay the groundwork for the development of future studies and testable interventions to address those behaviors which increase this community's risk of cardiovascular disease.
## Lessons for the international scientific community
This baseline health risk assessment for the Chamorro community was initiated by the leaders of the local Chamorro community to identify and address the factors that contribute to improving and diminishing the health of their community. It is a demonstration of a community-campus partnership in action. Through this collaboration with members of the local scientific community, the Chamorro leaders now have preliminary knowledge to help their members gain more personalized insight into their cardiovascular risk status and how to improve it. In turn, the University and the Chamorro community leaders now have a solid partnership that they can use as a foundation for future mutually beneficial undertakings.
# Conclusion
Healthcare providers play a crucial role in the crusade against cardiovascular disease. Their frequent interactions with patients give them the opportunity to alter the course of this deadly illness by stressing behavioral risk reductions and other prevention strategies. Increasing awareness of the health needs of the Chamorro community and providing culturally competent intervention will enhance health providers' effectiveness as counselors with this ethnic group. Chamorro leaders have an equally important role to play in guiding their community toward the adoption of optimally healthful life styles. This paper presents baseline cardiovascular disease-related health data specific to the continental U.S. Chamorro population. It also relates various Pacific Islander cultural values and practices that may affect Chamorro patients' responsiveness to health professionals.
Treltas, Thomas Guzman, Thomas Cruz, Joaquin Naputi, Jeanette Perez, Jess L.G. Perez, Julita Santos, Jess Quitugua, John Fejeran, Angelina Mummert, and Dennis Rodriguez.
Dr. Man Li Shi contributed to the conceptualization and data collection and organization for this project. Elizabeth Gilpin, M.A., director of the Biostatistics Unit of the Moores UCSD Cancer Center offered statistical guidance. In addition, the authors thank Drs. Allan Hubbell and Maria Reyes of UCI for their help with this project. Most importantly, the authors acknowledge the 228 men and women from the Chamorro community who generously gave of their time to help assess the health and well being of San Diego's Chamorro community.
[table] Table 1: Participants' Sociodemographic Data [/table]
[table] Table 2: Cardiovascular Disease Risk Factors [/table]
[table] Table 4: Weight Control [/table]
[table] Table 3: Chamorro Cumulative Risk Factors [/table]
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The effect of Melissa officinalis syrup on patients with mild to moderate psoriasis: a randomized, double-blind placebo-controlled clinical trial
Objective: Psoriasis is an immune-mediated inflammatory skin disease. It can involve any body skin area, particularly the scalp, lower back, elbows, and knees. There are several topical and systemic therapies for the treatment. Nowadays, herbal medicines are popular treatments for dermatologic conditions. This two-arm parallel, randomized placebo-controlled clinical trial was conducted to examine the hypothesis of the efficacy of Melissa officinalis syrup on patients with mild-to-moderate Plaque psoriasis.Result: Among 100 patients, 95 participants completed the trial and five of them withdrew. The mean pruritus intensity and PASI scores decreased significantly in the intervention group compared to the placebo group (P < 0.001). The DLQI score in the intervention group increased post-treatment compared to pre-treatment (P = 0.029); however, there was no significant difference between the intervention and control group at the end of the study (0.065).
# Introduction
Psoriasis is defined as an immune-mediated inflammatory disease. Based on the World Health Organization reports, it is estimated that at least 100 million people have psoriasis worldwide. Skin manifestations are more prominent symptom of psoriasis. It can affect any skin area, particularly the scalp, such as topical, systemic, and biologic therapies for psoriasis treatment [bib_ref] Systemic pharmacological treatments for chronic plaque psoriasis: a network meta-analysis, Sbidian [/bib_ref] [bib_ref] The inflammatory response in psoriasis: a comprehensive review, Deng [/bib_ref] [bib_ref] S3-guidelines on the treatment of psoriasis vulgaris (short version), Nast [/bib_ref]. Nowadays, many patients intend to use complementary and alternative medicine (CAM) due to low efficacy and side effects of conventional therapies [bib_ref] Complementary and alternative medicine therapies in acne, psoriasis, and atopic eczema: results..., Magin [/bib_ref]. It is estimated that 43-69% of patients with psoriasis use different modalities of CAM [bib_ref] Complementary and alternative medicine for psoriasis: a qualitative review of the clinical..., Smith [/bib_ref] [bib_ref] Use of alternative treatments by patients with psoriasis, Clark [/bib_ref] , which herbal medicines are the most popular [bib_ref] Complementary and alternative medicine for psoriasis: what the dermatologist needs to know, Talbott [/bib_ref].
Melissa officinalis is one of the herbs that are theoretically effective and safe. It has traditionally been applied in skin disorders such as eczema and skin inflammation. Melissa officinalis syrup is a mixture that contains four ingredients of Damask rose (Rosa damascena), Fennel (Foeniculum vulgare), Lemon balm (Melissa Officinalis), and honey. This syrup is formulated based on the Traditional Persian Medicine (TPM) texts.
While it is supposed that Melissa officinalis syrup is a compelling mixture for the improvement of psoriasis in theory, but the evidenced-based efficacy of it was unclear. Therefore, this randomized placebo-controlled clinical Yargholi et al. trial was designed to investigate the effectiveness of this formula on patients with psoriasis.
## Main text
# Methods
The present study is a 12-week, randomized, doubleblind, parallel control trial carried out in the Skin and Stem Cell Research Center affiliated to Tehran University of Medical Sciences between November 2019 and May 2020. The trial was registered at the Iranian registry of clinical trials on November 9th, 2019 (https:// www. irct. ir/ trial/ 43434; registration number: IRCT20191104045326N1). A written consent form was taken from eligible patients before recruitment in the trial.
## Participants
We recruited patients with mild to moderate psoriasis, aged over 18 years old. Participants with the following conditions were included: a Psoriasis Area and Severity Index (PASI) of < 30%, and a body surface area (BSA) of < 10%, clinically diagnosed Plaque psoriasis, controlled psoriasis during last two months, not using Psoralen and phototherapy during the past 4 weeks, not participating in other studies during the previous month and during the study. Patients were excluded if they (1) had different types of psoriasis such as psoriatic arthritis, pustular psoriasis, erythrodermic psoriasis, etc. (2) used corticosteroids and immunosuppressive drugs and systemic therapies during the last 4 weeks, (3) using some medications that may intensify psoriasis including beta-blockers, Calcium channel blockers, non-steroidal anti-inflammatory drugs, lithium, etc. (4) patients with cardiovascular and cancer diseases, (5) allergy to any components of syrup, (6) pregnancy and lactation, (7) Acute progressive psoriasis and erythroderma tendency.
## Sample size estimation
Based on previous studies and considering the following formula as the sample size equation for comparing two populations, p1 = 15%, p0 = 40%, α = 0.05, β = 20%, the sample size was estimated at 45 individuals. Finally, considering a 10% loss to follow-up, 50 individuals were recruited in each group.
## Randomization and intervention
Participants were randomly assigned to the intervention or control group using the random number sequence function in Excel and a 1:1 allocation ratio. Patients
[formula] N = 2 Z 1−α/2 + Z 1−β 2 × p(1 − p) (po − p1) 2 P = po + p1 2 . [/formula]
in the intervention group consumed two tablespoons (each tablespoon = 10 ml) of Melissa officinalis syrup three times per day (two tablespoons before each meal of breakfast/ lunch/ dinner) and patients in the control group received the same amount of placebo.
## Drug preparation
Melissa officinalis syrup contained 3 g of Lemon balm, 8 g of Damask rose, and 1 g of Fennel. Firstly, the decoction of these herbs was prepared. The herbs and 450 ml water were added to a pot and heated to boil gently. It boiled until the volume is reduced to 250 ml. While boiling, some honey was added to sweeten it. The placebo syrup contained sugar, caramel coloring additive, and 1% rose water. The pharmacy department of traditional medicine college prepared both Melissa officinalis syrup and placebo. There is no detectable difference between Melissa officinalis syrup and placebo syrup in color, odor, viscosity, and containers. Patients were monitored by a practitioner and they were able to ask their questions anywhere and anytime by telephone or face-to-face in the clinic. The duration of intervention was 12 weeks and patients were checked during this period for any potential side effects.
## Total phenolic and total flavonoid assay
The total phenolic content was determined by using the Folin-Ciocalteu assay and was expressed as mg Gallic acid Equivalents (GAE). Total flavonoid content was measured by the aluminium chloride colorimetric assay and was expressed as mg quercetin equivalents (QE) [bib_ref] Phytochemical profiling and ameliorative effects of Achillea cretica L. on rat model..., Bina [/bib_ref]. Total phenolic and flavonoid contents of each ml of syrup were estimated to be equivalent to 185.22 ± 0.71 mg gallic acid and 103.64 ± 1.32 mg quercetin, respectively.
## Acute toxicity study
Wistar rats were used for the assessment of acute toxicity of syrup [bib_ref] In vitro and in vivo antidiabetic activity of Tamarix stricta Boiss.: role..., Bahramsoltani [/bib_ref]. Considering the results, the syrup shows no acute toxicity up to the dose of 5 ml/kg. This dose is corresponding to 0.81 ml/kg in human [bib_ref] Dose translation from animal to human studies revisited, Reagan-Shaw [/bib_ref].
## Outcomes
The tools used for data collection were a socio-demographic questionnaire, Psoriasis Area and Severity Index (PASI), Life Quality Index (DLQI) questionnaire, and Visual Analogue Scale (VAS). Photographs of each subject was taken at the baseline, during, and end of study. PASI combines the severity of lesions and the body surface area involvement into one score. It has a range score from 0 (no disease) to 72 (maximum severity of the disease) [bib_ref] The reliability of three psoriasis assessment tools: psoriasis area and severity index,..., Bożek [/bib_ref]. DLQI questionnaire is designed for adult patients with skin diseases and is a 4-point Likert scale questionnaire that each question gets a score from zero (not at all) to three (very much). The total DLQI score range from 0 to 30, that higher scores represent higher impairment of quality of life [bib_ref] Dermatology life quality index (DLQI)-a simple practical measure for routine clinical use, Finlay [/bib_ref]. A VAS scale measured the pruritus intensity. Patients draw a line on 0-10 cm VAS scale to determine their itching level at the beginning and end of the study. It is a reliable and valid instrument that is most widely used in epidemiologic and clinical trials. It has a range score from 0 to 10, of which 0 expresses no pruritus and 10 depicts intense pruritus [bib_ref] Assessment of pruritus intensity: prospective study on validity and reliability of the..., Phan [/bib_ref].
## Statistical analyses
We used SPSS version 19 (SPSS Inc., Chicago, IL, USA) for windows to perform data analysis. P values under 0.05 were regarded as significant. Categorical data were expressed in percentage (%), while continuous data were expressed in mean ± SD in evaluating patients' demographic details. Chi-square or Fisher exact test was used for the categorical data and Independent Samples t-Test was used for continuous data to show the differences. For comparison of PASI, DLQI, and VAS parameters between intervention and placebo groups, an independent t-test was applied at the first and end of the study. A paired sample t-test was also used to compare the mean of variables at pre and post-treatment in each group.
# Results
Among 215 patients examined for eligibility, one hundred participants met the inclusion criteria that were randomized into intervention (n = 50) or placebo (n = 50) groups. At the end of follow-up, five patients (3 in the intervention group and 2 in the placebo group) withdrew and 95 individuals completed the trial [fig_ref] Figure 1: Flow diagram of the study in the placebo group at the end... [/fig_ref]. There was no significant difference between intervention and placebo groups in terms of baseline variables [fig_ref] Table 1: Demographics characteristics of participants at the baseline of study [/fig_ref]. Results indicated that no notable differences in any scales between the two groups were found at the study's baseline (P > 0.05). The mean pruritus intensity score decreased significantly from 6.34 ± 2.68 at the baseline of the study to 3.45 ± 2.18 at the end of followup (P < 0.001), which was considerably lower than the placebo group (P < 0.001). While pruritus intensity score did not change significantly in the placebo group (P = 0.886). Moreover, patients in the intervention group had significantly lower PASI score than patients [fig_ref] Table 2: Results of itch score, PASI score, and DLQI questionnaire between intervention and... [/fig_ref].
# Discussion
This trial indicated that Melissa officinalis syrup could significantly improve the treatment of psoriasis through reducing scores of PASI and itch in patients with mild to moderate severity.
The first-line of psoriasis treatment is usually topical treatments such as vitamin D analogues and corticosteroids [bib_ref] Topical treatments for scalp psoriasis, Schlager [/bib_ref]. Furthermore, some topical herbal medicines such as aloe vera, cayenne, chamomile, turmeric, and flaxseed oil can likely have beneficial effects on psoriasis management [bib_ref] Herbal remedies: A new era for psoriasis diseases, Sahu [/bib_ref]. besides, TPM recommends several topical remedies, including oil of wheat germ, flaxseed, black seed, and violet [bib_ref] Psoriasis and topical Iranian traditional medicine, Atyabi [/bib_ref].
In addition to topical use, some herbal medicines are used as oral administration. A review of clinical trials was conducted by May and colleagues to assess herbal medicines' systemic use in psoriasis. They identified 12 clinical trials, that eight of them were multi-ingredient herbal formulations [bib_ref] Oral herbal medicines for psoriasis: a review of clinical studies, May [/bib_ref]. Moreover, a meta-analysis by Zhang et al. indicated that oral Chinese herbal medicines could be useful in the treatment of psoriasis [bib_ref] Is oral Chinese herbal medicine beneficial for psoriasis vulgaris? A metaanalysis of..., Zhang [/bib_ref]. TPM recommend Melissa officinalis syrup, which contain three herbs of Lemon balm, Fennel, and Damask rose.
Lemon balm (Melissa officinalis) is one of the wellknown herbal medicines used in traditional medicine all over the world [bib_ref] Melissa officinalis L.-a review of its traditional uses, phytochemistry and pharmacology, Shakeri [/bib_ref]. Dimitris et al. carried out an experimental study to investigate the effects of Melissa officinalis on psoriasis in mice. They concluded that dichloromethane extract and decoction of Melissa officinalis could prevent epidermal hyperplasia and scaling, while methanol extract did not have a significant effect. A valuable finding was that the decoction was more effective as compared to dichloromethane extract that approved the traditional use of lemon balm decoction as an anti-psoriatic [bib_ref] ssp altissima extracts: a therapeutic approach targeting psoriasis in mice, Dimitris [/bib_ref]. Damask rose (Rosa damascena) is an herbal medicine used in TPM for different conditions, including abdominal pain, digestive disorders, wound healing, and skin health [bib_ref] Pharmacological effects of Rosa damascena, Boskabady [/bib_ref]. Shabikin et al. found that topical rose hip seed oil combined with oral fat-soluble vitamins could be useful in different inflammatory dermatitis such as eczema, neurodermatitis, and cheilitis [bib_ref] A polyvitamin preparation of fat-soluble vitamins (carotolin) and rose hip oil in..., Shabykin [/bib_ref].
Fennel (Foeniculum vulgare) is a well-known medicinal plant and have had a widespread application traditionally in different conditions such as respiratory and gastrointestinal problems, arthritis, and mouth ulcer in many parts of the world. An experimental study demonstrated that Fennel has anti-inflammatory and antimicrobial effects and enhance synthesis of collagen, which might be useful in wound healing. Furthermore, Fennel is a rich source of phytoestrogens, which are able to promote collagen and water content and protect the skin from oxidative stress [bib_ref] Recent advances in the anti-aging effects of phytoestrogens on collagen, water content,..., Liu [/bib_ref].
How this formulation could improve psoriasis symptoms is not entirely known. Psoriasis is a disease with inflammation origin [bib_ref] Systemic inflammation and cardiovascular comorbidity in psoriasis patients: causes and consequences, Boehncke [/bib_ref]. A number of previous studies indicated anti-oxidant, anti-inflammatory, and immunomodulatory activities of Melissa officinalis, damask rose, and fennel [bib_ref] Pharmacological effects of Rosa damascena, Boskabady [/bib_ref] [bib_ref] Therapeutic and pharmacological potential of Foeniculum vulgare Mill: a review, Kooti [/bib_ref]. These herbs are a rich source of phenolic compounds such as flavonoids, cumarin, tannin, caffeic acid, caffeic acid, and rosmarinic acid [bib_ref] Medicinal properties of Foeniculum vulgare Mill. in traditional Iranian medicine and modern..., Rahimi [/bib_ref] [bib_ref] Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and prevents UVB-induced oxidative..., Pérez-Sánchez [/bib_ref] [bib_ref] Antioxidant activity and ultraperformance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting..., Kumar [/bib_ref]. A study confirmed the therapeutic effects of rosmarinic acid on atopic dermatitis [bib_ref] Effect of rosmarinic acid on atopic dermatitis, Lee [/bib_ref]. Also, another study demonstrated that decoction of Melissa officinalis has a lot of caffeic acids and rosmarinic acid, whose synergistic effects resulted in significant improvement of psoriasis [bib_ref] ssp altissima extracts: a therapeutic approach targeting psoriasis in mice, Dimitris [/bib_ref].
In summary, treatment with Melissa officinalis syrup demonstrated a significant reduction in pruritus and PASI scores and improved quality of life. The findings of this study may be useful for patients with mild-to-moderate chronic plaque psoriasis.
# Limitations
This study has some inevitable limitations including (1) due to lack of post-trial follow-up, long term effects of Melissa officinalis syrup is not ascertained; (2) using questionnaire may lead to bias; (3) participants were only patients with mild to moderate plaque-type psoriasis and patients with severe psoriasis and other types of psoriasis were not included; (4) biochemical tests were not considered because of resources limitations and they are proposed to be measured in future trials.
[fig] Figure 1: Flow diagram of the study in the placebo group at the end of the study (1.65 ± 2.17 versus 3.36 ± 2.38; P < 0.001). Lesion improvement in the arm area of the patients are demonstrated in Additional file 1:Figure S1. The DLQI score was 67.63 ± 22.97 in the Melissa officinalis group and 64.43 ± 23.66 for the placebo group (P = 0.503). After a 12-week intervention, the scores were changed to 74.37 ± 23.89 and 65.29 ± 23.74, respectively [/fig]
[table] Table 1: Demographics characteristics of participants at the baseline of study [/table]
[table] Table 2: Results of itch score, PASI score, and DLQI questionnaire between intervention and control groups [/table]
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Whole-Genome Comparative Analysis of Two Carbapenem-Resistant ST-258 Klebsiella pneumoniae Strains Isolated during a North-Eastern Ohio Outbreak: Differences within the High Heterogeneity Zones
Klebsiella pneumoniae has become one of the most dangerous causative agents of hospital infections due to the acquisition of resistance to carbapenems, one of the last resort families of antibiotics. Resistance is usually mediated by carbapenemases coded for by different classes of genes. A prolonged outbreak of carbapenem-resistant K. pneumoniae infections has been recently described in northeastern Ohio. Most strains isolated from patients during this outbreak belong to MLST sequence type 258 (ST258). To understand more about this outbreak two isolates (strains 140 and 677), one of them responsible for a fatal infection, were selected for genome comparison analyses. Whole genome map and sequence comparisons demonstrated that both strains are highly related showing 99% average nucleotide identity. However, the genomes differ at the so-called high heterogeneity zone (HHZ) and other minor regions. This study identifies the potential value of the HHZ as a potential marker for K. pneumoniae clinical and epidemiological studies.
# Introduction
Klebsiella pneumoniae is the causative agent of numerous infectious diseases [bib_ref] Plasmid-encoded amikacin resistance in multiresistant strains of Klebsiella pneumoniae isolated from neonates..., Woloj [/bib_ref] [bib_ref] Antibiotic susceptibility of bacterial strains isolated from patients with community-acquired urinary tract..., Daza [/bib_ref] [bib_ref] Community-acquired pneumonia in patients requiring hospitalization, Liam [/bib_ref] [bib_ref] Klebsiella pneumoniae sepsis with unusual cutaneous presentation of generalized pustulosis, Huang [/bib_ref] [bib_ref] Septic arthritis subsequent to urosepsis caused by hypermucoviscous Klebsiella pneumoniae, Suzuki [/bib_ref] [bib_ref] Klebsiella pneumoniae and the pyogenic liver abscess: implications and association of the..., Alcantar-Curiel [/bib_ref] [bib_ref] Transfer of CMY-2 ephalosporinase from Escherichia coli to virulent Klebsiella pneumoniae causing..., Lin [/bib_ref] [bib_ref] Clinical and microbiological characteristics of Klebsiella pneumoniae liver abscess in East China, Qu [/bib_ref] [bib_ref] The emerging threat of multidrug-resistant Gram-negative bacteria in urology, Zowawi [/bib_ref] and can also act as triggering factor in the initiation and development of ankylosing spondylitis and Crohn's disease ; [bib_ref] Ankylosing spondylitis is linked to Klebsiellathe evidence, Rashid [/bib_ref] [bib_ref] The link between ankylosing spondylitis, Crohn's disease, Klebsiella, and starch consumption, Rashid [/bib_ref]. K. pneumoniae strains recently acquired genes coding for carbapenemases [bib_ref] The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria, Nordmann [/bib_ref] [bib_ref] Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases, Munoz-Price [/bib_ref] [bib_ref] Genome sequences of two Klebsiella pneumoniae isolates from different geographical regions, Argentina..., Xie [/bib_ref] [bib_ref] Genome sequences of two carbapenemaseresistant Klebsiella pneumoniae ST258 isolates, Ramirez [/bib_ref] , which increased the severity of the infections by complicating treatments [bib_ref] Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause..., Hirsch [/bib_ref] ; . As a consequence, numerous groups studied aspects of virulence, antibiotic resistance, and epidemiology of carbapenem-resistant K. pneumoniae (CRKp) infections [bib_ref] Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella..., Lin [/bib_ref] [bib_ref] Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the..., Endimiani [/bib_ref] [bib_ref] Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates, Shu [/bib_ref] [bib_ref] Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause..., Hirsch [/bib_ref] [bib_ref] Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ)..., Ramirez [/bib_ref] [bib_ref] Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella..., Deleo [/bib_ref]. In one study, a combination of whole genome mapping (WGM) and whole genome sequencing (WGS) led to the identification of a particular chromosome segment, known as high heterogeneity zone (HHZ), later also called region of divergence (rd). The HHZ shows high variability and includes a "hot spot", also known as attO hot spot, where DNA fragments of a different origin can be inserted, and the capsular polysaccharide biosynthesis gene cluster (CPC) [bib_ref] Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella..., Lin [/bib_ref] [bib_ref] Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates, Shu [/bib_ref] [bib_ref] Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ)..., Ramirez [/bib_ref] [bib_ref] Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella..., Deleo [/bib_ref].
An outbreak of CRKp infection recently occurred in northeastern Ohio with most strains belonging to MLST sequence type 258 (ST258) . To characterize KPCproducing strains causing this outbreak we analyzed two isolates that led to different outcomes after treatment. Our results showed high homogeneity at the genomic level, but significant differences occurred at one region within the HHZ.
# Results and discussion
## Cases description
Klebsiella pneumoniae Kb140 is a blood isolate from a 64-year-old man with a history of coronary artery disease, congestive heart failure, peptic ulcer disease, and chronic obstructive pulmonary disease (which was treated with chronic corticosteroids) that was transferred from an outside facility to a referral hospital to be treated for pneumonia [bib_ref] Genome sequences of two carbapenemaseresistant Klebsiella pneumoniae ST258 isolates, Ramirez [/bib_ref]. Despite antibiotic treatment (intravenous tigecycline) and other procedures the patient died on hospital day 8. K. pneumoniae Kb677 was isolated from of urine culture of an 87-year-old woman with a history of localized breast cancer without metastases who was admitted to a referral hospital from the community [bib_ref] Genome sequences of two carbapenemaseresistant Klebsiella pneumoniae ST258 isolates, Ramirez [/bib_ref]. She did not meet CDC/NHSN criteria for urinary tract infection, and was not treated with any antibiotics with in vitro activity against CRKp. She was discharged back to her home after a prolonged hospital stay of 28 days. The antimicrobial susceptibilities of both isolates are summarized in table 1. Significant differences are that Kb140 is susceptible to gentamicin, but not to tobramycin or amikacin; and Kb677 is susceptible to amikacin but not gentamicin or tobramycin.
## Whole genome map comparison
The HHZ can be divided into distinct regions of which 3 and 4 seem to exhibit the highest variability [bib_ref] Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ)..., Ramirez [/bib_ref]. Region 3 consists of a "hot spot", also known as attO [bib_ref] Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella..., Lin [/bib_ref] , for insertion of DNA fragments. In at least one strain, NTUH-K2044, the insertion in Region 3 is the ICEKp1, a ca. 76-kbp integrative and conjugative element that includes a high-pathogenicity island [bib_ref] Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella..., Lin [/bib_ref]. HHZ Region 4 contains the CPC [bib_ref] Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ)..., Ramirez [/bib_ref] , which shows high variability among strains [bib_ref] Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates, Shu [/bib_ref]. Comparison of WGMs of strains Kb140 and Kb677 revealed that, although not identical, their genomes are very similar (regions in blue, [fig_ref] FIG. 1: -Genomic comparisons [/fig_ref]. One of the areas of difference (white in the WGMs) is located within the HHZ [fig_ref] FIG. 1: -Genomic comparisons [/fig_ref] and b), more precisely, at Region 4 [fig_ref] FIG. 1: -Genomic comparisons [/fig_ref]. Further WGM comparisons showed that both strains are closely related to K. pneumoniae VA360, a strain isolated in the same region before the beginning of the surveillance study, strain Kb140 being the most similar [fig_ref] FIG. 1: -Genomic comparisons [/fig_ref] [bib_ref] Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ)..., Ramirez [/bib_ref] [bib_ref] Genome sequences of two Klebsiella pneumoniae isolates from different geographical regions, Argentina..., Xie [/bib_ref].
## Wgs: comparison of the hhzs
The genomes of strains Kb140 and Kb677 are 5,677,714 and 5,894,762-bp long, respectively. Analysis of the HHZs at the nucleotide level showed that the Region 3 of both strains, as well as that of strain VA360, includes the same DNA fragment, an indication of relatedness. A comparison of the Region 3 of all three strains and those of well-studied strains such as NTUH-K2044, KCTC2242, which include unique DNA fragments, and MGH 78578, which does not include an insert is shown in figure 2a.
Region 3 of strains Kb140, Kb677, and VA360 includes elements coding for functions involved in conjugation, a
[formula] Interpretation MIC (mg/mL) Interpretation Amikacin 32 I 4 S Aztreonam >16 R >16 R Ciprofloxacin >2 R >2 R Colistin 0.5 S 0.5 S Doripenem a >2 R 2 I Doxycycline 16 R >16 R Ertapenem >4 R >4 R Cefepime >16 R 8 I Cefotaxime >32 R 32 R Ceftazidime >16 R >16 R Gentamicin 1 S >8 R Imipenem >8 R 8 R Levofloxacin >8 R >8 R Meropenem >8 R 8 R Minocycline 16 R >16 R Piperacillin/ tazobactam >64/128 R >64/128 R Polymyxin B 0.5 S 0.5 S Trimethoprim/ sulfamethoxazole >4/76 R >4/76 R Tigecycline a 2 S 4 I Tobramycin >8 R >8 R a [/formula]
Values for doripenem and tigecycline are too close (one dilution) to assure that their phenotypes are actually different.
type III restriction endonuclease and the cognate methylase as well as a P4-like integrase and other hypothetical genes with unknown function [fig_ref] Table 2: Summary of main ORFs found in Region 3 ORFs are color coded... [/fig_ref]. The integrase is found in numerous genomes of Gram-negatives (supplementary [fig_ref] FIG. 1: -Genomic comparisons [/fig_ref] , Supplementary Material online) and its amino acid sequence shares 50% identity and 68% similarity with the ICEKp1 integrase [bib_ref] Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella..., Lin [/bib_ref] (supplementary [fig_ref] FIG. 2: -HHZ Regions 3 and 4 [/fig_ref] , Supplementary Material online). As it is the case in ICEKp1, the integrase could mediate insertion and excision of this DNA fragment. The Region 3 of strains Kb140, Kb677, and VA360 was also found in sixteen CRKp strains (supplementary , Supplementary Material online) [bib_ref] Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing, Snitkin [/bib_ref].
A comparison of the HHZ Region 4 among strains Kb140, Kb677, and VA360 showed that while the CPCs were identical in strains Kb140 and VA360, they were significantly different from that of strain Kb677 [fig_ref] FIG. 2: -HHZ Regions 3 and 4 [/fig_ref]. BLAST analyses of both versions of Region 4 indicated that only ten completed genomes include a CPC identical to that of strain Kb140 (supplementary , Supplementary Material online). Conversely, the CPC from strain Kb677 was found in 11 strains, 3 of them the KPNIH10, 30660/NJST258_1, and 30684/NJST258_2 that also have identical versions of HHZ Region 3 (supplementary , Supplementary Material online). There is a conspicuous GC% difference in value at the HHZ with respect to the rest of the genome at the locations corresponding to the Regions 3 and 4 (see .
## Wgs: multiple genome alignment and genomic features
The nucleotide sequences of both strains showed 99% average identity. MAUVE alignment including the K. pneumoniae VA360, NTUH-K2044, and MGH 78578 genomes identified seven locally collinear blocks (LCBs) and no inversions [fig_ref] FIG. 4: -Alignment of K [/fig_ref]. A 5,757-nucleotides deletion was observed in the Kb677 genome. The deleted fragment includes genes coding for homologs to a two-component regulatory system, a heat shock protein, a putative LuxR regulator, the competence damageinducible protein A, and a hypothetical protein. A 2,096-nucleotide deletion coding for the 23S ribosomal RNA was identified in the Kb140 genome. Furthermore, 5 and 14 unique regions were found in the Kb140 and Kb677 genomes, respectively. Three of them are prophage-like sequences and considerably larger than the rest [fig_ref] Table 3: Unique Region of K [/fig_ref].
In both genomes 73% of the genes were assigned to a functional category including carbohydrate and amino acid metabolism, transcription and energy production and conversion (table 4). As expected, there was no significant difference in functional categories between the five strains (table 4).
# Conclusions
The importance of K. pneumoniae as a pathogen has significantly grown in recent years [bib_ref] The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria, Nordmann [/bib_ref]. This is, at least in part, due the rise of strains that are becoming resistant to most antibiotics used in standard treatments. In particular, the relatively recent acquisition of carbapenemase genes led to dissemination of CRKp strains that are the hardest to treat [bib_ref] The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria, Nordmann [/bib_ref] [bib_ref] Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases, Munoz-Price [/bib_ref] [bib_ref] Genome sequences of two Klebsiella pneumoniae isolates from different geographical regions, Argentina..., Xie [/bib_ref] [bib_ref] Genome sequences of two carbapenemaseresistant Klebsiella pneumoniae ST258 isolates, Ramirez [/bib_ref]. These facts led us and other research groups to carry out comparative genomics studies that may help understanding the epidemiology and dissemination of CRKp strains.
Here, we compared the genomes of two strains isolated during an outbreak of CRKp infection in northeastern Ohio. The hot spot sequences, also known as attO [bib_ref] Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella..., Lin [/bib_ref] , are red and the sequences immediately adjacent to the hot spots are black. In the case of strain NTUH-K2044 the inserted sequenced is the ICEKp1 pathogenicity island. (b) Schematic comparison of the Region 4, which consists of the CPC, within the HHZs of K. pneumoniae strains Kb140, Kb677, and VA360. Grayed regions represent high homology. In those cases where there is no 100% identity, the identity percentage is shown. In the case of strain VA360, the Region 4 was put together using different contigs, which are identified by their accession numbers. Coordinate numbers are those in GenBank. Potential function of genes [bib_ref] Clonally diverse rfb gene clusters are involved in expression of a family..., Kelly [/bib_ref] [bib_ref] Biosynthesis and assembly of capsular polysaccharides in Escherichia coli, Whitfield [/bib_ref] [bib_ref] Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates, Shu [/bib_ref] : uge, UDP-galacturonate 4-epimerase; ugd, UDP-glucose dehydrogenase; ATF, acyl transferase; gnd, gluconate-6-phosphate dehydrogenase; wcaJ, undecaprenyl-phosphate glycosyltransferase; GTF, glycosyl transferase; POL, polysaccharide biosynthesis; int, integrase-like; tnp, transposase-like; cpsF, CMP-N-acetylneuraminic acid synthetase; tcd, glycosyltransferase; OAL, O-antigen ligase; etk, tyrosine-protein kinase; wza, polysaccharide export lipoprotein; wzi, integral outer membrane protein; PAP2, PAP2 superfamily; galF, modulator of GalU to elevate the cellular concentration of UDP-glucose; Hyp, hypothetical; epsG, putative capsular polysaccharide biosynthesis protein; rfbX (wzx), flippase; wcaG, GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/ reductase; rfbAB, ATP-binding cassette transporter; rfbC, involved in polysaccharide synthesis (epimerase), rfbD, involved in polysaccharide transport; wbaP, membrane protein that helps transfer a galactose residue from UDP-Gal to undecaprenol diphosphate to form Gal-p-UndP.
Although strains Kb677 and Kb140 differ in the carbapenemase gene they carry, only minor differences were detected between their genomes, in general due to insertions and deletions. This was not surprising because as others and we have previously observed comparing other K. pneumoniae strains [bib_ref] Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella..., Deleo [/bib_ref] [bib_ref] Genome sequences of two carbapenemaseresistant Klebsiella pneumoniae ST258 isolates, Ramirez [/bib_ref] [bib_ref] Whole-genome typing and characterization of blaVIM19-harbouring ST383 Klebsiella pneumoniae by PFGE, whole-genome..., Sabirova [/bib_ref] , the genomes of this bacterium seem to be quite homogeneous. However, in spite of the similarities between both genomes, strains Kb677 and Kb140 show significant differences at the HHZ. This region, also known as "rd", was shown to be one of the most variable in K. pneumoniae isolates [bib_ref] Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella..., Deleo [/bib_ref]. These results underscore the potential importance of the HHZ. This study shows that two strains isolated during the same outbreak could be quickly differentiated by their HHZs. We propose that this zone of the K. pneumoniae genome can be used as a signature for epidemiology analysis.
# Materials and methods
## Bacterial strains
Klebsiella pneumoniae Kb140 and Kb677 belong to ST-258 possess a different rep-PCR type (type-A for Kb140 and type-B for Kb677). Strain Kb140 harbors bla KPC-2 while Kb677 harbors bla KPC-3 . Their antimicrobial susceptibilities were determined by broth microdilution according to CLSI methods and interpretations (Clinical-Laboratory-Standard-Institute 2012).
## Genomic analyses
The available scaffolds for the strains Kb140 and Kb677 (accession number AQRD01000000 and AQPG01000000, respectively) were ordered and oriented with the MAUVE Contig Mover [bib_ref] progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement, Darling [/bib_ref] , using the K. pneumoniae NTUH-K2044 genome as reference (accession number AP006725). Genomes were aligned with the open-source MAUVE aligner version 2.3.1 using the progressive algorithm. The alignments were generated using the default settings (http://darlinglab.org/mauve/mauve.html, last accessed June 8, 2016). Conserved segments that appear to be internally free from genome rearrangements, known as LCBs, were identified using MAUVE. Functional categories groups for genes have been predicted by comparison with the Clusters of Orthologous Groups (COGs). The prediction of coding sequences (CDS) was made by Rast version 4.0 software and confirmed by BLAST [bib_ref] The RAST Server: rapid annotations using subsystems technology, Aziz [/bib_ref]. tRNA genes were identified using tRNAscan-SE (Lowe and Eddy
[fig] FIG. 1: -Genomic comparisons. (a) The K. pneumoniae Kb140 and Kb677 whole genome maps were compared using the MapSolver 3.2.0 software. The blue and white regions represent matching and nonmatching DNA fragments, respectively. The location of the HHZ is indicated. (b) Zoom-in the HHZ region. (c) Map similarity cluster showing K. pneumoniae Kb140 and Kb677 in relation to other completely sequenced K. pneumoniae strains. Dendogram was generated using Unweighted Pair Group Method with Arithmetic Mean. The strains Kb140, Kb670, and the closely related VA360 are boxed. [/fig]
[fig] FIG. 2: -HHZ Regions 3 and 4. (a) Schematic diagram of the Region 3 within the HHZs of several K. pneumoniae strains. The DNA sequences surrounding the hot spot and inserted fragments are represented in blue. The inserted regions are shown in thinner lines with different colors to indicate that the sequences are different. The sequences inserted in strains Kb140, Kb677, and VA360 are identical. The coordinates are those found in GenBank. [/fig]
[fig] FIG. 4: -Alignment of K. pneumoniae strains genomes using MAUVE. Each genome's panel contains a scale showing the sequence coordinates, colored blocks that represent regions of the genome sequence that aligned to part of the other genomes, and a single black horizontal center line where the blocks that lie above are in the forward orientation (in this case there are no blocks in reverse orientation, then the lines look to be at the bottom across the genome). [/fig]
[table] Table 1: Antimicrobial Susceptibility of K. pneumoniae Isolates Kb140 and Kb677 [/table]
[table] Table 2: Summary of main ORFs found in Region 3 ORFs are color coded based on COG classifications. Ring 3: Common regions and unique regions are represented in gray or colors, respectively. Pink and orange correspond to HHZ Regions 3 and 4. Ring 4: GC content. Ring 5: GC skew, calculated in Artemis. [/table]
[table] Table 3: Unique Region of K. pneumoniae Kb140 and Kb677 [/table]
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Urinary liver‐type fatty acid‐binding protein is independently associated with graft failure in outpatient kidney transplant recipients
This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Abbreviations: AIC, akaike information criterion; AUC, area under the curve; CI, confidence interval; CKD-EPI, chronic kidney disease-epidemiology collaboration equation; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; HDL, high-density lipoprotein; HLA, human leukocyte antigen; HR, hazard ratio; hs-CRP, high-sensitivity C-reactive protein; IQR, interquartile range; KTR, kidney transplant recipients; LDL, low-density lipoprotein; ROC, receiver operator characteristic; SBP, systolic blood pressure; SD, standard deviation; SQUASH, short questionnaire to assess health-enhancing physical activity; uL-FABP, urinary liver-type fatty acid-binding protein.Urinary liver-type fatty acid-binding protein (uL-FABP) is a biomarker of kidney hypoxia and ischemia, and thus offers a novel approach to identify early kidney insults associated with increased risk of graft failure in outpatient kidney transplant recipients (KTR). We investigated whether uL-FABP is associated with graft failure and whether it improves risk prediction. We studied a cohort of 638 outpatient KTR with a functional graft ≥1-year. During a median follow-up of 5.3 years, 80 KTR developed graft failure. uL-FABP (median 2.11, interquartile range 0.93-7.37 µg/24"/>h) was prospectively associated with the risk of graft failure (hazard ratio 1.75; 95% confidence interval 1.27-2.41 per 1-SD increment; P = .001), independent of potential confounders including estimated glomerular filtration rate and proteinuria. uL-FABP showed excellent discrimination ability for graft failure (c-statistic of 0.83) and its addition to a prediction model composed by established clinical predictors of graft failure significantly improved the c-statistic to 0.89 (P for F-test <.001). These results were robust to several sensitivity analyses. Further validation studies are warranted to evaluate the potential use of a risk-prediction model including uL-FABP to improve identification of outpatient KTR at high risk of graft failure in clinical care.K E Y W O R D Sclinical research/practice, graft survival, kidney transplantation/nephrology, outpatient care, risk assessment/risk stratification
## | introduc ti on
Short-term outcomes of kidney transplantation have seen great improvements over the last decades. [bib_ref] Long-term renal allograft survival: have we made significant progress or is it..., Meier-Kriesche [/bib_ref] [bib_ref] Long-term renal allograft survival in the United States: a critical reappraisal, Lamb [/bib_ref] In contrast, improving long-term kidney graft survival continues to be a major challenge with no comparable achievements during the same time frame. [bib_ref] Lack of improvement in renal allograft survival despite a marked decrease in..., Meier-Kriesche [/bib_ref] [bib_ref] Improved graft survival after renal transplantation in the United States, Hariharan [/bib_ref] Improvement of risk-prediction tools is the first step in advancing early risk management strategies post-kidney transplantation. [bib_ref] Risk prediction models for graft failure in kidney transplantation: a systematic review, Kaboré [/bib_ref] [bib_ref] Risk stratification for rejection and infection after kidney transplantation, Cippà [/bib_ref] However, current clinical parameters [bib_ref] Tissue transcriptome-driven identification of epidermal growth factor as a chronic kidney disease..., Ju [/bib_ref] [bib_ref] The prognostic importance of severity and type of post-transplant proteinuria, Yıldız [/bib_ref] [bib_ref] Post-transplant renal function in the first year predicts long-term kidney transplant survival, Hariharan [/bib_ref] are of limited potential to allow improvement of long-term outcomes, because their alteration usually reflects already advanced structural damage. [bib_ref] Monitoring and managing graft health in the kidney transplant recipient, Josephson [/bib_ref] Liver-type fatty acid-binding protein (L-FABP) is an intracellular lipid chaperon [bib_ref] Urinary fatty acid-binding protein 1: an early predictive biomarker of kidney injury, Noiri [/bib_ref] that in the kidney is exclusively expressed in the proximal tubule. [bib_ref] Renal L-type fatty acid-binding protein in acute ischemic injury, Yamamoto [/bib_ref] Early in the pathophysiology of chronic rejection, attenuated blood flow due to arterial intimal fibrosis leads to hypoxic challenge and graft ischemia. [bib_ref] Antibody-mediated rejection in renal allografts: lessons from pathology, Racusen [/bib_ref] It has been described that upon detection of lipid peroxidation increments, an hypoxia-responsive element upregulates L-FABP synthesis, which then allows binding of lipid peroxides for their urinary excretion [bib_ref] Urinary fatty acid-binding protein 1: an early predictive biomarker of kidney injury, Noiri [/bib_ref] and both expression and urinary excretion of L-FABP have been shown to be increased under tubular hypoxic conditions. [bib_ref] Renal L-type fatty acid-binding protein in acute ischemic injury, Yamamoto [/bib_ref] [bib_ref] Urine injury biomarkers and risk of adverse outcomes in recipients of prevalent..., Bansal [/bib_ref] Because kidney tubular epithelial cells are very rich in mitochondria, and therefore particularly vulnerable to hypoxic challenge, L-FABP may offer a novel interesting approach to identifying early graft tissue insult. [bib_ref] Urinary fatty acid-binding protein 1: an early predictive biomarker of kidney injury, Noiri [/bib_ref] In the kidney transplantation setting specifically, L-FABP measurement during hypothermic machine perfusion showed to be inversely associated with graft function at 6 months post-transplantation. [bib_ref] Associations of perfusate biomarkers and pump parameters with delayed graft function and..., Parikh [/bib_ref] Furthermore, an elegant study by that urinary L-FABP (uL-FABP) is directly correlated with graft ischemia time. [bib_ref] Renal L-type fatty acid-binding protein in acute ischemic injury, Yamamoto [/bib_ref] No study to date, however, has been devoted to investigating the biologically plausible association between uL-FABP and risk of graft failure in outpatient KTR.
In the current study, we aimed to investigate the prospective association of uL-FABP and graft failure in outpatient KTR. Furthermore, we aimed to explore its risk-predictive ability and whether addition of uL-FABP into a model of established risk factors could improve risk-predictive ability and model fit for kidney graft failure.
## | material s and me thods
## | study design and population
In this prospective cohort study, adult KTR who visited the outpatient clinic at the University Medical Center Groningen (the Netherlands) between November 2008 and May 2011 and had a functioning graft for at least 1-year were invited to participate. The invitation was restricted to patients with 1-year functional graft because the objective of the TransplantLines study was to identify risk factors that impacted long-term graft survival, where, contrary to the first-year post-transplantation, little improvement has been seen in the last decades. [bib_ref] Lack of improvement in renal allograft survival despite a marked decrease in..., Meier-Kriesche [/bib_ref] [bib_ref] Improved graft survival after renal transplantation in the United States, Hariharan [/bib_ref] Seven hundred and seven patients signed a written informed consent at a median of 5.8 (interquartile range 2.0-12.2) years post-transplantation. We excluded patients in whom uL-FABP measurements were missing (n = 69), resulting in 638 KTR, of whom the data are presented here. The current study was approved by the institutional review board (METc 2008/186) and adhered to the Declarations of Helsinki and Istanbul.
The primary endpoint of the current study was death-censored graft failure, defined as restart of dialysis or re-transplantation.
Follow-up was performed according to the American Society of Transplantation guidelines 16 until June, 2016. Collection of data was ensured by the continuous surveillance system of the outpatient clinic of our university hospital and close collaboration with affiliated hospitals. We contacted general practitioners or referring nephrologists in cases where the status of a patient was unknown. No participants were lost to follow-up.
## | data collection
Baseline data were collected during a visit to the outpatient clinic, following a detailed protocol described elsewhere. [bib_ref] Sodium intake and blood pressure in renal transplant recipients, Van Den Berg [/bib_ref] [bib_ref] Dietary protein, blood pressure and renal function in renal transplant recipients, Van Den Berg [/bib_ref] Anthropometric measurements were taken while participants wore indoor clothing without shoes. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using a semiautomatic device (Dinamap1846; Critikon) every minute for 15 minutes. [bib_ref] Sodium intake and blood pressure in renal transplant recipients, Van Den Berg [/bib_ref] Relevant donor, recipient and transplant information was extracted from the Groningen Renal Transplant Database, which has been described in detail before. [bib_ref] Metabolic syndrome is associated with impaired long-term renal allograft function; not all..., De Vries [/bib_ref]
## | laboratory measurements and calculations
At first visit, blood samples were taken after a fasting period of ap- [bib_ref] A new equation to estimate glomerular filtration rate, Levey [/bib_ref] The cumulative dose of prednisolone was calculated as the sum of the maintenance dose of prednisolone from transplantation until baseline.
According to a strict protocol, all KTR were asked to collect a 24-hour urine sample during the day before the same visit. uL-FABP was measured with an enzyme-linked immunosorbent assay (human uL-FABP assay kit 96 test; CMIC holdings Co). The test has a detection limit of 0.036 µg/L. The intra-assay variability calculated based on four replicate measurements on urine samples with uL-FABP concentrations of 2 and 40 µg/L, were 3.8% and 2.5%, respectively. Inter-assay variabilities, as assessed with repeated measurements in the same samples were 10.4% and 7.3%, respectively.
## | statistical analyses
## | association of ul-fabp with risk of graft failure
A restricted cubic spline regression, with three knots located at the 10th, 50th, and 90th percentile, was performed and graphed to visualize the association of uL-FABP with graft failure. Nonlinearity was tested by using the likelihood ratio test, comparing models with linear or linear and cubic spline terms. The association of uL-FABP with risk of graft failure was then analyzed using Cox proportional-hazards regression analyses. In model 1 we performed multivariable-adjusted analyses according to the Reference Model (determined as explained in the preceding section). Thereafter, we computed further models, with additive adjustments to Model 1 to avoid inclusion of too many variables for the number of events. Thus, we additionally adjusted for donor and transplantation characteristics (donor age, donor type [living, deceased after brain dead and deceased after cardiac dead], donor height, donor weight, donor diabetes and hypertension; and time since transplantation; Model 2); inflammation and immunosuppressive therapy (high-sensitivity C-reactive protein, use of calcineurin inhibitors, use of proliferation inhibitors, and cumulative prednisolone dose; Model 3); blood pressure and metabolismrelated variables (systolic blood pressure, use of antihypertensive medication, fasting plasma glucose, plasma HDL cholesterol, and triglycerides; Model 4) and a combination of the prior (systolic blood pressure, diastolic blood pressure, high-sensitivity C-reactive protein, and plasma HDL cholesterol; Model 5).
## | discrimination power and model riskprediction ability for graft failure
We explored uL-FABP risk-prediction ability by calculating the c-statistic of the Reference Model, and then the c-statistic after adding uL-FABP, to investigate whether adding uL-FABP to the Reference Model increased the model risk-prediction ability. We also performed an F-test to check whether the difference between both risk-prediction ability models was significant. Next, the Akaike information criterion (AIC) was used to evaluate model fit. Finally, a receiver operating characteristic (ROC) curve was generated for the reference model before and after inclusion of uL-FABP, the area under the curve was calculated for both curves.
## | secondary analyses and sensitivity analyses
In secondary analyses, we performed multivariable Cox proportional-hazards regression analyses, evaluation of model risk-prediction ability, and model fit analoguously to primary analyses, yet
## | association of ul-fabp with secondary outcomes
Exploratory univariable and eGFR-adjusted linear regression (for continuous variables), logistic regression (for dichotomic variables), and Cox regression (for time-dependent outcomes) analyses were performed to evaluate the association between uL-FABP and other outcomes of clinical importance for KTR (progressive proteinuria, clinical episodes of rejection, graft loss, cardiovascular events, cardiovascular mortality, and all-cause mortality).
## | re sults
## | baseline characteristics
Baseline characteristics of the study population are presented in Two hundred and sixteen (34%) of the organs were obtained from living donors and mean donor age was 43 ± 15 years. Patients in the highest tertile of uL-FABP, when compared to the other two tertiles, were in their majority male (p < .001), had lower eGFR (p < .001), higher urinary protein excretion (p < .001), and received a kidney from an older donor (p < .001), whom where most usually female (p = .02). As for their immunosuppressive regimen they more frequently used tacrolimus (p = .002). Patients in the third tertile also had higher SBP and DBP (p < .001), more frequently used any antihypertensive medication (p = .03), had lower HDL cholesterol (p < .001), and had higher triglycerides (p = .005) and plasma glucose (p = .01). Finally, patients in the highest tertile had more apparent inflammation shown by higher hs-CRP concentration (p = .01). patients whose values of uL-FABP were above the median, the most frequent cause also was chronic rejection (78%), followed by recurrence of primary disease (11%) and infection of the graft (4%); and within patients below the median the most common cause was chronic rejection, in a lower proportion (43%), followed by acute rejection (29%). The distributions of causes among subgroups was significantly different (p < .001; . .
## | reference model
## | ul-fabp and prediction of graft failure
## Ta b l e 1 baseline characteristics of the study population
## | ul-fabp and prospective association with the risk of other outcomes
The exploration of the association of uL-FABP with secondary outcomes showed that it was significantly prospectively associated with,
## | discuss ion
In stable KTR, this study shows that uL-FABP is positively and strongly associated with the risk of graft failure, independently of several established risk factors, including HLA mismatching, eGFR, and urinary protein excretion. Moreover we show that uL-FABP has a strong predictive value for this outcome and that inclusion of uL-FABP into a risk-prediction model composed by well-established risk factors of graft failure, seems to significantly improve risk-prediction value and model fit; although this findings would require validation in an external cohort.
Chronic graft failure remains a major challenge in kidney transplantation. [bib_ref] Predicting long-term outcome in renal transplantation, Keown [/bib_ref] A main characteristic of this phenomenon is arterial intimal fibrosis, which generates a progressive luminal narrowing of graft vessels, and therefore progressive ischemia of the transplanted kidney [bib_ref] Urine injury biomarkers and risk of adverse outcomes in recipients of prevalent..., Bansal [/bib_ref] The role of L-FABP is to eliminate lipid peroxides, produced under circumstances of hypoxia-induced oxidative stress, by transferring them into the tubular lumen for further urinary excretion. [bib_ref] Biomarkers of AKI: a review of mechanistic relevance and potential therapeutic implications, Alge [/bib_ref] Under hypoxic conditions, its synthesis is increased by the activation of an hypoxia-inducible factor 1α response element in the promotor region of the L-FABP gene and its enhanced genetic expression within the kidney has shown to be protective of ischemic injury in rat models. [bib_ref] Renal L-type fatty acid-binding protein in acute ischemic injury, Yamamoto [/bib_ref] [bib_ref] Biomarkers of delayed graft function as a form of acute kidney injury..., Malyszko [/bib_ref] This response to injury leads to an increase of uL-FABP, which is why it works as a marker of ongoing of renal hypoxia. [bib_ref] Biomarkers of AKI: a review of mechanistic relevance and potential therapeutic implications, Alge [/bib_ref] The same study showed that in the kidney post-transplantation setting during a short-follow up, uL-FABP was indeed increased by hypoxic conditions of the graft, with a direct correlation between uL-FABP and ischemia time during transplantation and also with outcomes with it being directly associated with longer hospital stay after the procedure. [bib_ref] Renal L-type fatty acid-binding protein in acute ischemic injury, Yamamoto [/bib_ref] Also, higher concentrations of L-FABP during hypothermic machine perfusion have been associated with lower eGFR in the short term after transplantation. [bib_ref] Associations of perfusate biomarkers and pump parameters with delayed graft function and..., Parikh [/bib_ref] are established, which is a significant advantage over markers like proteinuria and eGFR. [bib_ref] Monitoring and managing graft health in the kidney transplant recipient, Josephson [/bib_ref] Although tubular ischemia is a multifactorial process,the main recognized enhancer of this phenomenon is immunologic aggression of the host against the allograft. [bib_ref] Progression of renal damage in chronic rejection, Ponticelli [/bib_ref] Therefore, attention should be given to other strategies to salvage the graft such as the tailoring of immunosuppressive regimens. [bib_ref] Progression of renal damage in chronic rejection, Ponticelli [/bib_ref] [bib_ref] Chronic allograft failure: a disease we don't understand and can't cure?, Schratzberger [/bib_ref] However, we acknowledge that supporting these hypotheses requires further evidence. Our findings are a call for the performance of studies that allow for defining cut-off points for uL-FABP and assess its impact in real clinical practice.
A strength of this study is that collection of our data was ensured by the continuous surveillance system of the outpatient clinic of our university hospital and close collaboration with affiliated hospitals which provided us with complete information on endpoints during follow-up. Moreover, our extensively phenotyped cohort allowed us to evaluate several potential confounders, and the robustness of our findings was tested with multiple sensitivity analyses.
Because of its observational design, our study does not allow hard conclusions on causality, and reversed causation or residual confounding may occur. Furthermore, we did not have data on de novo DSA and nonadherence, so we could not explore associations with these outcomes. Next, the current study was performed in a single center with over-representation of Caucasian subjects, which calls prudence to extrapolation of our results to different populations regarding ethnicity. Finally, although the main source of uL-FABP is kidney tubular production, [bib_ref] Urinary fatty acid-binding protein as a new clinical marker of the progression..., Kamijo [/bib_ref] [bib_ref] Renal L-type fatty acid-binding protein mediates the bezafibrate reduction of cisplatin-induced acute..., Negishi [/bib_ref] it is also produced in other organs and can be filtered into urine, 11 therefore it cannot be considered a completely kidney-specific biomarker; however, studies performed on this matter show that: (a) in a mice model of acute kidney injury, the magnitude of the urine increase after kidney injury was much higher than that of the plasma, [bib_ref] Renal L-type fatty acid-binding protein mediates the bezafibrate reduction of cisplatin-induced acute..., Negishi [/bib_ref] Dr. Sotomayor is supported by a doctorate studies grant from CONICYT (F 72190118).
## D i sclos u r e
The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Takeshi
Sugaya is a researcher of the company that developed the assay used to measure L-FABP in the current study.
## Data ava i l a b i l i t y s tat e m e n t
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
## O rci d
Manuela Yepes-Calderón https://orcid. org/0000-0002-4693-5974
Camilo G. Sotomayor https://orcid.org/0000-0001-6835-6386
[fig] 3. 3: | uL-FABP and association with the risk of graft failure uL-FABP was univariately associated with the risk of graft failure as shown in Cox regression analyses (HR 3.37; 95% CI 2.66-4.29 per 1-SD increment; p < .001; Table S2) and restricted cubic spline regression (Figure 1). Multivariable-adjusted analyses showed that this association was independent of adjustment for variables of the Reference Model (HR 1.75; 95% CI, 1.27-2.41 per 1-SD increment; p = .001; Model 1), and independent of additional adjustment for donor and transplantation characteristics (Model 2), inflammation and immunosuppressive therapy (Model 3), blood pressure and metabolism-related characteristics (Model 4) and a combination of the prior (Model 5; [/fig]
[table] Table 1: In total 638 KTR (57% men, 53 ± 13 years old, 99% Caucasian) were included in the analyses. Median (IQR) uL-FABP was 2.11 (0.93-7.37) µg/24 -h. Mean eGFR was 52 ± 20 ml/min/1.73 m 2 , and median urinary protein excretion was 0.20 (0.02-0.39) g/24 h. Preemptive transplantation was performed in 102 (16%) patients. [/table]
[table] Table S1: In univariable Cox regression analyses of the associations between different literature-based established risk factors with the risk of graft failure, the presence of HLA antibodies class II showed the strongest association with outcome (HR 3.50; 95% CI 2.22-5.50; p < .001). Other variables significantly associated with the risk of graft failure and also included in the Reference Model computed by means of backwards selection were eGFR (HR 0.70; 95% CI 0.65-0.76 per 1-SD increment; p < .001), urinary protein excretion (HR 1.50; 95% CI 1.37-1.63 per 1-SD increment; p < .001), recipient age (HR 0.77; 95% CI 0.62-0.95 per 1-SD increment; p = .01), and preemptive transplantation (HR 0.39; 95% CI 0.17-0.89; p = .03)(Table S2). [/table]
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Correction: A Novel Antifungal Is Active against Candida albicans Biofilms and Inhibits Mutagenic Acetaldehyde Production In Vitro
## Supporting information
File S1. Originally published, uncorrected article.
[fig] File S2: Republished, corrected article. (PDF) Reference 1. Nieminen MT, Novak-Frazer L, Rautemaa V, Rajendran R, Sorsa T, et al. (2014) A Novel Antifungal Is Active against Candida albicans Biofilms and Inhibits Mutagenic Acetaldehyde Production In Vitro. PLoS ONE 9(5): e97864. doi:10.1371/journal.pone.0097864. Citation: The PLOS ONE Staff (2014) Correction: A Novel Antifungal Is Active against Candida albicans Biofilms and Inhibits Mutagenic Acetaldehyde Produc- [/fig]
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COVID-19 in the Age of Artificial Intelligence: A Comprehensive Review
The recent COVID-19 pandemic, which broke at the end of the year 2019 in Wuhan, China, has infected more than 98.52 million people by today (January 23, 2021) with over 2.11 million deaths across the globe. To combat the growing pandemic on urgent basis, there is need to design effective solutions using new techniques that could exploit recent technology, such as machine learning, deep learning, big data, artificial intelligence, Internet of Things, for identification and tracking of COVID-19 cases in near real time. These technologies have offered inexpensive and rapid solution for proper screening, analyzing, prediction and tracking of COVID-19 positive cases. In this paper, a detailed review of the role of AI as a decisive tool for prognosis, analyze, and tracking the COVID-19 cases is performed. We searched various databases including Google Scholar, IEEE Library, Scopus and Web of Science using a combination of different keywords consisting of COVID-19 and AI. We have identified various applications, where AI can help healthcare practitioners in the process of identification and monitoring of COVID-19 cases. A compact summary of the corona virus cases are first highlighted, followed by the application of AI. Finally, we conclude the paper by highlighting new research directions and discuss the research challenges. Even though scientists and researchers have gathered and exchanged sufficient knowledge over last couple of months, but this structured review also examined technological perspectives while encompassing the medical aspect to help the healthcare practitioners, policymakers, decision makers, policymakers, AI scientists and virologists to quell this infectious COVID-19 pandemic outbreak.Graphic abstract
# Introduction
COVID-19 is a highly contagious epidemic disease caused by novel coronavirus (SARS-CoV-2) which has been declared as pandemic by WHO. Researchers across the globe are working round the clock to find solutions and design strategies to control the pandemic and minimize its impact on human health and economy [bib_ref] A novel adaptive deep learning model of COVID-19 with focus on mortality..., Farooq [/bib_ref]. One of the large family of viruses is called Coronaviruses, which may affect and endanger humans' lives by causing acute ailment [bib_ref] Origin and evolution of pathogenic coronaviruses, Cui [/bib_ref]. A virus is an infectious microorganism constitutes a specific genome wrapped in a protein layer with the ability to replicate inside living cells. These billions of tiny but powerful viruses, smaller than human cells, can cause viral infection when entered in living creature by hijacking the host cells and forcefully turning those to virus-making factory. This may lead to severe health problems, such as blindness due to smallpox virus or fatal inflammation of the brain or spinal cord by rabies virus [bib_ref] Smallpox: the triumph over the most terrible of the ministers of death, Barquet [/bib_ref] [bib_ref] Perspectives in diagnosis and treatment of rabies viral encephalitis: insights from pathogenesis, Mahadevan [/bib_ref].
These viruses are either pandemic or epidemic that lasts over a definite duration of time [bib_ref] Defining epidemics in computer simulation models: How do definitions influence conclusions?, Orbann [/bib_ref]. A pandemic is an occurrence of huge morbidities and mortalities caused by the proliferation of infectious disease at vast geographical area. Contrarily, when a disease is spread in limited region over time. Many epidemics and pandemics occurred over a period, but the mortality rate shows that pandemics had effects that are more devastating in the history of human life. Such as a decade ago, SARS epidemic virus infected around 8096 humans causing deaths of more than 770 humans. Beside such small epidemic, years ago, a famous pandemic known as smallpox affected millions of lives and eventually ended up with 500 million fatalities across the globe. Few of the viruses that caused epidemic and pandemic over last 102 years are depicted in [fig_ref] Figure 1: Deadliest viruses over last 102 years [/fig_ref].
The novel Coronavirus is the recent pandemic, officially known as SARS-CoV-2 and member of broader family of infectious viruses, which can affect the respiratory system of humans [bib_ref] The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it..., Gorbalenya [/bib_ref]. In 2002, the first pathogen, SARS emerged in Guangdong (China) that caused mild infection in humans. Another pathogenic member of coronavirus family, known as MERS-CoV, discovered in 2012 around Middle East regions that caused panic due to high infection rate which affected 2494 humans with 858 deaths [bib_ref] Isolation of a novel coronavirus from a man with pneumonia in Saudi..., Zaki [/bib_ref]. Last December (2019), the deadly COVID-19, the seventh strain of coronavirus, originated in Huanan Seafood Market, Wuhan state of Hubei province, China, that quickly gained global attention due to fast transmission among species and caused respiratory problems [bib_ref] A novel coronavirus from patients with pneumonia in China, Zhu [/bib_ref]. COVID-19 being an infectious disease has high transmission rate among humans as respiratory droplets of infected patient are inhaled by humans around [bib_ref] Seasonality of respiratory viral infections, Moriyama [/bib_ref]. Tiredness, cough, fever, and loss of smell are considered as most common symptoms, while headache, aches, rash on skin, diarrhea and sore throat are less common symptoms observed in patients affected by COVID-19. Besides these normal looking symptoms, it can cause severe respiratory problems that can damage several human organs, which eventually causes death [bib_ref] The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak, Rothan [/bib_ref] [bib_ref] Clinical course and risk factors for mortality of adult inpatients with COVID-19..., Zhou [/bib_ref].
On January 30, 2020, due to high horizontal transmission rate among humans of 18 different countries, WHO announced it a PHEIC. Keeping in view the sharp incline of COVID-19 cases around the world in succeeding 2 months, health organizations stated it as global pandemic due to its hazardous effect on human life [bib_ref] The clinical dynamics of 18 cases of COVID-19 outside of Wuhan, China, Wang [/bib_ref]. Since the first outbreak of highly pathogenic in China, the pandemic has affected 213 countries and territories around the world according to figures compiled by Worldometer. As reported by John Hopkins University this particular virus has infected more than 98,529,820 humans around the globe, while the tally of confirmed deaths is over 2,116,101, thus becoming the greatest pandemic of all time. As per John Hopkins data, a total number of infections and deaths in top 10 disease burden countries are shown in [fig_ref] Figure 2: Top [/fig_ref].
The early identification of COVID-19 cases is momentous as it not only helps start treatment of the cases immediately, but also facilitates containing the virus by isolating the patient from other humans. Presently, RT-PCR is considered as the established procedure to identify the positive cases of COVID-19. To further speed up the identification operations, there is still room for the advancement of better auxiliary alternative diagnostic tools to enhance the identification and tracking at earliest and start the cure right away [bib_ref] Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19)..., Ai [/bib_ref]. As this deadly pathogen is spreading sustainably, easily, and exponentially among mankind, the healthcare workers and medical staff to quell this are severely limited. Due to this scarcity, radiologists are overwhelmed and in severe need of digital tools to take the workload off them. AI experts have suggested a more feasible solution to keep pace in battling this disease by developing ML and DL techniques. Such systems are founded on predicting and diagnosing pneumonia image modalities and scans of the chest thus aiding physicians.
AI-based techniques have shown promising results for various CV tasks, such as image classification, speech recognition, machine translation, object recognition etc. The recent progress in AI techniques is driven by advent of deeper network architectures, availability of powerful computation platforms and accessibility to large scale benchmark data sets [bib_ref] A survey on image data augmentation for deep learning, Shorten [/bib_ref]. The DL methods have produced more promising results for various complex CV tasks compared to traditional ML approaches due to their capabilities to learn and represent features automatically. This eliminates the need to manually engineer features based on human expertise and hence obtain higher accuracies for different classification and regression tasks.
Although DL-based methods have been successful in solving various problems, yet they suffer from two main problems: (1) they are extremely difficult to train, and (2) they require large amount of training data. The first problem is usually solved by implementing and running highly optimal code on powerful GPU-based computers. The later problem can also be alleviated by using data generation techniques, such as GANs. The main objective of GAN is to generate additional data that is similar as much as possible to the original training data. This data along with original data is then used to train the DL networks.
Motivated from CV community, the AI methods have also been adopted for medical image analysis.
In addition to the known established procedure to detect COVID-19-infected humans, there is urgency to develop auxiliary tools that can be exploited for identification and monitoring of positive cases. The availability of CT and CXR images of lungs provide certain characteristics linked with COVID-19 [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref]. The DL algorithms, such as CNN, incrementally learn the patterns in such images by passing the input data though a sequence of convolutional layers. Initial layers of the network capture low-level features, such as edges, lines, corners etc. while the later layers derive highly abstract features, which can help to capture the most prominent feature that can distinguish between COVID-19 and other cases.
Practicing AI systems for investigation, prediction and analysis of diseases is long-established. The first-ever adoption of such program was fashioned in 1976 called MYCIN which operated and prescribed antibiotics for a bacterial illness [bib_ref] Design considerations for MYCIN, Shortliffe [/bib_ref]. Many healthcare experts have been employing such methods not only to identify diseases but also for formulating drugs, analyzing medical images collected for clinical trials and pandemic prediction.
Many examples of ML and AI medical tools for diagnosis of non-infectious (diabetic, cancer, Parkinson's, heart diseases etc.) [bib_ref] A decision support system for diabetes prediction using machine learning and deep..., Yahyaoui [/bib_ref] [bib_ref] Type 2 diabetes data classification using stacked autoencoders in deep neural networks, Kannadasan [/bib_ref] [bib_ref] Classification models for heart disease prediction using feature selection and PCA, Gárate-Escamila [/bib_ref] [bib_ref] Application of adaptive back-propagation neural networks for Parkinson's disease prediction, Rasheed [/bib_ref] and contagious diseases (HIV, Ebola, SARS, and COVID-19) [bib_ref] Automated diagnosis of HIV-associated neurocognitive disorders using large-scale Granger causality analysis of..., Chockanathan [/bib_ref] [bib_ref] Machine-learning prognostic models from the 2014-16 Ebola outbreak: data-harmonization challenges, validation strategies,..., Colubri [/bib_ref] [bib_ref] A novel target detection method for SAR images based on shadow proposal..., Gao [/bib_ref] [bib_ref] Deep nonsmooth nonnegative matrix factorization network with semisupervised learning for SAR image..., Li [/bib_ref] were developed. In a recent series, ML methods have been successfully used for Ebola outbreak estimation. The purpose of obtaining a better outcome was achieved by conducting experiments on ten distinctive classifiers giving accuracy results of approximately 90.95% with 5.39% MAE and 42.41% RMSE value [bib_ref] Performance analysis of time series forecasting using Machine Learning algorithms for prediction..., Pandey [/bib_ref].
## Comparisons to similar surveys
The COVID-19 pandemic have turned the center of research activities as scientists and researchers are focusing more to mitigate this disease by proposing various methods in AIbased domain. Meanwhile, experts have presented various review and survey articles based on role of AI in COVID-19 to help policy makers and medical practitioners. These peer reviewed published surveys can be categorized into two inculcation; problem-based AI solutions, and AI-frameworks applied on different COVID-19 problems. Such as Pham et al. and Rasheed et al. [bib_ref] Artificial intelligence (AI) and big data for coronavirus (COVID-19) pandemic: a survey..., Pham [/bib_ref] [bib_ref] A survey on artificial intelligence approaches in supporting frontline workers and decision..., Rasheed [/bib_ref] presented a survey that categorizes the tasks in respond to COVID-19 pandemic by outlining the applications of big data and AI but mostly investigated the papers that have not been peer-reviewed.
Moreover, the open research challenges are neither mentioned nor discussed. Similarly, Bansal et al. [bib_ref] Utility of artificial intelligence amidst the COVID 19 pandemic: a review, Bansal [/bib_ref] briefly outlined the role of AI approaches used for identification, prediction and management of COVID-19. However, it did not cover all aspects, such as death rate and severity assessment. In addition, Kumar et al. [bib_ref] A review of modern technologies for tackling COVID-19 pandemic, Kumar [/bib_ref] succinctly generalized the role of DL-and ML-based networks to quell COVID-19 though it did not inspect the papers based on COVID-19 diagnosis through clinical data or respiratory waves. Besides, Lalmuanawma et al. [bib_ref] Applications of machine learning and artificial intelligence for COVID-19 (SARS-CoV-2) pandemic: a..., Lalmuanawma [/bib_ref] analyzed AI-based applications from various aspects but inspected few papers. Contrarily, Hussain et al. [bib_ref] AI techniques for COVID-19, Hussain [/bib_ref] overviewed basic AI-based frameworks and Big Data applications applied to combat COVID-19. It elaborated various AI classified learning techniques with cursory details of COVID-19 clinical data analysis and results. Similarly, Swapnarekha et al. [bib_ref] Role of intelligent computing in COVID-19 prognosis: a state-of-the-art review, Swapnarekha [/bib_ref] categorized the review into type of DL, ML and statistical models to quell COVID-19-related issues. The survey covered vast area, from origin of COVID-19 virus to AI-based models, but focused less on comparative analysis of implemented techniques. A very short survey on COVID-19 detection and prediction is presented in Ref. [bib_ref] Review on machine and deep learning models for the detection and prediction..., Salehi [/bib_ref] by analyzing work of only 10 articles.
Beside these, Jamshidi et al. [bib_ref] Artificial intelligence and COVID-19: deep learning approaches for diagnosis and treatment, Jamshidi [/bib_ref] only examined the publication based on advanced DL methods, such as GAN, Extreme Learning Machine, RNN and LSTM for COVID-19 diagnosis and treatment. It just presented the implemented models without comparative analysis. Likewise, Shinde et al. [bib_ref] Hassanien AE (2020) Forecasting models for coronavirus disease (COVID-19): a survey of..., Shinde [/bib_ref] delineates statistical and AI-based forecasting models only, whereas Albahri et al. and Ahmad et al. [bib_ref] Role of biological data mining and machine learning techniques in detecting and..., Albahri [/bib_ref] [bib_ref] The number of confirmed cases of COVID-19 by using machine learning: methods..., Ahmad [/bib_ref] reviewed only ML and data mining techniques for detection of COVID-19. Similarly, Monshi et al. [bib_ref] Deep learning in generating radiology reports: a survey, Monshi [/bib_ref] mainly focused on taxonomy of advanced DL-based methods for generating radiology reports. Some articles reviewed only specific type of data set, such as Jalaber et al. [bib_ref] Lederlin M (2020) Chest CT in COVID-19 pneumonia: a review of current..., Jalaber [/bib_ref] set forth with role of CT images to handle COVID-19 suspected patients at large, severity signs and presentation of lesions, and in the end inspected five articles to describe the role of AI for COVID-19 diagnosis. Shaikh et al. [bib_ref] Current landscape of imaging and the potential role for artificial intelligence in..., Shaikh [/bib_ref] investigated AI approaches and landscape of radiographic imaging modalities (CXR, CT and PET) in few articles with limited information about obtained results. Likewise, Dong et al. [bib_ref] The role of imaging in the detection and management of COVID-19: a..., Dong [/bib_ref] mainly highlighted CT and PET-CT imaging characteristics presented in different articles and later compared the AI techniques implemented for COVID-19 detection, while Shi et al. [bib_ref] Review of artificial intelligence techniques in imaging data acquisition, segmentation and diagnosis..., Shi [/bib_ref] accentuated AI approaches to diagnose COVID-19 that segments CXR and CT images. Articles like Refs. [bib_ref] Diagnostic methods and potential portable biosensors for coronavirus disease 2019, Cui [/bib_ref] [bib_ref] Application of cognitive Internet of Medical Things for COVID-19 pandemic, Swayamsiddha [/bib_ref] discussed the aspects of IoT and biosensors in COVID-19 management.
The study shows that most of above mentioned review articles either focused on single aspect of COVID-19 management or delineated one type of data set. In addition, majority of these surveys presented little comparative analysis and investigated less than fifty articles, which includes high number of papers that have not been peer-reviewed. Our paper mostly covers peer-reviewed articles that presented AI techniques to accomplish tasks, such as COVID-19 diagnosis, prediction, survival assessment and disease prediction, pandemic outbreak forecasting, and drug discovery. Following are the points, which differentiate this study from aforementioned review and survey papers.
- Covers majority of aspects and problems to manage COVID-19 pandemic, such as diagnosis, prediction, disease severity and survival assessment, outbreak forecasting, protein sequence formation and drug discovery. - Focuses on both ML-as well DL-based models and frameworks. - Incorporates all types of data, such as radiographic images (CXR, CT, and ultrasound images), clinical blood samples data, respiratory and coughing waves, time series and other textual data. - Detail comparative performance analysis of various AIbased techniques implemented to combat COVID-19. - A comprehensive analysis mostly based on peer-reviewed articles (90% are peer-reviewed published papers).
## Scope and contribution of the survey
The primary aim of this comprehensive study revolves around the in-depth analysis of AI-based approaches and models used to quell and combat COVID-19 pandemic by mitigating the virus in various prospects, such as prognosis and diagnosis, drug discovery and molecular structural formation. This review will provide a meaningful and compact knowledge both for medical computer scientist and experts to further broaden the research direction to deal with this deadly virus. The main contributions of this study are as follows:
- The short summary of history, patterns, and characteristics of infectious viruses including COVID-19 are presented. - AI techniques and tools adopted to mitigate COVID-pandemic in various prospects, such as prognosis and diagnosis of SARS-CoV-2 disease, drug discovery and molecular structural formation, are highlighted - An extensive information regarding approaches to diagnose COVID-19 using radiography images, breathing and coughing wave samples, and clinical blood samples are described in details. - A comprehensive summary on issues and recommendations to overcome this infectious virus is provided that can timely facilitate effective decision making. - A detailed discussion on open research challenges regarding COVID-19 is also provided.
The remaining paper is managed as follows. Section 2 focuses on the novel ML and DL techniques that are in practice for diagnosis through various modes. Furthermore, it explores the potential ML-and DL-based tools to predict survival and mortality rate, discover vaccine and forecast the COVID-19 pandemic outbreak. Section 3 presents the issues and recommendation to overcome virus, while Sect. [bib_ref] Perspectives in diagnosis and treatment of rabies viral encephalitis: insights from pathogenesis, Mahadevan [/bib_ref] outlines the open research challenges regarding COVID-19 and presents counter-measures to layout a firm groundwork 1 3 for further research. Finally, the conclusion to summarize the overview of this review is presented in Sect. 5.
## Ai-based applications to quell covid-19
The inevitable infectious pandemics are unpredictable and can inflict huge agonies and mortalities across the world. The newly emerged SARS-CoV-2 virus may just be a small capsid but too powerful that requires great efforts and better countermeasures by society to mitigate its negative impact. At the time of this COVID-19-related global emergency, AI researchers had responded the threat by strategically applying various ML and DL techniques in a wide range of applications that not only detect and classify the COVID-19 cases but also forecast the outbreak, tracks the transmission pattern, discovers the effective drugs, predicts the mortality rate and assess the disease's severity.
AI is a wide-ranging scientific area concerned with mimicking human intellectual processes by smart devices. ML is sub-domain of AI that uses statistical models to learn from examples (also known as instances) in data to predict future outcomes without prior knowledge and explicit programming [bib_ref] Artificial intelligence, machine learning and health systems, Panch [/bib_ref]. Whereas, DL is the most tangible manifestation of ML that exploits artificial neural networks for classification or detection task by discovering useful representations from raw data within the predefined space of possibilities. As COVID-19 pandemic is under the spotlight in medical research and AI-based technologies are one of panacea, this section encompasses various novel applications established on ML and DL methods to combat ongoing SARS-Cov-2 pandemic crisis. [fig_ref] Figure 3: Illustration of computer vision and AI based model for COVID-19 diagnosis and... [/fig_ref] unfolds the general approach used to incorporate AI techniques that requires clinical blood samples and radiography images for identification, classification and diagnosis of COVID-19. Various repository are built to store and share data sets regarding COVID-19. Later on, besides data mining, various pre-processing techniques, such as noise removal, data cleaning, feature extraction, segmentation and feature analysis, are mostly employed to enhance the data set and transform it to more meaningful and effective representation. Finally, AI-based techniques and tools are defined to utilize the data sets for COVID-19 segregate COVID-19 affected patients from others.
## Radiography image-based covid-19 diagnostic tools
Saving precious lives is the topmost priority in emergencies, but that requires early detection of disease. The outbreak of this pandemic created a new landscape and requirements of rapid diagnostic tools for early disease detection. Early ailment detection leads to immediate treatment, which can save many lives and helps in halting the pandemic spread. The standard RT-PCR technique limits the early detection of COVID-19 due to low sensitivity and high procedural and experimental time. Contrarily, AI-based health care systems provide outstanding support for efficient screening, early identification and fast diagnosis by analyzing Clinical blood sample data and radiology images, such as CT and CXR, thus providing a sigh of relief for radiologists. Researchers and scientists effectively adopted several ML approaches and techniques to curb the COVID-19 ailment, such as Sethy et al. [bib_ref] Detection of coronavirus disease (COVID-19) based on deep features and support vector..., Sethy [/bib_ref] , developed automatic tool that predicts COVID-19 infection in CXR images by employing To segregate the patients affected by COVID-19 among normal and other pneumonia affected patients, they exercised thirteen different pre-trained state-of-the-art models (VGG19, VGG16, AlexNet etc.) to extract features from 381 CXR. Each class/label (normal, COVID-19, bacterial pneumonia) has 127 CXR to balance the data set. Later, SVM classified COVID-19-infected patients by exploiting the extracted deep features. By comparative analysis, author demonstrated that SVM with ResNet50 model achieved 95.33% average accuracy with a data split ratio of 80:20% while training and testing, respectively. Moreover, it also accomplished better performance in terms of F1-score and sensitivity of 95.34% and 95.33%, respectively. A combination of data over-sampling, image augmentation techniques with ML-based classifier has been introduced in Ref. [bib_ref] Classification of Coronavirus (COVID-19) from X-ray and CT images using shrunken features, Öztürk [/bib_ref]. The researcher first extracted the features by GLCM and its variants, and then used SMOTE for balancing class distribution. The model consists of SVM classifier with stacked AE and PCA to exhibit an accuracy of 94.23%, precision of 96.73%, sensitivity of 91.88%, F1-score of 93.99%, and specificity of 98.54% on CXR.
An alternative technique has been proposed by Ref. [bib_ref] Diagnosis of coronavirus disease 2019 (COVID-19) with structured latent multi-view representation learning, Kang [/bib_ref] to show the effectiveness of multi-view representation learning that transform original features into latent representation of class space for COVID-19 diagnosis. In pre-processing step, V-Net model [bib_ref] V-Net: fully convolutional neural networks for volumetric medical image segmentation, Milletari [/bib_ref] extracted pulmonary segments and lung lobes from CT images to segment the infected lesions. They further divided the obtained 189-dimensional features into two radiomic features (Gray, and Texture) using GLCM and its variants, and five handcrafted features (histogram, number, intensity, volume, and surface). The CPM-Netslearnt the later features. Later, they trained Latent-representation Regressor model followed by several ML-based classifier models (LNR, SVM, Gaussian Naïve Bayes, KNN, and logistic regression) for COVID-19 prediction. With the incorporation of proposed model, the model achieved an overall accuracy, specificity and sensitivity of 95.5%, 93.2% and 96.6%, respectively, on 2522 CT images, among which 1495 samples belong to patients affected by COVID-19). [fig_ref] Table 1: Adopting radiography images for COVID-19 diagnostic applications based on machine-learning approaches P... [/fig_ref] represents ML-based approaches and classifiers, such as DT, KNN, LNR, and LD used to screen and detect COVID-19 cases by analyzing medical radiology images. The costly RT-PCR tests kits are short in supply; therefore, AI scientists have proposed various cost-effective solutions by attempting various DL models in prediction, diagnosis and prognosis of SARS-CoV-2 due to outstanding performance in handling and processing complex biological and medical data. Such as Apostolopoulos and Mpesiana [bib_ref] Covid-19: automatic detection from X-ray images utilizing transfer learning with convolutional neural..., Apostolopoulos [/bib_ref] exploited transfer learning technique with various state-of-the-art CNN-based frameworks including Inception, Inception ResNet v2, MobileNet v2, VGG19 and Xception to isolate SARS-CoV-2-infected patients among 1427 CXRs images. The analyzing, author concludes that MobileNet v2 surpassed other frameworks by securing sensitivity of 99.10%, specificity of 97.09%, accuracy of 97.04% on twoclass problem, while on three-class classification task, it achieved an accuracy of 92.85%. Moreover, they tested the implemented models on second data set that contains 224 COVID-19-infected cases images, 714 CXRs of patients with viral pneumonia, and 504 CXRs of healthy person. On this data set, for three-class problem, MobileNet v2 attained accuracy of 94.72%, while for two-class problem, it secured 98.66% sensitivity, 96.46% specificity, 96.78% accuracy. Similarly, Brunese et al. [bib_ref] Explainable deep learning for pulmonary disease and coronavirus COVID-19 detection from X-rays, Brunese [/bib_ref] implemented DTL approach with fine-tuned DL-based customized VGG16 framework to differentiate between pulmonary diseases patients and health person (model-1), and then figures out COVID-19-infected patients among discovered pulmonary diseases patients (model-2). The suggested model uses 6523 CXRs, among which 250 CXRs correspond to COVID-19-infected patients, 2753 CXRs belong to pulmonary diseases patients and 3520 images of healthy patients to diagnose COVID-19 while highlighting the potential-infected region due to SARS-CoV-2 virus. Model-1 accomplished a sensitivity, f1-score, specificity, and accuracy, of 96%, 94%, 98%, and 96%, respectively. The experimental finding yields that second model, disease classification model, attained sensitivity, specificity, accuracy and f1-score of 87%, 94%, 98%, and 89%, respectively. Apart from using the DTL approaches and pre-trained models, authors of Ref. [bib_ref] A machine learning-based framework for diagnosis of COVID-19 from chest X-ray images, Rasheed [/bib_ref] designed and trained a CNN-based network that utilizes features extracted through PCA. The authors further proposed a GAN model to eliminate the class imbalance issue and enhance the data set. The incorporation of PCA not only significantly reduced the computational time but also improved the accuracy to its maximum extent.
Besides CXRs, researchers also focused on CT images, such as Xu et al., designed a 3-D CNN-based framework to isolate patients affected by COVID-19 among healthy and IAVP in timely manner. They segmented 219 COVID-19-infected CT images, 224 IAVP CT images, and 175 normal cases CT images and extracted meaningful features by incorporating ResNet model. Finally, locationattention classification framework achieved an overall prediction accuracy of 86.7%. Jaiswal et al. [bib_ref] Classification of the COVID-19 infected patients using DenseNet201 based deep transfer learning, Jaiswal [/bib_ref] presented an alternate state-of-the-art CNN-based model to distinguish COVID-19-infected humans using chest CT images. It employed pre-trained DenseNet201 with DTL approach to analyze 1230 CT images of patients other than COVID-19, while 1262 CT images are of SARS-CoV-2 positive cases. The proposed model achieved precision, sensitivity, specificity, accuracy and f1-score of 96.29%, 96.29%, 96.21%, 96.25%, and 96.29%, respectively. [fig_ref] Table 2: Adopting radiography images for COVID-19 diagnostic applications based on deep-learning approaches [/fig_ref] lists performance details of COVID-19 diagnostic tools and applications based on DL-guided methods that may aid concerned personnel while selecting an appropriate architecture for SARS-CoV-2-infected patient's identification.
## Routine clinical data-based diagnostic tools
Due to expensive radiographic imaging machines, several developing countries and states lacks in CT, CXR and ultrasound machines but has basic blood testing facilities. Keeping in view such scenarios, scientists and programmers have developed AI-based applications and tools to screen COVID-19 positive case using Clinical blood reports. Batista et al.implemented five various ML classifiers, such as RF, NN, LR, SVM, and gradient boosting trees to segregate COVID-19-infected patients by collecting a 235 adult patients blood sample data from hospital in Brazil. The collected data set contains 125 samples of COVID-19 negative patients, while 110 samples belong to COVID-19-infected patients. Each data instance had 15 attributes that includes CRP, mean corpuscular hemoglobin, MCV, mean corpuscular hemoglobin concentration, age, gender, hemoglobin, RDW, red blood cells, leukocytes, monocytes, platelets, lymphocytes, basophils, and eosinophils. From experimental findings, it is noted that SVM outperformed other ML approaches by securing a sensitivity, specificity, accuracy, F1-score, NPV, PPV and brier score of 67.7%, 85.0%, 84.7%, 72.4%, 77.3%, 77.8% and 16.0%, respectively, when tested and trained under tenfold cross validation. [fig_ref] Table 3: Artificial intelligence-based diagnostic tools for COVID-19 using data related to clinical blood... [/fig_ref] lists COVID-19 diagnostic applications empowered by various AI approaches, which analyzes data related to routine clinical blood samples.
## Coughing waves and respiratory pattern-based diagnostic tools
Beside diagnostic applications for COVID-19 based on radiography images or clinical blood samples data, Wang et al.presented a classification network (BI-AT-GRU) which effectively utilizes the respiratory patterns of patients [bib_ref] Learning phrase representations using RNN encoder-decoder for statistical machine translation, Cho [/bib_ref]. In addition to the stimulated data, it adequately uses real-world data. The framework discovers and differentiates the respiratory pattern known as Tachypnea (an occurrence of more speedy respiration) among six other patterns of viral infections. Due to scarcity of respiratory data, authors used illustrates various ML-and DL-based COVID-19 diagnostic tools and applications that uses coughing or respiratory data.
## Disease severity and survival-mortality assessment models
The timely knowledge about the severity of disease facilitates the attending staff in dealing patients priority wise and utilizing the hospital facilities accordingly. A very rare work has been observed on lung's ultrasound data, such as Carrer et al. [bib_ref] Automatic pleural line extraction and COVID-19 scoring from lung ultrasound data, Carrer [/bib_ref] , proposed a unsupervised method based on Viterbi algorithm and Hidden Markov model for localization and detection of pleural line in LUS data. Later it evaluates the severity level of patient using SVM. From results, it is observed that pleura detection model achieved an accuracy of 94% and 84% for linear and convex probes, while SVM classifier evaluated the severity with an accuracy of 94% and 88% for linear and convex probes. Zhou et al. [bib_ref] A rapid, accurate and machine-agnostic segmentation and quantification method for CT-based COVID-19..., Zhou [/bib_ref] proposed machine-agnostic quantification and segmentation technique to cater 3D segmentation problem in CT images for severity identification of COVID-19-infected regions. The proposed simulator decreases the model parameters by using symmetry properties of lungs and tissues that decomposes 3D segmentation into three 2D ones (along y-z, x-y, and x-z planes) thus reduces time complexity. The three independent 2D U-Nets segmented infectious Bai et al. [bib_ref] Predicting COVID-19 malignant progression with AI techniques, Bai [/bib_ref] proposed a hybrid DL-based network with multivariate LR classifier to predict COVID-19 malignant progression. The model converts statistical instances (75 clinical data characteristics) to 40-D feature vector using MLP. Finally, it predicts high-risk patients using quantitative CT sequences, obtained at different time interval, along with these transformed multi-dimensional feature vectors with the help of LSTM. The proposed severity assessment tool secured an AUC of 95.4% and overall accuracy of 89.1% when evaluated on data set of 133 patients under fivefold cross-validation. Similarly, an ML-based MCDM has been proposed in Ref. [bib_ref] Helping doctors hasten COVID-19 treatment: towards a rescue framework for the transfusion..., Albahri [/bib_ref] to optimize the treatment strategy. The network detects the severely infected SARS-CoV-2 patients and prioritized them for relevant convalescent plasma transfusion. [fig_ref] Table 5: Mortality and survival rate prediction with disease severity assessment of patients using... [/fig_ref] shows the severity and fatality assessment models based on conventional ML and advanced DL techniques.
## Covid-19 outbreak forecasting models
The widespread of COVID-19 outbreak has created panic as a human community is still at risk, while hospitals are full, people are facing financial issues as governments are struggling to pass critical decisions, mortality rate is increasing exponentially, whereas social activities are halted. In this high uncertainty, experts have applied various DL and ML techniques to design outbreak-forecasting models that would help decision makers to recommend new preventive strategies and develop critical measures for future possibilities. Such as Carrillo-Larco and Castillo-Cara [bib_ref] Using countrylevel variables to classify countries according to the number of confirmed..., Carrillo-Larco [/bib_ref] suggested an unsupervised ML-based model that uses k-means clustering algorithm to classify the countries sharing same number of confirmed COVID-19 cases. In this study, researchers considered different attributes, such as prevalence of tuberculosis and HIV/AIDS diseases in 156 countries, social-economic parameters, such as gross domestic production as social-economic parameters and other health system metric, such as air quality along with COVID-19 prevalence data (confirmed cases, death etc.). It concludes that the integration of PCA with k-means-based model successfully stratify countries into five and six groups. The researchers conclude that model works well for countries stratification based on confirmed SARS-CoV-2 cases but not able to classify in terms of SARS-CoV-2 fatality cases. To assist policymaker, Kavadi et al. [bib_ref] Partial derivative nonlinear global pandemic machine learning prediction of COVID 19, Kavadi [/bib_ref] presented a PDR-NML framework that predicts SARS-CoV-2 transmission patterns in India. The proposed statistical model, PPDLR, normalizes it by searching the best features, which are then fed to a support Kuhn-Tucker-based nonlinear global pandemic ML model to forecast the future outbreak of COVID-19 pandemic cases. The presented model outperformed LNR and other famous AI-base models by securing 99.7% accuracy. Hu et al. [bib_ref] Forecasting and evaluating multiple interventions for COVID-19 worldwide, Hu [/bib_ref] attempted modified AE that uses real-time data for pandemic outbreak forecasting in large geographical area by considering the interventions and measures to curb the pandemic. The framework use data of confirmed COVID-19 cases happened between January 20, 2020 to March 16, 2020 in 152 countries to forecast future cases, as well as pandemic peak and end time. It consists of 2 single AEs each comprised of 3 feed-forward NN layers that performed well while estimating the daily new cases in China as compared to SEIR model. The model attained error of 0.00134 and recommended that early precautionary measures would eliminate 99.4% of potential cases.
Other than the conventional ML algorithm, experts also used advanced DL techniques to determine the unseen forthcoming cases. Authors in Ref.forecasted global pandemic outbreak by using multivariate spatiotemporal model based on convolutional LSTM framework. They used the data of Italy and USA, and transformed spatial features into clusters. The proposed forecasting tool predicted number of potential cases for the next 5 days with an MAPE of 5.57% and 0.3% for USA and Italy, respectively. [fig_ref] Table 6: COVID-19 outbreak prediction and risk assessment using artificial intelligence-based applications [/fig_ref] illustrates more AI-based applications/tools to assess the risk and predict the pandemic outbreak.
## Covid-19 protein sequence formation and drug discovery models
In this panic-stricken era of COVID-19, rapid drug discovery in accordance with the exact virus genome is crucial to saving thousands of lives. Still many genomes and peptides of this noxious virus are being identified on a regular basis.
To bring the effective drug-making process up to speed, [bib_ref] Modified SEIR and AI prediction of the epidemics trend of COVID-19 in..., Yang [/bib_ref] Customized SEIR with LSTM Analyze and predict COVID-19 pandemic curve for China TS many ML models are in process to master the viral structural analysis. In the interest of seeking probable vaccine possibilities for SARS-CoV-2, Ong et al. [bib_ref] COVID-19 coronavirus vaccine design using reverse vaccinology and machine learning, Ong [/bib_ref] established a machine learning-based Vaxign-ML reverse vaccinology system. Their research based on a system equipped with five conventional ML algorithms (XGB, SVM, RF, kNN and logistic regression) applied on extracted protein data set, subsequent to fivefold cross-validation, defined further with biological and physicochemical characteristics,. From findings, it can be noted that XGB model indicated an F1-measure of 94%. The results of the system indicated conservancy of SARS-CoV-2 N protein sequence with SARS-CoV and MERS-CoV only. While discussing the crucial matters of virus attacking and attaching itself to the host, the responsible adhesions proteins found were the S protein along with non-structural proteins; nsp3, 3CL-pro, and nsp8-10. Moreover, high protective antigenicity causing inferred by the designed Vaxign-ML system was attributed to three proteins namely S, nsp3, and nsp8 as potential vaccine candidate based on high protegenicity score. This particularly tailored vaccine aptitudes for designing a reliable and competent COVID-19 vaccine.
Magar et al.founded a machine learning idea envisioned to identify synthetic COVID-19 inhibitory antibodies. The ML strategies segregated the data of virus-antibody sequences via graphical diagrams and reported 8 stable antibodies with the ability to perform as COVID-19 inhibitors. The way that led the COVID-19 antibody identification starts with gathering and maintaining the data for the devised system. Then comes the featurization, embedding and benchmarking ML designs and screening for the best model available. Later on, a hypothetical antibody group is assembled and ML screening is done for obliteration. Lastly, the validity of the suggested antibodies is evaluated. Such generalized flowchart makes fast and facile screening of probable antibodies having an immense potential to defeat COVID-19. depicts other AI-empowered applications used to detect protein sequence and discover to tackle COVID-19 pandemic.
# Discussion
In this survey, we not only presented an analysis of clinically utilized AI tools providing assistance against COVID-19 but also presented a detailed historical account of the virus and related family. The detailed investigation reveals several ML and DL approaches so far lending help to a great extent, initiating from image diagnostics and going up to the presentation of prospective models for methodical anticipation of the epidemics outbreaks. Therefore, this study can facilitate healthcare and research operatives to efficiently and effectively cope the COVID-19 pandemic. With timely and precise analysis, approach at hand for the desired solution an immediate response against the disease can be lead. This survey concentrates mainly on the obstacles faced so far while executing AI and ML-based arrangements for COVID-19 prognosis. Moreover, some suggested plans and quick fixes to resolve those issues are also covered.
The foremost inaccuracy fails to arise from not constituting diagnostic programs, which segregates on the basis of symptoms. Thus, to have robust and well-generalized predicting tools, various other aliments (liver, heart, diabetes, etc.) and patient's information (gender, age, etc.) must be considered. In comparison with known community acquired and viral pneumonia, these aspects (patient's information and aliments) have significantly influences the severity of COVID-19 disease in a human. Thus, leading to an instantaneous identification of the virus. An enormous amount of data is laid out there by several countries severely hit by the pandemic. If those statistics are attained in the right way, schemes for the prediction can efficiently generate smart and uncomplicated results.
Then, there comes the issue of urgency. Since the government needs statistics right away to regulate quarantine laws for constraining the disease expansion. Therefore, the scientific data sets are constructed on hasty calculations. Thus, providing ineffective and non-reproducible structures. In pursuance of productive and quick implementation of policy models, the data set must be fixed and generate reproducible Protein sequence detection and drug discovery using artificial intelligence-based applications References Name of algorithm/model Problem/assignment [bib_ref] COVID-19 coronavirus vaccine design using reverse vaccinology and machine learning, Ong [/bib_ref] DTL-based Vaxign-machine-learning reverse vaccinology tool Candidate vaccine prediction for SARS-CoV-2 virusLSTM and semi-supervised VAE Discover drug by detecting SMILE fingerprint of moleculesGAN Designing the drug compound (non-CoV) [bib_ref] Predicting commercially available antiviral drugs that may act on the novel coronavirus..., Beck [/bib_ref] A pre-trained network based on AI approach, called Molecule-Transformer-Drug-Target-Interaction
Determine the availability of antiviral drug to tackle SARS-COV-2SVM, RF, MLP, LR, and XGBoost Potential antibodies discovery for COVID-19MLP and ANFIS Detect nucleic acid based on CRISPRGAN Develop formation of drug compound for COVID outcomes and solve the urgent problem as well. In designing the analysis applications based on statistical approach, one of the most important and challenging task is availability of high-quality samples at real time. In circumstances, where data are rapidly changing at faster pace, the reliability of produced tools are minimized. Therefore, to tackle this problem, regular updating the data based on region-specific values are decisive. For planning any systems for prediction studies, attaining large data is the crucial requirement that needs to be fulfilled. Deep learning-based systemization needs big data sets of clinical images and statistical numerals. A large amount of data can be produced for better training of deep learning approaches if obtained sets are not divided on the basis of geography. Regulation of data sources needs to be addressed as well while handling massive data to avoid misunderstandings. Yet, another challenge that arises while training and designing such AI systems is due to little or almost no association of researchers with medical experts. While assessing the medical data, such as X-ray or scans relatable medical personnel, must be present to make a definitive opinion.
A better and definitive estimation of potential patients and the number of deaths that are going to happen in future is a top priority in the current situation. This can be achieved by gathering and structuring the data of heavily affected countries and utilizing it with accurate and robust AI-based models as better equipment against an upcoming alarming situation.
## Open research challenges
The COVID-19 pandemic has unequivocally affected both individuals and economies everywhere in the world. This led to various systemic challenges in various field, such as health, governance, trade, education, technology etc. This section highlights some of the challenges to combat the current pandemic.
## Research collaboration and data sharing
The COVID-19 is a family of viruses that has various forms and has different behavior on people living in various regions around the globe. This requires a cross examination of the cases in multi-folded perspectives. There must be some agreements reached among various entities including civil society, private and public sectors to share data and conduct research to expedite the process for finding an ultimate solution. This envisions the authorities about expected abiding transformations, and motivates them to revamp the condition of the world by taking advantages of this moments.
It is critical for combating the disease to do research collaboration to accelerate the process towards normalizing the life and overcome the pandemic. This requires high-level decision making to remove obstacles in free flow of research ideas and make availability of research data for more critical decision making. Infrastructure should be developed to share and collaborate the research data globally, which is still a challenging task. This also requires policy to be defined for integrity of the patient data by introducing some defined set of rules and measures, interoperability and co-ordination, in conjunction with quality and interpretation of data.
## Lack of technology infrastructure
Availability of IT infrastructure is crucial for early detection, tracking and monitoring of the patients. In China, ubiquitous availability of IT infrastructure help finding hotspots and crowd gathering, thus assisted the administrators to take faster decision making and implementation. Developing countries are suffering from serious lack of infrastructures to fully utilize the ability of AI for both detection and previous strategies.
## Development of vaccines
AI is inevitable in almost all fields of life. Multidisciplinary research should be conducted for development of vaccine for COVID-19. The use of AI for drug delivery, design, development and efficacy will eventually speed up the drug testing in real time. Policies should be designed to take advantage of this.
## Increased pressure on hospitals
There has been sharp increase of patient admissions in hospitals across the globe that overwhelmed the healthcare professionals due to high workload. To increase the efficiency and properly managing the critical patients, AI should be used from patients' admission, testing and monitoring. This will reduce the pressure and workload for medical staff.
## Adopting models from developed countries
Some countries, such as South Korea, flattened the curve of COVID-19 by adopting digital technology and combining it with other strategies, such as early detection, free treatment, and isolation of COVID-19 cases. No severe lockdowns were placed; however, they used technology to monitor trends and hotspots to take early action. In addition, the public awareness and participation was very good. Adopting such model in the developing countries need these three important components: digital infrastructure, healthcare infrastructure, and public awareness.
## 3
## Transparent disclosing all information
Accurate information sharing is crucial for public safety and crucial decision making. Governments should develop strategies for sharing the accurate information about the COVID-19 cases, through press briefing, to make awareness among mankind about ways to minimize the transmission of virus, and the impact of the social distancing.
Finally, the developing countries must cautiously and gently relaxes the restrictions related to immigration and quarantine keeping in view the global transmission rate of disease and corresponding healthcare situation/facilities of the country. Countries should also aim for assistance and capacity building in communal institutions. International associations and societies must collaborate and join the developing countries to cope with this pandemic.
## Lesson learning from this pandemic
This may not be the last pandemic, as world has witnessed many similar challenges in the past. Learning some lessons from current pandemics and preparing for future challenges is a crucial step now. The models and preparedness should be done keeping future perspective in mind. Creating awareness among people and balanced distribution of wealth can highly reduce the risks of spread of such diseases.
# Conclusion
The recent advancements in AI techniques have played an important role in biomedical sciences providing a handy role in diagnosis and monitoring of various diseases. For COVID-19 pandemic, it is essential to detect it as early as possible by collecting and analyzing related information to predict, where this virus will affect in the future. In this paper, we report the recent active role of AI for combating the COVID-19 and highlighted new research directions and main challenges in adopting a robust solution for this pandemic. The paper spans over two main domains, medical and technological. In the first domain, this review covered the global transmission patterns of COVID-19 and brief history of various other viruses. Then, it described the advances in AI tools to diagnose, assess the severity of disease, predict the mortality rate, and discover the drug compounds. Furthermore, an analysis of recently applied techniques for various biological and computing approaches have been presented. This work will help decision makers to better understand the role of AI for combating COVID-19, so that better decisions can be taken to properly handle and take precautionary steps by designing instructions in pandemic stricken regions. Subsequently, computer-aided platforms are in operation for smart utilization of medical facilities on a priority basis.
# Declarations
## Conflict of interest
The authors declare that they have no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Informed Consent For this type of study, formal consent is not required.
## Statement of human and animal rights
This paper does not contain any studies with human participants or animals performed by any of the authors.
[fig] Figure 1: Deadliest viruses over last 102 years (as of [/fig]
[fig] Figure 2: Top [/fig]
[fig] Figure 3: Illustration of computer vision and AI based model for COVID-19 diagnosis and prediction SVM. [/fig]
[table] Table 1: Adopting radiography images for COVID-19 diagnostic applications based on machine-learning approaches P precision, Sp specificity, Se sensitivity, F1 f1-measure, A accuracy, CXR chest X-ray images, CT computed tomography images *Values related to classification of COVID-19 class only [/table]
[table] Table 2: Adopting radiography images for COVID-19 diagnostic applications based on deep-learning approaches [/table]
[table] Table 3: Artificial intelligence-based diagnostic tools for COVID-19 using data related to clinical blood samplesTable 4Artificial intelligence-based diagnostic tools for COVID-19 using respiratory data P precision, Sp specificity, Se sensitivity, A accuracy [/table]
[table] Table 5: Mortality and survival rate prediction with disease severity assessment of patients using artificial intelligence-based application CXR chest X-ray images, CT computed tomography images, CD clinical data, TS time series [/table]
[table] Table 6: COVID-19 outbreak prediction and risk assessment using artificial intelligence-based applications [/table]
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The SUMO-specific isopeptidase SENP2 associates dynamically with nuclear pore complexes through interactions with karyopherins and the Nup107-160 nucleoporin subcomplex
The association of small, ubiquitin-related modifier-specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat-containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway.
# Introduction
Small, ubiquitin-related modifiers (SUMOs) are ∼100-amino acid proteins that covalently and reversibly attach to lysine residues in substrate proteins to modulate their localization, activity, and/or stability [bib_ref] Protein modification by SUMO, Johnson [/bib_ref] [bib_ref] Concepts in sumoylation: a decade on, Geiss-Friedlander [/bib_ref]. Genetic and proteomic studies have identified hundreds of SUMO substrates, implicating sumoylation as an essential regulator of a wide array of cellular processes, including transcription, chromatin structure, DNA repair, chromosome segregation, stress response, and nucleocytoplasmic transport [bib_ref] Differential regulation of sentrinized proteins by a novel sentrin-specific protease, Golebiowski [/bib_ref] [bib_ref] Global map of SUMO function revealed by protein-protein interaction and genetic networks, Makhnevych [/bib_ref]. Sumoylation is achieved through a three-step enzymatic cascade involving an ATP-dependent E1-activating enzyme (the Aos1/Uba2 heterodimer), an E2-conjugating enzyme (Ubc9), and in many cases, one of several E3 ligases [bib_ref] Protein modification by SUMO, Johnson [/bib_ref]. Reversal of this modification is achieved by a family of SUMO-specific isopeptidases (referred to as sentrin-specific proteases, or SENPs, in vertebrates; [bib_ref] Modification in reverse: the SUMO proteases, Mukhopadhyay [/bib_ref].
While there are two SUMO-specific isopeptidases in yeasts, Ulp1 and Ulp2, mammalian genomes encode six: SENP1, SENP2, SENP3, SENP5, SENP6, and SENP7. Each of these enzymes contains a highly conserved C-terminal catalytic domain but is distinguished by a distinct N-terminal domain that targets it to a unique subcellular location [bib_ref] Modification in reverse: the SUMO proteases, Mukhopadhyay [/bib_ref]. Ulp1, SENP1, and SENP2 are localized to nuclear pore complexes (NPCs), while Ulp2, SENP6, and SENP7 localize throughout the nucleoplasm [bib_ref] A new protease required for cell-cycle progression in yeast, Li [/bib_ref] [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref] [bib_ref] Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1, Bailey [/bib_ref] [bib_ref] Modification in reverse: the SUMO proteases, Mukhopadhyay [/bib_ref] [bib_ref] Essential role of nuclear localization for yeast Ulp2 SUMO protease function, Kroetz [/bib_ref]. Vertebrates contain a unique pair of nucleolar-specific isopeptidases, SENP3 and SENP5 [bib_ref] A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at..., Nishida [/bib_ref] [bib_ref] Characterization of a family of nucleolar SUMOspecific proteases with preference for SUMO-2..., Gong [/bib_ref]. Studies in yeast have demonstrated clearly that the regulated localization of SUMO-specific isopeptidases serves to define their functions by restricting access to protein substrates [bib_ref] The role of karyopherins in the regulated sumoylation of septins, Makhnevych [/bib_ref] [bib_ref] Essential role of nuclear localization for yeast Ulp2 SUMO protease function, Kroetz [/bib_ref]. How subcellular localization affects the functions of each of the six mammalian SUMO-specific isopeptidases, however, remains to be fully characterized.
The conserved localization of SUMO-specific isopeptidases at NPCs implies a functional link between sumoylation and nucleocytoplasmic transport [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref] [bib_ref] The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization,..., Li [/bib_ref] [bib_ref] Unconventional tethering of Ulp1 to the transport channel of the nuclear pore..., Panse [/bib_ref] [bib_ref] Drosophila Ulp1, a nuclear pore-associated SUMO protease, prevents accumulation of cytoplasmic SUMO..., Smith [/bib_ref] [bib_ref] NUCLEAR PORE ANCHOR, the Arabidopsis homolog of Tpr/Mlp1/Mlp2/megator, is involved in mRNA..., Xu [/bib_ref]. Consistent with this link, the SUMO E2-conjugating enzyme Ubc9 and the E3 ligase RanBP2/Nup358 also localize to NPCs [bib_ref] The nucleoporin RanBP2 has SUMO1 E3 ligase activity, Pichler [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref]. NPCs are massive protein complexes that permeate the nuclear envelope and provide a selective passageway for the transport of proteins and RNAs into and out of the nucleus [bib_ref] The nuclear pore complex has entered the atomic age, Brohawn [/bib_ref] [bib_ref] The nuclear pore complex: bridging nuclear transport and gene regulation, Strambio-De-Castillia [/bib_ref]. Signal-mediated transport through NPCs is dependent on a family of soluble transport receptors called karyopherins (or importins and exportins) that bind to nuclear localization or export sequences (NLSs or NESs) in cargo proteins and facilitate their translocation through NPCs via interactions with FG repeat-containing nucleoporins [bib_ref] Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport, Terry [/bib_ref]. In the budding yeast, mutants deficient for sumoylation exhibit defects in the nuclear import of proteins containing classical NLSs due to impaired recycling of Kap60 (karyopherin-α; [bib_ref] A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast, Stade [/bib_ref]. Exactly how sumoylation affects karyopherin recycling and/or NPC function remains to be fully understood.
In addition to affecting nucleocytoplasmic transport in yeast, sumoylation at NPCs has also been determined to be important for proper regulation of mRNA export and DNA repair. In particular, mutations affecting the localization of Ulp1 at NPCs also affect the retention of unspliced mRNAs in the nucleus and NPC-coupled DNA repair processes [bib_ref] A SUMO ligase is part of a nuclear multiprotein complex that affects..., Zhao [/bib_ref] [bib_ref] A nuclear envelope protein linking nuclear pore basket assembly, SUMO protease regulation,..., Lewis [/bib_ref] [bib_ref] Nucleoporins prevent DNA damage accumulation by modulating Ulp1-dependent sumoylation processes, Palancade [/bib_ref] [bib_ref] Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase, Nagai [/bib_ref]. The localization of Ulp1 with NPCs has been studied extensively and shown to involve a complex network of interactions. Two domains in the N-terminus of Ulp1 mediate unconventional interactions with the nuclear transport receptors Kap60/Kap95 (karyopherin-α/β) and Kap121 (Pse1; [bib_ref] The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization,..., Li [/bib_ref] [bib_ref] Unconventional tethering of Ulp1 to the transport channel of the nuclear pore..., Panse [/bib_ref]. These receptors target Ulp1 to NPCs, where its association is dependent upon a subset of nucleoporins and NPC-associated proteins that include Nup60, the Nup84 nucleoporin subcomplex, Mlp1/Mlp2, and Esc1 [bib_ref] Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent..., Zhao [/bib_ref] [bib_ref] A nuclear envelope protein linking nuclear pore basket assembly, SUMO protease regulation,..., Lewis [/bib_ref] [bib_ref] Nucleoporins prevent DNA damage accumulation by modulating Ulp1-dependent sumoylation processes, Palancade [/bib_ref]. Although the proper localization of Ulp1 to NPCs is critical for mRNA surveillance and DNA repair, the roles that sumoylation and desumoylation play in these processes remain poorly understood. Also, whether the localization of SUMO-specific isopeptidases in mammalian cells is similarly important for these processes remains to be determined.
While Ulp1 is localized to NPCs during interphase, it is released and relocalizes to the septin ring during mitosis, a targeting event dependent on interactions with Kap121 [bib_ref] The role of karyopherins in the regulated sumoylation of septins, Makhnevych [/bib_ref]. In addition, Ulp1 is released from NPCs in response to ethanol stress and relocalizes to nucleoli, a process mediated in part by interactions with Kap60/Kap95 [bib_ref] A novel mechanism for SUMO system control: regulated Ulp1 nucleolar sequestration, Sydorskyy [/bib_ref]. The localization of Ulp1 and its access to distinct subsets of sumoylated proteins are therefore subject to regulation and affected by its association with nuclear transport receptors.
Unlike Ulp1, relatively little is known about the association and functions of its vertebrate counterparts, SENP1 and SENP2, at NPCs. Previous work demonstrated that SENP2 is essential for embryonic development in mice, underscoring the importance of understanding its cellular functions. In cultured cells, SENP2 has been shown to shuttle between the nucleus and the cytoplasm and to concentrate at the nucleoplasmic face of NPCs through interactions with Nup153 [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref] [bib_ref] Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2, Itahana [/bib_ref]. Whether interaction with Nup153 is the sole determinant of SENP2 localization at NPCs, and whether this interaction is direct or indirect, was unknown. In this study, we provide a more detailed analysis of the molecular interactions between SENP2 and NPCs. Our findings reveal that SENP2 contains a high-affinity NLS that mediates unconventional interactions with nuclear transport receptors and tethering to FG-repeat nucleoporins. We also demonstrate that SENP2 contains a second NPCtargeting signal that mediates interactions with the Nup107-160 nucleoporin subcomplex, a subcomplex with essential functions in interphase NPC assembly and mitotic chromosome segregation. Our findings indicate that a complex network of interactions define the steady-state association of SENP2 with NPCs and suggest possible new functions for SENP2, as well as mechanisms for regulating its substrate selectivity.
# Results
Endogenous SENP2 localizes to NPCs, the nucleoplasm, and the cytoplasm Exogenously expressed vertebrate SENP2 has been described as localizing to the nucleoplasm, cytoplasm, and NPC [bib_ref] Characterization of a novel mammalian SUMO-1/Smt3-specific isopeptidase, a homologue of rat axam,..., Nishida [/bib_ref] [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref] [bib_ref] Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2, Itahana [/bib_ref]. To characterize the localization of endogenous SENP2, we produced rabbit polyclonal antibodies and demonstrated specificity by immunoblot analysis of untransfected and green fluorescent protein (GFP)-SENP2 transfected cell lysates. The antibody recognized GFP-SENP2, as well as multiple lower-molecular-weight bands in lysates of transfected and untransfected HeLa cells . Four of the lower-molecular-weight bands were reduced upon transfection of three different RNA interference (RNAi) oligos directed against SENP2 in HEK293 and HeLa cells: one major band at 50 kDa and three additional bands at 60, 45, and 44 kDa ; unpublished data). We confirmed that these bands corresponded to different molecular-weight forms of SENP2 by treating lysates with a SUMO-2 vinyl sulfone derivative (SUMO-2-VS; . SUMO-2-VS forms a covalent intermediate with SUMO isopeptidases, resulting in a ∼20-kDa shift in molecular weight [bib_ref] SUSP1 antagonizes formation of highly SUMO2/3-conjugated species, Mukhopadhyay [/bib_ref]. All four specific bands shifted upon treatment with SUMO2-VS, indicating that each protein contains isopeptidase activity. The different forms of SENP2 may arise through alternative splicing, as documented in mouse cells [bib_ref] Characterization of a novel mammalian SUMO-1/Smt3-specific isopeptidase, a homologue of rat axam,..., Nishida [/bib_ref] [bib_ref] SUMO-1 protease-1 regulates gene transcription through PML, Best [/bib_ref].
We next examined the localization of endogenous SENP2 by indirect immunofluorescence microscopy. Endogenous SENP2 was concentrated at the nuclear envelope in interphase cells, where it colocalized with FG-repeat nucleoporins recognized by mAb 414 [bib_ref] Identification and characterization of a nuclear pore complex protein, Davis [/bib_ref]. In addition, diffuse staining was detected throughout the cytoplasm and nucleoplasm. NPC staining, as well as much of the diffuse nucleoplasmic and cytoplasmic signals, was markedly decreased in cells transfected with SENP2 small interfering RNAs (siRNAs; . Consistent with previous findings [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref] , transiently expressed, full-length GFP-SENP2 localized predominantly to NPCs (see later in the paper). Our findings reveal that human cells express multiple isoforms of SENP2 and suggest that specific isoforms may differentially distribute among NPCs, the nucleus, and the cytoplasm.
## Senp2 contains multiple npctargeting signals
The N-terminal domain of SENP2 is necessary and sufficient for NPC localization [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref]. To further define and characterize NPC-targeting elements, we generated a series of N-and C-terminal SENP2 truncation expression constructs . Localization of GFP-SENP2 truncation mutants at NPCs was examined by colocalization with mAb 414 in HeLa cells. As previously reported, fusion of the N-terminal 63 amino acids of SENP2 to GFP was sufficient for NPC localization ; [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref]. A notable increase in nucleoplasmic signal was also detected relative to full-length SENP2, suggesting that residues in addition to 1-63 may contribute to NPC interactions. Consistent with this, deletion of the N-terminal 63 amino acids from SENP2 (NΔ63) resulted in a notable increase in cytoplasmic and nucleoplasmic staining without completely abolishing NPC localization . Deletion of the Nterminal 143 amino acids from SENP2 (NΔ143) had a similar effect . Expression of the catalytic domain of SENP2 alone fused to GFP (NΔ367), however, resulted in predominantly diffuse nucleoplasmic localization with no apparent NPC targeting .
These results indicate that residues 1-63, as well as a second signal located between residues 143-367, specify NPC targeting. Supporting the presence of a second signal, fusion of residues 143-350 to GFP resulted in NPC localization, as well as diffuse nucleoplasmic, nucleolar, and cytoplasmic localization . A SENP2 mutant containing the first 63 amino acids but lacking FIGURE 1: Characterization of endogenous SENP2. (A) Lysates of untransfected or GFP-SENP2-transfected HeLa cells were resolved by SDS-PAGE and immunoblot analysis was performed using SENP2-specific polyclonal antibodies. *, GFP-SENP2. (B) HEK293 cells were transfected with scramble (Control) or one of three SENP2-specific siRNA oligos. Cell extracts were incubated in the absence (−) or presence (+) of HA-tagged SUMO-2-VS for 30 min. Reactions were terminated by addition of SDS sample buffer and resolved by SDS-PAGE. Immunoblot analysis was performed using SENP2-specific antibodies. Red dots, RNAi-sensitive bands; blue dots, SUMO-2-VS shifted bands; *, nonspecific bands. (C) HeLa cells were stained with antibodies specific for SENP2 and nucleoporins (mAb 414) and analyzed by indirect immunofluorescence microscopy. Scale bar: 10 μm. (D) HeLa cells were transfected with scramble (Control) or SENP2-specific siRNA oligos and examined by indirect immunofluorescence microscopy using SENP2specific polyclonal antibodies. Images were taken using identical exposure settings. Scale bar: 10 μm.
residues 144-349 (Δ144-349) localized to NPCs, indicating that both NPC-targeting signals are self-sufficient .
## Senp2 interacts with karyopherins and nucleoporins
To identify SENP2-interacting proteins involved in SENP2's association with NPCs we used an affinity purification-mass spectrometry (AP-MS) approach [bib_ref] Analysis of protein complexes using mass spectrometry, Gingras [/bib_ref]. Tetracycline-inducible stable cell lines expressing full-length, Flag-tagged SENP2 were established. Following induction in two independently isolated lines, Flag-tagged SENP2 was immunopurified under nondenaturing purification conditions. SENP2-interacting proteins were analyzed by liquid chromatography, which was followed by tandem mass spectrometry (LC-MS/MS). Cells expressing the Flag tag alone or unrelated proteins were analyzed simultaneously, and only those proteins found specifically in Flag-tagged SENP2-expressing cells are reported [fig_ref] TABLE 1: SENP2 interacts with soluble nuclear transport receptors and nucleoporins [/fig_ref]. Spectral counts, corresponding to the number of times a peptide from each protein was observed, are indicated, providing a rough approximation of relative abundance. Multiple nuclear transport receptors, including karyopherin-β 1 , -β 2 ; karyopherin-α 1 , -α 2 , α 3 , -α 4 , and -α 6 ; and the karyopherin-α recycling factor CSE1L, were identified. In addition, a subset of FG repeat-containing nucleoporins (Nup153, Nup50, and Nup358/RanBP2) and members of the Nup107-160 subcomplex copurified specifically with Flag-SENP2. A variety of other putative interacting proteins were also identified, including proteins involved in splicing, transcription, and the cytoskeletal functions.
To define interacting partners specific to each of the two identified NPC-localization signals, we generated stable cell lines expressing SENP2 truncation mutants, including the N-terminus alone (amino acids [aa], 1-350), the catalytic domain alone (aa 350-589), and an internal deletion mutant lacking residues acids 144-349. As with full-length SENP2, proteins interacting with these mutants were identified by AP-MS. Proteins interacting with the N-terminal domain of SENP2 were similar to those observed with the full-length protein. Consistent with NPC-targeting signals being located within the N-terminal domain, associations with karyopherins and nucleoporins were retained [fig_ref] TABLE 1: SENP2 interacts with soluble nuclear transport receptors and nucleoporins [/fig_ref]. Analysis of the catalytic domain alone revealed interactions with the subset of assorted "other" proteins found to interact with full-length SENP2. The internal deletion mutant, SENP2 Δ144-349, retained its interactions with karyopherins and FG repeat-containing nucleoporins but not with members of the Nup107-160 subcomplex.
Previous studies identified a functional NLS in the N-terminus of SENP2 between amino acids 28 and 52 [bib_ref] Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2, Itahana [/bib_ref]. To address whether this NLS mediates SENP2-karyopherin and nucleoporin interactions, we mutated two critical residues in this signal (R29A/R49A) and identified binding partners of the resulting protein using AP-MS. This mutant did not copurify with karyopherins, indicating that the NLS is required either directly or indirectly for karyopherin binding [fig_ref] TABLE 1: SENP2 interacts with soluble nuclear transport receptors and nucleoporins [/fig_ref]. The NLS mutant also failed to copurify with FG repeat-containing nucleoporins, indicating that SENP2 interactions with these nucleoporins are likely to be karyopherinmediated. Peptides derived from two members of the Nup107-160 subcomplex were, however, still detected.
The N-terminal NLS of SENP2 is a high-affinity, karyopherin-α-binding NLS The N-terminal NLS in SENP2 is a bipartite signal with an unusually long and negatively charged spacer segment. At a basic level, this sequence bears resemblance to a subclass of NLSs found in proteins called karyopherin-α-releasing factors (KaRFs; . These proteins, which include Nup2 in yeast and Nup50 in vertebrates, are characterized by a high-affinity bipartite NLS capable of displacing cargo containing a classical NLS [bib_ref] Accelerating the rate of disassembly of karyopherin.cargo complexes, Gilchrist [/bib_ref] [bib_ref] Structural basis for Nup2p function in cargo release and karyopherin recycling in..., Matsuura [/bib_ref] [bib_ref] Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling, Matsuura [/bib_ref].
To investigate the nature of the interactions between the Nterminal NLS of SENP2 and karyopherin-α, recombinant glutathione S-transferase (GST)-tagged SENP2 1-63 or the NLS mutant, SENP2 1-63(R29A/R49A), were immobilized on glutathione beads and incubated with increasing concentrations of purified Histagged karyopherin-α 2 . The N-terminal 63 amino acids of SENP2 interacted robustly with karyopherin-α alone in an NLS-dependent manner . To investigate whether SENP2 could function as a KaRF, binding competition assays were performed. Biotinylated karyopherin-α was preincubated with an excess of maltosebinding protein (MBP)-tagged SV40 NLS and subsequently with increasing concentrations of GST-tagged SENP2 1-63 (ranging from a 0.5:1 to a 4:1 M ratio of SENP2:NLS). Karyopherin-α/NLS complexes were captured using streptavidin-agarose beads and analyzed by immunoblot analysis. A reciprocal experiment was also performed in which karyopherin-α was preincubated with excess SENP2 1-63 and increasing concentrations of the SV40 NLS were titrated into the reaction. Consistent with having a high-affinity NLS with KaRF-like activity, SENP2 1-63 displaced the SV40 NLS from karyopherin-α. Excess SV40 NLS, however, did not displace SENP2 1-63 in the reciprocal experiment . We also investigated the Ran-GTP sensitivity of a karyopherin-α/β heterodimer complexed with SENP2. Unlike the Ran-GTP-insensitive association of yeast Ulp1 with both Kap95 (karyopherin-β) and Kap60 (karyopherin-α; [bib_ref] Unconventional tethering of Ulp1 to the transport channel of the nuclear pore..., Panse [/bib_ref] , Ran-GTP displaced karyopherin-β from SENP2. Karyopherin-α binding, however, was unaffected .
To address whether individual karyopherins and nucleoporins associate directly with SENP2 or indirectly through extended protein complexes, we repeated the AP-MS analysis of wild-type SENP2, this time performing the purifications in both the presence and absence of nonhydrolyzable Ran-GTP. We anticipated that Ran-GTP would displace karyopherins associated indirectly with SENP2, but not karyopherins bound with high affinity to the N-terminal NLS, as shown above. Using spectral counts as an indicator of relative abundance, we detected significant reductions in the copurification of karyopherin-β 1 and -α 2 in the presence of Ran-GTP, as well as the FIGURE 2: Schematic representations of SENP2 truncation mutants. The indicated SENP2 truncation mutants were generated using PCR and subcloned into the pEGFP-C1 vector. Features and domains of SENP2, including the catalytic domain (green), NLS, and NES motifs and defined NPC-targeting regions (yellow) are indicated.
FG-repeat nucleoporins, Nup358 and Nup153 . We detected no significant reductions in the levels of karyopherin-α 3 and -α 4 , suggesting SENP2 associates directly with a distinct subfamily of karyopherins. Levels of Nup133 remained unaffected, consistent with SENP2 forming a direct association with the Nup107 complex.
## Nuclear transport receptors mediate interactions between senp2 and nucleoporins
To further verify and characterize SENP2-interacting proteins, immunopurification and immunoblotting experiments were performed. GFP-tagged, full-length SENP2 and SENP2 1-63, as well as the NLS mutants SENP2(R29A/R49A) and SENP2 1-63(R29A/R49A), FIGURE 3: SENP2 contains multiple N-terminal NPC-targeting signals. HeLa cells were transfected with constructs coding for wild-type GFP-SENP2 or the indicated GFP-SENP2 deletion mutants. Colocalization with FG repeatcontaining nucleoporins was determined by labeling cells with mAb 414 and by indirect immunofluorescence microscopy analysis. DNA was detected by staining with DAPI. Scale bar: 10 μm.
Flag-tagged SENP2 wild-type and indicated mutants were expressed in Flp-In T-REx 293 cells. Cells were lysed under nondenaturing conditions and Flag-tagged SENP2 and associated proteins were immunopurified using Flag-M2 agarose beads. Immunopurified proteins were eluted with ammonium hydroxide, digested with trypsin, and analyzed by LC-MS/MS. For protein identification, Thermo
.RAW files were converted to the .mzXML format using Proteowizard, then searched using X!Tandem against the human (Human RefSeq Version 37) database. Spectral counts, corresponding to the number of times a peptide from each protein was observed, are indicated. a SUMO-2/3 identifications were combined. Both SUMO-2 and SUMO-3-specific peptides were observed. Searched with GPM X!Tandem and analyzed with ProHits. GPM peptide cuttoff value = −2.0.
[formula] SENP2_FL_poolB_pelletA_run1 SENP2_FL_poolB_pelletA_run2 SENP2_FL_poolB_pelletB_run1 SENP2_FL_poolB_pelletB_run2 SENP2_FL_poolB_pelletC_run1 SENP2_FL_poolB_pelletC_run2 SENP2_FL_poolC_pelletB_run1 SENP2_FL_poolC_pelletB_run2 SENP2_1-350_poolB_pelletA_run1 SENP2_1-350_poolB_pelletA_run2 SENP2_1-350_poolC_pelletA_run1 SENP2_1-350_poolC_pelletA_run2 SENP2_350-589_poolB_pelletA_run1 SENP2_350-589_poolB_pelletA_run2 SENP2_350-589_poolC_pelletA_run1 SENP2_350-589_poolC_pelletA_run2 SENP2_∆143-350_poolB_pelletA_run1 SENP2_∆143-350_poolB_pelletA_run2 SENP2_∆143-350_poolC_pelletA_run1 SENP2_∆143-350_poolC_pelletA_run2 SENP2_∆143-350_poolC_pelletB_run1 SENP2_∆143-350_poolC_pelletB_run2 SENP2_1-350mNLS_poolB_pelletA_run1 SENP2_1-350mNLS_poolB_pelletA_run2 SENP2_1-350mNLS_poolC_pelletA_run1 SENP2_1-350mNLS_poolC_pelletA_run2 [/formula]
Bait 59343 were transiently expressed in HeLa cells. The SENP2 proteins were immunopurified and blots were probed with antibodies specific for karyopherin-α 2 , karyopherin-β 1 , FG-repeat nucleoporins recognized by mAb 414, or Nup107. Consistent with the AP-MS results, fulllength SENP2 copurified with karyopherin-α and -β, the FG repeatcontaining nucleoporin Nup153, and also with Nup107 [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref] , lane 7). Interactions with FG repeat-containing nucleoporins again appeared to be dependent on karyopherin binding, as mutating the NLS resulted in a loss of not only karyopherin-α and -β, but also Nup153 [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref] , lane 8). Also consistent with the AP-MS analysis, interactions between SENP2 and Nup107 were unaffected by the NLS mutation [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref] , lane 8).
Analysis of SENP2 1-63 and SENP2 1-63(R29A/R49A) revealed that the N-terminal 63 amino acids of SENP2 were sufficient for interactions with karyopherin-α and -β, and that these interactions were dependent upon the NLS [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref]. Interactions with the FG-repeat nucleoporins Nup153 and Nup62 were also detected with the N-terminal 63 amino acids of SENP2. Interactions with Nup62 were not detected with full-length SENP2 [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref] , lane 7), suggesting differences in NPC association compared with residues 1-63 alone. Nup107 did not copurify with the N-terminal 63 amino acids of SENP2, consistent with its interactions being mediated by a second targeting signal.
To further demonstrate the role of karyopherins in mediating interactions between SENP2 and FG-repeat nucleoporins, we performed in vitro binding assays using purified recombinant SENP2 1-63 and the FG-repeat domain of Nup153. GST-tagged SENP2 1-63 was immobilized on glutathione beads and incubated with the FG-repeat domain of Nup153 alone, together with karyopherin-α, or with both karyopherin-α and -β [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref]. Interaction between SENP2 1-63 and the Nup153 FG-repeat domain was detected only in the presence of both karyopherin-α and -β, indicating that the NLS-dependent interaction between SENP2 and FG-repeat nucleoporins is mediated through association with nuclear transport receptors.
In addition to having an N-terminal NLS, SENP2 also contains a CRM1-dependent nuclear export signal (NES) between residues 317 and 332 [bib_ref] Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2, Itahana [/bib_ref]. To investigate whether both the NLS and the NES function cooperatively to affect SENP2 localization, we transfected cells with either wild-type SENP2, SENP2 NΔ66, or SENP2 NΔ66/NES (which contains leucine to alanine substitutions at NES residues 329 and 331; NΔ66 constructs were obtained from [bib_ref] Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2, Itahana [/bib_ref]. Cells were then treated or not treated with the CRM1 inhibitor, leptomycin B (LMB). Whereas CRM1 inhibition caused no notable differences in the localization of wild-type SENP2, it caused a clear redistribution of SENP2 NΔ66 from the cytoplasm and NPCs to the nucleoplasm [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref]. The effect of LMB on SENP2 NΔ66 was direct, as a similar change in localization was observed in cells expressing the SENP2 NΔ66/NES mutant. Thus the steady-state distribution of SENP2 among NPCs, the nucleus, and the cytoplasm is determined by both nuclear import and export receptors.
A second NPC-targeting signal mediates interactions between SENP2 and the Nup107-160 nucleoporin subcomplex AP-MS analysis revealed that a SENP2 mutant lacking amino acids 143-350 interacted with karyopherins and FG repeat-containing nucleoporins, but not with members of the Nup107-160 subcomplex [fig_ref] TABLE 1: SENP2 interacts with soluble nuclear transport receptors and nucleoporins [/fig_ref]. To further examine whether the residues within this region of SENP2 might mediate interactions with the Nup107-160 subcomplex, plasmids encoding GFP-tagged SENP2, SENP2 143-350, or SENP2 Δ144-349 were transiently expressed in HeLa cells, and interacting proteins were immunopurified. Immunopurified complexes were probed using antibodies specific for two members FIGURE 4: SENP2 interacts with karyopherin-α through a high-affinity NLS. (A) Alignment of the N-terminal nuclear localization sequences of SENP2, Nup2, and Nup50 with positively and negatively charged residues indicated in red and blue, respectively. (B) GST-tagged SENP2 1-63 wild-type (lanes 1-5) or NLS mutant SENP2 1-63(R29A/ R49A) (lanes 6-10) were immobilized on glutathione beads and incubated with increasing concentrations of purified His-tagged karyopherin-α. Bound protein was eluted with SDS-sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting using GST-or His-specific antibodies. (C) MBP-tagged SV40 NLS was preincubated with biotinylated karyopherin-α. Increasing concentrations of GST-tagged SENP2 1-63 were subsequently incubated with the preformed karyopherin-α/NLS complexes and incubated for 1 h. Karyopherin-α and associated proteins were isolated using streptavidin beads and analyzed by immunoblotting with anti-MBP and -GST specific antibodies [fig_ref] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160... [/fig_ref]. The reciprocal experiment, in which GST-SENP2 1-63 was preincubated with biotinylated karyopherin-α and binding was subsequently challenged with increasing concentrations of MBP-SV40 NLS, was also performed (lanes 7-12). (D) GST-SENP2 or GST alone were immobilized on glutathione beads and incubated with His-tagged karyopherin-α and -β in the absence or presence of Ran-GTP, as indicated. Binding reactions were analyzed by immunoblotting with His-tag-specific antibodies. Karyopherin-α and -β were run alone as controls. Molecular-weight markers are indicated. (E) Immunoprecipitation MS analysis was performed using cell lysate prepared from cells expressing Flag-tagged, wild-type SENP2. Spectral counts obtained for each of the indicated interacting proteins obtained from immunopurifications performed in either the presence or absence of nonhydrolyzable Ran-GTP are plotted. Error bars represent the SE from four independent reactions (*p < 0.001; **p < 0.05).
of the Nup107-160 subcomplex, Nup107 and Nup96. Nup107 and Nup96 both copurified with full-length SENP2, and their association required residues 143-350 of SENP2, as deletion of these residues abrogated the interactions [fig_ref] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160... [/fig_ref]. Moreover, the fragment of SENP2 containing residues 143-350 alone was sufficient for binding to the Nup107-160 subcomplex [fig_ref] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160... [/fig_ref]. Thus the association of SENP2 with the NPC is achieved through both karyopherin-mediated interactions with FG repeat-containing nucleoporins and association with the Nup107-160 subcomplex, as summarized in [fig_ref] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160... [/fig_ref].
## Senp2-targeting signals individually affect dynamic associations with npcs
To investigate the dynamics of SENP2 interactions with NPCs, cells were transfected with a plasmid encoding full-length GFP-SENP2, and fluorescence recovery after photobleaching (FRAP) experiments were performed. A defined region of nuclear envelope was photobleached, images were collected at 2-s intervals following the initial bleaching event, and the percentage of initial fluorescence was plotted versus time to a obtain FRAP recovery curve . A rapid recovery to ∼20% of the original signal was observed within the first minute, with little further recovery observed over an additional 2 min. This result suggests the existence of two pools of SENP2 at NPCs, one that is dynamic and one that is more stably bound. To investigate the contributions of the individual N-terminal targeting signals in determining SENP2 mobility, we performed FRAP analysis on cells expressing GFP-tagged SENP2 1-63 (containing just the NLS) or 1-350 (containing both the NLS and the Nup170-160 binding domain; . SENP2 1-63 recovered more quickly and more fully compared with SENP2 1-350, consistent with the targeting domains acting in a combinatorial manner to affect NPC association. SENP2 1-63 also recovered more quickly and fully relative to full-length SENP2, whereas the recovery of SENP2 1-350 was reduced.
To further investigate the contributions that the NLS and Nup107-160-binding domain make toward SENP2 association with NPCs, under nondenaturing conditions, and GFP-tagged proteins were immunopurified using a GFP-specific antibody. Cell lysates (lanes 1-5) and immunopurified proteins (lanes 6-10) were resolved by SDS-PAGE, and immunoblot analysis was performed with antibodies specific for karyopherin-α 2 , karyopherin-β 1 , Nup358/Nup153/Nup62 (mAb 414), Nup107, and GFP, as indicated. (B) Recombinant GST-tagged SENP2 1-63 was immobilized on glutathione beads and incubated with a His-tagged C-terminal FG-repeat domain of Nup153 (Nup153C) either alone, together with His-tagged karyopherin-α, or together with His-tagged karyopherin-α and -β. Bound proteins were eluted with SDS sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting using His-tag-specific antibodies. (C) HeLa cells transfected with GFP-SENP2, GFP-SENP2 NΔ66, or GFP-SENP2 NΔ66/ NES (in which the NES between residues 317 and 332 was mutated), were treated with LMB or carrier (Control) for 40 min. Cells were analyzed by fluorescence microscopy.
we characterized the effects of ATP depletion on the localization of SENP2 1-63 and 1-350. ATP depletion results in a loss of Ran-GTP and a sequestration of karyopherin-α receptors in the nucleus [bib_ref] The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion, Schwoebel [/bib_ref]. With this in mind, we anticipated that the karyopherin-α-dependent association of SENP2 1-63 with NPCs might be particularly sensitive to ATP depletion, whereas SENP2 1-350 might be less sensitive, due to associations with the Nup107-160 subcomplex. Cells were transiently transfected with constructs coding for GFP-tagged SENP2 1-63 and 1-350 and their localizations were determined in untreated cells and in cells treated with 2-deoxy-d-glucose and sodium azide to deplete ATP . The localization of karyopherin-α 3 was examined as an indicator of the efficiency of ATP depletion. In control cells, SENP2 1-63 was detected at NPCs with a faint signal also visible in the nucleoplasm. Karyopherin-α 3 was detected throughout the nucleoplasm and cytoplasm and also concentrated at NPCs. As anticipated, karyopherin-α 3 shifted to the nucleoplasm in cells depleted of ATP. A shift in the distribution of SENP2 1-63 was also observed in ATP-depleted cells, with a notable increase in nucleoplasmic localization. The significance of this shift was verified by quantifying the ratio of NPC to nucleoplasmic signal in control and ATP-depleted cells . Notably, NPC localization was not fully eliminated. In comparison with SENP2 1-63, SENP2 1-350 was concentrated approximately twofold higher at NPCs in untreated cells. A shift in distribution from NPCs was also detected upon ATP depletion; however, the ratio between NPCs and nucleoplasm remained nearly twice as high compared with SENP2 1-63 . Together with the FRAP analysis, these results are consistent with the N-terminal NLS in SENP2 mediating a more dynamic association with NPCs, and with the Nup107-160 binding domain facilitating more stable associations.
## Senp2 npc-targeting signals function to restrict substrate accessibility
Mutations in yeast Ulp1 that affect its association with NPCs also affect its ability to access and desumoylate specific proteins [bib_ref] The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization,..., Li [/bib_ref]. To investigate the roles of NPC tethering on SENP2 substrate accessibility, we performed Western blot analysis on uninduced and induced stable cell lines expressing Flag-tagged, wild-type SENP2; SENP2 variants in which one or the other of the NPC-targeting signals was disrupted; and the catalytic domain alone . Immunofluorescence microscopy and anti-Flag Western blotting verified the induced expression and localization of each protein. In comparison with uninduced cells, we observed no significant changes in overall sumoylation levels in cells induced to express wild-type SENP2. In contrast, we observed global decreases in high-molecular-weight SUMO-1 and SUMO-2/3 conjugates in cells induced to express SENP2 in which the Nup107-160 binding domain was deleted (Δ143-349) and in which the entire N-terminal domain was deleted (NΔ367). Thus NPC-targeting signals function to restrict substrate specificity through their effects on the steady-state association of SENP2 with NPCs.
# Discussion
SUMO-specific isopeptidases are found concentrated at NPCs in organisms ranging from yeast to human [bib_ref] Modification in reverse: the SUMO proteases, Mukhopadhyay [/bib_ref]. These isopeptidases are believed to define a functionally distinct subclass of desumoylating enzymes; however, their precise functions remain to be fully understood. In the current study, we have characterized the molecular interactions that mediate the association of SENP2 with NPCs in mammalian cells. We have demonstrated that the association of SENP2 with NPCs is mediated by a combination of interactions between soluble nuclear import and export receptors, peripherally associated nuclear basket proteins, and a core structural subcomplex of the NPC [fig_ref] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160... [/fig_ref]. These interactions are mediated by three targeting signals in SENP2: a high-affinity NLS, a Nup107-160-binding element, and an NES. Our findings indicate that the precise location of SENP2 within cells and at NPCs is determined by the combined action of these targeting signals [fig_ref] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160... [/fig_ref].
## Senp2 interactions with karyopherins
Among the most remarkable factors that we discovered affecting SENP2 association with NPCs is the association with soluble nuclear transport receptors. Our analysis revealed that SENP2 interacts with multiple members of the karyopherin family, including several karyopherin-αs (α 1 , α 2 , α 3 , α 4 , and α 6 ), as well as multiple karyopherin-βs (β 1 , β 2 , β 2B , importin-7, and CSE1L). Interactions with each of these transport receptors was dependent on the N-terminal NLS of SENP2, which we found to have an unusually high affinity for karyopherin-α. AP-MS analysis of SENP2 immunopurified from cell lysates in the presence of Ran-GTP revealed that only karyopherin-α 3 and -α 4 were resistant to dissociation. This suggests that, in vivo, karyopherin-α 3 and -α 4 selectively associate with SENP2 to affect its localization. Whether these two receptors, which form a distinct phylogenetic branch of the karyopherin-α family [bib_ref] Evolution of the metazoan-specific importin α gene family, Mason [/bib_ref] , bind more tightly to SENP2 relative to other receptors remains to be determined. The dissociation of other karyopherins by FIGURE 7: SENP2 is dynamically associated with NPCs. (A) A defined region of the nuclear envelope of GFP-SENP2transfected cells was photobleached and images were collected every 2 s for 200 s. Fluorescence intensity after bleaching was normalized to 0%, and the relative recovery was plotted as a function of time. Average SE from 10 experiments was ± 3%. (B) FRAP analysis was performed as in (A), with cells transfected with GFP-SENP2 1-63 and 1-350. Average SEs from five experiments were ± 5% and ± 7% for cells expressing GFP-SENP2 1-63 and GFP-SENP2 1-350, respectively. (C) HeLa cells were transiently transfected with constructs encoding GFP-tagged SENP2 1-63 and 1-350. Cells were either untreated or treated with 2-deoxy-d-glucose and sodium azide to deplete ATP and stained with karyopherin-α 3 -specific antibodies. Cells were analyzed by fluorescence microscopy. Scale bar: 10 μm. (D) The ratio of fluorescence intensities between NPCs and nucleoplasm was determined and plotted. Error bars represent the SE (n = 25).
Ran-GTP suggests either an indirect association with SENP2, possibly through complexes of FG-repeat nucleoporins, or lower-affinity direct interactions. In addition to its N-terminal NLS, SENP2 also contains an NES that operates to limit accumulation in the nucleus and more generally to affect the distribution of SENP2 among NPCs, the nucleus, and the cytoplasm. Although this NES appears to be recognized by CRM1, based on LMB sensitivity [fig_ref] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC [/fig_ref] , we did not detect CRM1 in our AP-MS analysis, suggesting a transient or unstable interaction under the purification conditions used.
Like SENP2, the localization of yeast Ulp1 at NPCs is also dependent on unconventional interactions with multiple karyopherins, including karyopherin-α (Kap60) and karyopherin-β (Kap95), as well as karyopherin-121 (Kap121; [bib_ref] Unconventional tethering of Ulp1 to the transport channel of the nuclear pore..., Panse [/bib_ref]. In the case of Ulp1, these interactions are insensitive to dissociation by Ran-GTP. Similarly, we found that karyopherin-α interactions with SENP2 are unaffected by Ran-GTP, although karyopherin-β binding is affected. Insights into the exact molecular nature of the unusual interactions between karyopherins and Ulp1 and SENP2 will require further studies. Nonetheless, it appears that both Ulp1 and SENP2 form unusually stable associations with karyopherins that prevent their dissociation upon entry into the nucleus and thus enabling concentrations at NPCs. Consistent with this, SENP2 concentrates at the nucleoplasmic face of NPCs through interactions with Nup153 [bib_ref] Association of the human SUMO-1 protease SENP2 with the nuclear pore, Hang [/bib_ref] [bib_ref] Enzymes of the SUMO modification pathway localize to filaments of the nuclear..., Zhang [/bib_ref] , and our findings demonstrate that this interaction is karyopherin-α/β dependent.
In addition to facilitating associations with NPCs, our findings also indicate that the N-terminal NLS of SENP2 is able to displace more traditional, lower-affinity NLS sequences from karyopherin-α. SENP2 could therefore function as a KaRF. KaRFs are proposed to function at the nucleoplasmic face of the NPC to facilitate dissociation of nuclear import cargoes from karyopherin-α and Ran-GTP initiated release of karyopherin-β [bib_ref] Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin..., Gilchrist [/bib_ref] [bib_ref] Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling, Matsuura [/bib_ref]. KaRF proteins include yeast Nup2 and mammalian Nup50, proteins that, like SENP2, are dynamically associated with NPCs [bib_ref] Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export, Guan [/bib_ref] [bib_ref] Nup2p dynamically associates with the distal regions of the yeast nuclear pore..., Dilworth [/bib_ref] [bib_ref] Npap60/ Nup50 is a tri-stable switch that stimulates importin-α:β-mediated nuclear protein import, Lindsay [/bib_ref] [bib_ref] Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin..., Gilchrist [/bib_ref]. This consideration raises the question of the precise functional significance of the interactions between nuclear transport receptors and Ulp1 and SENP2. Our studies provide clear evidence that karyopherin interactions affect the association of SENP2 with NPCs. However, it is possible that the interactions between SENP2 and karyopherins may also play a role in affecting karyopherin function. This is a particularly intriguing possibility, given the large number of different karyopherins associated directly or indirectly with SENP2. In addition to affecting substrate release through KaRF activity, SENP2 could also mediate the desumoylation of karyopherins and/or associated cargo proteins, thereby affecting their functions or transport. In the budding yeast, inhibition of sumoylation affects the nuclear import of proteins bearing classical NLSs and this inhibition is due in part to defects in the recycling of karyopherin-α (Kap60) from the nucleus to the cytoplasm [bib_ref] A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast, Stade [/bib_ref]. We have observed a similar effect on karyopherin-α recycling upon RNAi-mediated knockdown of Ubc9 in HeLa cells (unpublished data). The specific protein targets of sumoylation important for karyopherin-α recycling are not known; FIGURE 8: The NPC-targeting signals in SENP2 function to restrict substrate accessibility. Inducible stable cell lines for expressing Flag-tagged, wild-type SENP2, SENP2 Δ144-349, and SENP2 NΔ367 were cultured either in the absence or in the presence of doxycycline (Dox) to induce protein expression. (A) Induced cells were analyzed by immunofluorescence microscopy using anti-Flag-specific antibodies. Uninduced cells showed negligible background staining, as exemplified in the control. Scale bar: 10 μm. (B) Lysates were prepared from uninduced (−) and induced (+) cells, and equal quantities of total protein were analyzed by immunoblotting with antibodies specific for the Flag-tag, tubulin, SUMO-2/3, or SUMO-1. Molecular mass markers are indicated.
however, it is intriguing to speculate that these targets are subject to regulation by SENP2-and Ulp1-mediated desumoylation.
## Senp2 on and off the npc
FRAP analysis revealed that a fraction of overexpressed GFP-SENP2 associates dynamically with NPCs. This mobility, and the previously reported finding that SENP2 shuttles between the nucleus and the cytoplasm [bib_ref] Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2, Itahana [/bib_ref] , indicates that targets of SENP2 desumoylation may include proteins in both the cytoplasm and the nucleoplasm. In addition to the shuttling ability of full-length SENP2, we also detected multiple lower-molecular-weight isoforms of SENP2 potentially derived from alternatively spliced mRNAs. Based on their reactivity with SUMO-2-VS, each of the lower-molecularweight forms of SENP2 have been determined to contain the Cterminal catalytic domain and are therefore likely to be missing elements from the N-terminal targeting domain. Consistent with this, alternative splicing in mouse cells generates multiple SENP2 isoforms with N-terminal deletions and notably distinct subcellular localizations to the cytoplasm and nucleus [bib_ref] Characterization of a novel mammalian SUMO-1/Smt3-specific isopeptidase, a homologue of rat axam,..., Nishida [/bib_ref] [bib_ref] SUMO-1 protease-1 regulates gene transcription through PML, Best [/bib_ref]. Two active human SENP2 variants are also predicted by the Celera Genomics genome sequencing project (GenBank accession numbers EAW78215 and EAW78216). These predicted isoforms include full-length SENP2 and a protein lacking the N-terminal 80 amino acids. The latter would be expected to share a localization pattern similar to our NΔ63 mutant, which was found at NPCs and in both the nucleus and cytoplasm . Consistent with the expression of multiple SENP2 isoforms with distinct localizations, endogenous SENP2 was detected at NPCs, as well as in the nucleoplasm and cytoplasm .
The association of Ulp1 with NPCs in yeast is subject to regulation. In mitosis, Ulp1 dissociates from NPCs and relocates in a Kap121-dependent manner to the septin ring, where it mediates desumoylation of the septin proteins [bib_ref] The role of karyopherins in the regulated sumoylation of septins, Makhnevych [/bib_ref]. Similarly, Ulp1 dissociates from NPCs in response to ethanol stress and relocates to nucleoli in a manner dependent on Kap95 and Kap60 [bib_ref] A novel mechanism for SUMO system control: regulated Ulp1 nucleolar sequestration, Sydorskyy [/bib_ref]. Our findings revealed that combinatorial effects of interactions with nuclear export and import factors and nucleoporins determine the precise localization of SENP2. Although inducible changes in SENP2 localization in interphase cells have not been reported, it is interesting to speculate that regulation of interactions between SENP2 and any one of the factors affecting NPC targeting could also affect substrate specificity. Consistent with this, our analysis of individual SENP2 mutants lacking the Nup107-160-binding domain or the entire N-terminus showed clear effects on cellular sumoylation levels and thus a connection between localization and substrate accessibility.
## Senp2 interactions with the nup107-160 nucleoporin subcomplex
In addition to their roles in NPC-related processes during interphase, nucleoporins have been implicated in mitotic processes related to chromosome segregation by a growing body of evidence [bib_ref] Nuclear transport and the mitotic apparatus: an evolving relationship, Wozniak [/bib_ref]. Most notably, a fraction of the Nup107-160 nucleoporin subcomplex relocalizes to spindle fibers and to the outer-kinetochore plate during prophase, where it affects spindle assembly and establishment of microtubule/kinetochore attachments [bib_ref] An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in..., Belgareh [/bib_ref] [bib_ref] The entire Nup107-160 complex, including three new members, is targeted as one..., Loiodice [/bib_ref] [bib_ref] The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly, Orjalo [/bib_ref] [bib_ref] Nuclear transport and the mitotic apparatus: an evolving relationship, Wozniak [/bib_ref]. Our finding that SENP2 interacts with the Nup107-160 subcomplex therefore has potentially important implications for understanding the molecular basis for the effects of this subcomplex on spindle and kinetochore function. Interestingly, we have previously found that overexpression of SENP2 leads to defects in microtubule/ kinetochore attachments, due to defects in the recruitment of CENP-E to the outer kinetochore [bib_ref] SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and..., Zhang [/bib_ref]. Thus, it is intriguing to speculate that some functions of the Nup107-160 subcomplex in mitosis may be affected through its association with SENP2.
The evolutionary conservation of SUMO-specific isopeptidases localized at NPCs implies important evolutionarily conserved functions for this subclass of isopeptidases. This implication is further underscored by our findings that the molecular mechanisms regulating the NPC associations of human SENP2 and yeast Ulp1 are also highly conserved. An important step toward understanding the critical functions of these isopeptidases lies in defining the specific subset of sumoylated proteins they regulate. Intriguingly, many of the nucleoporins and karyopherins that interact with SENP2 are also known to be sumoylated, suggesting that these proteins themselves may be substrates [bib_ref] Differential regulation of sentrinized proteins by a novel sentrin-specific protease, Golebiowski [/bib_ref]. Characterization of these potential SENP2 substrates, and other substrates both on and off the NPC, will be an important quest for future studies.
## Materials and methods antibodies
temperature. Increasing concentrations of the competing protein were then added directly to the preformed complexes and incubated for an additional 1 h at room temperature. Streptavidin-agarose beads (Pierce) were added to each reaction and incubated for 30 min at room temperature. Beads were washed six times with binding buffer, and bound proteins were eluted with SDS sample buffer.
To assay SENP2 reactivity with SUMO-vinyl sulfones, untransfected cells or cells transfected with the indicated plasmids were resuspended in reaction buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.5% Triton-X 100, 1 μg/ml leupeptin and pepstatin A, 20 μg/ml aprotinin) and sonicated for 20 s. Lysates were centrifuged at 16,000 × g for 10 min, and the protein concentration of the lysate was determined empirically. Protein concentration was maintained between 0.5 μg/ml and 2.0 μg/ml. HA-tagged SUMO-2-vinyl sulfone (Boston Biochem, Boston, MA) was diluted to 10 ng/μl in a buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 10% glycerol. Diluted HA-SUMO-2-VS was added to the appropriate volume of cell lysate at a final concentration of 0.3 ng/μl and incubated for 30 min at room temperature. Reactions were terminated by addition of SDS sample buffer.
## Stable cell lines
Tetracycline-inducible, Flag-tagged SENP2 proteins were expressed in human Flp-In T-REx 293 cells (Invitrogen). Protein expression was induced by adding 1 μg/ml tetracycline to the culture medium (DMEM + 10% fetal calf serum) for 24 h.
## Cell culture, transfection, and rna interference
HeLa or HEK293 cells were maintained at 37°C in DMEM supplemented with 10% fetal bovine serum, 10 mM HEPES (pH 8.0), and 1% penicillin-streptomycin. Cells were transfected using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). siRNA oligos were used at a final concentration of 20 nM, unless otherwise indicated. SENP2 RNA oligo #1 (5′-GCCCAUG-GUAACUUCUGCUUGUAAU-3′) was obtained from Invitrogen. SENP2 RNA oligo #2 (5′-AUAUCUGGAUUCUAUGGGAUU-3′) was obtained from Ambion (Austin, TX). SENP2 RNA oligo #3 (5′-GC-CUAUUCAUCGGAAGGUAtt-3′), a Silencer Select Pre-designed siRNA, was obtained from Ambion.
For ATP-depletion experiments, HeLa cells were cultured in the presence of 6 mM 2-deoxy-d-glucose and 10 mM Na-azide for 1 h, as previously described [bib_ref] The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion, Schwoebel [/bib_ref]. For LMB treatment, HeLa cells were transfected for 24 h with SENP2, SENP2 NΔ66, or SENP2 NΔ66/ΔNES, and then treated with 10 nM LMB (Sigma-Aldrich) for 40 min at 37°C. Cells were fixed and processed for fluorescence microscopy as described in Immunofluorescence Microscopy.
## Immunoblotting and immunopurification
Immunoblot analysis was performed using enzyme-linked chemiluminescence ECL-Plus reagent (GE Healthcare). For immunopurification experiments involving GFP-tagged proteins, cells were lysed in buffer containing 50 mM TrisHCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 5 mM EDTA, 1% Triton-X 100, 2 mM PMSF, and 10 mM NEM. Lysates were placed on ice for 5 min and then sonicated for 30 s and centrifuged at 16,000 × g for 15 min. Lysate was incubated with GFP antibody-bound protein-A agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 6 h, and then beads were washed six times in PBS and bound proteins were eluted directly in SDS sample buffer. leupeptin and pepstatin A). Lysozyme (1 mg/ml) was added, and the mixture was incubated on ice for 15 min. A final concentration of 10 mM dithiothreitol (DTT) and 1.4% N-lauryl sarcosine (Sigma-Aldrich, St. Louis, MO) were added immediately prior to sonication. Lysate was sonicated four times at 15-s intervals and then centrifuged at 30,000 × g for 20 min at 4°C. The supernatant was diluted 1:2 in fresh STE buffer and Triton-X 100 was added at a final concentration of 2%. The sample was incubated with glutathione-Sepharose beads (GE Healthcare), and bound protein was eluted in buffer containing 50 mM TrisHCl (pH 9.0) and 50 mM glutathione (Sigma-Aldrich). His-tagged mouse karyopherin-α 2 , human karyopherin-β 1 , and a C-terminal fragment of human Nup153 were purified using standard nickel-agarose affinity column chromatography, as recommended by the manufacturer (Qiagen). The plasmid encoding GSTtagged MBP-SV40 NLS was transformed into E. coli BL21 (Codon-Plus) cells; lysed in phosphate-buffered saline (PBS) containing 1 mM DTT, 0.5% Triton-X 100, 10% glycerol, 1 mg/ml lysozyme, 1:3000 dilution of Benzonase nuclease (Novagen, San Diego, CA), 1 mM PMSF, 1 μg/ml leupeptin and pepstatin A; and purified by affinity chromatography using glutathione-Sepharose beads (GE Healthcare). Protein was eluted by thrombin cleavage (GE Healthcare).
RanQ69L was purified essentially as described in [bib_ref] RanGAP1 induces GTPase activity of nuclear Ras-related Ran, Bischoff [/bib_ref] , with several modifications. Briefly, the clarified E. coli extract in lysis buffer (50 mM NaCl, 5 mM EDTA, 50 mM HEPES, 2 mM DTT, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, pH 7.6) was passed through a 10-ml diethylaminoethyl cellulose column equilibrated with buffer 1 (25 mM HEPES, 1 mM MgCl 2 , 2 mM DTT, pH 7.6, 50 mM NaCl), and the flow-through was collected. This was then subjected to a 45% ammonium sulfate precipitation, which was followed by a 60% ammonium sulfate saturation of the 45% supernatant. The protein pellet from the 60% cut was dissolved in buffer 1 and separated on a Sephacryl S-100 column. The Ran-containing fractions were incubated on ice with 5 mM EDTA and 10 mM GTP for 1 h, then MgCl 2 was added at a final concentration of 20 mM. The GTP-loaded RanQ69L was then purified using a 25-800 mM linear NaCl gradient on a 25-ml SP-Sepharose FF column (GE Healthcare) equilibrated with buffer 1 containing 25 mM NaCl.
## In vitro binding, nls competition assays, and sumo-vinyl sulfone reactions
To characterize interactions with karyopherins and Nup153, GSTtagged SENP2 1-63 or SENP2 1-63(R29A/R49A) was immobilized on glutathione-Sepharose beads (GE Healthcare) and nonspecific protein-binding sites were blocked by incubation in PBS containing 2% bovine serum albumin (BSA) for 20 min at 4°C. For karyopherin-α 2 binding, beads were incubated with increasing concentrations of purified His-tagged karyopherin-α 2 in binding buffer (0.1% Tween-20 in PBS) at room temperature for 1 h. Beads were washed six times with binding buffer, and bound proteins were eluted with SDS sample buffer. To assay for Nup153 binding, beads were incubated with a His-tagged, C-terminal fragment of Nup153 (amino acids 1287-1475) alone, in the presence of Histagged karyopherin-α 2 , or in the presence of His-karyopherin-α 2 and -β 1 in binding buffer (20 mM TrisHCl. pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 30 min at room temperature. The beads were washed six times with binding buffer and the bound proteins were eluted with SDS sample buffer.
For NLS competition assays, His-tagged karyopherin-α 2 was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin according to the manufacturer's protocol (Pierce, Rockford, IL). Recombinant MBP-SV40 NLS or GST-SENP2 1-63 was preincubated with the biotinylated karyopherin-α 2 in binding buffer (1% NP-40 in PBS) for 1 h at room Triton-X 100. Immunostaining was carried out as previously described [bib_ref] A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1..., Matunis [/bib_ref]. Images were collected using a Zeiss ObserverZ1 fluorescence microscope with an Apotome VH optical sectioning grid. Images were processed using AxioVision Software Release 4.8.1 (Zeiss, Jena, Germany).
For quantitative analysis of fluorescence intensities, acquired images were analyzed using ImageJ . Two rectangular regions of equal dimensions were drawn at either the nuclear envelope or within the nucleoplasm of a cell. The pixel intensities were measured, and ratios were determined for 25 individual cells. Average ratios and SEs were graphed.
## Frap
Cells were cultured in 35-mm glass-bottom dishes (MatTek, Ashland, MA). At 24 h following transfection, FRAP experiments were performed on a Zeiss LSM510 confocal microscope using a Plan-Apochromat 63×/1.4 oil objective and a pinhole open to 134 μm. A defined region of the nuclear envelope was photobleached using a 488-nm laser at 1.5% power, and then images were acquired every 2 s postbleaching. Fluorescence intensity within the defined region, as well as an equivalent-sized region in an adjacent cell, was quantified using the LSM Image browser software. No significant background photobleaching was detected in the neighboring cells. The signal intensity after photobleaching within the defined region was normalized to 0%, and the relative recovery of fluorescence intensity was plotted versus time. Data were obtained for 5-10 cells for each sample, and the normalized fluorescence intensities were averaged and plotted. An average SE of ± 3% was obtained for the full-length SENP2, while average standard errors of ± 5% and ± 7% were obtained for SENP2 1-63 and 1-350, respectively.
For MS analysis of SENP2-interacting proteins, 6 × 150 cm 2 dishes of subconfluent (75-85%), stable, Flag-tagged SENP2-expressing cells were scraped into PBS, pooled, washed twice in 25 ml PBS, and collected by centrifugation at 1000 × g for 5 min at 4°C. Cell pellets were stored at −80°C. The cell pellet was weighed, and 1:4 pellet weight:volume lysis buffer was added. Lysis buffer consisted of 50 mM HEPES-NaOH (pH 8.0), 100 mM KCl, 2 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM PMSF, 1 mM DTT, and 1:500 protease inhibitor cocktail (Sigma-Aldrich). On resuspension, cells were incubated on ice for 10 min, subjected to one additional freezethaw cycle, and then centrifuged at 27,000 × g for 20 min at 4°C. Supernatant was transferred to a fresh 15-ml conical tube, and 1:1000 benzonase nuclease (Novagen) plus 30 μl packed, preequilibrated Flag-M2 agarose beads (Sigma-Aldrich) were added. The mixture was incubated for 2 h at 4°C with end-over-end rotation. Beads were pelleted by centrifugation at 1000 × g for 1 min and transferred with 1 ml of lysis buffer to a fresh centrifuge tube. Beads were washed once with 1 ml lysis buffer and twice with 1 ml ammonium bicarbonate rinsing buffer (50 mM ammonium bicarbonate, pH 8.0, 75 mM KCl). Elution was performed by incubating the beads with 150 μl of 125 mM ammonium hydroxide (pH 11.0). The elution step was repeated twice. Eluate was centrifuged at 1000 × g for 1 min, transferred to a fresh centrifuge tube, and lyophilized. For Ran sensitivity assays, cleared lysates were spiked with 2 μM RanQ69L protein or an equal volume of vehicle (PBS pH 7.6), and affinity purification was conducted as above.
## Ms
One microgram MS-grade TPCK trypsin (Promega, Madison, WI) dissolved in 70 μl of 50 mM ammonium bicarbonate (pH 8.3) was added to the Flag eluate and incubated at 37°C overnight. The sample was lyophilized and brought up in buffer A (0.1% formic acid). LC analytical columns (75-mm inner diameter) and precolumns (100-mm inner diameter) were made in-house from fused silica capillary tubing from InnovaQuartz (Phoenix, AZ) and packed with 100 Å C 18 -coated silica particles (Magic, Michrom Bioresources, Auburn, CA). Peptides were subjected to LC-electrospray ionization-MS/MS, using a 120-min reverse-phase LC (95% water-95% acetonitrile, 0.1% formic acid) buffer gradient running at 250 nl/min on a Proxeon EASY-nLC pump in-line with a hybrid LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). A parent ion scan was performed in the Orbitrap using a resolving power of 30,000, then the six most intense peaks were selected for MS/MS (minimum ion count of 1000 for activation), using standard collision-induced dissociation fragmentation. Fragment ions were detected in the LTQ. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of −0.1 to +2.1 Thomson units; exclusion list size = 500) detected three times within 45 s were excluded from analysis for 60 s. For protein identification, Thermo .RAW files were converted to the .mzXML format using Proteowizard [bib_ref] ProteoWizard: open source software for rapid proteomics tools development, Kessner [/bib_ref] , then searched using X!Tandem [bib_ref] TANDEM: matching proteins with tandem mass spectra, Craig [/bib_ref] against the human (Human RefSeq Version 37) database. X!Tandem search parameters were: complete modifications, none; cysteine modifications, none; potential modifications, +16@M and W, +32@M and W, +42@N-terminus, +1@N and Q. Each immunopurification sample was analyzed using multiple technical replicates. Data were analyzed using the ProHits software tool [bib_ref] ProHits: integrated software for mass spectrometrybased interaction proteomics, Liu [/bib_ref]. Proteins identified with an X!Tandem expect score of −2.0 or lower and detected only in the SENP2 AP-MS analyses are reported.
## Immunofluorescence microscopy
HeLa cells were cultured on glass coverslips, fixed in 2% formaldehyde for 30 min at room temperature, and permeabilized in 0.5%
[fig] FIGURE 5: Nuclear transport receptors mediate interactions between SENP2 and the NPC. (A) HeLa cells were transfected with constructs encoding GFP, GFP-SENP2 (WT), GFP-SENP2(R29A/R49A), GFP-SENP2 1-63, or GFP-SENP2 1-63(R29A/R49A) for 36 h. Cells were lysed [/fig]
[fig] FIGURE 6: SENP2 contains a second NPC-targeting signal that mediates interactions with the Nup107-160 nucleoporin subcomplex. (A) HeLa cells were transfected with constructs encoding GFP, GFP-SENP2 (WT), GFP-SENP2 143-350, or GFP-SENP2 Δ144-349. Cells were lysed under nondenaturing conditions and proteins were immunopurified using GFP-specific antibodies. Cell lysates (lanes 1-4) and immunopurified proteins (lanes 5-8) were resolved by SDS-PAGE and analyzed by immunoblotting with Nup107-, Nup96-or GFPspecific antibodies. (B) Schematic representation summarizing the interactions affecting the association of SENP2 with NPCs. [/fig]
[table] TABLE 1: SENP2 interacts with soluble nuclear transport receptors and nucleoporins. [/table]
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Should capnography be used as a guide for choosing a ventilation strategy in circulatory shock caused by severe hypothermia? Observational case-series study
Background: Severe accidental hypothermia can cause circulatory disturbances ranging from cardiac arrhythmias through circulatory shock to cardiac arrest. Severity of shock, pulmonary hypoperfusion and ventilation-perfusion mismatch are reflected by a discrepancy between measurements of CO 2 levels in end-tidal air (EtCO 2 ) and partial CO 2 pressure in arterial blood (PaCO 2 ). This disparity can pose a problem in the choice of an optimal ventilation strategy for accidental hypothermia victims, particularly in the prehospital period. We hypothesized that in severely hypothermic patients capnometry should not be used as a reliable guide to choose optimal ventilatory parameters. Methods: We undertook a pilot, observational case-series study, in which we included all consecutive patients admitted to the Severe Hypothermia Treatment Centre in Cracow, Poland for VA-ECMO in stage III hypothermia and with signs of circulatory shock. We performed serial measurements of arterial blood gases and EtCO 2 , core temperature, and calculated a PaCO 2 /EtCO 2 quotient. Results: The study population consisted of 13 consecutive patients (ten males, three females, median 60 years old). The core temperature measured in esophagus was 20.7-29.0°C, median 25.7°C. In extreme cases we have observed a Pa-EtCO 2 gradient of 35-36 mmHg. Median PaCO 2 /EtCO 2 quotient was 2.15. Discussion and Conclusion: Severe hypothermia seems to present an example of extremely large Pa-EtCO 2 gradient. EtCO 2 monitoring does not seem to be a reliable guide to ventilation parameters in severe hypothermia.
# Background
While end-tidal carbon dioxide (EtCO 2 ) monitoring is one of the objective standards set in the Intensive Care Society guidelines [bib_ref] End tidal carbon dioxide monitoring in prehospital and retrieval medicine: a review, Donald [/bib_ref] and is of particular use for verification of endotracheal tube placement, it does not seem to be a reliable guide to ventilation in profound shock states.
It was noted that abnormal EtCO 2 measurements on initial emergency department presentation correlate with bad prognosis both in adults and children. Since cerebral blood vessels are sensitive to changes in partial pressure of CO 2 (PaCO 2 ), and hypocapnia induced by hyperventilation can lead to vasoconstriction and as a consequence worsening of secondary brain injury, it is advocated that ventilation parameters should be aimed at achieving "normocapnia".
Pulmonary hypoperfusion and pulmonary ventilationperfusion mismatch seem to play an important role among many factors determining extremely large Pa-EtCO 2 gradient observed in severe hypothermia victims. This discrepancy is further aggravated by a drop in blood temperature itself.
There is no published data on Pa-EtCO 2 gradient and reliability of EtCO 2 measurement in severe hypothermia. Based on our experience we hypothesize that in severely hypothermic patients capnometry should not be used as a reliable guide to choose optimal ventilatory parameters.
# Methods
We carried out a retrospective observational case-series study. All patients admitted to the Severe Hypothermia Treatment Centre (SHTC-Cracow, Poland) with stage III hypothermia, that still had a circulation and features of shock, were enrolled [bib_ref] Severe Accidental Hypothermia Center, Darocha [/bib_ref]. All data analyzed was collected on admission. The measurement of the central temperature (Tc) was taken in the lower third esophagus, using single-use Smiths Medical 12Fr probes, coupled with a SpaceLab cardiomonitor. The value of EtCO 2 was estimated from the main stream with the use of a capnometer from the SpaceLab monitoring system. Blood tests were assayed by routine automated laboratory techniques (Radiometer Copenhagen model ABL80). Blood gas analyses according to alpha-stat (blood gases measured at 37°C ) were performed in the central hospital laboratory, certified with a program by RIQAS (Randox Quality Assessment Scheme, UK). Simple plotting of PaCO 2 against EtCO 2 was performed. The study was approved by the Local Ethical Committee of the John Paul II Hospital in Cracow.
# Results
The study population consisted of 13 patients (ten males, three females, median 60 age years). The core temperature measured in the oesophagus was 20.7-29°C, median 25.7°C. PaCO 2 values varied between 17 to 53,1 mmHg (median 25.5 mmHg), and EtCO 2 from 12 to 19 mmHg (median 17 mmHg).
In extreme cases we have observed a Pa-EtCO 2 gradient of 35-36 mmHg. Median PaCO 2 /EtCO 2 quotient was 2.15 (blood gases measured at 37°C). [fig_ref] Figure 1: Pa-EtCO 2 gradient parameters of patients in stage III hypothermia treated as... [/fig_ref] summarizes the parameters of patients in stage III hypothermia who still had a circulation.
# Discussion
General guidelines for ventilatory support do not cover special population of severe hypothermia patients (Swiss Stage III and IV, [fig_ref] Table 1: Swiss Stage of Hypothermia [/fig_ref] [bib_ref] The medical on-site treatment of hypothermia: ICAR-MEDCOM recommendation, Durrer [/bib_ref]. Some experts recommend that the respiratory rate of mechanical ventilation should be lower, others prefer the ventilation rate to be normal [bib_ref] Accidental hypothermia, Lloyd [/bib_ref]. The Wilderness Medical Society guidelines state that in intubated patients, without the possibility of EtCO 2 control, it is recommended to decrease the respiratory rate by half in relation to the value in normothermia. At the same time, in patients in which capnometry is available, it is recommended to maintain EtCO 2 in normal range [bib_ref] Wilderness Medical Society practice guidelines for the out-of-hospital evaluation and treatment of..., Zafren [/bib_ref]. In the latest review of the current knowledge about hypothermia, there is an emphasis on the maintenance of normocapnia in order to prevent arrhythmia related to hyper-or hypoventilation [bib_ref] Accidental hypothermia-an update, Paal [/bib_ref].
Maintenance of normoventillation and normocapnia in patients in hypothermia is not an easy task. In mild, therapeutic hypothermia, such as in the ICU, normocapnia is achieved and maintained in only about 55% [bib_ref] Incidence of iatrogenic dyscarbia during mild therapeutic hypothermia after successful resuscitation from..., Falkenbach [/bib_ref]. Unfortunately, even the EtCO 2 does not solve the problem. It has been ascertained that in mild, therapeutic hypothermia (36 -32°C), the gradient between PaCO 2 and EtCO 2 may increase 2,5-fold and be as high as 18.7 mmHg [bib_ref] The arterial to end-tidal carbon dioxide gradient increases with uncorrected but not..., Sitzwohl [/bib_ref].
During the prehospital period, the only practical way to assess PaCO 2 is by indirect measurement of end-tidal CO 2 (EtCO 2 ). In normotermia, the Pa-EtCO 2 gradient is usually 4-6 mmHg, so the EtCO 2 values may be easily However, in our opinion, in significantly decreased core temperatures this is an unreliable guide to ventilation because of the profound metabolic, circulatory and respiratory disturbances, especially within the ventilation -perfusion mismatch, which accompanies severe hypothermia. This has been confirmed by the results obtained in our patients.
The impact of hypothermia on the partial pressure of CO 2 in arterial blood and acid-base balance has been known for years, especially in cardiac surgery. Two methods of interpreting blood gas results have been proposed. One is the pH-stat strategy in which ventilation is adjusted to maintain PaCO 2 at 40 mmHg at the patient's current body temperature. Such a correction is difficult to calculate, and is hardly ever used in the adult population [bib_ref] Is pH-stat or alpha-stat the best technique to follow in patients undergoing..., Aziz [/bib_ref]. Most centres, including ours, do not use this method, and prefer to use the alpha-stat strategy instead. In this approach, ventilation is adjusted to maintain PaCO 2 at 40 mmHg at 37°C, meaning that PaCO 2 will be <40 mm Hg in hypothermia. The alpha-stat strategy is recommended nowadays for patients with hypothermia [bib_ref] Accidental hypothermia-an update, Paal [/bib_ref] [bib_ref] The evaluation and management of accidental hypothermia, Kempainen [/bib_ref].
The difference between EtCO 2 and PaCO 2 (blood gases measured at 37°C) are generally consistent with the observations conducted by Sitzwohl et al., although this study only looked at patients with mild intraoperative hypothermia (32-36°C) [bib_ref] The arterial to end-tidal carbon dioxide gradient increases with uncorrected but not..., Sitzwohl [/bib_ref]. The small number of observations obtained so far does not allow us to carry out statistical analysis. Nevertheless, there is a definite trend of increasing Pa-EtCO 2 gradient above 1 as the core temperature falls, although the correlation is not linear, as presented on the [fig_ref] Figure 1: Pa-EtCO 2 gradient parameters of patients in stage III hypothermia treated as... [/fig_ref]. In our opinion, such a large gradient is a result of both increased CO 2 solubility as temperature decreases and the increase in ventilation-perfusion disorders, including low cardiac output. Interestingly, Sitzwohl et al. found that the mode of ventilation did not have a significant effect on the Pa-EtCO 2 gradient. Unfortunately, no data concerning the actual parameters of ventilation in the pre-hospital phase has been recorded in our patients, though we hope to obtain this data in a future study.
At present, it is unclear whether the recommendation to mildly hypoventilate patients with hypothermia has clinical significance [bib_ref] Wilderness Medical Society practice guidelines for the out-of-hospital evaluation and treatment of..., Zafren [/bib_ref]. It is worth noting that based on local guidelines, SHTC coordinators routinely recommend the use of normoventilation during transport as part of a lung-protective ventilation strategy (Vt = 6-7 ml/kg of ideal body weight, PEEP 5 mmHg and ventilatory rate = 10/min), and it should be regarded as the optimal ventilation strategy in adults. We also advocate for avoidance of manual bag-valve ventilation due to its tendency to hyperventilate. EtCO 2 monitoring in hypothermic victims should be used not only as proof of correct positioning of the endotracheal tube, but also, as a sign of preserved pulmonary flow, while circulatory instability is reflected by a fall of EtCO 2 . Critically low values suggest cardiac arrest. Such observation may be particularly valuable in the event of cardiac arrest with pulseless electrical activity (PEA), whose confirmation can be very difficult in the pre-hospital phase, where no ultrasound and invasive blood pressure measurement are available.
# Conclusions
A very high Pa-EtCO 2 gradient is found in patients with severe hypothermia.
In patients with severe hypothermia, the EtCO 2 values should not be used as the main criterion for the selection of ventilatory parameters.
The optimal ventilatory technique in patients with hypothermia should be mechanical lung protective ventilation.
Authors' contributions TD: designed the study, supervised data collection, took part in manuscript preparation, contributed substantially to the revision of the manuscript, takes responsibility for the paper as a whole. SK: supervised data collection, took part in manuscript preparation, contributed substantially to the revision of the manuscript. AJ, PP, MZ, contributed substantially to the revision of the manuscript. TS, RG, JP, JKK, RD: provided advice on study design, contributed to the revision of the manuscript. All authors read and approved the final manuscript.
## Competing interests
The authors declare that they have no competing interests.
## Consent for publication
Written informed consent was obtained from the patients for publication of their individual details in this manuscript. The consent form is held by the authors and is available for review by the Editor-in-Chief.
[fig] Figure 1: Pa-EtCO 2 gradient parameters of patients in stage III hypothermia treated as a baseline for establishing parameters of normoventilation. [/fig]
[table] Table 1: Swiss Stage of Hypothermia [/table]
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Disruption of the RNA exosome reveals the hidden face of the malaria parasite transcriptome
Antisense transcription emerges as a key regulator of important biological processes in the human malaria parasite Plasmodium falciparum. RNA-processing factors, however, remain poorly characterized in this pathogen. Here, we purified the multiprotein RNA exosome complex of malaria parasites by affinity chromatography, using HA-tagged PfRrp4 and PfDis3 as the ligands. Seven distinct core exosome subunits (PfRrp41, PfMtr3, PfRrp42, PfRrp45, PfRrp4, PfRrp40, PfCsl4) and two exoribonuclease proteins PfRrp6 and PfDis3 are identified by mass spectrometry. Western blot analysis detects Dis3 and Rrp4 predominantly in the cytoplasmic fraction during asexual blood stage development. An inducible gene knock out of the PfDis3 subunit reveals the upregulation of structural and coding RNA, but the vast majority belongs to antisense RNA. Furthermore, we detect numerous types of cryptic unstable transcripts (CUTs) linked to virulence gene families including antisense RNA in the rif gene family. Our work highlights the limitations of steady-state RNA analysis to predict transcriptional activity and link the RNA surveillance machinery directly with post-transcriptional control and gene expression in malaria parasites.ARTICLE HISTORY
# Introduction
Malaria remains one of the most devastating infectious diseases in the world with an estimated 400.000 malaria deaths each year globally. The causative agent is a unicellular protozoan pathogen that belongs to the genus Plasmodium (phylum Apicomplexa). In humans, the most virulent form of malaria is caused by P. falciparum. Complex regulatory pathways control parasite development, pathogenesis and transmission during the life cycle stages in the human and mosquito host. In addition to transcription factor networks, previous studies revealed that epigenetic factors control transcription initiation of P. falciparum virulence genes [bib_ref] Epigenetic regulation of virulence gene expression in parasitic protozoa, Duraisingh [/bib_ref]. In the past years, however, post-transcriptional regulation has emerged as another important pathway downstream of the transcriptional control level [bib_ref] Genome-wide analysis in Plasmodium falciparum reveals early and late phases of RNA..., Rai [/bib_ref] [bib_ref] Translational regulation in blood stages of the malaria parasite Plasmodium spp.: systems-wide..., Vembar [/bib_ref]. This includes translational repression in sexual stages [bib_ref] Regulation of sexual development of Plasmodium by translational repression, Mair [/bib_ref] and degradation of nascent mRNA by a 3ʹexoribonuclease called PfRNase II [bib_ref] Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria, Zhang [/bib_ref]. Antisense transcript production was reported for about 25% of genes [bib_ref] Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts..., Siegel [/bib_ref] and recently, it was demonstrated that the developmentally controlled elimination of antisense RNA for a gametocyte development protein 1 gene (GDV1) initiated expression and commitment to sexual stages [bib_ref] GDV1 induces sexual commitment of malaria parasites by antagonizing HP1-dependent gene silencing, Filarsky [/bib_ref]. The key regulator of RNA processing and turnover in eukaryotic cells, the RNA exosome complex [bib_ref] The many pathways of RNA degradation, Houseley [/bib_ref] has not been well-studied in malaria parasites and the role of the two exosome associated RNases in post-transcriptional regulation remains elusive.
The RNA exosome is a ubiquitous multi-subunit ribonuclease complex controlling RNA metabolism including RNA maturation and RNA quality surveillance [bib_ref] The eukaryotic RNA exosome, Januszyk [/bib_ref]. Moreover, it is emerging as a regulator of expression levels of specific mRNAs and maintenance of genome stability [bib_ref] The regulation and functions of the nuclear RNA exosome complex, Kilchert [/bib_ref]. The architecture and composition of RNA exosomes is highly conserved from archaea to human. In archaea, two subunits with bacterial phosphorolytic exoribonuclease RNase PH-like domain form a hexameric ring-like structure by combination of three Rrp41-Rrp42 heterodimers, whereas three copies of one or both Rrp4 and Csl4, containing S1/KH domains, form a 'cap' locating on top of the ring for structure stabilization and extension of the exosome entry tunnel [bib_ref] The exosome and RNA quality control in the nucleus, Vanacova [/bib_ref] [bib_ref] Reconstitution, activities, and structure of the eukaryotic RNA exosome, Liu [/bib_ref]. Though the eukaryotic exosome core shares structure similarity with archaea, the subunit composition varies among them. In the well-studied S. cerevisiae and human exosome cores, there are six different RNase PH domain-containing subunits in the basal ring structure known as Rrp41-Ski6, Rrp42, Rrp43-OIP2, Rrp45-PM/ Scl75, Rrp46, and Mtr3. These RNase PH subunits are assembled into a toroidal hexamer with three RNA-binding subunits on the top side, Rrp4, Rrp40, and Csl4 to accommodate target RNAs by a RNA entry channel inside the exosome core [bib_ref] The many pathways of RNA degradation, Houseley [/bib_ref] [bib_ref] The eukaryotic RNA exosome, Januszyk [/bib_ref] [bib_ref] The regulation and functions of the nuclear RNA exosome complex, Kilchert [/bib_ref] [bib_ref] The exosome and RNA quality control in the nucleus, Vanacova [/bib_ref]. The eukaryotic RNA exosome is observed in both nuclear and cytoplasmic subcellular compartments, but the two forms of RNA exosome execute different biological functions by interaction with specific co-factors such as the nuclear TRAMP (Trf4-Air2-Mtr4 polyadenylation complex) or the cytoplasmic Ski (superkiller) complex [bib_ref] The many pathways of RNA degradation, Houseley [/bib_ref] [bib_ref] Identification of a nuclear exosome decay pathway for processed transcripts, Meola [/bib_ref] [bib_ref] The yeast ski complex: crystal structure and RNA channeling to the exosome..., Halbach [/bib_ref]. Usually, the nuclear RNA exosome processes immature precursors of structured RNAs such as rRNA and controls turnover of non-coding RNAs, cryptic unstable transcripts (CUTs), and rarely nuclear mRNA, whereas the cytoplasmic RNA exosome is mainly implicated in mRNA surveillance, e.g. Nonsense-mediated decay (NMD) pathway that degrades mRNAs containing premature termination codons [bib_ref] Threading the barrel of the RNA exosome, Schneider [/bib_ref] and general mRNA turnover. Despite the presence of six RNase PH domain-contained proteins, it is now widely accepted that the yeast and human exosome cores are catalytically inactive due to the loss of key catalytic residues of their bacterial homologue. The function of RNA processing and degradation are executed by the two RNA exosome-associated catalytically active subunits, Dis3/ Rrp44 and Rrp6. The association of these two exoribonucleases with the RNA exosome core and the targeted substrates vary in different organisms, though evolutionary conserved functions such as rRNA maturation are maintained [bib_ref] The eukaryotic RNA exosome: same scaffold but variable catalytic subunits, Lykke-Andersen [/bib_ref]. For example, Rrp6 is only incorporated into the nuclear exosome in S. cerevisiae but is an integral part of the nuclear and cytoplasmic forms in African trypanosomes [bib_ref] The roles of intersubunit interactions in exosome stability, Estévez [/bib_ref]. Human hDIS3 is mainly a nuclear protein contributing to shaping the RNA polymerase II transcriptome by degrading unwanted transcripts [bib_ref] DIS3 shapes the RNA polymerase II transcriptome in humans by degrading a..., Szczepińska [/bib_ref] , while its homolog hDIS3L is restricted to cytoplasmic exosome complexes.
The study of protozoan eukaryotes such as Tetrahymena, Trichomonas, Trypanosome, and Giardia have revealed slightly distinct types of organizations. In P. falciparum, only eight putative exosome subunits, PfRrp41, PfRrp42, PfRrp45, PfRrp4, PfRrp40, PfCsl4, PfRrp44/PfDis3, and PfRrp6 were identified in silico with three exosome core subunits, Rrp43, Prp46, and Mtr3 missing in the parasites [bib_ref] Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria, Zhang [/bib_ref]. In this study we sought to experimentally identify and functionally characterize the plasmodial RNA exosome. We generated HA-tagged transfectant lines for two putative subunits (Rrp4 and Rpr44/Dis3). Affinity purification of the RNA exosome complex followed by mass spectrometry analysis consistently detected eight exosome subunits, but also identifies a putative additional subunit, PfMtr3. We observe a predominant cytoplasmic location of the RNA exosome in asexual blood stage parasites. Conditional knock out of PfDis3 revealed dramatically more RNA transcripts then predicted by steady state RNA analysis. We identify the upregulation of more than 1000 antisense RNAs and detect cryptic transcripts made from virulence gene loci. This finding opens new experimental avenues into our understanding of transcriptionally active genome regions and directly links exosome-mediated RNA decay to gene regulation in this fatal pathogen.
# Results
## Characterization of p. falciparum exosome subunit rrp4 and dis3
Our first aim was to identify the composition of the P. falciparum exosome complex, including core exosome subunits and associated catalytic factors, by Co-immunoprecipitation (Co-IP) followed by Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. As antibodies generated against recombinant proteins or synthetic peptides of P. falciparum exosome subunits displayed non-specific background signal, we decided to generate transgenic parasite lines with terminal epitope-tags (3xHA-3xTy1), both for the putative core exosome component Rrp4 (called here PfRrp4, PF3D7_0410400) and the associated catalytic subunit Dis3/Rrp44 (called here PfDis3, PF3D7_1359300). Utilizing the CRISPR/Cas9 gene editing technique to achieve fast endogenous integration of the tag, plasmids targeting either the N-or C-terminus of the PfRrp4 or PfDis3 gene were constructed and co-transfected with Cas9 expression vector pUF1-cas9 [fig_ref] Figure 1: Cytoplasmic localization of RNA exosome in P [/fig_ref]. An episomal expression construct (pARL-gfp-HA-Ty1) was used as negative control for the Co-IP assay [fig_ref] Figure 1: Cytoplasmic localization of RNA exosome in P [/fig_ref] ). Three weeks after transfection, parasites carrying pL6-Ty1-HA-PfRrp4/pUF1-cas9, pL6-PfDis3-HA-Ty1/pUF1-cas9, and pARL-gfp-HA-Ty1 control were obtained by drug selection with both WR and BSD. C-terminal tagging of the PfRrp4 gene and N-terminal tagging of the PfDis3 gene was not successful after three attempts of transfection, suggesting that the C-terminal and N-terminal sequences are important for conformation-dependent functions of PfRrp4 and PfDis3, respectively. However, failure to genetically modify these genes may also be due to technical reasons. Clones of the Ty1-HA-PfRrp4, PfDis3-HA-Ty1 and GFP control transfectant were obtained by limiting dilution, and the integration verified by PCR and Western blot. Epitope-tagged proteins were detected as a single band by western blot assay, confirming the specificity of the antibodies against Ty1 or HA-tagged proteins in P. falciparum parasite asexual blood stage extracts [fig_ref] Figure 1: Cytoplasmic localization of RNA exosome in P [/fig_ref].
Subsequently, we analyzed the expression pattern of the core exosome subunit PfRrp4 and the exosome associated factor PfDis3 across the asexual blood stage by western blot. We prepared nuclear and cytoplasmic protein extracts from highly synchronized ring, trophozoite, and schizont-stage parasites of epitope-tagged transfectant lines. Western blot analysis shows that PfRrp4 and PfDis3 proteins are expressed in all intraerythrocytic stages (ring, trophozoite and schizont). PfDis3, however, was only detected in the cytoplasmic fraction even after overexposure of the blot [fig_ref] Figure 1: Cytoplasmic localization of RNA exosome in P [/fig_ref]. PfRrp4 was also mainly in the cytoplasmic fraction but a very minor fraction was detected in the nuclear extract only after overexposure [fig_ref] Figure 1: Cytoplasmic localization of RNA exosome in P [/fig_ref]. Antibodies against histone H3 and aldolase were used as markers of the two cellular compartments. Our cellular fractionation data suggest a predominant cytoplasmic role of the exosome. This observation was further validated by IFA with synchronized parasites [fig_ref] Figure 1: Cytoplasmic localization of RNA exosome in P [/fig_ref]. In rings and trophozoites the antibody signal for both tagged proteins is clearly distinct from the nucleus. In the schizont stage the IFA images are more difficult to interpretate due to the high parasite load at this stage.
## Identification of p. falciparum rna exosome composition
Although the exosome structure is highly conserved from Archaea to eukaryotic organisms, its subunit composition varies, as Archaea only possesses two subunits in the hexameric ring structure, Rrp41 and Rrp42, whereas studied model organisms such as yeast, human or drosophila use six distinct RNase PH-domain containing proteins. Putative orthologues of two exosome-associated catalytic subunits (Rrp6 and Dis3) and three structure stabilization subunits of the core exosome 'cap' (Rrp4, Rrp40, Csl4) were annotated in the P. falciparum genome database (PlasmoDB.org) by bioinformatic analysis according to their conserved functional domains such as RNase II, RNase D, S1, and KH. However, only three candidate subunits of the hexameric RNase-PH ring structure were predicted, i.e. Rrp41, Rrp42, and Rrp45. To experimentally establish the composition of the P. falciparum exosome complex, we performed co-immunoprecipitation (Co-IP) coupled to LC-MS/MS analysis utilizing total extracts from the Ty1-HA-tagged transfectants of PfRrp4 and PfDis3 with GFP-HA-Ty1 as negative control [fig_ref] Figure 2: Composition identification of P [/fig_ref] and S2). The mass spectrometry results included peptides for virtually the entire predicted exosome complex. We also observed peptides of abundant proteins such as histones, heat-shock proteins (HSP) and ribosomal proteins, however these proteins were also present in both replicates of our GFP unspecific control and excluded by further statistical analysis (p < 0.05) (see Dataset S1 and Materials and Methods section). Importantly, no exosome-related peptides were detected in the GFP-HA-Ty1 pull down.
As summarized in [fig_ref] Figure 2: Composition identification of P [/fig_ref] , in the two independent biological replicates we detect three annotated exosome RNase PH-ring structure subunits (Rrp41, Rrp42, Rrp45) and all predicted cap structure subunits (Rrp4, Rrp40, Csl4) of the core exosome, as well as two exosome-associated catalytic enzymes (Dis3 and Rrp6). Interestingly, a protein annotated as putative 3ʹ exoribonuclease (PF3D7_0209200), was also isolated in all replicates, but absent in the GFP control (p < 0.05). It possesses a RNase PH domain (InterPro domain IPR001247) and had previously been informatically predicted as putative exosome subunit [bib_ref] Identification and analysis of the RNA degrading complexes and machinery of Giardia..., Williams [/bib_ref]. Sequence homology indicates that this protein belongs to the Rrp41-subtype of the RNase PH protein family (comprising eukaryotic Rrp41, Mtr3 and Rrp46 proteins) and is a putative orthologue of Mtr3 (see [fig_ref] Figure 3: Generation of DiCre recombinase-mediated conditional PfDis3 KO line [/fig_ref] , suggesting two Rrp41-like proteins, called here (a) Schematic representation of constructs for generation of epitope-tagging PfRrp4 (left) and PfDis3 (right) transfectant lines by the CRISPR-Cas9 system. For both strains, the plasmids carrying a single guide RNA (sgRNA) and the Cas9 endonuclease were co-transfected. bsd, blasticidin S deaminase. hdhfr, human dihydrofolate reductase. HR, homology region.(b) Western blot analysis of Ty1-HA-PfRrp4 and PfDis3-HA-Ty1 lines with nuclear and cytoplasmic extracts, respectively. Aldolase and Histone 3 were used as loading control. CE, cytoplasmic extract. NE, nuclear extract. R, ring. T, trophozoite. S, schizont.(c) Immunofluorescence assay (IFA) of Ty1-HA-PfRrp4 and PfDis3-HA-Ty1 lines in Ring, Trophozoite, Schizont, respectively. The nuclei were stained by DAPI. The bar represents 1 μm.
Rrp41and Mtr3, exist in the genome of P. falciparum. Surprisingly, the bioinformatically predicted Rrp42 subunit was only detected by mass spectrometry analysis in one assay with Dis3-HA-Ty1 line. In all replicates, the three cap structure subunits and PfDis3 were detected whereas PfRrp6 was absent in one assay with the PfRrp4-HA-Ty1 line.
However, since Rrp6 copurified with Dis3 in both replicates, and this interaction is thought to be mediated via the core in other organisms [bib_ref] The regulation and functions of the nuclear RNA exosome complex, Kilchert [/bib_ref] , this possibility has to be further investigated. Since we only identified four core members of the hexameric ring structure, this finding suggests an intermediate form of this complex in malaria parasites compared to that of Archaea, S.cerevisiae and human [fig_ref] Figure 2: Composition identification of P [/fig_ref] ).
## Inducible gene knock out shows that pfdis3 is essential
To assess the physiological role of the RNA exosome in P. falciparum, we wanted to delete its catalytic subunit PfDis3. We failed to obtain mutant parasites with a traditional knock-out approach and mutagenesis of the putative critical aspartate residues, D607A, D613A, D615A, and D616A, accounting for the catalytic activity of PfDis3 [bib_ref] Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria, Zhang [/bib_ref] [bib_ref] Structural basis for processivity and single-strand specificity of RNase II, Zuo [/bib_ref] (data not shown), indicating an essential function of this gene. For this reason, a conditional knockout strategy that allows targeting of essential genes was utilized [bib_ref] A versatile strategy for rapid conditional genome engineering using loxP sites in..., Jones [/bib_ref] [bib_ref] Generating conditional gene knockouts in Plasmodium -a toolkit to produce stable DiCre..., Knuepfer [/bib_ref]. Using parasites expressing inducible DiCre recombinase (3D7A DC) we introduced a cassette containing artificial introns which contain loxP sites (loxPint) flanking the exonuclease domain of PfDis3 CDS [fig_ref] Figure 3: Generation of DiCre recombinase-mediated conditional PfDis3 KO line [/fig_ref] ) [bib_ref] A versatile strategy for rapid conditional genome engineering using loxP sites in..., Jones [/bib_ref]. This modification should be phenotypically silent, as after splicing of the introns the correct coding sequence is reconstituted. The integration of the cassette was verified by PCR [fig_ref] Figure 3: Generation of DiCre recombinase-mediated conditional PfDis3 KO line [/fig_ref] and two clones were isolated using the limited dilution technique. We used synchronized ring stage parasites to induce Pfdis3 gene deletion by addition of 100 nM rapamycin for 4 hours. In the next cycle the genotype of the induced KO culture was compared to the wildtype and uninduced KO by PCR [fig_ref] Figure 3: Generation of DiCre recombinase-mediated conditional PfDis3 KO line [/fig_ref]. We observed the slightly higher band in the uninduced KO parasites compared to the wild type, corresponding to the integration of two loxP introns. Upon induction by rapamycin, a lower band corresponding to the expected excision product is detected. Sanger sequencing of the PCR amplified fragment confirmed the correct recombination event. Since the Pfdis3 deletion was not complete in the culture after 4 hours, we increased the rapamycin concentration and the incubation time. These changes did not increase the deletion efficiency. Even repeating the induction in the next cycle after the initial rapamycin treatment did fail to give complete excision, as the undeleted genotype was reappearing in the following cycles [fig_ref] Figure 3: Generation of DiCre recombinase-mediated conditional PfDis3 KO line [/fig_ref] ). Furthermore, our attempts to obtain a Pfdis3 knock out clone by limiting dilution technique directly after the initial rapamycin induction, failed. We performed the growth rate analysis with and without rapamycin induction as well as cell cycle progression analysis. No major difference was observed for the first two cycles (see [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref]. Taken together, these data further indicate that the Pfdis3 KO has an important growth defect that is overgrown by parasites with intact Pfdis3 after several blood stage cycles.
## Pfdis3 degrades mrna but targets predominantly antisense rna
To evaluate the effect of exosome disruption by depletion of Pfdis3, the global RNA transcriptome was analyzed by RNA-seq. We examined poly (A) enriched RNA from two clones (E2/F10) of the conditional Dis3-loxPint KO. RNA from uninduced (DMSO treated) and induced (rapamycin treated) KO cultures was isolated in the cycle after induction (day 2, late ring stage parasites, 20 ± 5 hours post-infection, see [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref] ). Differential gene expression was evaluated by combining the results from the two [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref]. In Dataset S2 all transcripts with at least 2-fold differential expression between the two conditions are listed and the raw data is accessible on NCBI BioProject (accession: PRJNA453694).
We were also interested to see the expression of the Pfdis3 transcript itself. When focusing on the loxP flanked region of the Pfdis3 gene the decrease of read coverage is evident in the induced KO, but as could be expected from the previous PCR result, the deletion is not complete and a few reads remain for the floxed part [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref]. Nevertheless, the KO reveals a major effect on the RNA homeostasis of the parasite.
When the reads are separated into sense and antisense alignments, we observe a striking global increase of antisense transcripts (n = 1046) and to a lesser extend sense transcripts (n = 131) (Dataset S2, visualized in [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref]. Many of these are cryptic transcripts not detectable in parasites before disruption of Pfdis3.
We detected increased transcripts in mRNAs but also in different species of pseudogenes and noncoding RNAs (snRNAs, snoRNAs, RNA of unknown function RUF1, RUF2). Some representative examples taken from the integrative genome viewer (IGV) are shown in [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref] to illustrate the different types of transcriptional changes in Pfdis3 mutant parasites.
When analyzing the antisense dataset, we noticed a remarkable number of highly expressed rif antisense transcripts. Rifins represent a class of surface proteins that are encoded by a multicopy gene family organized in the genome in arrays often associated tail to tail with neighboring var genes, coding for another P. falciparum virulence factor [bib_ref] Genome sequence of the human malaria parasite Plasmodium falciparum, Gardner [/bib_ref]. In many cases the rif antisense RNA is barely detectable before the KO, whereas its expression is strongly increased in the Pfdis3 mutant, often to a higher expression level than the corresponding rif sense transcript [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref]. Furthermore, we observe bidirectional promoter activity from the var intron that has been reported previously [bib_ref] Plasmodium falciparum var genes are regulated by two regions with separate promoters,..., Calderwood [/bib_ref] and has been associated to transcribed var genes [bib_ref] Antisense long noncoding RNAs regulate var gene activation in the malaria parasite..., Amit-Avraham [/bib_ref] [bib_ref] PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum, Jiang [/bib_ref]. The intron derived antisense RNA is significantly increased in 22 silent var genes upon Pfdis3 disruption (see Dataset S2 and example in [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref] ), indicating that the var antisense steady state level is controlled by PfDis3.
# Discussion
Malaria parasites use post-transcriptional regulation to fine tune gene expression and to transition between different developmental stages. The mechanism by which RNA is targeted for turnover is still not understood in this pathogen. Here we present functional studies that reveal canonical and parasite specific contributions of the plasmodial RNA exosome to RNA surveillance and establishment of antisense RNA levels. By bioinformatic prediction, six members of the exosome core and two exoribonuclease members, PfDis3 and PfRrp6, were previously identified in the P. falciparum genome. The 3ʹ-5ʹ exoribonuclease activity of PfDis3 had previously been validated by using a recombinant Dis3 [bib_ref] Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria, Zhang [/bib_ref]. In this work, we generated tagged exosome members to investigate experimentally the composition of the RNA exosome complex. We detected all predicted plasmodium members of the exosome complex and identified an additional member being part of the complex. We call this protein Mtr3-like core member (PfMtr3).
Eukaryotic exosomes of studied model organisms, from trypanosomes, yeast, fly and human, canonically possess at least six distinct RNase PH domain proteins, three of the Rrp41-subtype (Rrp41, Rrp46 and Mtr3) and three of the Rrp42-subtype [fig_ref] Figure 2: Composition identification of P [/fig_ref]. Even including the newly identified exosome protein PfMtr3, P. falciparum is still short two RNase PH domain proteins and no additional proteins could be identified in the published genome. Furthermore, genome database searches reveal the same situation for all other published Plasmodium species (PlasmoDB.org). Interestingly, the apicomplexan phylum seems heterogenous in this regard, as the genomes of some species like Toxoplasma gondii and Cryptosporidium parvum encode six distinct proteins, while others, for example Giardia, Theileria parva or Gregarina niphandrodes, like Plasmodium, encode only four . The consequences on the stoichiometry of the different subunits present in the exosome complex or possible specialized subcomplexes will have to be examined in future studies.
P. falciparum shows another unusual feature. We observed an uncommon RNA exosome complex distribution between cytoplasmic and nuclear fraction in asexual blood stage parasites. Only a very minor fraction was detected in the nucleus. This may indicate that the cytoplasmic exosome activity has been amplified in P. falciparum to perform extensive RNA turnover in blood stage parasites. This idea is supported by our finding, that the majority of antisense RNA (> 1000) expression levels are affected by the depletion of PfDis3. The general role of the widespread abundance of antisense in malaria parasites is still obscure. However, for a few genes experimental evidence supports a role in the control of expression. For example, the var gene family, that encodes the major virulence factor of P. falciparum, produces antisense lncRNA from its intron, that have been suggested to play a role in the activation process [bib_ref] Antisense long noncoding RNAs regulate var gene activation in the malaria parasite..., Amit-Avraham [/bib_ref] [bib_ref] PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum, Jiang [/bib_ref]. Another recent study on the GDV1 gene provided solid experimental evidence that the abolition of antisense RNA of the GDV1 gene leads to the expression of the protein with subsequent activation of the sexual stages [bib_ref] GDV1 induces sexual commitment of malaria parasites by antagonizing HP1-dependent gene silencing, Filarsky [/bib_ref]. Furthermore, several studies have shown extensive dynamics in nascent mRNA transcription, mRNA stabilization and mRNA decay during asexual development [bib_ref] Genome-wide real-time in vivo transcriptional dynamics during Plasmodium falciparum blood-stage development, Painter [/bib_ref] [bib_ref] Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening..., Shock [/bib_ref]. In line with that, the RNA exosome might be one effector complex mediating mRNA decay as observed by the increase of mRNA transcripts following PfDis3 KO in addition to its role in antisense transcript degradation. Overall, we observe an important increase of the steady state level of antisense RNA. We hypothesize that the RNA exosome plays a prominent role in P. falciparum gene regulation via its role in the control of antisense RNA degradation. Future studies will explore if the increase of antisense RNA in PfDis3 mutants translates into different levels of protein expression.
Another important observation in PfDis3 mutant parasites, is the detection of cryptic transcripts that are not observed in steady state RNA samples of the corresponding uninduced cultures. For example, the pseudo var gene (PF3D7_1480100) with an incomplete 3ʹ end produces sense transcripts and the members of the rif gene family make antisense transcripts either in the presence or absence of sense transcripts [fig_ref] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts [/fig_ref]. Our work demonstrates that the actual reported genome-wide transcription profile for P. falciparum is an underestimation of the actively transcribed regions.
The mutant PfDis3 also reveals changes in levels of RNA molecules that normally are processed in the nucleus in other organisms (eg snoRNAs). This is counter intuitive, given that at best only traces of PfDis3 could be detected in nuclear extracts by Western blot. We hypothesize that a feedback loop from the disrupted exosome in the cytoplasm could affect nuclear processing, for example by affecting correct processing of spliceosomal RNAs (eg snRNA U4) in the cytoplasm that are also required for nuclear processing events [bib_ref] The exosome controls alternative splicing by mediating the gene expression and assembly..., Zhang [/bib_ref].
In conclusion, here we experimentally validate the RNA exosome composition and its cellular localization in malaria parasites. An inducible knock out of Dis3 reveals canonical functions reported in other organisms and the control of antisense RNA in malaria parasites. The latter are likely to have important implications because they link the RNA surveillance machinery directly with transcriptional control and gene expression. The next big step in understanding the regulatory function of RNA exosome will be to identify the proteins that target the different types of RNA classes to the exosome either for processing or degradation.
# Materials and methods
## Plasmid construction for transfection
To generate Rrp4-and Dis3-HA-Ty1 tagged lines, we modified the plasmid pL6-gfp by replacing the eGFP box with a 1-kb homologue sequence flanking the N-or C-terminus of the targeted genes which contained two epitopes (HA-Ty1) each in three copies in tandem, targeting the N-or C-terminal end, and inserting a guide RNA sequence specific to the PfRrp4 gene (PF3D7_0410400) or PfDis3 gene (PF3D7_1359300) by In-Fusion PCR Cloning System, respectively (see [bib_ref] Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9..., Ghorbal [/bib_ref]. The resulting plasmids were pL6-PfRrp4-HA-Ty1, pL6-Ty1-HA-PfRrp4, pL6-PfDis3-HA-Ty1, and pL6-Ty1-HA-PfDis3. The plasmid pUF1-Cas9-infusion carrying Cas9 expression cassette was modified by replacing the original ydhodh gene with hdhfr.
For the Dis3-loxPint conditional knock out construct, a synthetic sequence including the homology boxes, artificial introns containing loxP sites and a recoded exonuclease domain was ordered from Genscript with the Dis3 gRNA sequence (GTAGATGTATAAGTGTGTTA) to give pL7-Dis3-loxPint.
Parasite culture and transfection P. falciparum 3D7 strain was maintained in culture in vitro and synchronized as described previously [bib_ref] A critical role of perinuclear filamentous actin in spatial repositioning and mutually..., Zhang [/bib_ref]. For transfection, synchronized ring-stage parasites at~5% parasitemia were transfected with~100 μg of plasmid sgRNA and Cas9 by electroporation as described previously. Transfected parasites were selected by blasticidin S deaminase drug and WR99210. After approximately 1 month, the established culture with rrp4or dis3-tag integration were cloned by limitation cloning, and the integration events were validated by PCR following sequencing.
For the the conditional Dis3 KO, pL7-Dis3-loxPint and pUF1-Cas9 were co-transfected as described above in P. falciparum 3D7A DiCre background [bib_ref] A versatile strategy for rapid conditional genome engineering using loxP sites in..., Jones [/bib_ref] [bib_ref] Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum..., Collins [/bib_ref] Transfected parasites were selected with 2.5nM WR99210 and 1µM DSM1. Integration was verified and the culture cloned out by limiting dilution.
## Western blot
Total parasite extracts were prepared by 0.15% saponin treatment and resuspended in 1x SDS-loading buffer (Bio-Rad), then separated on 8~12% SDS-PAGE gel according to the molecular weight of targeted proteins, and subjected to Western blot analysis. The commercial antibodies used in this study were mouse anti-Ty1 (1:500, Sigma), mouse anti-HA (1:2000, Roche), rabbit anti-PfAldolase (1:1000, Abcam), and rabbit anti-Histone 3 (1:1000, Abcam). ECL western blotting kit (GE healthcare) was used to develop blots.
## Immunofluorescence
Immunofluorescence assay was performed on synchronous parasites fixed with 4% paraformaldehyde in 1xPBS as described previously [bib_ref] A critical role of perinuclear filamentous actin in spatial repositioning and mutually..., Zhang [/bib_ref]. Antibody dilutions for mouse anti-Ty1 was 1:300, and second antibodies of Alexa-Fluor-488-conjugated anti-rabbit were 1:2000.
## Co-immunoprecipitation (co-ip)
The Co-IP was performed as described previously [bib_ref] Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria, Zhang [/bib_ref]. Briefly, asynchronized asexual-stage parasite cultures were harvested and treated with 0.15% saponin. The released parasites were washed with 1xPBS for three times, then resuspended in six volumes of lysis buffer (25 mM Tris-Cl, pH 7.5, 100 mM KCl, 2 mM EDTA, 0.5 mM PMSF, 0.05% NP-40, 1x protease inhibitor cocktail (Thermo)), and subjected to sonication at highest power for 3 min at 30 sec intervals with a sonicator (Bioruptor). The supernatants of the lysates were isolated and immediately incubated with magnetic beads pre-coupled with HA antibody (Thermo) for 2 h at 4 ℃. After washing the beads twice with IPP500 (500 mM NaCl, 10 mM Tris-Cl, pH 8.0, 0.05% NP-40) and once with 1x PBS, bound proteins were either eluted with sample buffer for SDS-PAGE followed by silver-staining, coomassie-staining, and western blot analysis. After validation of the enrichment of targeting proteins by western blot with anti-Ty1 antibody, the full gel lanes loaded with elution fractions were subject to mass spectrometry (LC-MS/MS) analysis with at least two technical replicates.
## Lc-ms/ms
After filtration through 0.22 µm membrane the clear solution was subjected to nano LC-MS/MS separation. A volume of 3.0 µL of each sample was desalted by loading on a Thermo C18 PepMap100 precolumn (300 µm × 5 mm) and eluted on a Thermo Acclaim PepMap RSLC analytical column (75 µm × 15 cm). Mobile phase A (0.1% formic acid in H 2 O) and mobile phase B (0.1% formic acid in acetonitrile) were used to establish the 120 min gradient comprised of 85 min of 4−30% B, 15 min of 30−50% B, and 5 min of 90% B, followed by re-equilibrating at 4% B for 15 min. The flow rate was 0.3 µL/min. Peptides were then analyzed on a Q-Exactive proteomic mass spectrometer (Thermo Scientific) in a data-dependent manner, with automatic switching between MS and MS/MS scans using a top 20 method. MS spectra were acquired at a resolution of 70,000 with a target value of 3 × 10 6 ions or a maximum integration time of 50 ms. The scan range was limited from 375 to 1400 m/z. Peptide fragmentation was performed via higher-energy collision dissociation (HCD) with the energy set at 32 NCE. The MS/MS spectra were acquired at a resolution of 35,000 with a target value of 1 × 10 5 ions or a maximum integration time of 100 ms. The fixed first m/z was 100, and the isolation window was 1.2 m/z.
The data were processed with MaxQuant version 1.5.3.30, and the peptides were identified from the MS/MS spectra searched against the protein database (PlasmoDB 38 Released, 19 June 2018) using the Andromeda search engine. The precursor mass tolerance was set to 10 ppm and fragment ion mass tolerance to 0.02 Da. One missed cleavage site of trypsin was allowed. Carbamidomethyl (C) was used as a fixed modification and oxidation (M) was used as variable modifications. All spectra were searched against protein database using a target false discovery rate (FDR) of 1%. Other parameters were used as pre-set in the software. LFQ experiments in MaxQuant were performed using the built-in label-free quantification algorithm (MaxLFQ) [bib_ref] Accurate proteome-wide labelfree quantification by delayed normalization and maximal peptide ratio extraction,..., Cox [/bib_ref]. Missing values were imputed to mimic low abundance values (width 0.2, downshift 2.1, total matrix). Statistical analysis was performed with Perseus 1.5.1.6. LFQ ratios were transformed with log 2-(x) and then normalized using Z-score, and -log 10-(pvalue) of all proteins were obtained by a two-sided one sample t-test over two biological replicates. Only proteins identified have average ratios > 2.0 and p-values < 0.05 were considered statistical significant identification.
## Conditional knockout induction and sample preparation
For the induction of the Dis3-loxPint KO the cultures were synchronized by Sorbitol/Plasmion treatment and at the ring stage incubated with 100 nM rapamycin (induced) or the same volume of DMSO (uninduced) for 4 hours. The culture was washed and left to the next cycle. On day 2 after induction, gDNA (NucleoSpin® Tissue kit, Macherey-Nagel) and RNA (miRNeasy kit, Qiagen) was harvested from Saponin lysed cells pellets. Oligonucleotides used in PCR to verify integration and recombination were p1, p2, and p3 as shown in .
For RNAseq two independent Dis3-loxPint clones (E2/ F10) were analyzed, from repeated induction rounds (2x E2 +/-RAP; 2x F10+/-RAP). Total RNA was poly(A) selected (Dynabeads® mRNA DIRECT™ Kit, Thermo Fischer Scientific) before library preparation with the TruSeq Stranded mRNA kit (Illumina) and paired end sequencing was performed on the Illumina Nextseq 550 machine.
# Rna sequencing analysis
Raw Illumina data were converted to fastq files and demultiplexed using Illumina's bcl2fastq (v2.19.1.403). The paired sequencing reads were mapped to the P. falciparum genome (plasmoDB.org, v3 release 28) [bib_ref] Genome sequence of the human malaria parasite Plasmodium falciparum, Gardner [/bib_ref] using 'bwa mem' [bib_ref] Fast and accurate short read alignment with Burrows-Wheeler transform, Li [/bib_ref] allowing only unique read mappings to the genome (option -c 1). Optical read mapping duplicates were removed using samtools 'rmdup' [bib_ref] The sequence alignment/ map format and SAMtools, Li [/bib_ref] and only alignments with a mapping quality ≥ 20 were retained using samtools 'view'.
Read counts for genic regions were obtained using 'htseqcount' [bib_ref] HTSeq-a Python framework to work with high-throughput sequencing data, Anders [/bib_ref] for both the sense and antisense direction of transcription. The two files (i.e. sense + antisense read counts) were merged for library normalization between replicates and conditions. Differential expression analysis was performed in R (http://www. r-project.org/) using the package edgeR [bib_ref] edgeR: a Bioconductor package for differential expression analysis of digital gene expression..., Robinson [/bib_ref] with an FDR value cutoff ≤ 0.05.
For visualization of the RNA-seq data were, bedgraph formatted files were generated from the BAM alignment files using deepTools2 'bamCoverage' [bib_ref] deepTools2: a next generation web server for deep-sequencing data analysis, Ramírez [/bib_ref] with a binsize of 1 nucleotide and RPKM ('reads pere kilobase per one million mapped reads') library normalization enabled (option: -normalizeToRPKM) for comarpsion between samples. The data were visualized using the Integrated Genomics Viewer (version 2.3.78) [bib_ref] Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration, Thorvaldsdóttir [/bib_ref].
[fig] Figure 1: Cytoplasmic localization of RNA exosome in P.falciparum parasites. [/fig]
[fig] Figure 2: Composition identification of P.falciparum RNA exosome subunits. (a) Schematic representation of exosome isolation by Co-IP with antibody against HA fused to PfRrp4 and PfDis3 respectively.(b) Comparative summary of the RNA exosome composition in Archaea, S.cerevisiae, human, and P.falciparum identified by Co-IP and LC-MS/MS in this study.(c) Putative model of RNA exosome complex of P.falciparum parasites compared with that of Archaea, S.cerevisiae, and human. clones in replicate experiments (2x E2+/-and 2x F10+/-). Analysis of expression in the induced KO vs uninduced culture revealed a large number of transcripts with increased expression and only very few transcripts displayed a significantly decreased abundance [/fig]
[fig] Figure 3: Generation of DiCre recombinase-mediated conditional PfDis3 KO line. (a) Schematic representation of the PfDis3-loxPint conditional KO strategy. Co-transfection of the plasmid pL7-dis3-loxPint with pUF1-Cas9 leads to integration of the loxPint containing cassette in the endogenous locus. Activation of DiCre recombinase by rapamycin induces the excision of the loxP flanked CDS region that contains the exonuclease domain (asterisk) and by frameshift mutation to a premature termination codon (PTC, red diamond). The sgRNA target sequence is marked with SEED.(b) PCR verification of the conditional KO using oligonucleotides in homology box1 and the 3ʹCDS (p1-p2). Bands of the expected size were observed (wildtype-1020bp, undeleted Pfdis3-loxPint 1250bp, deleted Pfdis3-loxPint 610bp; gDNA harvested day 2 after induction).(c) Repeated induction and outgrowth of KO cultures. On day 2 after the first induction, rapamycin treatment was repeated and gDNA from the following days analyzed by PCR (p1-p3, expected size undeleted: 855bp, deleted: 217bp). [/fig]
[fig] Figure 4: Differential gene expression and accumulation of virulence gene-associated cryptic unstable transcripts (CUTs) in Dis3-loxPint KO line. (a) Scatter plot of differential gene expression in the Pfdis3-loxPint KO. Log2 RPKM values of the uninduced Pfdis3-KO are plotted against the log2 RPKM values of the induced KO (combined results from two clones in replicate). The yellow and dark green dots mark differentially expressed (≥ 1.75 fold) antisense and sense transcripts, respectively. The grey dots mark genes that are not significantly changed (combined sense and antisense).(b) Expression profile for the Pfdis3 transcript, with the black box highlighting the KO region (bedgraph coverage illustration of relative expression from IGV). The uninduced expression profile is marked in green and the Pfdis3-KO in red; upper part of the panel show sense (s) and the lower panel antisense (as) expression for both conditions, respectively.(c)Other representative examples of differentially expressed genes: increased sense transcript was observed for Sir2a (PF3D7_1328800), PfEMP1 pseudogene (PF3D7_1480100), snoRNA07 (PF3D7_1109000), while for alpha tubulin 2 (PF3D7_0422300) antisense RNA is increased.(d) Representative expression profiles of two silent var genes and their neighboring rif (PF3D7_1200500 and PF3D7_1300200). As these genes are coded on different strands, sense direction for the var gene and antisense for the rif are in the top part of the panel; antisense for the var gene and sense for the rif in the lower part. [/fig]
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